Synthetic wollastonite

Endpoint summary

Description of key information

There are no repeated dose toxicity studies on synthetic wollastonite, all data are from the read-across substance, Kieselguhr soda ash or amoprhous or crystalline silica.

A 90 day oral study in rats NOAEL 3737.9 mg/kg (highest concentration tested) carried out on the analogue substance Kieselguhr, soda ash flux-calcined did not produce any treatment related effects.

No dermal repeat dose studies have been considered neccssary.

Repeat dose inhalation studies performed using either amorphous or crystalline silica demonstrate that although amorphous silica does cause a transient inflammatory response, crystalline silica at lower time - or dose-exposure leads to much more persistent (and consequently much more severe) pulmonary toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Septmber 24 1991 - December 30 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 6 weeks
- Weight at study initiation: 163 - 220 g for males and 136 - 171 g for females
- Housing: Stainless steel, hanging wire-mesh cages
- Diet: Pelleted Purina Certified Rodent Chow #5002 available ad libitum during acclimation period
- Water: Tapwater via an automatic watering system was available ad libitum during both acclimation and study periods
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 6°F
- Humidity: 50 ± 20%
- Photoperiod: 12hrs dark / 12 hrs light:

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test materials were formulated into pellets with a single lot of Purina Certified Rodent Chow #5002. Purina Certified Rodent Chow #5002 was also formulated into pellets to serve as the control material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The total silica and crystalline silica (quartz and cristobalite) were determined in the feed mixtures priorto the study and in the pellets at weeks 1, 7 and 13. The percent total silica in the 5% feed mixtures and pellets of Natural Diatomaceous Earth was 4.8% with 0.44% quartz and no cristobalite. The percent total silica in the 1% feed mixtures and pellets of Flux Calcined Diatomaceous Earth was 1.2% with 0.24% quartz and 0.26& cristobalite. The percent total silica in the 5% feed mixtures and pellets of Flux Calcined Diatomaceous Earth was 5.1% with 0.43% quartz and 1.70% cristobalite.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
Natural Diatomaceous Earth 5% (average of 3765.5 mg/kg/day)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
Flux Calcined Diatomaceous Earth 1% ( average of 698.3 mg/kg/day)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
Flux Calcined Diatomaceous Earth 5% ( average of 3737.9 mg/kg/day)
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Time schedule: Twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Once daily


BODY WEIGHT: Yes
Time schedule for examinations: Measured and recorded at randomization, prior to treatment and weekly, thereafter

WATER CONSUMPTION: No


FOOD CONSUMPTION AND COMPOUND INTAKE (: Yes
Time schedule for examinations: Measured and recorded weekly for weeks 1-13

HAEMATOLOGY: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: first ten animals/sex/group
Parameters checked: cell morphology, corrected leukocyte count (COR WBC), erythrocyte count (RBC), haematocrit (HCT), haemoglobin(HGB), leukocyte count (WBC), leukocyte differential, platelet count

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: first ten animals/sex/group
Parameters checked: alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium, chloride, creatine , gamma glutamyltransferase (GGT), glucose, inorganic phosphorous (IN PHOS), potassium, sodium, total bilirubin (T BILI), total protein (T PROT), creatinine, total protein (T PROT),

URINALYSIS: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: last five animals/sex/group
Parameters checked: Urine samples were analyzed for total silica content

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: An indirect opthalmoscopic examination was performed on each animal prior to treatment and on all group 1 and 4 animals during week 13

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY:
Performed on all animals. The necropsy included: adrenals, aorta, brain, cervical spinal cord, colon, cecum, rectum, duodenum, jejunum,ileum, esophagus, eyes, heart, kidneys, lesions, liver, lumbar spinal cord, lung, mammary gland, mesenteric lymph node, mid-thoracic spinal cord, ovaries, pancreas, pituitary, prostat, sciatic nerve, spleen, salivary glands, sternum with bone marrow, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus with vagina and cervix.

HISTOPATHOLOGY:
Histopathology performed on all preserved tissue from groups 1, 2 and 4

ORAN WEIGHTS:
Adrenals, kidneys, liver, testes with epididymides

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived to study termination. Alopecia and sores were the clinical signs most frequenly observed but occurred sporadically at a low frequency and were not considered related to treatment.


BODY WEIGHT AND WEIGHT GAIN
During the study there were no statistically significant differences noted in the mean body weight or mean body weight change data between the treated and control groups of either sex

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
mean food consumption for the group 3 males was significantly less than the respective control value for weeks 1-4, 1-8, and 1-13. No other statistically significant differences were noted in food consumption data for either sex at the intervals analyzed. Mean overall compound consumption (mg/kg/day) for weeks 1-13 was 3533.3, 655.3 and 3543.4 for the group 2, 3 and 4 males, respectively, and 3997.8, 740.7, and 3932.3 for the group 2, 3 and 4 females, respectively.

FOOD EFFICIENCY
In the males, mean overall food efficiency (%) for weeks 1-13 was 14.3, 14.6, 15.5 and 14.8 for groups 1 through 4, respectively, and in females was 9.1, 8.8, 9.3 and 9.0 forgroups 1 through 4 respectively.


OPHTHALMOSCOPIC EXAMINATION
During the 13 week ophthalmic examinations, a group 4 female exhibited chromodacryorrhea of the right eye. This finding was not considered treatment related. No other ophthalmic lesions were observed at the week 13 examinations.

HAEMATOLOGY
There were no significant findings in the hematology data

CLINICAL CHEMISTRY
There were no significant findings in the serum chemistry data

URINALYSIS
Refer to table 2 for data

ORGAN WEIGHTS
Statistical analysis of mean organ weight data revealed no significant differences between treated and control groups.


GROSS PATHOLOGY
There were few gross pathology findings at terminal scarifice and the findings were of the type routinely observed in rats. None were considered treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
No compound related histomorphological findings were observed after 13 weeks of treatment. Similar frequency and severity of commonly seen spontaneous disease lesions and incidental findings were noted in control and experimental rats of both sexes.

Dose descriptor:

NOAEL

Remarks:

Flux Calcined DE

Effect level:

3 737.9 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No treatment related effects

Critical effects observed:

not specified

Table 2: Mean total silica content in urine ( µg/g)

Group	Week 1		Week 7		Week 13
	Male	Female	Male	Female	Male	Female
1	31	28	29	34	28	58
2	36	40	48	52	42	91
3	34	24	23	30	37	43
4	35	45	30	46	32	49

Results were obtained from 5 animals/group/sex

Within one standard deviation the silica content in the urine of both males and females fed the control diet, the 5% natural DE pellets and the 1% flux calcined pellets or the 5% flux calcined pellets did not changed between 1 and 13 weeks during the study. Within one standard deviation, the silica content in the urine of the males and females ded the 5% natural DE pellets, the 1% flux calcined pellets or the 5% flux calcined pellets did not differ from the silica content in males and females fed the control diet.

Conclusions:

In conclusion, administration of Natural DE at a dose level of 5% (average of 3765.6 mg/kg/day) or Flux Calcined DE at dose levels of 1% (average of 698.3 mg/kg/day) and 5% (average of 3737.9 mg/kg/day) did not produce any treatment related effects.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Septmber 24 1991 - December 30 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 6 weeks
- Weight at study initiation: 163 - 220 g for males and 136 - 171 g for females
- Housing: Stainless steel, hanging wire-mesh cages
- Diet: Pelleted Purina Certified Rodent Chow #5002 available ad libitum during acclimation period
- Water: Tapwater via an automatic watering system was available ad libitum during both acclimation and study periods
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 6°F
- Humidity: 50 ± 20%
- Photoperiod: 12hrs dark / 12 hrs light:

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test materials were formulated into pellets with a single lot of Purina Certified Rodent Chow #5002. Purina Certified Rodent Chow #5002 was also formulated into pellets to serve as the control material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The total silica and crystalline silica (quartz and cristobalite) were determined in the feed mixtures priorto the study and in the pellets at weeks 1, 7 and 13. The percent total silica in the 5% feed mixtures and pellets of Natural Diatomaceous Earth was 4.8% with 0.44% quartz and no cristobalite. The percent total silica in the 1% feed mixtures and pellets of Flux Calcined Diatomaceous Earth was 1.2% with 0.24% quartz and 0.26& cristobalite. The percent total silica in the 5% feed mixtures and pellets of Flux Calcined Diatomaceous Earth was 5.1% with 0.43% quartz and 1.70% cristobalite.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
Natural Diatomaceous Earth 5% (average of 3765.5 mg/kg/day)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
Flux Calcined Diatomaceous Earth 1% ( average of 698.3 mg/kg/day)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
Flux Calcined Diatomaceous Earth 5% ( average of 3737.9 mg/kg/day)
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Time schedule: Twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Once daily


BODY WEIGHT: Yes
Time schedule for examinations: Measured and recorded at randomization, prior to treatment and weekly, thereafter

WATER CONSUMPTION: No


FOOD CONSUMPTION AND COMPOUND INTAKE (: Yes
Time schedule for examinations: Measured and recorded weekly for weeks 1-13

HAEMATOLOGY: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: first ten animals/sex/group
Parameters checked: cell morphology, corrected leukocyte count (COR WBC), erythrocyte count (RBC), haematocrit (HCT), haemoglobin(HGB), leukocyte count (WBC), leukocyte differential, platelet count

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: first ten animals/sex/group
Parameters checked: alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium, chloride, creatine , gamma glutamyltransferase (GGT), glucose, inorganic phosphorous (IN PHOS), potassium, sodium, total bilirubin (T BILI), total protein (T PROT), creatinine, total protein (T PROT),

URINALYSIS: Yes
Time schedule for collection of blood: During week 13 of the study
Animals fasted: Yes
How many animals: last five animals/sex/group
Parameters checked: Urine samples were analyzed for total silica content

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: An indirect opthalmoscopic examination was performed on each animal prior to treatment and on all group 1 and 4 animals during week 13

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY:
Performed on all animals. The necropsy included: adrenals, aorta, brain, cervical spinal cord, colon, cecum, rectum, duodenum, jejunum,ileum, esophagus, eyes, heart, kidneys, lesions, liver, lumbar spinal cord, lung, mammary gland, mesenteric lymph node, mid-thoracic spinal cord, ovaries, pancreas, pituitary, prostat, sciatic nerve, spleen, salivary glands, sternum with bone marrow, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus with vagina and cervix.

HISTOPATHOLOGY:
Histopathology performed on all preserved tissue from groups 1, 2 and 4

ORAN WEIGHTS:
Adrenals, kidneys, liver, testes with epididymides

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived to study termination. Alopecia and sores were the clinical signs most frequenly observed but occurred sporadically at a low frequency and were not considered related to treatment.


BODY WEIGHT AND WEIGHT GAIN
During the study there were no statistically significant differences noted in the mean body weight or mean body weight change data between the treated and control groups of either sex

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
mean food consumption for the group 3 males was significantly less than the respective control value for weeks 1-4, 1-8, and 1-13. No other statistically significant differences were noted in food consumption data for either sex at the intervals analyzed. Mean overall compound consumption (mg/kg/day) for weeks 1-13 was 3533.3, 655.3 and 3543.4 for the group 2, 3 and 4 males, respectively, and 3997.8, 740.7, and 3932.3 for the group 2, 3 and 4 females, respectively.

FOOD EFFICIENCY
In the males, mean overall food efficiency (%) for weeks 1-13 was 14.3, 14.6, 15.5 and 14.8 for groups 1 through 4, respectively, and in females was 9.1, 8.8, 9.3 and 9.0 forgroups 1 through 4 respectively.


OPHTHALMOSCOPIC EXAMINATION
During the 13 week ophthalmic examinations, a group 4 female exhibited chromodacryorrhea of the right eye. This finding was not considered treatment related. No other ophthalmic lesions were observed at the week 13 examinations.

HAEMATOLOGY
There were no significant findings in the hematology data

CLINICAL CHEMISTRY
There were no significant findings in the serum chemistry data

URINALYSIS
Refer to table 2 for data

ORGAN WEIGHTS
Statistical analysis of mean organ weight data revealed no significant differences between treated and control groups.


GROSS PATHOLOGY
There were few gross pathology findings at terminal scarifice and the findings were of the type routinely observed in rats. None were considered treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
No compound related histomorphological findings were observed after 13 weeks of treatment. Similar frequency and severity of commonly seen spontaneous disease lesions and incidental findings were noted in control and experimental rats of both sexes.

Dose descriptor:

NOAEL

Remarks:

Flux Calcined DE

Effect level:

3 737.9 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No treatment related effects

Critical effects observed:

not specified

Table 2: Mean total silica content in urine ( µg/g)

Group	Week 1		Week 7		Week 13
	Male	Female	Male	Female	Male	Female
1	31	28	29	34	28	58
2	36	40	48	52	42	91
3	34	24	23	30	37	43
4	35	45	30	46	32	49

Results were obtained from 5 animals/group/sex

Within one standard deviation the silica content in the urine of both males and females fed the control diet, the 5% natural DE pellets and the 1% flux calcined pellets or the 5% flux calcined pellets did not changed between 1 and 13 weeks during the study. Within one standard deviation, the silica content in the urine of the males and females ded the 5% natural DE pellets, the 1% flux calcined pellets or the 5% flux calcined pellets did not differ from the silica content in males and females fed the control diet.

Conclusions:

In conclusion, administration of Natural DE at a dose level of 5% (average of 3765.6 mg/kg/day) or Flux Calcined DE at dose levels of 1% (average of 698.3 mg/kg/day) and 5% (average of 3737.9 mg/kg/day) did not produce any treatment related effects.

Reference 3

Endpoint:

chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 737.9 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Published data not containing full information available in the original study report

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and regional lymph nodes. Post-exposure recovery period up to one year.

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline silica).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO
- Age at study initiation: 6 weeks
- Weight at study initiation: males 107 +/- 1 g - 109 +/- 2 g; females 105 +/- 1 g - 109 +/- 1 g
- Fasting period before study: no
- Housing: single in stainless steel wire cages during exposure. 5 males/5 females/ stainless steel cage after the exposure period
- Diet: no access during exposure
- Water:unflouridated tap water ad libitum (no access during exposure)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-1
- Humidity (%):65 - 75
- Air changes (per hr): 12x/h (airflow approximately 40 m3/h)
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H 1000 inhalation chambers
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator composed of a dust feed mechanism and an atomizer operated by compressed air
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C/65 - 75 % rel. humidity
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: Primary particle size calculated using arithmetic mean of transmission electron micrograph magnifications. These particles form agglomerates and aggregates for which it was not possible to determine the aerodynamic size distribution in the test atmosphere due to the weakness of the bonds and the electrostatic charge fo the particles.
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry - Air samples are drawn through glass fiber filters (Sartorius SM 13430) and weighed (3 - 4 times per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test material in the test atmospheres were determined by gravimetry. Samples of the test atmospheres were drawn through glass fibre filters (Sartorius SM 13430). The filters were weighed just before and after sampling. No further details are available in the literature paper.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
1.3, 5.9 or 31 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
1, 6 and 30 mg/m3 (target concentrations)
Basis:
nominal conc.

No. of animals per sex per dose:

70 animals/sex/dose
Aerosil 200: assigned dose groups B, C, and D, each sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica, 58 mg/m3) included (assigned Group G)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females



URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes



NEUROBEHAVIOURAL EXAMINATION: No


OTHER: ---

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Respiration rate: concentration-related increase
No mortality

BODY WEIGHT AND WEIGHT GAIN
No effect in females at all dose levels
Depressive effect in males:
1 mg/m3: slightly at day 14 of exposure only (~ -5%)
6 mg/m3: slightly from day 49 to day 77 of exposure (~ - 6 to <5 %)
no more significant by end of exposure (day 91)
30 mg/m3: significantly throughout exposure: ~ -7 - -10 %, day 91: -7 %
Recovery: no difference from control at day 455 (52 weeks post-exposure)


HAEMATOLOGY
1 mg/m3: no effects
6 mg/m3: White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
but concentration-response relationship was poor.
After 3 months recovery, these blood parameters normalized in males and females.
30 mg/m3: Red blood cell count and hemoglobin were statistically higher in males, but not in females.
White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
at 3 months of recovery (days 176/177), but concentration-response relationship was poor.
In females, a slight increase above the control group apparently still existed after 6 months of recovery (day 275).


CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, thymus, adrenals, testes, brain, spleen, kidney
Treatment-related degrees of severity: swollen lungs and enlarged mediastinal lypmph nodes at the end of exposure
LUNG
1 mg/m3: no significant increase
6 mg/m3: mean increase in relative weight 1.7x (males), 1.4x (females)
30 mg/m3: mean increase in relative weight 2.3x (males), 2.0x (females)
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes, the degree of severity being treatment-related.
At 6 and 30 mg/m3, collagen content in the lungs was clearly increased, most pronounced in males.

The above-mentioned effects gradually subsided after the exposure period,
but in males exposed to 6 and 30 mg/m3 the collagen content was still above control values at the end of the study.


HISTOPATHOLOGY: NON-NEOPLASTIC
Accumulation of alveolar macrophages and granular material, cellular debris, polymorphonuclear leucocytes, increased septal cellularity.
Alveolar bronchialisation, focal interstitial fibrosis, cholesterol clefts and granuloma-like lesions in the lung.
The granuloma-like lesions were seen in a few animals at the end of exposure period and after 13 weeks of recovery. They did not show fibroblastic activity and hyalinization and regressed during recovery [not progressive, i.e. no silicogenic nodules formed (no silicosis)].

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as focal necrosis and slight atrophy of the olfactory epithelium.

Interstitial fibrosis was not noted directly after the exposure period, but appeared with a delay,
for the first time observed after 13 wks post-exposure: increasing incidence especially in 30-mg rats, and a few in the 6-mg group,
but decreased in severity and frequency until the end of the study (Report p. 51).

All types of pulmonary lesions were more marked in males than in females.


The level of 1.3 mg/m3 induced only slight changes after 13-wk exposure, which generally recovered quickly (no differences from control after 13-wk post-exposure).

Morphological changes after 13-wk exposure, that were considered statistically significant at 1.3 mg/m3:

males females ! males females
treated ! untreated controls
------------------------------------------------------------------------------------------------------------------------------
Accumulation of alveolar macrophages: slight in 10/10 (very) slight in 10/10 ! (very) slight 4/10 slight in 1/10
Intra-alveolar polymorphonuclear !
leukocytes: (very) slight in 6/10 (very) slight in 8/10 ! 0/10 0/10
Increased septal cellularity: ( very) slight in 10/10 (very) slight in 9/10 ! very slight 1/10 very slight 1/10
Olfactory epithelial atrophy: (very) slight in 5/10 (very) slight in 8/10 ! 0/10 0/10
Intracytoplasmic proteinaceous droplets !
-respiratory epithelium: in 8/10 in 9/10 ! 1/10 0/10
Mediastinal lymph node !
-macrophage accumulation: (very) slight in 8/10 (very) slight in 8/10 ! 0/10 0/10
------------------------------------------------------------------------------------------------------------------------------



HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable): no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period:
on the average 0.1 - 0.2 mg per lung of male animal groups (not dose-related), 0.05 - 0.21 mg per lung of female groups (dose-related).
Only one male exposed to 30 mg/m3 showed a small amount of silica in the regional lymph node.
90 days after termination of exposure (day 188), no silica could be recovered from any animal.

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the slight and fully reversible pulmonary response noted at this exposure level (inflammation reaction) (see details below "Details on results")

Key result

Dose descriptor:

LOAEC

Effect level:

5.9 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: (haematology); organ weights (hypertrophy lung); histopathology (collagen increase, sporadic focal fibrosis)

Key result

Dose descriptor:

NOEC

Effect level:

< 1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' for more information on the results from this study

Findings for quartz-exposed rats:

Clinical signs:None

Body weight:The body weight of quartz-exposed rats was not affected during the exposure period. The rats showed a slightly progressive reduction in weight gain throughout the post exposure period.

Haematology:There was a statistically significant increase in neutrophilic leucocyte counts during the exposure period. The counts increased during the first 13 weeks after the end of the exposures and remained high during the whole post exposure period. Red blood cell counts, haemoglobin content and packed cell volumes had slightly increased in males exposed to quartz by the end of the exposure period. The males continued to show high red blood cell values throughout the observation period. The remaining haematological parameters that were examined did not show differences that could be related to treatment.

Clinical chemistry:From 13 weeks after exposure alanine aminotransferase activities increased in quartz-exposed rats. In males the increase was approximately 50-90% (p<0.01) in comparison with controls. Alkaline phosphatase activity had increased in rats exposed to quartz 52 weeks after the exposure period. The remaining biochemical parameters that were examined did not show differences that could be related to treatment.

Organ weights:At the end of the exposure period both absolute and relative lung weight were statistically significantly increased compared to controls. The increase was greater in males than in females. Lung weight increased progressively during the post-exposure period to levels three or more times higher than those of control animals. The only other organ that showed increased weight was the thymus. Both the absolute and relative thymus weights were significantly increased (by about 140% of control weight in males at day 188). This increase was most pronounced in males and had disappeared 39 weeks after the end of exposure.

Lung collagen contents:At the end of the exposure period, the lung collagen content of quartz exposed rats was slightly higher than in controls, but in the course of the post-exposure period it had increased markedly.

Silicon in the lungs and associated lymph nodes:Silicon levels in the lungs of males were higher 26 weeks after the end of exposure than at 13 weeks after the end of exposure. Males rats exposed to quartz exhibited even higher values at week 39 than week 26 after exposure. This finding was not observed in females, but this could not be explained by the authors.

Pathology:Most of the rats exposed to quartz and killed at the end of the exposure period had swollen and spotted lungs with a spongy consistency and/or irregular surface and enlarged lung-associated lymph nodes. These changes were more pronounced than in the amorphous silica-exposed rats. The gross lesions in the lungs and lung-associated lymph nodes remained present during the whole post-exposure period. Microscopic changes were mainly observed in the lungs. Changes in rats killed at the end of the exposure period comprised slight to severe accumulation of alveolar macrophages, intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes in the alveolar spaces and increased septal cellularity, seen as an increase in the number of type II pneumocytes and macrophages within the alveolar walls. During the post-exposure period no recovery from lung lesions was observed in quartz-exposed rats. Accumulation of intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes were found in all quartz-exposed rats during the post-exposure period. Alveolar broncholization persisted mainly in quartz-exposed animals. Focal interstitial fibrosis, seen as amorphous eosinophilic, collagen-containing thickenings of the septa, was first observed 13 weeks after exposure and became more severe during the post exposure period. Alveolar cholesterol clefts were observed for the first time after 26 weeks of non-exposure. During the remaining recovery period this lesion became more pronounced. Granulomas, seen as aggregates of macrophage-like cells were observed in nearly all rats exposed to quartz at the end of the exposure period. Slight fibrosis was demonstrated in the granulomas in animals of the quartz group. One year after the end of the exposure period, one male rat had a focus of squamous metaplasia in the periphery of the lung. In addition, in one female a small but unequivocal squamous cell carcinoma was found in the lung parenchyma.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Published data not containing full information available in the original study report

Justification for type of information:

The use of data derived for Silicon dioxide are justified for read-across to synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Silicon dioxide and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and regional lymph nodes. Post-exposure recovery period up to one year.

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline silica).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO
- Age at study initiation: 6 weeks
- Weight at study initiation: males 107 +/- 1 g - 109 +/- 2 g; females 105 +/- 1 g - 109 +/- 1 g
- Fasting period before study: no
- Housing: single in stainless steel wire cages during exposure. 5 males/5 females/ stainless steel cage after the exposure period
- Diet: no access during exposure
- Water:unflouridated tap water ad libitum (no access during exposure)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-1
- Humidity (%):65 - 75
- Air changes (per hr): 12x/h (airflow approximately 40 m3/h)
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H 1000 inhalation chambers
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator composed of a dust feed mechanism and an atomizer operated by compressed air
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C/65 - 75 % rel. humidity
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: Primary particle size calculated using arithmetic mean of transmission electron micrograph magnifications. These particles form agglomerates and aggregates for which it was not possible to determine the aerodynamic size distribution in the test atmosphere due to the weakness of the bonds and the electrostatic charge fo the particles.
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry - Air samples are drawn through glass fiber filters (Sartorius SM 13430) and weighed (3 - 4 times per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test material in the test atmospheres were determined by gravimetry. Samples of the test atmospheres were drawn through glass fibre filters (Sartorius SM 13430). The filters were weighed just before and after sampling. No further details are available in the literature paper.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
1.3, 5.9 or 31 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
1, 6 and 30 mg/m3 (target concentrations)
Basis:
nominal conc.

No. of animals per sex per dose:

70 animals/sex/dose
Aerosil 200: assigned dose groups B, C, and D, each sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica, 58 mg/m3) included (assigned Group G)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females



URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes



NEUROBEHAVIOURAL EXAMINATION: No


OTHER: ---

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Respiration rate: concentration-related increase
No mortality

BODY WEIGHT AND WEIGHT GAIN
No effect in females at all dose levels
Depressive effect in males:
1 mg/m3: slightly at day 14 of exposure only (~ -5%)
6 mg/m3: slightly from day 49 to day 77 of exposure (~ - 6 to <5 %)
no more significant by end of exposure (day 91)
30 mg/m3: significantly throughout exposure: ~ -7 - -10 %, day 91: -7 %
Recovery: no difference from control at day 455 (52 weeks post-exposure)


HAEMATOLOGY
1 mg/m3: no effects
6 mg/m3: White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
but concentration-response relationship was poor.
After 3 months recovery, these blood parameters normalized in males and females.
30 mg/m3: Red blood cell count and hemoglobin were statistically higher in males, but not in females.
White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
at 3 months of recovery (days 176/177), but concentration-response relationship was poor.
In females, a slight increase above the control group apparently still existed after 6 months of recovery (day 275).


CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, thymus, adrenals, testes, brain, spleen, kidney
Treatment-related degrees of severity: swollen lungs and enlarged mediastinal lypmph nodes at the end of exposure
LUNG
1 mg/m3: no significant increase
6 mg/m3: mean increase in relative weight 1.7x (males), 1.4x (females)
30 mg/m3: mean increase in relative weight 2.3x (males), 2.0x (females)
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes, the degree of severity being treatment-related.
At 6 and 30 mg/m3, collagen content in the lungs was clearly increased, most pronounced in males.

The above-mentioned effects gradually subsided after the exposure period,
but in males exposed to 6 and 30 mg/m3 the collagen content was still above control values at the end of the study.


HISTOPATHOLOGY: NON-NEOPLASTIC
Accumulation of alveolar macrophages and granular material, cellular debris, polymorphonuclear leucocytes, increased septal cellularity.
Alveolar bronchialisation, focal interstitial fibrosis, cholesterol clefts and granuloma-like lesions in the lung.
The granuloma-like lesions were seen in a few animals at the end of exposure period and after 13 weeks of recovery. They did not show fibroblastic activity and hyalinization and regressed during recovery [not progressive, i.e. no silicogenic nodules formed (no silicosis)].

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as focal necrosis and slight atrophy of the olfactory epithelium.

Interstitial fibrosis was not noted directly after the exposure period, but appeared with a delay,
for the first time observed after 13 wks post-exposure: increasing incidence especially in 30-mg rats, and a few in the 6-mg group,
but decreased in severity and frequency until the end of the study (Report p. 51).

All types of pulmonary lesions were more marked in males than in females.


The level of 1.3 mg/m3 induced only slight changes after 13-wk exposure, which generally recovered quickly (no differences from control after 13-wk post-exposure).

Morphological changes after 13-wk exposure, that were considered statistically significant at 1.3 mg/m3:

males females ! males females
treated ! untreated controls
------------------------------------------------------------------------------------------------------------------------------
Accumulation of alveolar macrophages: slight in 10/10 (very) slight in 10/10 ! (very) slight 4/10 slight in 1/10
Intra-alveolar polymorphonuclear !
leukocytes: (very) slight in 6/10 (very) slight in 8/10 ! 0/10 0/10
Increased septal cellularity: ( very) slight in 10/10 (very) slight in 9/10 ! very slight 1/10 very slight 1/10
Olfactory epithelial atrophy: (very) slight in 5/10 (very) slight in 8/10 ! 0/10 0/10
Intracytoplasmic proteinaceous droplets !
-respiratory epithelium: in 8/10 in 9/10 ! 1/10 0/10
Mediastinal lymph node !
-macrophage accumulation: (very) slight in 8/10 (very) slight in 8/10 ! 0/10 0/10
------------------------------------------------------------------------------------------------------------------------------



HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable): no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period:
on the average 0.1 - 0.2 mg per lung of male animal groups (not dose-related), 0.05 - 0.21 mg per lung of female groups (dose-related).
Only one male exposed to 30 mg/m3 showed a small amount of silica in the regional lymph node.
90 days after termination of exposure (day 188), no silica could be recovered from any animal.

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the slight and fully reversible pulmonary response noted at this exposure level (inflammation reaction) (see details below "Details on results")

Key result

Dose descriptor:

LOAEC

Effect level:

5.9 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: (haematology); organ weights (hypertrophy lung); histopathology (collagen increase, sporadic focal fibrosis)

Key result

Dose descriptor:

NOEC

Effect level:

< 1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' for more information on the results from this study

Findings for quartz-exposed rats:

Clinical signs:None

Body weight:The body weight of quartz-exposed rats was not affected during the exposure period. The rats showed a slightly progressive reduction in weight gain throughout the post exposure period.

Haematology:There was a statistically significant increase in neutrophilic leucocyte counts during the exposure period. The counts increased during the first 13 weeks after the end of the exposures and remained high during the whole post exposure period. Red blood cell counts, haemoglobin content and packed cell volumes had slightly increased in males exposed to quartz by the end of the exposure period. The males continued to show high red blood cell values throughout the observation period. The remaining haematological parameters that were examined did not show differences that could be related to treatment.

Clinical chemistry:From 13 weeks after exposure alanine aminotransferase activities increased in quartz-exposed rats. In males the increase was approximately 50-90% (p<0.01) in comparison with controls. Alkaline phosphatase activity had increased in rats exposed to quartz 52 weeks after the exposure period. The remaining biochemical parameters that were examined did not show differences that could be related to treatment.

Organ weights:At the end of the exposure period both absolute and relative lung weight were statistically significantly increased compared to controls. The increase was greater in males than in females. Lung weight increased progressively during the post-exposure period to levels three or more times higher than those of control animals. The only other organ that showed increased weight was the thymus. Both the absolute and relative thymus weights were significantly increased (by about 140% of control weight in males at day 188). This increase was most pronounced in males and had disappeared 39 weeks after the end of exposure.

Lung collagen contents:At the end of the exposure period, the lung collagen content of quartz exposed rats was slightly higher than in controls, but in the course of the post-exposure period it had increased markedly.

Silicon in the lungs and associated lymph nodes:Silicon levels in the lungs of males were higher 26 weeks after the end of exposure than at 13 weeks after the end of exposure. Males rats exposed to quartz exhibited even higher values at week 39 than week 26 after exposure. This finding was not observed in females, but this could not be explained by the authors.

Pathology:Most of the rats exposed to quartz and killed at the end of the exposure period had swollen and spotted lungs with a spongy consistency and/or irregular surface and enlarged lung-associated lymph nodes. These changes were more pronounced than in the amorphous silica-exposed rats. The gross lesions in the lungs and lung-associated lymph nodes remained present during the whole post-exposure period. Microscopic changes were mainly observed in the lungs. Changes in rats killed at the end of the exposure period comprised slight to severe accumulation of alveolar macrophages, intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes in the alveolar spaces and increased septal cellularity, seen as an increase in the number of type II pneumocytes and macrophages within the alveolar walls. During the post-exposure period no recovery from lung lesions was observed in quartz-exposed rats. Accumulation of intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes were found in all quartz-exposed rats during the post-exposure period. Alveolar broncholization persisted mainly in quartz-exposed animals. Focal interstitial fibrosis, seen as amorphous eosinophilic, collagen-containing thickenings of the septa, was first observed 13 weeks after exposure and became more severe during the post exposure period. Alveolar cholesterol clefts were observed for the first time after 26 weeks of non-exposure. During the remaining recovery period this lesion became more pronounced. Granulomas, seen as aggregates of macrophage-like cells were observed in nearly all rats exposed to quartz at the end of the exposure period. Slight fibrosis was demonstrated in the granulomas in animals of the quartz group. One year after the end of the exposure period, one male rat had a focus of squamous metaplasia in the periphery of the lung. In addition, in one female a small but unequivocal squamous cell carcinoma was found in the lung parenchyma.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 1

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

Comparable to guideline study with acceptable restrictions in synopsis of the whole testing programme (see Reusal et al (1991) Aero200)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and lymph nodes. Post-exposure recovery period up to one year. One high exposure level only within a combined study (in contrast to Aerosil 200, see other entry).

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline).

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO, Zeist/NL
- Age at study initiation: 4 weeks
- Weight at study initiation: 50 - 70 g
- Fasting period before study: no
- Housing: single during exposure
- Diet: no access during exposure
- Water: no access during exposure
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-1
- Humidity (%): 50 - 70
- Air changes (per hr): 12x/h
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C, minimum 19.1, max. 25.4 °C /
65 - 75 % rel. humidity, during extreme weather occasionally up to 95.5 % or down to 48 %.
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: due to electrostatic charge of the particles not measured:
technical failure of the 10-stage Mercer cascade impactor and the QCM cascade
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically - Air samples are drawn through glass fiber filters (Sartorius)
and weighed (3 - 4 time per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see Report Tables (Part 2), Table 2: Daily mean concentrations are documented:
based on 254 measurements: 34.91 (SEM 0.49) mg/m3

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
35 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
30 mg/m3 (target concentration)
Basis:
nominal conc.

No. of animals per sex per dose:

70
Sipernat 22S: assigned dose groups F, sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica) included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females


URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No particular observations
No mortality

BODY WEIGHT AND WEIGHT GAIN
Slightly decreased body weight, ~ -5 % by 13 wks exposure
Recovery: no significant difference from control at day 455, still ~ -4 % (52 weeks post-exposure)


HAEMATOLOGY
No significant effects, but white blood cell count elevated in both males and females at the end of exposure period ,
but not clearly attributable to increases in the numbers of neutrophilic leukocytes.
After 13 weeks of recovery (day 176/177), neutrophil count still tended to be higher than the control in males and females,
and normalized by 26 weeks of recovery (day 274/275).

CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, adrenals, testes, brain, spleen, kidney
Increased organ weights of lung and thymus at the end of exposure.
Swollen lungs and enlarged mediastinal lypmph nodes
LUNG
Slight mean increase in relative weight: 1.3x (males, females) as compared to control
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes.
The effects gradually subsided after the exposure period:
Lung weight normalised after 13 weeks post-exposure in males and females.


HISTOPATHOLOGY: NON-NEOPLASTIC
In the lung: Accumulation of alveolar macrophages, intra-alveolar polymorphonuclear leukocytes,
and increased septal cellularityin malesand females.
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as very slight focal necrosis and slight atrophy of the olfactory epithelium, intracytoplasmic proteinaceous droplets.

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).

Collagen content in the lungs was slightly increased at the end of exposure.
During the recovery period all changes disappeared mostly within 13 to 26 week.


HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable) no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period (Tables 59):
on the average 0.5 mg per lung of male animal groups, 0.35 mg per lung of female groups, decreasing over time and
no longer measurable after 39 weeks post exposure (day 370).

Dose descriptor:

NOAEC

Basis for effect level:

other: The study was performed using a single dose level and adverse effects were noted at this level

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' in Read Across SAS 01_Degussa 87-0004-DGT_ Aerosil 200_inhal, 13 wk, rat for more information on the results from this study.

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 1

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Comparable to guideline study with acceptable restrictions in synopsis of the whole testing programme (see Reusal et al (1991) Aero200)

Justification for type of information:

The use of data derived for Silicon dioxide are justified for read-across to synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Silicon dioxide and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and lymph nodes. Post-exposure recovery period up to one year. One high exposure level only within a combined study (in contrast to Aerosil 200, see other entry).

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline).

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO, Zeist/NL
- Age at study initiation: 4 weeks
- Weight at study initiation: 50 - 70 g
- Fasting period before study: no
- Housing: single during exposure
- Diet: no access during exposure
- Water: no access during exposure
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-1
- Humidity (%): 50 - 70
- Air changes (per hr): 12x/h
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C, minimum 19.1, max. 25.4 °C /
65 - 75 % rel. humidity, during extreme weather occasionally up to 95.5 % or down to 48 %.
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: due to electrostatic charge of the particles not measured:
technical failure of the 10-stage Mercer cascade impactor and the QCM cascade
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically - Air samples are drawn through glass fiber filters (Sartorius)
and weighed (3 - 4 time per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see Report Tables (Part 2), Table 2: Daily mean concentrations are documented:
based on 254 measurements: 34.91 (SEM 0.49) mg/m3

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
35 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
30 mg/m3 (target concentration)
Basis:
nominal conc.

No. of animals per sex per dose:

70
Sipernat 22S: assigned dose groups F, sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica) included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females


URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No particular observations
No mortality

BODY WEIGHT AND WEIGHT GAIN
Slightly decreased body weight, ~ -5 % by 13 wks exposure
Recovery: no significant difference from control at day 455, still ~ -4 % (52 weeks post-exposure)


HAEMATOLOGY
No significant effects, but white blood cell count elevated in both males and females at the end of exposure period ,
but not clearly attributable to increases in the numbers of neutrophilic leukocytes.
After 13 weeks of recovery (day 176/177), neutrophil count still tended to be higher than the control in males and females,
and normalized by 26 weeks of recovery (day 274/275).

CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, adrenals, testes, brain, spleen, kidney
Increased organ weights of lung and thymus at the end of exposure.
Swollen lungs and enlarged mediastinal lypmph nodes
LUNG
Slight mean increase in relative weight: 1.3x (males, females) as compared to control
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes.
The effects gradually subsided after the exposure period:
Lung weight normalised after 13 weeks post-exposure in males and females.


HISTOPATHOLOGY: NON-NEOPLASTIC
In the lung: Accumulation of alveolar macrophages, intra-alveolar polymorphonuclear leukocytes,
and increased septal cellularityin malesand females.
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as very slight focal necrosis and slight atrophy of the olfactory epithelium, intracytoplasmic proteinaceous droplets.

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).

Collagen content in the lungs was slightly increased at the end of exposure.
During the recovery period all changes disappeared mostly within 13 to 26 week.


HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable) no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period (Tables 59):
on the average 0.5 mg per lung of male animal groups, 0.35 mg per lung of female groups, decreasing over time and
no longer measurable after 39 weeks post exposure (day 370).

Dose descriptor:

NOAEC

Basis for effect level:

other: The study was performed using a single dose level and adverse effects were noted at this level

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' in Read Across SAS 01_Degussa 87-0004-DGT_ Aerosil 200_inhal, 13 wk, rat for more information on the results from this study.

Reference 5

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Only one exposure concentration used. Target organ effects focused on specific investigations based on pulmonary effects (lung lavage cytology and biochemistry) therefore, histopathology is limited and clinical chemistry/haematology is not included.

Principles of method if other than guideline:

- Principle of test: Comparative study examining pulmonary inflammatory cell responses to synthetic amorphous and crystalline silica

- Short description of test conditions: Rats were exposed to amorphous and crystalline silica
for up to 13 weeks. Effects to the lung were examined at 6.5 and 13 weeks of exposure and 3 and 8 months recovery. The study utilized lung burden analysis, Bronchoalveolar Lavage Fluid Analysis (BAL), histopathology of the lung tissue,  MIP-2 gene expression, TUNEL staining and Type II cell isolation and HPRT assays to examine the effects of exposure.

- Parameters analysed / observed: Cellular and biochemical lung damage and inflammation.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 200 - 250 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Mass median aerodynamic diameter (MMAD):

0.81 µm

Remarks on MMAD:

MMAD / GSD: mass median diameter - crystalline silica: 1.3 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: compartmentalized, 300-liter horizontal laminar flow chamber
- Source and rate of air: no data
- Method of conditioning air: The aersol was brought to Boltzman equilibrium by passing the airbourne particles across a 20-mCi 85K source
- System of generating particulates/aerosols: screw-feed mechanism (ACCURate, Whitewater, WI) in combination with a Venturi type dust feeder
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: no data
- Samples taken from breathing zone: no data

VEHICLE: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentration: The average aerosol concentration for the amorphous silica groups was 50.4 mg/m3, SD +-19 mg/m3.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6h/d, 5d/wk

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

amorphous silica

No. of animals per sex per dose:

no data, for single analytical endpoint: n =4

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: 3 and 8 months

Positive control:

Quartz (crystalline silica): Cristobalite, 3 mg/m3
The average aerosol concentration for the Crystalline silica groups was 3 mg/m3 with +-1.0 mg/m3.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: Lactate dyhydrogenase (LDH), protein, glucuronidase

LUNG BURDEN: Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: ug SiO2/Lung (Amorphous/crystalline)

OTHER: Histopathology
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: alveolitis, severity of inflammation in bronchioles and ronchi, fibrosis and relative amount of lung parenchyma.

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes - Scored and ranked on severity: 0.0 (no significant lesions) - 4.0 (very severe process). Results were summed to determine a summary toxicity score.

Other examinations:

Expression of MIP-2 mRNA in lungs was assessed. RNA transcript levels were assessed by PCR amplification of the MIP-2 cDNA.
Immunohistochemistry - Terminal transferase dUTP nick-end-labelling (TUNEL)-staining was carried out on lung sections to identify the extent of potential apoptosis/necrosis resulting from DNA damage.

Statistics:

Analysis of variance, Dunnett´s test for determination of differences between control and treated groups.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased numbers of neutrophils and macrophages were noted at 45 days of treatment and 90 days treatment. Numbers then decreased over the post-exposure period.
Fibrosis detected by Gormer's trichome staining was present in the alveolar septa of the silica treated lungs. Fibrosis subsided during recovery in the case of synthetic amorphous silica.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL Anyalysis:
Significant changes were noted in all BAL parameters with total cell numbers. PMN, protein and glucuronidase levels in BAL greater in the amorphous silica exposed groups than the crystalline silica exposure groups. Changes in the amorphous silica groups returned close to control levels by the end of the 3 month recovery period and were not significantly different by the end of the 8 month recovery period. BAL changes in the crystalline silica groups persisted throughout the post exposure period.
Table 1. presents the results of the BAL fluid analysis.

LUNG SILICA BURDENS:
Group-mean lung burdens were 882.7 mg/lung for amorphous silica. At 3 months post exposure, amorphous silica burdens were significantly decreased in comparison to the end of exposure and decreased further by 8 months of exposure. Crystalline silica burdens post-exposure were relatively unchanged from the end of exposure. Table 2. provides a summary of the silica lung burden results.

GENE EXPRESSION of MIP-2 (inflammatory cytokine gene):
MIP-2 was evident at the end of the 13 weeks exposure (amorphous and crystalline silica). At the end of the 8 month recovery period MIP-2 mRNA was detected in the lungs of rats exposed to crystalline silica. There was minimal detection of MIP-2 mRNA in the lungs of rats exposed to amorphous silica after the same period of recovery. Minimal or no detectable MIP-2 m-RNA expression was found in control rats.

IMMUNOHISTOPATHOLOGY:
After 13-wk exposure to synthetic amorphous silica, intensely stained TUNEL-positive cells were detected throughout the terminal bronchiolar epithelium and throughout the parenchyma of rat lungs. Only a small amount of staining was seen after exposure to crystalline silica. TUNEL-staining was indistinguishable between the amorphous-treated and the control group during the recovery period. In contrast, lungs exposed to crystalline silica showed intense staining localized to cell debris and macrophages in hypertrophic areas of the parenchyma cells.

Details on results:

Table 1.
Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

Remarks on result:

not determinable

Critical effects observed:

not specified

Table 1.

Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

	Total cells x 10^7	% AM	% PMN	% Lymph	% Viable	Protein (µg/ml)	LDH (nmol/min/ml)	Β Glucuronidase (nmol/min/ml)
Sham exposure
Week 6.5	1.09 +/- 0.23	99.7 +/- 0.36	0.24 +/- 0.23	0.77 +/- 0.13	93.8 +/- 2.74	0.16 +/- 0.6	56.2 +/- 14.7	0.47 +/- 0.11
Week 13	2.89 +/- 0.5	98.4 +/- 1.9	0.26 +/- 0.24	0.64 +/- 0.53	94.6 +/- 0.63	0.26 +/- 0.05	11.7 +/- 46.0	0.53 +/- 0.01
Recovery
Week 12	1.76 +/- 0.24	98.1 +/- 0.47	0.65 +/- 0.52	1.22 +/- 0.33	92.9 +/- 1.1	0.21 +/- 0.04	47.9 +/- 9.5	0.53 +/- 0.07
Week 32	1.20 +/- 0.11	98.8 +/- 0.43	0.73 +/- 0.34	0.48 +/- 0.18	94.0 +/- 2.4	0.175 +/- 0.02	41.6 +/- 6.5	0.26 +/- 0.06
Crystalline SiO2 exposure
Week 6.5	8.5 +/- 0.54*	64.7 +/- 2.5*	32.7 +/- 2.7*	2.6 +/- 1.4	95.2 +/- 1.3	0.513 +/- 0.04*	314.6 +/- 16.9*	4.9 +/- 0.8*
Week 13	13.9 +/- 1.2*	50.7 +/- 5.9*	46.9 +/- 5.4*	2.4 +/- 1.6	94.6 +/- 1.9	1.18 +/- 0.09*	798.5 +/- 165.6*	21.3 +/- 2.8*
Recovery
Week 12	19.2 +/- 2.3*	58.7 +/- 8.7*	38.1 +/- 8.5*	3.2 +/- 1.4	95.7 +/- 0.9	1.03 +/- 0.12*	809.5 +/- 196.3*	20.4 +/- 3.0*
Week 32	16.2 +/- 1.7*	64.9 +/- 2.4*	30.8 +/- 2.6*	4.3 +/- 1.6	94.5 +/- 1.4	0.93 +/- 0.14*	1188.2 +/- 163.5*	11.6 +/- 1.85*
Amorphous SiO2
Week 6.5	16.8 +/- 0.54*	42.8 +/- 4.9*	55.2 +/- 4.8*	2.0 +/- 0.8	97.0 +/- 0.9	0.94 +/- 0.08*	709.4 +/- 101.0*	19.3 +/- 3.0*
Week 13	16.9 +/- 2.2*	42.6 +/- 2.9*	55.3 +/- 2.2*	2.1 +/- 1.2	94.2 +/- 1.4	1.59 +/- 0.08*	1808.0 +/- 631.6*	29.2 +/- 2.5*
Recovery
Week 12	2.7 +/- 0.82	88.8 +/- 0.9	9.3 +/- 0.9*	1.9 +/- 0.4	93.8 +/- 1.5	0.386 +/- 0.06	192.1 +/- 57.6*	1.1 +/- 0.19
Week 32	2.2 +/- 0.08	94.9 +/- 1.1	2.6 +/- 1.3	2.5 +/- 0.5	94.2 +/- 2.4	0.339 +/- 0.02	152.0 +/- 68.8	0.46 +/- 0.06

* Significantly different from age-matched control group; p # 0.05.

 

Table 2.

Lung Burdens after Subchronic Exposure of Rats to Crystalline and Amorphous Silicas (µg SiO₂/lung)

		Weeks of Exposure				Weeks of Exposure
Treatment Group		6.5		13		12		32
Control		55.9 +/- 40.4		42.5 +/- 16.9		28.1 +/- 13		39.8 +/- 8.7
Crystalline, 3 mg/m^3		335.6 +/- 28.3*		819.0 +/- 83.3*		657.6 +/- 28*		743.0 +/- 14.5*
Amorphous, 50 mg/m^3		755.9 +/- 22.9*		882.7 +/- 83.1*		156.0 +/- 38.6*		92.6 +/- 38.6

 

Note. Values represent the ⴟ +/- SD; n = 4 rats/treatment.

* Significantly different from age-matched control group; p # 0.05.

Reference 6

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The use of data derived for Silicon dioxide are justified for read-across to synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Silicon dioxide and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Only one exposure concentration used. Target organ effects focused on specific investigations based on pulmonary effects (lung lavage cytology and biochemistry) therefore, histopathology is limited and clinical chemistry/haematology is not included.

Principles of method if other than guideline:

- Principle of test: Comparative study examining pulmonary inflammatory cell responses to synthetic amorphous and crystalline silica

- Short description of test conditions: Rats were exposed to amorphous and crystalline silica
for up to 13 weeks. Effects to the lung were examined at 6.5 and 13 weeks of exposure and 3 and 8 months recovery. The study utilized lung burden analysis, Bronchoalveolar Lavage Fluid Analysis (BAL), histopathology of the lung tissue,  MIP-2 gene expression, TUNEL staining and Type II cell isolation and HPRT assays to examine the effects of exposure.

- Parameters analysed / observed: Cellular and biochemical lung damage and inflammation.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 200 - 250 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Mass median aerodynamic diameter (MMAD):

0.81 µm

Remarks on MMAD:

MMAD / GSD: mass median diameter - crystalline silica: 1.3 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: compartmentalized, 300-liter horizontal laminar flow chamber
- Source and rate of air: no data
- Method of conditioning air: The aersol was brought to Boltzman equilibrium by passing the airbourne particles across a 20-mCi 85K source
- System of generating particulates/aerosols: screw-feed mechanism (ACCURate, Whitewater, WI) in combination with a Venturi type dust feeder
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: no data
- Samples taken from breathing zone: no data

VEHICLE: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentration: The average aerosol concentration for the amorphous silica groups was 50.4 mg/m3, SD +-19 mg/m3.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6h/d, 5d/wk

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

amorphous silica

No. of animals per sex per dose:

no data, for single analytical endpoint: n =4

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: 3 and 8 months

Positive control:

Quartz (crystalline silica): Cristobalite, 3 mg/m3
The average aerosol concentration for the Crystalline silica groups was 3 mg/m3 with +-1.0 mg/m3.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: Lactate dyhydrogenase (LDH), protein, glucuronidase

LUNG BURDEN: Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: ug SiO2/Lung (Amorphous/crystalline)

OTHER: Histopathology
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: alveolitis, severity of inflammation in bronchioles and ronchi, fibrosis and relative amount of lung parenchyma.

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes - Scored and ranked on severity: 0.0 (no significant lesions) - 4.0 (very severe process). Results were summed to determine a summary toxicity score.

Other examinations:

Expression of MIP-2 mRNA in lungs was assessed. RNA transcript levels were assessed by PCR amplification of the MIP-2 cDNA.
Immunohistochemistry - Terminal transferase dUTP nick-end-labelling (TUNEL)-staining was carried out on lung sections to identify the extent of potential apoptosis/necrosis resulting from DNA damage.

Statistics:

Analysis of variance, Dunnett´s test for determination of differences between control and treated groups.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased numbers of neutrophils and macrophages were noted at 45 days of treatment and 90 days treatment. Numbers then decreased over the post-exposure period.
Fibrosis detected by Gormer's trichome staining was present in the alveolar septa of the silica treated lungs. Fibrosis subsided during recovery in the case of synthetic amorphous silica.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL Anyalysis:
Significant changes were noted in all BAL parameters with total cell numbers. PMN, protein and glucuronidase levels in BAL greater in the amorphous silica exposed groups than the crystalline silica exposure groups. Changes in the amorphous silica groups returned close to control levels by the end of the 3 month recovery period and were not significantly different by the end of the 8 month recovery period. BAL changes in the crystalline silica groups persisted throughout the post exposure period.
Table 1. presents the results of the BAL fluid analysis.

LUNG SILICA BURDENS:
Group-mean lung burdens were 882.7 mg/lung for amorphous silica. At 3 months post exposure, amorphous silica burdens were significantly decreased in comparison to the end of exposure and decreased further by 8 months of exposure. Crystalline silica burdens post-exposure were relatively unchanged from the end of exposure. Table 2. provides a summary of the silica lung burden results.

GENE EXPRESSION of MIP-2 (inflammatory cytokine gene):
MIP-2 was evident at the end of the 13 weeks exposure (amorphous and crystalline silica). At the end of the 8 month recovery period MIP-2 mRNA was detected in the lungs of rats exposed to crystalline silica. There was minimal detection of MIP-2 mRNA in the lungs of rats exposed to amorphous silica after the same period of recovery. Minimal or no detectable MIP-2 m-RNA expression was found in control rats.

IMMUNOHISTOPATHOLOGY:
After 13-wk exposure to synthetic amorphous silica, intensely stained TUNEL-positive cells were detected throughout the terminal bronchiolar epithelium and throughout the parenchyma of rat lungs. Only a small amount of staining was seen after exposure to crystalline silica. TUNEL-staining was indistinguishable between the amorphous-treated and the control group during the recovery period. In contrast, lungs exposed to crystalline silica showed intense staining localized to cell debris and macrophages in hypertrophic areas of the parenchyma cells.

Details on results:

Table 1.
Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

Remarks on result:

not determinable

Critical effects observed:

not specified

Table 1.

Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

	Total cells x 10^7	% AM	% PMN	% Lymph	% Viable	Protein (µg/ml)	LDH (nmol/min/ml)	Β Glucuronidase (nmol/min/ml)
Sham exposure
Week 6.5	1.09 +/- 0.23	99.7 +/- 0.36	0.24 +/- 0.23	0.77 +/- 0.13	93.8 +/- 2.74	0.16 +/- 0.6	56.2 +/- 14.7	0.47 +/- 0.11
Week 13	2.89 +/- 0.5	98.4 +/- 1.9	0.26 +/- 0.24	0.64 +/- 0.53	94.6 +/- 0.63	0.26 +/- 0.05	11.7 +/- 46.0	0.53 +/- 0.01
Recovery
Week 12	1.76 +/- 0.24	98.1 +/- 0.47	0.65 +/- 0.52	1.22 +/- 0.33	92.9 +/- 1.1	0.21 +/- 0.04	47.9 +/- 9.5	0.53 +/- 0.07
Week 32	1.20 +/- 0.11	98.8 +/- 0.43	0.73 +/- 0.34	0.48 +/- 0.18	94.0 +/- 2.4	0.175 +/- 0.02	41.6 +/- 6.5	0.26 +/- 0.06
Crystalline SiO2 exposure
Week 6.5	8.5 +/- 0.54*	64.7 +/- 2.5*	32.7 +/- 2.7*	2.6 +/- 1.4	95.2 +/- 1.3	0.513 +/- 0.04*	314.6 +/- 16.9*	4.9 +/- 0.8*
Week 13	13.9 +/- 1.2*	50.7 +/- 5.9*	46.9 +/- 5.4*	2.4 +/- 1.6	94.6 +/- 1.9	1.18 +/- 0.09*	798.5 +/- 165.6*	21.3 +/- 2.8*
Recovery
Week 12	19.2 +/- 2.3*	58.7 +/- 8.7*	38.1 +/- 8.5*	3.2 +/- 1.4	95.7 +/- 0.9	1.03 +/- 0.12*	809.5 +/- 196.3*	20.4 +/- 3.0*
Week 32	16.2 +/- 1.7*	64.9 +/- 2.4*	30.8 +/- 2.6*	4.3 +/- 1.6	94.5 +/- 1.4	0.93 +/- 0.14*	1188.2 +/- 163.5*	11.6 +/- 1.85*
Amorphous SiO2
Week 6.5	16.8 +/- 0.54*	42.8 +/- 4.9*	55.2 +/- 4.8*	2.0 +/- 0.8	97.0 +/- 0.9	0.94 +/- 0.08*	709.4 +/- 101.0*	19.3 +/- 3.0*
Week 13	16.9 +/- 2.2*	42.6 +/- 2.9*	55.3 +/- 2.2*	2.1 +/- 1.2	94.2 +/- 1.4	1.59 +/- 0.08*	1808.0 +/- 631.6*	29.2 +/- 2.5*
Recovery
Week 12	2.7 +/- 0.82	88.8 +/- 0.9	9.3 +/- 0.9*	1.9 +/- 0.4	93.8 +/- 1.5	0.386 +/- 0.06	192.1 +/- 57.6*	1.1 +/- 0.19
Week 32	2.2 +/- 0.08	94.9 +/- 1.1	2.6 +/- 1.3	2.5 +/- 0.5	94.2 +/- 2.4	0.339 +/- 0.02	152.0 +/- 68.8	0.46 +/- 0.06

* Significantly different from age-matched control group; p # 0.05.

 

Table 2.

Lung Burdens after Subchronic Exposure of Rats to Crystalline and Amorphous Silicas (µg SiO₂/lung)

		Weeks of Exposure				Weeks of Exposure
Treatment Group		6.5		13		12		32
Control		55.9 +/- 40.4		42.5 +/- 16.9		28.1 +/- 13		39.8 +/- 8.7
Crystalline, 3 mg/m^3		335.6 +/- 28.3*		819.0 +/- 83.3*		657.6 +/- 28*		743.0 +/- 14.5*
Amorphous, 50 mg/m^3		755.9 +/- 22.9*		882.7 +/- 83.1*		156.0 +/- 38.6*		92.6 +/- 38.6

 

Note. Values represent the ⴟ +/- SD; n = 4 rats/treatment.

* Significantly different from age-matched control group; p # 0.05.

Reference 7

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The initial sample used in this study contained 45% of cristobalite and approximately 55% of amorphous silica. The respirable fraction was 3.8% including 1.8% crystalline silica. In order to be compliant with the OECD TG 412 (which recommends the generation of aerosols with mass median aerodynamic diameters ranging from 1 to 3 μm), the test material was micronized to allow that the majority of material/particles could reach the deep lung (alveoli). The test method of processing of the material resulted in the content of respirable crystalline silica being increased from 1.8% to approximately 45%, which is approximately double the worse-case level found in commercially produced material. Under these conditions, the toxicity observed in the study material reflects and confirms the health hazards of extremely high concentrations of the crystalline silica. However, as stated above, the study material is not reflective of the toxicity of, or risk factor associated with, the much lower concentration of crystalline silica contained in commercially available product, the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: males: 8 weeks; females 9 weeks (at delivery)
- Weight at study initiation: males: 216.7-294.3 g; females 169.1-209.1 g (at acclimatization)
- Fasting period before study: n/a
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment.
- Diet: Pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations..
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except during the periods when the animals were restrained in the
exposure tubes.
- Acclimation period: Eight or nine days under test conditions after clinical health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2 and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The the group means of the chemically determined mass median aerodynamic diameters (MMAD) were within the target range of 1 to 3 μm. Single measurements slightly above the upper limit were considered to be acceptable. The corresponding geometric standard deviations (GSD) were also within the target range of 1.5 to 3. The results of the gravimetric determinations were considered to be comparable.
Overall, deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. Hence, the particle size distribution and MMADs obtained were considered to be appropriate.
The values for gravimetrically and chemically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) for Soda-ash flux-calcined kieselguhr are shown in Tables 1 & 2 below.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system. Ports for animal exposure are positioned radially around the nose-only, flow-past exposure chamber on several different levels.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR 3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. The generated test aerosol was diluted as necessary with compressed air to achieve the concentrations required for this study.
- Temperature in air chamber: 22.6/-0.2 - 23.2 +/- 0.2 deg C
- Relative humidity: 1.2 +/- 0.9 - 5.0 +/- 1.7 %
- Air flow rate: The flow of air at each tube was 1.0 L/min
- Method of particle size determination: The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. The airflow rate through the impactor was 1 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentrations were determined gravimetrically and chemically (please refer to detailed description below)

VEHICLE
Compressed air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric determination of the aerosol concentration was performed six times during each exposure for groups 2 to 4. Test aerosol samples were collected onto Millipore®durapore filters, Type HVLP using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port. The duration of sampling was sufficient to ensure reliable results. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
To determine the concentration of the test item based on the Silicon in the generated aerosol chemical analyses of test aerosol samples were performed twice per week and on two occasions of these selected exposures for groups 2 to 4. Filters sampled for gravimetric determination were used for chemical determination. An appropriate number of samples collected were transferred into labeled appropriate vials, forwarded at ambient temperature to the scientist responsible for formulation analysis and stored at room temperature (range of 20 ± 5 °C) until analysis. They were analyzed for Silicon by Harlan Laboratories Ltd. using an AAS method developed by Harlan Laboratories Ltd.

Duration of treatment / exposure:

6 hours/exposure

Frequency of treatment:

5 days/week at approximately 24 hour intervals for four consecutive weeks.
Following the treatment period there was a 9 week recovery period.

Remarks:

Doses / Concentrations:
0.044 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0.207 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0.700 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

20 animals/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale:Target Dose Levels were selected based on data from the 5 day dose range finding inhalation toxicity study. In the 5-day inhalation toxicity study, rats were dosed with target concentrations of 0, 0.20, 0.60 and 2.0 mg/L of the test item. A statistical significant increase in absolute lungs weights was observed at the mid dose in females and at the high dose in males and females. At the other doses, males and females already showed an increase in lung weight although not statistically significant compared to controls. According to the results of this preliminary study,
the dose-levels of 0.050, 0.20 and 0.80 mg/L were selected for the current study.
- Rationale for animal assignment: random

Positive control:

No positive control was used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily during acclimatization and recovery and twice dily during treatment.
- Cage side observations: mortality, viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly during acclimatization and recovery. On exposure days, once before, during and after the daily treatment, and once daily on non-exposure days of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatization and recovery, and twice weekly during treatment.

FOOD CONSUMPTION:
- Food consumption: Recorded (per cage) weekly during acclimatization, treatment and recovery.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatization (main study and recovery animals) and week 4 of treatment (main study animals).
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 18 hours before sampling
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a
- Battery of functions tested: sensory activity / grip strength / motor activity / other: n/a

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Other examinations:

BRONCHO-ALVEOLAR LAVAGE:

5 animals/sex/group were sacrificed approximately 24 h after the last exposure and a further 5 animals/sex/group were sacrificed approximately 24 h after 9-week recovery. The lungs of each dose group were washed six times with approximately 4 mL physiological saline at room temperature by slow instillation and withdrawal of fluid, with thoracic massage.
The following parameters were determined:
- Enzymatic activities of lactate dehydrogenase and alkaline phosphatase which were used as indicators of possible plasma membrane damage and/or cell lysis.
- Total protein which was indicative of inflammatory processes and damage to the alveolar capillary barrier.
- Phospholipids which was indicative of disturbances in the metabolic activity of type II epithelial cells.

Statistics:

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios, ophthalmoscopic examination as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for
the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied to the ophthalmoscopic and macroscopic findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled treatment and recovery periods. No clinical signs were noted during the course of this study.

BODY WEIGHT AND WEIGHT GAIN
Marginal mean body weight loss was noted between day 1 and day 4 of treatment in high dose group males attaining statistical significance for the body weight gain.
These animals gained weight thereafter and a dose relation was no longer observed from week 3 onwards. Accordingly, further statistical significant differences for the body weight development for low and high dose group males were considered to be incidental. Body weight gain in females was
similar across all groups.

FOOD CONSUMPTION
There were no effects on the food consumption that were considered to be related to treatment. Statistically significant decreased food intake on some occasions for males of the low and mid dose groups during the treatment period were considered to be incidental in the absence of a dose relationship. A statistically significantly increased food intake on same occasions for males of the mid and high dose groups was also considered to be fortuitous.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
No ophthalmoscopic findings were observed which were considered to be related to the treatment.
Corneal opacity observed at the end of the treatment period in some animals across all groups was already present before exposure start.

HAEMATOLOGY
At the end of the treatment period the absolute and relative neutrophil levels were increased in males and females of all three groups treated with the test substance. Statistical significance was attained in the mid and high dose groups for the absolute or relative levels in females and males, respectively.
At the end of the recovery period the effects on the neutrophils increased and a clear dose relation ship was recorded for the absolute levels. Statistical significant differences to controls were recorded for the relative levels in males in all groups treated with the test substance and for the absolute levels in males
and females in all groups treated with the test substance (except for low dose males). In addition, the absolute monocyte levels were statistically significantly increased in mid dose females and in both sexes at the high dose. Accordingly, the total leukocyte count was also increased in males and females in all groups treated with the test substance, attaining statistical significance in the high dose group.

CLINICAL CHEMISTRY
Aspartate aminotransferase was increased in both sexes in the high dose group at the end of the recovery period. Statistical significance was attained for males. Statistically significant differences in cholesterol, triglycerides and phospholipids at either the end of the treatment period or the 9 week recovery period were within the range of historical control data and therefore not considered to be related to treatment.

URINALYSIS
There were no findings in urinalysis that were considered to be test item-related.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Absolute and related lung weights were dose-dependently increased in both sexes in all groups treated with the test substance at the end of treatment. Statistical significance was obtained for the mid and high dose groups.
After the end of recovery, the there was a further dose-dependent increase in absolute and relative lungs weights and statistical significance was obtained for both sexes in all groups treated with the test substance (except for the absolute lung weight and the lung weight to body ratio for low dose females). In addition, the absolute and relative spleen weights were increased in both sexes in the high dose group, attaining statistical significance for males. Absolute and relative adrenal weights were increased in high dose males, attaining statistical significance for the absolute weight and the adrenal to brain weight ratio. Absolute and relative liver weights were increased in high dose females, attaining statistical significance for the liver to body weight ratio.
There were no further changes in organ weights that were considered to be test item-related. Single statistically significant differences between treated groups and controls were considered to be incidental as there was no dose-relationship and/or alterations were not consistently present in both sexes.

GROSS PATHOLOGY
The tracheobronchial/mediastinal lymph nodes were recorded as enlarged in two mid dose females and one male and three females in the high dose group at the end of the treatment period and in all animals treated with the test substance after the end of the recovery period.
All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
After the end of treatment, diffuse alveolar histiocytosis in the lungs was noted at a dose dependently increased severity in all treated animals. Amorphous material (considered to be the test item) was noted at a dose-dependently increased incidence and severity in mid and high dose males and females. In addition, perivascular/peribronchial lymphoid hyperplasia was noted some high dose animals.
In the tracheobronchial lymph nodes, histiocytic granulomas were noted in most treated animals at a dose-dependent severity. This finding was generally accompanied by lymphoid hyperplasia.
After the end of recovery period diffuse alveolar histiocytosis, the amorphous material and the perivascular/peribronchial lymphoid hyperplasia were still present in the lungs. However, the severity grade for the alveolar histiocytosis increased in all treatment groups and the incidences and the severity for the amorphous material were similar but this finding was additionally noted in two low dose females. In addition, the severity grade incidence for the lymphoid hyperplasia increased in the high dose group and this finding was noted in one low dose male and some mid dose animals. This hyperplasia was accompanied by histiocyte granulomas, partly with dose-dependent fibrosis. Finally, microgranulomas at the bronchiolar-alveolar junction at a dose-dependent severity were
noted in most treated animals.
In the tracheobronchial lymph nodes, histiocytic granulomas and lymphoid hyperplasia were still present but the severity increased as well as the incidence to all animals. In addition, there was fibrosis present in all these lymph nodes at a generally dose-dependently increased severity.
The other findings noted were within the range of incidental background lesions commonly recorded in animals of this strain and age.

HISTORICAL CONTROL DATA
Available for Haematology, Clinical chemistry and urinalysis. attached as file historical data.pdf

BRONCHO-ALVEOLAR LAVAGE
Lactate dehydrogenase was increased in the broncho-alveolar lavage fluid at the end of treatment in both sexes of all treated groups, attaining statistical significance in high dose males and in low and mid dose females. In addition, phospholipids and alkaline phosphatase were also increased in males and females but only attaining statistical significance on some occasions in mid and high dose groups. Finally, total protein was increased in the high dose group attaining statistical significance in females.
In addition, the number of cells in the broncho-alveolar lavage fluid increased dose-dependently in all reated groups attaining statistical significance in both sexes of the mid and high dose groups. This increase resulted mainly from macrophages and neutrophils but the absolute number of lymphocytes were also increased. The statistical significant increase of the cell viability in mid and high dose males was considered to be a consequence of these increased neutrophil levels.
After the end of the recovery period, completed recovery was noted for alkaline phosphatase and partial recovery was noted for phospholipids and lactate dehydrogenase: phospholipids were increased at the end of treatment in mid dose males and in both sexes at the high dose, attaining
statistical significance in the high dose group; lactate dehydrogenase remained increased in low and mid dose males and both sexes at the high dose, attaining statistical significance in this group.
A progression of the effects was noted for total protein and cell counts in the broncho-alveolar lavage fluid: Total protein was dose-dependently increased in males in all treated groups and in mid and high dose females attaining statistical significance in high dose males and in mid and high dose females. The number of cells increased in a dose-dependent manner and reached statistical significance in all reatment groups with a similar differential cell count as at the end of treatment.

Some further inter-group variations in hematology, clinical biochemistry, urinalysis, and broncho-alveolar lavage parameters occasionally achieved statistical significance reflecting changes that were considered to be in the normal range, displayed no dose-relationship and/or were not consistently present in both sexes. These changes were considered not to be test item related.
In addition, statistical significant decreases for differential cell counts were a consequence of the increase of neutrophils.

Dose descriptor:

NOAEC

Basis for effect level:

other: On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Summary tables for the results of examinations are attached as Summary tables Part 1.pdf and Summary tables Part 2.pdf

Executive summary:

In this 28-day repeat dose inhalation study performed according to OECD TG 412 under GLP, kieselguhr soda ash flux-calcined was administered to Wistar rats (20 animals/sex/dose) by nose-only, flow-past inhalation for a period of 5 days/week (6 hours/day) for 4 consecutive weeks at aerosol concentrations of 0.044, 0.207 and 0.700 mg/L air.

 

The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 9-week treatment-free recovery period following the treatment period in selected animals of all groups. Sub-groups of male and female animals served for determination of broncho-alveolar lavage fluid (BALF) parameters on day 28 of treatment or were kept for a 9-week off-dose period. Mortality, clinical signs, body weight, food consumption, organ weights, macroscopic and microscopic findings were recorded and ophthalmoscopic examinations and clinical laboratory investigations were performed. BALF samples were collected after the end of treatment and after recovery.

 

No premature deaths and no clinical signs were observed except for a slight and transient effect on body weight gain at the high dose. A dose-dependent increase in lung weights was recorded from the low dose to the high dose at the end of the treatment. A further increase in lung weights and lymph nodes had increased in size at the end of the recovery period. An increase in spleen, adrenals and liver weights at the high-dose at the end of the recovery period was observed but the relevance of these findings was not certain. In the alveoli, histiocytosis was observed increasing in incidence and severity from the low dose to the high dose at the end of the treatment. A progression in incidence and severity was also observed to occur after the recovery period. The presence of the test item was detected in the alveoli of the animals of the mid and high dose groups at the end of the treatment period. This was observed to persist at least until the end of the recovery period and additionally it was observed in 2/10 animals of the low dose recovery group.

 

In the observations on the peribronchial/perivascular zone, lymphoid hyperplasia was observed in the high dose group animals at the end of the treatment period. There was a progression in severity after the recovery period and additionally it was observed in some mid-dosed animals of the recovery group. The occurrence of microgranuloma and fibrosis was observed at the end of the treatment-free period. For the tracheobronchial lymph nodes, histiocytic granuloma was observed increasing in severity from the low dose to the high dose at the end of the treatment. There was a progression in incidence and severity as shown by the occurrence of fibrosis after the recovery period.

There were no adverse effects detected in organs other than the lungs. Some adaptive responses may have been induced in the liver, adrenals and spleen in response to the irritation and damage to the lung tissue.

 

On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Reference 8

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The initial sample used in this study contained 45% of cristobalite and approximately 55% of amorphous silica. The respirable fraction was 3.8% including 1.8% crystalline silica. In order to be compliant with the OECD TG 412 (which recommends the generation of aerosols with mass median aerodynamic diameters ranging from 1 to 3 μm), the test material was micronized to allow that the majority of material/particles could reach the deep lung (alveoli). The test method of processing of the material resulted in the content of respirable crystalline silica being increased from 1.8% to approximately 45%, which is approximately double the worse-case level found in commercially produced material. Under these conditions, the toxicity observed in the study material reflects and confirms the health hazards of extremely high concentrations of the crystalline silica. However, as stated above, the study material is not reflective of the toxicity of, or risk factor associated with, the much lower concentration of crystalline silica contained in commercially available product, the registered substance.

Justification for type of information:

The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: males: 8 weeks; females 9 weeks (at delivery)
- Weight at study initiation: males: 216.7-294.3 g; females 169.1-209.1 g (at acclimatization)
- Fasting period before study: n/a
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment.
- Diet: Pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations..
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except during the periods when the animals were restrained in the
exposure tubes.
- Acclimation period: Eight or nine days under test conditions after clinical health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2 and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The the group means of the chemically determined mass median aerodynamic diameters (MMAD) were within the target range of 1 to 3 μm. Single measurements slightly above the upper limit were considered to be acceptable. The corresponding geometric standard deviations (GSD) were also within the target range of 1.5 to 3. The results of the gravimetric determinations were considered to be comparable.
Overall, deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. Hence, the particle size distribution and MMADs obtained were considered to be appropriate.
The values for gravimetrically and chemically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) for Soda-ash flux-calcined kieselguhr are shown in Tables 1 & 2 below.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system. Ports for animal exposure are positioned radially around the nose-only, flow-past exposure chamber on several different levels.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR 3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. The generated test aerosol was diluted as necessary with compressed air to achieve the concentrations required for this study.
- Temperature in air chamber: 22.6/-0.2 - 23.2 +/- 0.2 deg C
- Relative humidity: 1.2 +/- 0.9 - 5.0 +/- 1.7 %
- Air flow rate: The flow of air at each tube was 1.0 L/min
- Method of particle size determination: The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. The airflow rate through the impactor was 1 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentrations were determined gravimetrically and chemically (please refer to detailed description below)

VEHICLE
Compressed air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric determination of the aerosol concentration was performed six times during each exposure for groups 2 to 4. Test aerosol samples were collected onto Millipore®durapore filters, Type HVLP using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port. The duration of sampling was sufficient to ensure reliable results. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
To determine the concentration of the test item based on the Silicon in the generated aerosol chemical analyses of test aerosol samples were performed twice per week and on two occasions of these selected exposures for groups 2 to 4. Filters sampled for gravimetric determination were used for chemical determination. An appropriate number of samples collected were transferred into labeled appropriate vials, forwarded at ambient temperature to the scientist responsible for formulation analysis and stored at room temperature (range of 20 ± 5 °C) until analysis. They were analyzed for Silicon by Harlan Laboratories Ltd. using an AAS method developed by Harlan Laboratories Ltd.

Duration of treatment / exposure:

6 hours/exposure

Frequency of treatment:

5 days/week at approximately 24 hour intervals for four consecutive weeks.
Following the treatment period there was a 9 week recovery period.

Remarks:

Doses / Concentrations:
0.044 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0.207 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

20 animals/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale:Target Dose Levels were selected based on data from the 5 day dose range finding inhalation toxicity study. In the 5-day inhalation toxicity study, rats were dosed with target concentrations of 0, 0.20, 0.60 and 2.0 mg/L of the test item. A statistical significant increase in absolute lungs weights was observed at the mid dose in females and at the high dose in males and females. At the other doses, males and females already showed an increase in lung weight although not statistically significant compared to controls. According to the results of this preliminary study,
the dose-levels of 0.050, 0.20 and 0.80 mg/L were selected for the current study.
- Rationale for animal assignment: random

Positive control:

No positive control was used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily during acclimatization and recovery and twice dily during treatment.
- Cage side observations: mortality, viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly during acclimatization and recovery. On exposure days, once before, during and after the daily treatment, and once daily on non-exposure days of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatization and recovery, and twice weekly during treatment.

FOOD CONSUMPTION:
- Food consumption: Recorded (per cage) weekly during acclimatization, treatment and recovery.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatization (main study and recovery animals) and week 4 of treatment (main study animals).
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 18 hours before sampling
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a
- Battery of functions tested: sensory activity / grip strength / motor activity / other: n/a

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Other examinations:

BRONCHO-ALVEOLAR LAVAGE:

5 animals/sex/group were sacrificed approximately 24 h after the last exposure and a further 5 animals/sex/group were sacrificed approximately 24 h after 9-week recovery. The lungs of each dose group were washed six times with approximately 4 mL physiological saline at room temperature by slow instillation and withdrawal of fluid, with thoracic massage.
The following parameters were determined:
- Enzymatic activities of lactate dehydrogenase and alkaline phosphatase which were used as indicators of possible plasma membrane damage and/or cell lysis.
- Total protein which was indicative of inflammatory processes and damage to the alveolar capillary barrier.
- Phospholipids which was indicative of disturbances in the metabolic activity of type II epithelial cells.

Statistics:

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios, ophthalmoscopic examination as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for
the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied to the ophthalmoscopic and macroscopic findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled treatment and recovery periods. No clinical signs were noted during the course of this study.

BODY WEIGHT AND WEIGHT GAIN
Marginal mean body weight loss was noted between day 1 and day 4 of treatment in high dose group males attaining statistical significance for the body weight gain.
These animals gained weight thereafter and a dose relation was no longer observed from week 3 onwards. Accordingly, further statistical significant differences for the body weight development for low and high dose group males were considered to be incidental. Body weight gain in females was
similar across all groups.

FOOD CONSUMPTION
There were no effects on the food consumption that were considered to be related to treatment. Statistically significant decreased food intake on some occasions for males of the low and mid dose groups during the treatment period were considered to be incidental in the absence of a dose relationship. A statistically significantly increased food intake on same occasions for males of the mid and high dose groups was also considered to be fortuitous.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
No ophthalmoscopic findings were observed which were considered to be related to the treatment.
Corneal opacity observed at the end of the treatment period in some animals across all groups was already present before exposure start.

HAEMATOLOGY
At the end of the treatment period the absolute and relative neutrophil levels were increased in males and females of all three groups treated with the test substance. Statistical significance was attained in the mid and high dose groups for the absolute or relative levels in females and males, respectively.
At the end of the recovery period the effects on the neutrophils increased and a clear dose relation ship was recorded for the absolute levels. Statistical significant differences to controls were recorded for the relative levels in males in all groups treated with the test substance and for the absolute levels in males
and females in all groups treated with the test substance (except for low dose males). In addition, the absolute monocyte levels were statistically significantly increased in mid dose females and in both sexes at the high dose. Accordingly, the total leukocyte count was also increased in males and females in all groups treated with the test substance, attaining statistical significance in the high dose group.

CLINICAL CHEMISTRY
Aspartate aminotransferase was increased in both sexes in the high dose group at the end of the recovery period. Statistical significance was attained for males. Statistically significant differences in cholesterol, triglycerides and phospholipids at either the end of the treatment period or the 9 week recovery period were within the range of historical control data and therefore not considered to be related to treatment.

URINALYSIS
There were no findings in urinalysis that were considered to be test item-related.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Absolute and related lung weights were dose-dependently increased in both sexes in all groups treated with the test substance at the end of treatment. Statistical significance was obtained for the mid and high dose groups.
After the end of recovery, the there was a further dose-dependent increase in absolute and relative lungs weights and statistical significance was obtained for both sexes in all groups treated with the test substance (except for the absolute lung weight and the lung weight to body ratio for low dose females). In addition, the absolute and relative spleen weights were increased in both sexes in the high dose group, attaining statistical significance for males. Absolute and relative adrenal weights were increased in high dose males, attaining statistical significance for the absolute weight and the adrenal to brain weight ratio. Absolute and relative liver weights were increased in high dose females, attaining statistical significance for the liver to body weight ratio.
There were no further changes in organ weights that were considered to be test item-related. Single statistically significant differences between treated groups and controls were considered to be incidental as there was no dose-relationship and/or alterations were not consistently present in both sexes.

GROSS PATHOLOGY
The tracheobronchial/mediastinal lymph nodes were recorded as enlarged in two mid dose females and one male and three females in the high dose group at the end of the treatment period and in all animals treated with the test substance after the end of the recovery period.
All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
After the end of treatment, diffuse alveolar histiocytosis in the lungs was noted at a dose dependently increased severity in all treated animals. Amorphous material (considered to be the test item) was noted at a dose-dependently increased incidence and severity in mid and high dose males and females. In addition, perivascular/peribronchial lymphoid hyperplasia was noted some high dose animals.
In the tracheobronchial lymph nodes, histiocytic granulomas were noted in most treated animals at a dose-dependent severity. This finding was generally accompanied by lymphoid hyperplasia.
After the end of recovery period diffuse alveolar histiocytosis, the amorphous material and the perivascular/peribronchial lymphoid hyperplasia were still present in the lungs. However, the severity grade for the alveolar histiocytosis increased in all treatment groups and the incidences and the severity for the amorphous material were similar but this finding was additionally noted in two low dose females. In addition, the severity grade incidence for the lymphoid hyperplasia increased in the high dose group and this finding was noted in one low dose male and some mid dose animals. This hyperplasia was accompanied by histiocyte granulomas, partly with dose-dependent fibrosis. Finally, microgranulomas at the bronchiolar-alveolar junction at a dose-dependent severity were
noted in most treated animals.
In the tracheobronchial lymph nodes, histiocytic granulomas and lymphoid hyperplasia were still present but the severity increased as well as the incidence to all animals. In addition, there was fibrosis present in all these lymph nodes at a generally dose-dependently increased severity.
The other findings noted were within the range of incidental background lesions commonly recorded in animals of this strain and age.

HISTORICAL CONTROL DATA
Available for Haematology, Clinical chemistry and urinalysis. attached as file historical data.pdf

BRONCHO-ALVEOLAR LAVAGE
Lactate dehydrogenase was increased in the broncho-alveolar lavage fluid at the end of treatment in both sexes of all treated groups, attaining statistical significance in high dose males and in low and mid dose females. In addition, phospholipids and alkaline phosphatase were also increased in males and females but only attaining statistical significance on some occasions in mid and high dose groups. Finally, total protein was increased in the high dose group attaining statistical significance in females.
In addition, the number of cells in the broncho-alveolar lavage fluid increased dose-dependently in all reated groups attaining statistical significance in both sexes of the mid and high dose groups. This increase resulted mainly from macrophages and neutrophils but the absolute number of lymphocytes were also increased. The statistical significant increase of the cell viability in mid and high dose males was considered to be a consequence of these increased neutrophil levels.
After the end of the recovery period, completed recovery was noted for alkaline phosphatase and partial recovery was noted for phospholipids and lactate dehydrogenase: phospholipids were increased at the end of treatment in mid dose males and in both sexes at the high dose, attaining
statistical significance in the high dose group; lactate dehydrogenase remained increased in low and mid dose males and both sexes at the high dose, attaining statistical significance in this group.
A progression of the effects was noted for total protein and cell counts in the broncho-alveolar lavage fluid: Total protein was dose-dependently increased in males in all treated groups and in mid and high dose females attaining statistical significance in high dose males and in mid and high dose females. The number of cells increased in a dose-dependent manner and reached statistical significance in all reatment groups with a similar differential cell count as at the end of treatment.

Some further inter-group variations in hematology, clinical biochemistry, urinalysis, and broncho-alveolar lavage parameters occasionally achieved statistical significance reflecting changes that were considered to be in the normal range, displayed no dose-relationship and/or were not consistently present in both sexes. These changes were considered not to be test item related.
In addition, statistical significant decreases for differential cell counts were a consequence of the increase of neutrophils.

Dose descriptor:

NOAEC

Basis for effect level:

other: On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Summary tables for the results of examinations are attached as Summary tables Part 1.pdf and Summary tables Part 2.pdf

Executive summary:

In this 28-day repeat dose inhalation study performed according to OECD TG 412 under GLP, kieselguhr soda ash flux-calcined was administered to Wistar rats (20 animals/sex/dose) by nose-only, flow-past inhalation for a period of 5 days/week (6 hours/day) for 4 consecutive weeks at aerosol concentrations of 0.044, 0.207 and 0.700 mg/L air.

 

The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 9-week treatment-free recovery period following the treatment period in selected animals of all groups. Sub-groups of male and female animals served for determination of broncho-alveolar lavage fluid (BALF) parameters on day 28 of treatment or were kept for a 9-week off-dose period. Mortality, clinical signs, body weight, food consumption, organ weights, macroscopic and microscopic findings were recorded and ophthalmoscopic examinations and clinical laboratory investigations were performed. BALF samples were collected after the end of treatment and after recovery.

 

No premature deaths and no clinical signs were observed except for a slight and transient effect on body weight gain at the high dose. A dose-dependent increase in lung weights was recorded from the low dose to the high dose at the end of the treatment. A further increase in lung weights and lymph nodes had increased in size at the end of the recovery period. An increase in spleen, adrenals and liver weights at the high-dose at the end of the recovery period was observed but the relevance of these findings was not certain. In the alveoli, histiocytosis was observed increasing in incidence and severity from the low dose to the high dose at the end of the treatment. A progression in incidence and severity was also observed to occur after the recovery period. The presence of the test item was detected in the alveoli of the animals of the mid and high dose groups at the end of the treatment period. This was observed to persist at least until the end of the recovery period and additionally it was observed in 2/10 animals of the low dose recovery group.

 

In the observations on the peribronchial/perivascular zone, lymphoid hyperplasia was observed in the high dose group animals at the end of the treatment period. There was a progression in severity after the recovery period and additionally it was observed in some mid-dosed animals of the recovery group. The occurrence of microgranuloma and fibrosis was observed at the end of the treatment-free period. For the tracheobronchial lymph nodes, histiocytic granuloma was observed increasing in severity from the low dose to the high dose at the end of the treatment. There was a progression in incidence and severity as shown by the occurrence of fibrosis after the recovery period.

There were no adverse effects detected in organs other than the lungs. Some adaptive responses may have been induced in the liver, adrenals and spleen in response to the irritation and damage to the lung tissue.

 

On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Reference 9

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: 5 day dose range finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Principles of method if other than guideline:

The purpose of this 5-day dose range finding inhalation study was to obtain preliminary information about the toxicity of Soda-ash flux-calcined kieselguhr when administered by nose only, flow-past inhalation exposure for a period of 5 days (6 hours/day). The results of this study were used to select the dose levels for further toxicity studies.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at delivery): Males: 8 weeks, Females: 9 weeks
- Weight (at acclimatization): Males: 209.7 to 236.9 g (± 6%), Females: 163.1 to 181.9 g (± 6%)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 82/09) rat maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: Eight days under test conditions after clinical health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2 and 4 hours, respectively.



ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: relative humidity range: (30 – 70%)
- Air changes: 10 – 15 air changes per hour
- Photoperiod: There was a 12-hour fluorescent light/12- hour dark cycle with music during the light period

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Remarks on MMAD:

MMAD / GSD: The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the chemical results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA). The target ranges were 1 to 3 μm for the MMAD and 1.5 to 3 for the GSD.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR 3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer
- Temperature, humidity, pressure in air chamber:
- Air flow rate: The flow of air at each tube was 1.0 L/min
- Method of particle size determination: The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor.


TEST ATMOSPHERE
- Brief description of analytical method used: To determine the concentration of the test item based on the Silicon content in the generated aerosol, chemical analyses of test aerosol samples were performed twice daily for groups 2 to 4. The filters taken for gravimetric determination were used for chemical analysis.They were analyzed for Silicon by Harlan Laboratories Ltd. using an AAS method developed by Harlan Laboratories Ltd.

Analytical verification of doses or concentrations:

yes

Frequency of treatment:

6-hours daily, at approximately 24-hour intervals.

Remarks:

Doses / Concentrations:
0.18, 0.58 and 1.57 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

5

Details on study design:

- Dose selection rationale: Target Concentrations were selected by the Sponsor based on data from the acute inhalation toxicity study performed at Harlan Laboratories Ltd. (study C88248). In addition, the target concentration for group 4 was considered to be the technical limit based on a 6 hour exposure).

Observations and examinations performed and frequency:

Viability / Mortality: Twice daily during treatment, once daily during acclimatization.

Clinical Signs: Were recorded once daily during treatment (after exposure) and on days 1 and 5 of acclimatization

Food Consumption: Was recorded (per cage) on days 1 and 5 of acclimatization and on days 1, 3 and 5 during the treatment period.

Body Weights: Were recorded on days 1 and 5 (each individual animal) of acclimatization and on days 1, 3 and 5 during treatment.

Sacrifice and pathology:

All animals were weighed and necropsied (samples taken are detailed in table 1)

Statistics:

laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test [(many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: All animals survived the scheduled treatment period. No clinical signs were noted during the course of the study.

BODY WEIGHT AND WEIGHT GAIN: Mean body weight loss was recorded in both male and female animals of group 4 over the five day treatment period. In male animals a mean of 11.9 g was lost between day 1 and 5. Over the same time period the female animals lost a mean of 2.7 g. Mean group body weights of all groups including control did not return to before treatment values. Statistically significant reduced body weight gain was observed in male rats of group 4 only when compared with the control group.

FOOD CONSUMPTION: A reduced mean food consumption was recorded on treatment days 1 to 3 and 3 to 5 for groups 4 animals when compared to days 5 to 10 for acclimatization.


ORGAN WEIGHTS: Absolute lung weights of group 4 males and group 3 and 4 females were significantly increased. Furthermore, significant increases were recorded for lung/body weight and lung/brain weight ratios of male and female animals of groups 3 and 4 and males of groups 3 and 4 and females of group 4, respectively.


MACROSCOPIC FINDINGS: No macroscopic findings were recorded which were considered to be related to treatment with the test item


MICROSCOPIC FINDINGS: There was a dose dependent increase in the incidence of alveolar histiocytosis. This finding was seen in one male of group 1, two males and females of group 2 and all animals of groups 3 and 4. In addition, alveolitis was observed in one male of group 3 and in all males and females of group 4. Furthermore, microgranulomas were noted in one male and female of group 3 and all males and females of group 4 and amorphous material in alveoli was found in four males and three females of group 4. These findings in the lungs are considered to represent an irritant effect of the test item. All other findings noted were considered to be incidental findings commonly noted in animals of this strain and age.

Critical effects observed:

not specified

The chemically determined mean aerosol concentrations were close to target for groups 2 and 3 and below the target for group 4. lower concentrations for group 4 were considered to be acceptable in view of the significant effects on body weight. Details on aerosol concentrations are summarized below:

Group	Achieved gravimetric aerosol concentration [mg/L]	Achieved chemical aerosol concentration [mg/L]	Target chemical aerosol concentration [mg/L]	Chemical aerosol concentration relative to target [%]
2	0.21 ± 0.00 (n=5, CV=2.4%)	0.18 ± 0.03 (n=5, CV=14.0%)	0.20	91.4%
3	0.61 ± 0.02 (n=5, CV=3.1%)	0.58 ± 0.05 (n=5, CV=9.4%)	0.60	96.2%
4	1.91 ± 0.11 (n=5, CV=5.7%)	1.57 ± 0.18 (n=5, CV=11.5%)	2.0	78.5%

The values for gravimetrically and chemically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) were as follows:

	Gravimetric determination
Group	MMAD [μm] and (mean GSD)	Mean percentage of particles < 3 μm
2	2.46 (2.93)	57.3%
3	2.47 (2.65)	58.0%
4	2.81 (1.91)	54.1%

The gravimetric determined Mass Median Aerodynamic Diameters (MMAD) were within the target range of 1 to 3 μm. Overall, deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. Hence, the particle size distribution and MMADs obtained were considered to be appropriate. The results of the chemical determined Mass Median Aerodynamic Diameters (MMAD) obtained by atomic absorption spectrometry (AAS) were considered to be unreliable, due to dissolving impactor stages which contained silicon.

The group mean values for temperature, relative humidity and oxygen concentration measured during the exposures were as follows:

Group	Temperature (°C)	Relative humidity (%)	Oxygen concentration (%)
1	22.8 ± 0.1 (n=5)	1.8 ± 0.2 (n=5)	20.6 ± 0.0 (n=5)
2	23.1 ± 0.1 (n=5)	1.6 ± 1.0 (n=5)	20.3 ± 0.0 (n=5)
3	23.0 ± 0.1 (n=5)	2.8 ± 0.2 (n=5)	20.7 ± 0.0 (n=5)
4	22.5 ± 0.1 (n=5)	4.9 ± 1.2 (n=5)	20.6 ± 0.1 (n=5)

All parameters were very consistent during the treatment period and similar for all groups. Therefore, they were considered to be satisfactory for this type of study.

Conclusions:

Exposure to Soda-ash flux-calcined kieselguhr at chemically determined aerosol concentrations of 0.18, 0.58 and 1.57 mg/L air resulted in effects on food consumption, body weight and lung weight as well as microscopic findings in the lung.

Reduced food consumption as well as reduced body weight gain and body weight loss were observed in animals of group 4.

Dose-related alveolar histiocytosis was observed in animals of all groups. Furthermore, alveolitis was seen in one male of group 3 and all animals of group 4 as well as increased absolute and relative lung weights was observed in the test item-treated groups 3 and 4. Additionally, microgranulomas were found in one male and female of group 3 and in all animals of group 4 and amorphous material in the alveoli was observed in most animals of group 4. These findings in the lungs were considered to represent an irritant effect of the test item. On the basis of the microscopic findings in the lung, a NOEL could not be established. Based on the results of this study, aerosol concentrations of 0.050, 0.20 and 0.80 mg/L air were considered to be suitable target concentrations for a 4-week inhalation study.

Reference 10

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study does not fulfill the requirements of a current OECD guideline study and has not been conducted according to GLP.

Principles of method if other than guideline:

Dogs, guinea pigs and rats were exposed daily, 6 hours per day, 5 days per week for periods up to 2.5 years to flux-calcined diatomaceous earth of 61% cristobalite content and 0.7 µ mass medium particle size. The concentrations were 2, 5 and 50 million particles per cubic foot of air (mppcf). Dogs were given an additional 10 month period of observation to study possible progression of tissue reaction.

GLP compliance:

no

Remarks:

Study predates GLP

Species:

other: Dog, guinea pig and rat

Strain:

other: Dogs: mongrels, guinea pigs: not stated, rats: wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dogs were obtained froma local supplier, guinea pigs were obtained from Rockland Farms (New City, New York); and the rats were from Hamilton Laboratory Animals (Cincinnati, Ohio)
- Fasting period before study: No food or water was available to animals during the daily six hour exposures.
- Diet: While in their housing vages th dogs were fed one daily ration of Bordens Dog Meal moistened with a mixture of equal amounts of water and powdered skim milk; guinea pigs and rats had available food and tap water ad libitum. Guinea pigs had Purina Rabbit Pellets supplemented twice weekly with greens, the rats had Rockland Rat Diet.
- Acclimation period: All animals, both those to be exposed and controls were pre-exposed to the control chamber to normal atmospheres of filtered and temperature and humidity controlled chambers for 6 hours, five days per week for 3 weeks.

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
In an effort to simulate the stress of work associated with industrial environment on the possible toxicity of low level, flux-calcined diatomaceous earth, separate groups of 10 control and 10, 5 mppcf exposed rats were placed in large motor driven rotating cages located in their respective exposure chambers. The cages were rotated at 7 rpm, 5 minutes each hour, starting after the first hour during a continuous 6 hour exposure. The rats were exposed daily in this manner for one year.

The dynamic type exposure chmabers were cubical units of 145 cubic feet capacity with conical galvanized-iron tops and aluminium double wall and floor construction. Control animals were exposed in a similar manner but recieved only filtered temperature controlled air.

TEST ATMOSPHERE
Chamber air samples were collected by two methods; the midget impringer and a semi continuous sampling analysis and recording method utilizing the Sinclair-Phoenix Aerosol and Smoke Photometer. A summary of the tabulated monthly cumulative chamber dust concentrations for each level for the 30 months of operation is shown in table 2.

Details on analytical verification of doses or concentrations:

Refer to table 2

Duration of treatment / exposure:

Up to 2.5 years

Frequency of treatment:

Daily, six hours per day

Remarks:

Doses / Concentrations:
2 mppcf
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
5 mppcf
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
50 mppcf
Basis:
nominal conc.

No. of animals per sex per dose:

Refer to table 1 for total number of animals

Control animals:

yes, concurrent no treatment

Details on study design:

Separate groups of animals were exposed at 2 and 5 million particles of flux-calcined diatomaceous earth per cubic foot of air 6 hours per day, 5 days per week on a 2 shift basis. The dual shift was necessitated by a shortage of exposure chambers, but was discontinued after 8 weeks when additional chamber became available. Table 1 shows that in addition to the groups of dogs, guinea pigs and rats exposed daily at the 2 and 5 mppcf levels, separate groups of rats and guinea pigs were exposed to an intermittent peak concentration of 50 mppcf for one hour, 3 times per week; and other group of rats and guinea pigs were exposed intermittently at the same peak 50 mppcf exposure.

Sacrifice and pathology:

Lungs, hilar lymph nodes, heart, liver, kidney, spleen, adrenal, small intestine (duodenum) and hepatic lymph nodes were preapred for histologic examination. In addition, sections of the trachea, pancreas and urinary bladder of the dogs were saved.

In an effort to determine the extent of retention of cristobalite in the lung tissue from each scheduled, serially sacrificed control, 2 mppcf and 5 mppcf dog was submitted for x-ray diffraction analysis.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

BODY WEIGHTS
All groups of dogs gained as much and sometimes more than controls. Guinea pigs showed no depressed growth at any level of exposure; the final body weights at the end of 12 and 18 months compared favourably with the controls. The terminal average body weights of rats exposed at the 5+50 mppcf level, however were considerably below those of the corresponding controls, as were those exposed at the 5 mppcf level.

HEMATOLOGY
There were no changes in the hematologic varaibles, hematocrit, hemoglobin and red and white cell counts that were determined on dogs trimonthly throughout the 2.5 year exposure nor in dogs that were retained another 10 months for observation.

X RAY DIFFRACTION OF THE LUNG
Spectrometric x-ray diffraction analyses of lungs of dogs exposed at dust levels of 2 and 5 mppcf indicated a cumulative deposition of cristobalite that plateaued after about 1800 hours at the lower level and after 3000 hours at the higher. The average value of the cristobalite content in dry lung tissue for the 2 mppcf level was 0.18% (range 0.14% - 0.27%) and 0.40% (range 0.37%-0.43%) for those exposed at 5 mppcf. No amount of cristobalite was detected in control animals.

PATHOLOGY
Detailed histologic evaluation of tissues of serially sacrificed animals showed that no fibrosis of the pulmonary parencgyma resulted in any of the species under any condition of exposure. However, there was no dust level which did not exhibit a histiocytic and giant cell infiltration in the pulmonary parenchyma, and moderate numbers of hyalinized fibrotic nodules developed in hilar lymph nodes in dogs at the 5 mppcf level and to a lesser degree at the 2 mppcf level. No recognizable pathologic abnormalities were observed in any other organs.

Dose descriptor:

NOAEL

Effect level:

5 other: mppcf

Sex:

male/female

Basis for effect level:

other: Based on no observed fibrotic changes in dogs

Critical effects observed:

not specified

Table 2: Thirty month average cumulative chamber dust concentration data

Nominal conc.	Actual average conc.	Deviation		Average range	Exposure days	Exposure hours	Calendar days	Gtvalue (mppcf-hr)
mppcf	mppcf	mppcf	%	mppcf				Actual	nominal
2	2.17	+0.45	20.7	1.6 – 3.1	630	3770	927	7,917	7540
5	5.27	+0.81	13.3	3.7 – 7.1	632	3766	927	19,734	18,830
50	46.8	-9.9		33.5 – 65.2	372	422	920	19,800	21,100

Conclusions:

The NOAEL from the study is 5 mppcf (equivalent to 0.14 mg/kg) based on no observed fibrotic changes while a NOEL was not found in the study as effects were already present at 2 mppcf

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1.3 mg/m³

Study duration:

subchronic

System:

other: Based on a slight and fully reversible inflammatory response in the respiratory tract, in particular the lung, at low concentration.

Organ:

alveoli

lungs

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Published data not containing full information available in the original study report

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and regional lymph nodes. Post-exposure recovery period up to one year.

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline silica).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO
- Age at study initiation: 6 weeks
- Weight at study initiation: males 107 +/- 1 g - 109 +/- 2 g; females 105 +/- 1 g - 109 +/- 1 g
- Fasting period before study: no
- Housing: single in stainless steel wire cages during exposure. 5 males/5 females/ stainless steel cage after the exposure period
- Diet: no access during exposure
- Water:unflouridated tap water ad libitum (no access during exposure)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-1
- Humidity (%):65 - 75
- Air changes (per hr): 12x/h (airflow approximately 40 m3/h)
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H 1000 inhalation chambers
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator composed of a dust feed mechanism and an atomizer operated by compressed air
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C/65 - 75 % rel. humidity
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: Primary particle size calculated using arithmetic mean of transmission electron micrograph magnifications. These particles form agglomerates and aggregates for which it was not possible to determine the aerodynamic size distribution in the test atmosphere due to the weakness of the bonds and the electrostatic charge fo the particles.
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry - Air samples are drawn through glass fiber filters (Sartorius SM 13430) and weighed (3 - 4 times per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test material in the test atmospheres were determined by gravimetry. Samples of the test atmospheres were drawn through glass fibre filters (Sartorius SM 13430). The filters were weighed just before and after sampling. No further details are available in the literature paper.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
1.3, 5.9 or 31 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
1, 6 and 30 mg/m3 (target concentrations)
Basis:
nominal conc.

No. of animals per sex per dose:

70 animals/sex/dose
Aerosil 200: assigned dose groups B, C, and D, each sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica, 58 mg/m3) included (assigned Group G)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females



URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes



NEUROBEHAVIOURAL EXAMINATION: No


OTHER: ---

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Respiration rate: concentration-related increase
No mortality

BODY WEIGHT AND WEIGHT GAIN
No effect in females at all dose levels
Depressive effect in males:
1 mg/m3: slightly at day 14 of exposure only (~ -5%)
6 mg/m3: slightly from day 49 to day 77 of exposure (~ - 6 to <5 %)
no more significant by end of exposure (day 91)
30 mg/m3: significantly throughout exposure: ~ -7 - -10 %, day 91: -7 %
Recovery: no difference from control at day 455 (52 weeks post-exposure)


HAEMATOLOGY
1 mg/m3: no effects
6 mg/m3: White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
but concentration-response relationship was poor.
After 3 months recovery, these blood parameters normalized in males and females.
30 mg/m3: Red blood cell count and hemoglobin were statistically higher in males, but not in females.
White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
at 3 months of recovery (days 176/177), but concentration-response relationship was poor.
In females, a slight increase above the control group apparently still existed after 6 months of recovery (day 275).


CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, thymus, adrenals, testes, brain, spleen, kidney
Treatment-related degrees of severity: swollen lungs and enlarged mediastinal lypmph nodes at the end of exposure
LUNG
1 mg/m3: no significant increase
6 mg/m3: mean increase in relative weight 1.7x (males), 1.4x (females)
30 mg/m3: mean increase in relative weight 2.3x (males), 2.0x (females)
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes, the degree of severity being treatment-related.
At 6 and 30 mg/m3, collagen content in the lungs was clearly increased, most pronounced in males.

The above-mentioned effects gradually subsided after the exposure period,
but in males exposed to 6 and 30 mg/m3 the collagen content was still above control values at the end of the study.


HISTOPATHOLOGY: NON-NEOPLASTIC
Accumulation of alveolar macrophages and granular material, cellular debris, polymorphonuclear leucocytes, increased septal cellularity.
Alveolar bronchialisation, focal interstitial fibrosis, cholesterol clefts and granuloma-like lesions in the lung.
The granuloma-like lesions were seen in a few animals at the end of exposure period and after 13 weeks of recovery. They did not show fibroblastic activity and hyalinization and regressed during recovery [not progressive, i.e. no silicogenic nodules formed (no silicosis)].

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as focal necrosis and slight atrophy of the olfactory epithelium.

Interstitial fibrosis was not noted directly after the exposure period, but appeared with a delay,
for the first time observed after 13 wks post-exposure: increasing incidence especially in 30-mg rats, and a few in the 6-mg group,
but decreased in severity and frequency until the end of the study (Report p. 51).

All types of pulmonary lesions were more marked in males than in females.


The level of 1.3 mg/m3 induced only slight changes after 13-wk exposure, which generally recovered quickly (no differences from control after 13-wk post-exposure).

Morphological changes after 13-wk exposure, that were considered statistically significant at 1.3 mg/m3:

males females ! males females
treated ! untreated controls
------------------------------------------------------------------------------------------------------------------------------
Accumulation of alveolar macrophages: slight in 10/10 (very) slight in 10/10 ! (very) slight 4/10 slight in 1/10
Intra-alveolar polymorphonuclear !
leukocytes: (very) slight in 6/10 (very) slight in 8/10 ! 0/10 0/10
Increased septal cellularity: ( very) slight in 10/10 (very) slight in 9/10 ! very slight 1/10 very slight 1/10
Olfactory epithelial atrophy: (very) slight in 5/10 (very) slight in 8/10 ! 0/10 0/10
Intracytoplasmic proteinaceous droplets !
-respiratory epithelium: in 8/10 in 9/10 ! 1/10 0/10
Mediastinal lymph node !
-macrophage accumulation: (very) slight in 8/10 (very) slight in 8/10 ! 0/10 0/10
------------------------------------------------------------------------------------------------------------------------------



HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable): no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period:
on the average 0.1 - 0.2 mg per lung of male animal groups (not dose-related), 0.05 - 0.21 mg per lung of female groups (dose-related).
Only one male exposed to 30 mg/m3 showed a small amount of silica in the regional lymph node.
90 days after termination of exposure (day 188), no silica could be recovered from any animal.

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the slight and fully reversible pulmonary response noted at this exposure level (inflammation reaction) (see details below "Details on results")

Key result

Dose descriptor:

LOAEC

Effect level:

5.9 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: (haematology); organ weights (hypertrophy lung); histopathology (collagen increase, sporadic focal fibrosis)

Key result

Dose descriptor:

NOEC

Effect level:

< 1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' for more information on the results from this study

Findings for quartz-exposed rats:

Clinical signs:None

Body weight:The body weight of quartz-exposed rats was not affected during the exposure period. The rats showed a slightly progressive reduction in weight gain throughout the post exposure period.

Haematology:There was a statistically significant increase in neutrophilic leucocyte counts during the exposure period. The counts increased during the first 13 weeks after the end of the exposures and remained high during the whole post exposure period. Red blood cell counts, haemoglobin content and packed cell volumes had slightly increased in males exposed to quartz by the end of the exposure period. The males continued to show high red blood cell values throughout the observation period. The remaining haematological parameters that were examined did not show differences that could be related to treatment.

Clinical chemistry:From 13 weeks after exposure alanine aminotransferase activities increased in quartz-exposed rats. In males the increase was approximately 50-90% (p<0.01) in comparison with controls. Alkaline phosphatase activity had increased in rats exposed to quartz 52 weeks after the exposure period. The remaining biochemical parameters that were examined did not show differences that could be related to treatment.

Organ weights:At the end of the exposure period both absolute and relative lung weight were statistically significantly increased compared to controls. The increase was greater in males than in females. Lung weight increased progressively during the post-exposure period to levels three or more times higher than those of control animals. The only other organ that showed increased weight was the thymus. Both the absolute and relative thymus weights were significantly increased (by about 140% of control weight in males at day 188). This increase was most pronounced in males and had disappeared 39 weeks after the end of exposure.

Lung collagen contents:At the end of the exposure period, the lung collagen content of quartz exposed rats was slightly higher than in controls, but in the course of the post-exposure period it had increased markedly.

Silicon in the lungs and associated lymph nodes:Silicon levels in the lungs of males were higher 26 weeks after the end of exposure than at 13 weeks after the end of exposure. Males rats exposed to quartz exhibited even higher values at week 39 than week 26 after exposure. This finding was not observed in females, but this could not be explained by the authors.

Pathology:Most of the rats exposed to quartz and killed at the end of the exposure period had swollen and spotted lungs with a spongy consistency and/or irregular surface and enlarged lung-associated lymph nodes. These changes were more pronounced than in the amorphous silica-exposed rats. The gross lesions in the lungs and lung-associated lymph nodes remained present during the whole post-exposure period. Microscopic changes were mainly observed in the lungs. Changes in rats killed at the end of the exposure period comprised slight to severe accumulation of alveolar macrophages, intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes in the alveolar spaces and increased septal cellularity, seen as an increase in the number of type II pneumocytes and macrophages within the alveolar walls. During the post-exposure period no recovery from lung lesions was observed in quartz-exposed rats. Accumulation of intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes were found in all quartz-exposed rats during the post-exposure period. Alveolar broncholization persisted mainly in quartz-exposed animals. Focal interstitial fibrosis, seen as amorphous eosinophilic, collagen-containing thickenings of the septa, was first observed 13 weeks after exposure and became more severe during the post exposure period. Alveolar cholesterol clefts were observed for the first time after 26 weeks of non-exposure. During the remaining recovery period this lesion became more pronounced. Granulomas, seen as aggregates of macrophage-like cells were observed in nearly all rats exposed to quartz at the end of the exposure period. Slight fibrosis was demonstrated in the granulomas in animals of the quartz group. One year after the end of the exposure period, one male rat had a focus of squamous metaplasia in the periphery of the lung. In addition, in one female a small but unequivocal squamous cell carcinoma was found in the lung parenchyma.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Published data not containing full information available in the original study report

Justification for type of information:

The use of data derived for Silicon dioxide are justified for read-across to synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Silicon dioxide and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and regional lymph nodes. Post-exposure recovery period up to one year.

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline silica).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO
- Age at study initiation: 6 weeks
- Weight at study initiation: males 107 +/- 1 g - 109 +/- 2 g; females 105 +/- 1 g - 109 +/- 1 g
- Fasting period before study: no
- Housing: single in stainless steel wire cages during exposure. 5 males/5 females/ stainless steel cage after the exposure period
- Diet: no access during exposure
- Water:unflouridated tap water ad libitum (no access during exposure)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-1
- Humidity (%):65 - 75
- Air changes (per hr): 12x/h (airflow approximately 40 m3/h)
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H 1000 inhalation chambers
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator composed of a dust feed mechanism and an atomizer operated by compressed air
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C/65 - 75 % rel. humidity
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: Primary particle size calculated using arithmetic mean of transmission electron micrograph magnifications. These particles form agglomerates and aggregates for which it was not possible to determine the aerodynamic size distribution in the test atmosphere due to the weakness of the bonds and the electrostatic charge fo the particles.
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry - Air samples are drawn through glass fiber filters (Sartorius SM 13430) and weighed (3 - 4 times per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test material in the test atmospheres were determined by gravimetry. Samples of the test atmospheres were drawn through glass fibre filters (Sartorius SM 13430). The filters were weighed just before and after sampling. No further details are available in the literature paper.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
1.3, 5.9 or 31 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
1, 6 and 30 mg/m3 (target concentrations)
Basis:
nominal conc.

No. of animals per sex per dose:

70 animals/sex/dose
Aerosil 200: assigned dose groups B, C, and D, each sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica, 58 mg/m3) included (assigned Group G)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females



URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes



NEUROBEHAVIOURAL EXAMINATION: No


OTHER: ---

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Respiration rate: concentration-related increase
No mortality

BODY WEIGHT AND WEIGHT GAIN
No effect in females at all dose levels
Depressive effect in males:
1 mg/m3: slightly at day 14 of exposure only (~ -5%)
6 mg/m3: slightly from day 49 to day 77 of exposure (~ - 6 to <5 %)
no more significant by end of exposure (day 91)
30 mg/m3: significantly throughout exposure: ~ -7 - -10 %, day 91: -7 %
Recovery: no difference from control at day 455 (52 weeks post-exposure)


HAEMATOLOGY
1 mg/m3: no effects
6 mg/m3: White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
but concentration-response relationship was poor.
After 3 months recovery, these blood parameters normalized in males and females.
30 mg/m3: Red blood cell count and hemoglobin were statistically higher in males, but not in females.
White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
at 3 months of recovery (days 176/177), but concentration-response relationship was poor.
In females, a slight increase above the control group apparently still existed after 6 months of recovery (day 275).


CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, thymus, adrenals, testes, brain, spleen, kidney
Treatment-related degrees of severity: swollen lungs and enlarged mediastinal lypmph nodes at the end of exposure
LUNG
1 mg/m3: no significant increase
6 mg/m3: mean increase in relative weight 1.7x (males), 1.4x (females)
30 mg/m3: mean increase in relative weight 2.3x (males), 2.0x (females)
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes, the degree of severity being treatment-related.
At 6 and 30 mg/m3, collagen content in the lungs was clearly increased, most pronounced in males.

The above-mentioned effects gradually subsided after the exposure period,
but in males exposed to 6 and 30 mg/m3 the collagen content was still above control values at the end of the study.


HISTOPATHOLOGY: NON-NEOPLASTIC
Accumulation of alveolar macrophages and granular material, cellular debris, polymorphonuclear leucocytes, increased septal cellularity.
Alveolar bronchialisation, focal interstitial fibrosis, cholesterol clefts and granuloma-like lesions in the lung.
The granuloma-like lesions were seen in a few animals at the end of exposure period and after 13 weeks of recovery. They did not show fibroblastic activity and hyalinization and regressed during recovery [not progressive, i.e. no silicogenic nodules formed (no silicosis)].

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as focal necrosis and slight atrophy of the olfactory epithelium.

Interstitial fibrosis was not noted directly after the exposure period, but appeared with a delay,
for the first time observed after 13 wks post-exposure: increasing incidence especially in 30-mg rats, and a few in the 6-mg group,
but decreased in severity and frequency until the end of the study (Report p. 51).

All types of pulmonary lesions were more marked in males than in females.


The level of 1.3 mg/m3 induced only slight changes after 13-wk exposure, which generally recovered quickly (no differences from control after 13-wk post-exposure).

Morphological changes after 13-wk exposure, that were considered statistically significant at 1.3 mg/m3:

males females ! males females
treated ! untreated controls
------------------------------------------------------------------------------------------------------------------------------
Accumulation of alveolar macrophages: slight in 10/10 (very) slight in 10/10 ! (very) slight 4/10 slight in 1/10
Intra-alveolar polymorphonuclear !
leukocytes: (very) slight in 6/10 (very) slight in 8/10 ! 0/10 0/10
Increased septal cellularity: ( very) slight in 10/10 (very) slight in 9/10 ! very slight 1/10 very slight 1/10
Olfactory epithelial atrophy: (very) slight in 5/10 (very) slight in 8/10 ! 0/10 0/10
Intracytoplasmic proteinaceous droplets !
-respiratory epithelium: in 8/10 in 9/10 ! 1/10 0/10
Mediastinal lymph node !
-macrophage accumulation: (very) slight in 8/10 (very) slight in 8/10 ! 0/10 0/10
------------------------------------------------------------------------------------------------------------------------------



HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable): no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period:
on the average 0.1 - 0.2 mg per lung of male animal groups (not dose-related), 0.05 - 0.21 mg per lung of female groups (dose-related).
Only one male exposed to 30 mg/m3 showed a small amount of silica in the regional lymph node.
90 days after termination of exposure (day 188), no silica could be recovered from any animal.

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the slight and fully reversible pulmonary response noted at this exposure level (inflammation reaction) (see details below "Details on results")

Key result

Dose descriptor:

LOAEC

Effect level:

5.9 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: (haematology); organ weights (hypertrophy lung); histopathology (collagen increase, sporadic focal fibrosis)

Key result

Dose descriptor:

NOEC

Effect level:

< 1.3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' for more information on the results from this study

Findings for quartz-exposed rats:

Clinical signs:None

Body weight:The body weight of quartz-exposed rats was not affected during the exposure period. The rats showed a slightly progressive reduction in weight gain throughout the post exposure period.

Haematology:There was a statistically significant increase in neutrophilic leucocyte counts during the exposure period. The counts increased during the first 13 weeks after the end of the exposures and remained high during the whole post exposure period. Red blood cell counts, haemoglobin content and packed cell volumes had slightly increased in males exposed to quartz by the end of the exposure period. The males continued to show high red blood cell values throughout the observation period. The remaining haematological parameters that were examined did not show differences that could be related to treatment.

Clinical chemistry:From 13 weeks after exposure alanine aminotransferase activities increased in quartz-exposed rats. In males the increase was approximately 50-90% (p<0.01) in comparison with controls. Alkaline phosphatase activity had increased in rats exposed to quartz 52 weeks after the exposure period. The remaining biochemical parameters that were examined did not show differences that could be related to treatment.

Organ weights:At the end of the exposure period both absolute and relative lung weight were statistically significantly increased compared to controls. The increase was greater in males than in females. Lung weight increased progressively during the post-exposure period to levels three or more times higher than those of control animals. The only other organ that showed increased weight was the thymus. Both the absolute and relative thymus weights were significantly increased (by about 140% of control weight in males at day 188). This increase was most pronounced in males and had disappeared 39 weeks after the end of exposure.

Lung collagen contents:At the end of the exposure period, the lung collagen content of quartz exposed rats was slightly higher than in controls, but in the course of the post-exposure period it had increased markedly.

Silicon in the lungs and associated lymph nodes:Silicon levels in the lungs of males were higher 26 weeks after the end of exposure than at 13 weeks after the end of exposure. Males rats exposed to quartz exhibited even higher values at week 39 than week 26 after exposure. This finding was not observed in females, but this could not be explained by the authors.

Pathology:Most of the rats exposed to quartz and killed at the end of the exposure period had swollen and spotted lungs with a spongy consistency and/or irregular surface and enlarged lung-associated lymph nodes. These changes were more pronounced than in the amorphous silica-exposed rats. The gross lesions in the lungs and lung-associated lymph nodes remained present during the whole post-exposure period. Microscopic changes were mainly observed in the lungs. Changes in rats killed at the end of the exposure period comprised slight to severe accumulation of alveolar macrophages, intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes in the alveolar spaces and increased septal cellularity, seen as an increase in the number of type II pneumocytes and macrophages within the alveolar walls. During the post-exposure period no recovery from lung lesions was observed in quartz-exposed rats. Accumulation of intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes were found in all quartz-exposed rats during the post-exposure period. Alveolar broncholization persisted mainly in quartz-exposed animals. Focal interstitial fibrosis, seen as amorphous eosinophilic, collagen-containing thickenings of the septa, was first observed 13 weeks after exposure and became more severe during the post exposure period. Alveolar cholesterol clefts were observed for the first time after 26 weeks of non-exposure. During the remaining recovery period this lesion became more pronounced. Granulomas, seen as aggregates of macrophage-like cells were observed in nearly all rats exposed to quartz at the end of the exposure period. Slight fibrosis was demonstrated in the granulomas in animals of the quartz group. One year after the end of the exposure period, one male rat had a focus of squamous metaplasia in the periphery of the lung. In addition, in one female a small but unequivocal squamous cell carcinoma was found in the lung parenchyma.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 1

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

Comparable to guideline study with acceptable restrictions in synopsis of the whole testing programme (see Reusal et al (1991) Aero200)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and lymph nodes. Post-exposure recovery period up to one year. One high exposure level only within a combined study (in contrast to Aerosil 200, see other entry).

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline).

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO, Zeist/NL
- Age at study initiation: 4 weeks
- Weight at study initiation: 50 - 70 g
- Fasting period before study: no
- Housing: single during exposure
- Diet: no access during exposure
- Water: no access during exposure
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-1
- Humidity (%): 50 - 70
- Air changes (per hr): 12x/h
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C, minimum 19.1, max. 25.4 °C /
65 - 75 % rel. humidity, during extreme weather occasionally up to 95.5 % or down to 48 %.
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: due to electrostatic charge of the particles not measured:
technical failure of the 10-stage Mercer cascade impactor and the QCM cascade
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically - Air samples are drawn through glass fiber filters (Sartorius)
and weighed (3 - 4 time per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see Report Tables (Part 2), Table 2: Daily mean concentrations are documented:
based on 254 measurements: 34.91 (SEM 0.49) mg/m3

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
35 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
30 mg/m3 (target concentration)
Basis:
nominal conc.

No. of animals per sex per dose:

70
Sipernat 22S: assigned dose groups F, sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica) included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females


URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No particular observations
No mortality

BODY WEIGHT AND WEIGHT GAIN
Slightly decreased body weight, ~ -5 % by 13 wks exposure
Recovery: no significant difference from control at day 455, still ~ -4 % (52 weeks post-exposure)


HAEMATOLOGY
No significant effects, but white blood cell count elevated in both males and females at the end of exposure period ,
but not clearly attributable to increases in the numbers of neutrophilic leukocytes.
After 13 weeks of recovery (day 176/177), neutrophil count still tended to be higher than the control in males and females,
and normalized by 26 weeks of recovery (day 274/275).

CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, adrenals, testes, brain, spleen, kidney
Increased organ weights of lung and thymus at the end of exposure.
Swollen lungs and enlarged mediastinal lypmph nodes
LUNG
Slight mean increase in relative weight: 1.3x (males, females) as compared to control
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes.
The effects gradually subsided after the exposure period:
Lung weight normalised after 13 weeks post-exposure in males and females.


HISTOPATHOLOGY: NON-NEOPLASTIC
In the lung: Accumulation of alveolar macrophages, intra-alveolar polymorphonuclear leukocytes,
and increased septal cellularityin malesand females.
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as very slight focal necrosis and slight atrophy of the olfactory epithelium, intracytoplasmic proteinaceous droplets.

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).

Collagen content in the lungs was slightly increased at the end of exposure.
During the recovery period all changes disappeared mostly within 13 to 26 week.


HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable) no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period (Tables 59):
on the average 0.5 mg per lung of male animal groups, 0.35 mg per lung of female groups, decreasing over time and
no longer measurable after 39 weeks post exposure (day 370).

Dose descriptor:

NOAEC

Basis for effect level:

other: The study was performed using a single dose level and adverse effects were noted at this level

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' in Read Across SAS 01_Degussa 87-0004-DGT_ Aerosil 200_inhal, 13 wk, rat for more information on the results from this study.

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 1

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Comparable to guideline study with acceptable restrictions in synopsis of the whole testing programme (see Reusal et al (1991) Aero200)

Justification for type of information:

The use of data derived for Silicon dioxide are justified for read-across to synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Silicon dioxide and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Special modifications as compared with standard study:  Focus upon lung, respiratory  tract, and lymph nodes. Post-exposure recovery period up to one year. One high exposure level only within a combined study (in contrast to Aerosil 200, see other entry).

Principles of method if other than guideline:

Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline).

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Breeding of Laboratory Animals TNO, Zeist/NL
- Age at study initiation: 4 weeks
- Weight at study initiation: 50 - 70 g
- Fasting period before study: no
- Housing: single during exposure
- Diet: no access during exposure
- Water: no access during exposure
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-1
- Humidity (%): 50 - 70
- Air changes (per hr): 12x/h
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: no monitoring data due to technical difficulties (see above "Details on inhalation exposure")

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m3
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- Method of conditioning air: no data
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Temperature, humidity, pressure in air chamber: av. 21 - 23 °C, minimum 19.1, max. 25.4 °C /
65 - 75 % rel. humidity, during extreme weather occasionally up to 95.5 % or down to 48 %.
- Air flow rate: approx. 40 m3/h
- Air change rate: 40 / 2.3 = ~17/h
- Method of particle size determination: due to electrostatic charge of the particles not measured:
technical failure of the 10-stage Mercer cascade impactor and the QCM cascade
- Treatment of exhaust air: filtered before release


TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically - Air samples are drawn through glass fiber filters (Sartorius)
and weighed (3 - 4 time per day)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see Report Tables (Part 2), Table 2: Daily mean concentrations are documented:
based on 254 measurements: 34.91 (SEM 0.49) mg/m3

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
35 mg/m3 (mean analytical values)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
30 mg/m3 (target concentration)
Basis:
nominal conc.

No. of animals per sex per dose:

70
Sipernat 22S: assigned dose groups F, sub-divided in 7 sub-groups a, b, c , d, e, f, and g
10 each (sacrificed after 13 wks),
50 each kept for a recovery period of at most 52 wks (13, 26, 39, and 52 wks). 

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks

Positive control:

Quartz (crystalline silica) included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: 2x/day, 1x/d (weekends)
- Cage side observations (mortalities) were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see body weight


BODY WEIGHT: Yes
- Time schedule for examinations: start, weekly during exposure, 1x/wk during recovery

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26, 39, 52, 65 (i.e. including recovery period)
- Anaesthetic used for blood collection: No (data)
- Animals fasted: No data
- How many animals: 10 males, 10 females


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14, 27, 40, 53, and 66
- Animals fasted: Yes overnight
- How many animals: 10 males, 10 females


URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26, 40/41, 52, and 65
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Relative organ weights
HISTOPATHOLOGY: Yes, in particular lung and lymph nodes
in addition:
Si contents of lung and lymph nodes
Collagen content in lung

Other examinations:

Relative organ weights

Statistics:

Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No particular observations
No mortality

BODY WEIGHT AND WEIGHT GAIN
Slightly decreased body weight, ~ -5 % by 13 wks exposure
Recovery: no significant difference from control at day 455, still ~ -4 % (52 weeks post-exposure)


HAEMATOLOGY
No significant effects, but white blood cell count elevated in both males and females at the end of exposure period ,
but not clearly attributable to increases in the numbers of neutrophilic leukocytes.
After 13 weeks of recovery (day 176/177), neutrophil count still tended to be higher than the control in males and females,
and normalized by 26 weeks of recovery (day 274/275).

CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, adrenals, testes, brain, spleen, kidney
Increased organ weights of lung and thymus at the end of exposure.
Swollen lungs and enlarged mediastinal lypmph nodes
LUNG
Slight mean increase in relative weight: 1.3x (males, females) as compared to control
LYMPH NODE: no weight data


PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes.
The effects gradually subsided after the exposure period:
Lung weight normalised after 13 weeks post-exposure in males and females.


HISTOPATHOLOGY: NON-NEOPLASTIC
In the lung: Accumulation of alveolar macrophages, intra-alveolar polymorphonuclear leukocytes,
and increased septal cellularityin malesand females.
Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period,
such as very slight focal necrosis and slight atrophy of the olfactory epithelium, intracytoplasmic proteinaceous droplets.

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).

Collagen content in the lungs was slightly increased at the end of exposure.
During the recovery period all changes disappeared mostly within 13 to 26 week.


HISTOPATHOLOGY: NEOPLASTIC
No particular findings


HISTORICAL CONTROL DATA (if applicable) no data


OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period (Tables 59):
on the average 0.5 mg per lung of male animal groups, 0.35 mg per lung of female groups, decreasing over time and
no longer measurable after 39 weeks post exposure (day 370).

Dose descriptor:

NOAEC

Basis for effect level:

other: The study was performed using a single dose level and adverse effects were noted at this level

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Please refer to attached document 'Tables and figures' in Read Across SAS 01_Degussa 87-0004-DGT_ Aerosil 200_inhal, 13 wk, rat for more information on the results from this study.

Reference 5

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Only one exposure concentration used. Target organ effects focused on specific investigations based on pulmonary effects (lung lavage cytology and biochemistry) therefore, histopathology is limited and clinical chemistry/haematology is not included.

Principles of method if other than guideline:

- Principle of test: Comparative study examining pulmonary inflammatory cell responses to synthetic amorphous and crystalline silica

- Short description of test conditions: Rats were exposed to amorphous and crystalline silica
for up to 13 weeks. Effects to the lung were examined at 6.5 and 13 weeks of exposure and 3 and 8 months recovery. The study utilized lung burden analysis, Bronchoalveolar Lavage Fluid Analysis (BAL), histopathology of the lung tissue,  MIP-2 gene expression, TUNEL staining and Type II cell isolation and HPRT assays to examine the effects of exposure.

- Parameters analysed / observed: Cellular and biochemical lung damage and inflammation.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 200 - 250 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Mass median aerodynamic diameter (MMAD):

0.81 µm

Remarks on MMAD:

MMAD / GSD: mass median diameter - crystalline silica: 1.3 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: compartmentalized, 300-liter horizontal laminar flow chamber
- Source and rate of air: no data
- Method of conditioning air: The aersol was brought to Boltzman equilibrium by passing the airbourne particles across a 20-mCi 85K source
- System of generating particulates/aerosols: screw-feed mechanism (ACCURate, Whitewater, WI) in combination with a Venturi type dust feeder
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: no data
- Samples taken from breathing zone: no data

VEHICLE: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentration: The average aerosol concentration for the amorphous silica groups was 50.4 mg/m3, SD +-19 mg/m3.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6h/d, 5d/wk

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

amorphous silica

No. of animals per sex per dose:

no data, for single analytical endpoint: n =4

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: 3 and 8 months

Positive control:

Quartz (crystalline silica): Cristobalite, 3 mg/m3
The average aerosol concentration for the Crystalline silica groups was 3 mg/m3 with +-1.0 mg/m3.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: Lactate dyhydrogenase (LDH), protein, glucuronidase

LUNG BURDEN: Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: ug SiO2/Lung (Amorphous/crystalline)

OTHER: Histopathology
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: alveolitis, severity of inflammation in bronchioles and ronchi, fibrosis and relative amount of lung parenchyma.

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes - Scored and ranked on severity: 0.0 (no significant lesions) - 4.0 (very severe process). Results were summed to determine a summary toxicity score.

Other examinations:

Expression of MIP-2 mRNA in lungs was assessed. RNA transcript levels were assessed by PCR amplification of the MIP-2 cDNA.
Immunohistochemistry - Terminal transferase dUTP nick-end-labelling (TUNEL)-staining was carried out on lung sections to identify the extent of potential apoptosis/necrosis resulting from DNA damage.

Statistics:

Analysis of variance, Dunnett´s test for determination of differences between control and treated groups.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased numbers of neutrophils and macrophages were noted at 45 days of treatment and 90 days treatment. Numbers then decreased over the post-exposure period.
Fibrosis detected by Gormer's trichome staining was present in the alveolar septa of the silica treated lungs. Fibrosis subsided during recovery in the case of synthetic amorphous silica.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL Anyalysis:
Significant changes were noted in all BAL parameters with total cell numbers. PMN, protein and glucuronidase levels in BAL greater in the amorphous silica exposed groups than the crystalline silica exposure groups. Changes in the amorphous silica groups returned close to control levels by the end of the 3 month recovery period and were not significantly different by the end of the 8 month recovery period. BAL changes in the crystalline silica groups persisted throughout the post exposure period.
Table 1. presents the results of the BAL fluid analysis.

LUNG SILICA BURDENS:
Group-mean lung burdens were 882.7 mg/lung for amorphous silica. At 3 months post exposure, amorphous silica burdens were significantly decreased in comparison to the end of exposure and decreased further by 8 months of exposure. Crystalline silica burdens post-exposure were relatively unchanged from the end of exposure. Table 2. provides a summary of the silica lung burden results.

GENE EXPRESSION of MIP-2 (inflammatory cytokine gene):
MIP-2 was evident at the end of the 13 weeks exposure (amorphous and crystalline silica). At the end of the 8 month recovery period MIP-2 mRNA was detected in the lungs of rats exposed to crystalline silica. There was minimal detection of MIP-2 mRNA in the lungs of rats exposed to amorphous silica after the same period of recovery. Minimal or no detectable MIP-2 m-RNA expression was found in control rats.

IMMUNOHISTOPATHOLOGY:
After 13-wk exposure to synthetic amorphous silica, intensely stained TUNEL-positive cells were detected throughout the terminal bronchiolar epithelium and throughout the parenchyma of rat lungs. Only a small amount of staining was seen after exposure to crystalline silica. TUNEL-staining was indistinguishable between the amorphous-treated and the control group during the recovery period. In contrast, lungs exposed to crystalline silica showed intense staining localized to cell debris and macrophages in hypertrophic areas of the parenchyma cells.

Details on results:

Table 1.
Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

Remarks on result:

not determinable

Critical effects observed:

not specified

Table 1.

Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

	Total cells x 10^7	% AM	% PMN	% Lymph	% Viable	Protein (µg/ml)	LDH (nmol/min/ml)	Β Glucuronidase (nmol/min/ml)
Sham exposure
Week 6.5	1.09 +/- 0.23	99.7 +/- 0.36	0.24 +/- 0.23	0.77 +/- 0.13	93.8 +/- 2.74	0.16 +/- 0.6	56.2 +/- 14.7	0.47 +/- 0.11
Week 13	2.89 +/- 0.5	98.4 +/- 1.9	0.26 +/- 0.24	0.64 +/- 0.53	94.6 +/- 0.63	0.26 +/- 0.05	11.7 +/- 46.0	0.53 +/- 0.01
Recovery
Week 12	1.76 +/- 0.24	98.1 +/- 0.47	0.65 +/- 0.52	1.22 +/- 0.33	92.9 +/- 1.1	0.21 +/- 0.04	47.9 +/- 9.5	0.53 +/- 0.07
Week 32	1.20 +/- 0.11	98.8 +/- 0.43	0.73 +/- 0.34	0.48 +/- 0.18	94.0 +/- 2.4	0.175 +/- 0.02	41.6 +/- 6.5	0.26 +/- 0.06
Crystalline SiO2 exposure
Week 6.5	8.5 +/- 0.54*	64.7 +/- 2.5*	32.7 +/- 2.7*	2.6 +/- 1.4	95.2 +/- 1.3	0.513 +/- 0.04*	314.6 +/- 16.9*	4.9 +/- 0.8*
Week 13	13.9 +/- 1.2*	50.7 +/- 5.9*	46.9 +/- 5.4*	2.4 +/- 1.6	94.6 +/- 1.9	1.18 +/- 0.09*	798.5 +/- 165.6*	21.3 +/- 2.8*
Recovery
Week 12	19.2 +/- 2.3*	58.7 +/- 8.7*	38.1 +/- 8.5*	3.2 +/- 1.4	95.7 +/- 0.9	1.03 +/- 0.12*	809.5 +/- 196.3*	20.4 +/- 3.0*
Week 32	16.2 +/- 1.7*	64.9 +/- 2.4*	30.8 +/- 2.6*	4.3 +/- 1.6	94.5 +/- 1.4	0.93 +/- 0.14*	1188.2 +/- 163.5*	11.6 +/- 1.85*
Amorphous SiO2
Week 6.5	16.8 +/- 0.54*	42.8 +/- 4.9*	55.2 +/- 4.8*	2.0 +/- 0.8	97.0 +/- 0.9	0.94 +/- 0.08*	709.4 +/- 101.0*	19.3 +/- 3.0*
Week 13	16.9 +/- 2.2*	42.6 +/- 2.9*	55.3 +/- 2.2*	2.1 +/- 1.2	94.2 +/- 1.4	1.59 +/- 0.08*	1808.0 +/- 631.6*	29.2 +/- 2.5*
Recovery
Week 12	2.7 +/- 0.82	88.8 +/- 0.9	9.3 +/- 0.9*	1.9 +/- 0.4	93.8 +/- 1.5	0.386 +/- 0.06	192.1 +/- 57.6*	1.1 +/- 0.19
Week 32	2.2 +/- 0.08	94.9 +/- 1.1	2.6 +/- 1.3	2.5 +/- 0.5	94.2 +/- 2.4	0.339 +/- 0.02	152.0 +/- 68.8	0.46 +/- 0.06

* Significantly different from age-matched control group; p # 0.05.

 

Table 2.

Lung Burdens after Subchronic Exposure of Rats to Crystalline and Amorphous Silicas (µg SiO₂/lung)

		Weeks of Exposure				Weeks of Exposure
Treatment Group		6.5		13		12		32
Control		55.9 +/- 40.4		42.5 +/- 16.9		28.1 +/- 13		39.8 +/- 8.7
Crystalline, 3 mg/m^3		335.6 +/- 28.3*		819.0 +/- 83.3*		657.6 +/- 28*		743.0 +/- 14.5*
Amorphous, 50 mg/m^3		755.9 +/- 22.9*		882.7 +/- 83.1*		156.0 +/- 38.6*		92.6 +/- 38.6

 

Note. Values represent the ⴟ +/- SD; n = 4 rats/treatment.

* Significantly different from age-matched control group; p # 0.05.

Reference 6

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The use of data derived for Silicon dioxide are justified for read-across to synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Silicon dioxide and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Only one exposure concentration used. Target organ effects focused on specific investigations based on pulmonary effects (lung lavage cytology and biochemistry) therefore, histopathology is limited and clinical chemistry/haematology is not included.

Principles of method if other than guideline:

- Principle of test: Comparative study examining pulmonary inflammatory cell responses to synthetic amorphous and crystalline silica

- Short description of test conditions: Rats were exposed to amorphous and crystalline silica
for up to 13 weeks. Effects to the lung were examined at 6.5 and 13 weeks of exposure and 3 and 8 months recovery. The study utilized lung burden analysis, Bronchoalveolar Lavage Fluid Analysis (BAL), histopathology of the lung tissue,  MIP-2 gene expression, TUNEL staining and Type II cell isolation and HPRT assays to examine the effects of exposure.

- Parameters analysed / observed: Cellular and biochemical lung damage and inflammation.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 200 - 250 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Mass median aerodynamic diameter (MMAD):

0.81 µm

Remarks on MMAD:

MMAD / GSD: mass median diameter - crystalline silica: 1.3 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: compartmentalized, 300-liter horizontal laminar flow chamber
- Source and rate of air: no data
- Method of conditioning air: The aersol was brought to Boltzman equilibrium by passing the airbourne particles across a 20-mCi 85K source
- System of generating particulates/aerosols: screw-feed mechanism (ACCURate, Whitewater, WI) in combination with a Venturi type dust feeder
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: no data
- Samples taken from breathing zone: no data

VEHICLE: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentration: The average aerosol concentration for the amorphous silica groups was 50.4 mg/m3, SD +-19 mg/m3.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6h/d, 5d/wk

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

amorphous silica

No. of animals per sex per dose:

no data, for single analytical endpoint: n =4

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: 3 and 8 months

Positive control:

Quartz (crystalline silica): Cristobalite, 3 mg/m3
The average aerosol concentration for the Crystalline silica groups was 3 mg/m3 with +-1.0 mg/m3.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: Lactate dyhydrogenase (LDH), protein, glucuronidase

LUNG BURDEN: Yes
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: ug SiO2/Lung (Amorphous/crystalline)

OTHER: Histopathology
- Time schedule for analysis: 6.5 and 13 weeks exposure; 12 and 32 weeks recovery
- Dose groups that were examined: All groups
- Number of animals: Not specified
- Parameters checked: alveolitis, severity of inflammation in bronchioles and ronchi, fibrosis and relative amount of lung parenchyma.

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes - Scored and ranked on severity: 0.0 (no significant lesions) - 4.0 (very severe process). Results were summed to determine a summary toxicity score.

Other examinations:

Expression of MIP-2 mRNA in lungs was assessed. RNA transcript levels were assessed by PCR amplification of the MIP-2 cDNA.
Immunohistochemistry - Terminal transferase dUTP nick-end-labelling (TUNEL)-staining was carried out on lung sections to identify the extent of potential apoptosis/necrosis resulting from DNA damage.

Statistics:

Analysis of variance, Dunnett´s test for determination of differences between control and treated groups.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased numbers of neutrophils and macrophages were noted at 45 days of treatment and 90 days treatment. Numbers then decreased over the post-exposure period.
Fibrosis detected by Gormer's trichome staining was present in the alveolar septa of the silica treated lungs. Fibrosis subsided during recovery in the case of synthetic amorphous silica.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL Anyalysis:
Significant changes were noted in all BAL parameters with total cell numbers. PMN, protein and glucuronidase levels in BAL greater in the amorphous silica exposed groups than the crystalline silica exposure groups. Changes in the amorphous silica groups returned close to control levels by the end of the 3 month recovery period and were not significantly different by the end of the 8 month recovery period. BAL changes in the crystalline silica groups persisted throughout the post exposure period.
Table 1. presents the results of the BAL fluid analysis.

LUNG SILICA BURDENS:
Group-mean lung burdens were 882.7 mg/lung for amorphous silica. At 3 months post exposure, amorphous silica burdens were significantly decreased in comparison to the end of exposure and decreased further by 8 months of exposure. Crystalline silica burdens post-exposure were relatively unchanged from the end of exposure. Table 2. provides a summary of the silica lung burden results.

GENE EXPRESSION of MIP-2 (inflammatory cytokine gene):
MIP-2 was evident at the end of the 13 weeks exposure (amorphous and crystalline silica). At the end of the 8 month recovery period MIP-2 mRNA was detected in the lungs of rats exposed to crystalline silica. There was minimal detection of MIP-2 mRNA in the lungs of rats exposed to amorphous silica after the same period of recovery. Minimal or no detectable MIP-2 m-RNA expression was found in control rats.

IMMUNOHISTOPATHOLOGY:
After 13-wk exposure to synthetic amorphous silica, intensely stained TUNEL-positive cells were detected throughout the terminal bronchiolar epithelium and throughout the parenchyma of rat lungs. Only a small amount of staining was seen after exposure to crystalline silica. TUNEL-staining was indistinguishable between the amorphous-treated and the control group during the recovery period. In contrast, lungs exposed to crystalline silica showed intense staining localized to cell debris and macrophages in hypertrophic areas of the parenchyma cells.

Details on results:

Table 1.
Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

Remarks on result:

not determinable

Critical effects observed:

not specified

Table 1.

Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery (ⴟ +/- SD; n = 4)

	Total cells x 10^7	% AM	% PMN	% Lymph	% Viable	Protein (µg/ml)	LDH (nmol/min/ml)	Β Glucuronidase (nmol/min/ml)
Sham exposure
Week 6.5	1.09 +/- 0.23	99.7 +/- 0.36	0.24 +/- 0.23	0.77 +/- 0.13	93.8 +/- 2.74	0.16 +/- 0.6	56.2 +/- 14.7	0.47 +/- 0.11
Week 13	2.89 +/- 0.5	98.4 +/- 1.9	0.26 +/- 0.24	0.64 +/- 0.53	94.6 +/- 0.63	0.26 +/- 0.05	11.7 +/- 46.0	0.53 +/- 0.01
Recovery
Week 12	1.76 +/- 0.24	98.1 +/- 0.47	0.65 +/- 0.52	1.22 +/- 0.33	92.9 +/- 1.1	0.21 +/- 0.04	47.9 +/- 9.5	0.53 +/- 0.07
Week 32	1.20 +/- 0.11	98.8 +/- 0.43	0.73 +/- 0.34	0.48 +/- 0.18	94.0 +/- 2.4	0.175 +/- 0.02	41.6 +/- 6.5	0.26 +/- 0.06
Crystalline SiO2 exposure
Week 6.5	8.5 +/- 0.54*	64.7 +/- 2.5*	32.7 +/- 2.7*	2.6 +/- 1.4	95.2 +/- 1.3	0.513 +/- 0.04*	314.6 +/- 16.9*	4.9 +/- 0.8*
Week 13	13.9 +/- 1.2*	50.7 +/- 5.9*	46.9 +/- 5.4*	2.4 +/- 1.6	94.6 +/- 1.9	1.18 +/- 0.09*	798.5 +/- 165.6*	21.3 +/- 2.8*
Recovery
Week 12	19.2 +/- 2.3*	58.7 +/- 8.7*	38.1 +/- 8.5*	3.2 +/- 1.4	95.7 +/- 0.9	1.03 +/- 0.12*	809.5 +/- 196.3*	20.4 +/- 3.0*
Week 32	16.2 +/- 1.7*	64.9 +/- 2.4*	30.8 +/- 2.6*	4.3 +/- 1.6	94.5 +/- 1.4	0.93 +/- 0.14*	1188.2 +/- 163.5*	11.6 +/- 1.85*
Amorphous SiO2
Week 6.5	16.8 +/- 0.54*	42.8 +/- 4.9*	55.2 +/- 4.8*	2.0 +/- 0.8	97.0 +/- 0.9	0.94 +/- 0.08*	709.4 +/- 101.0*	19.3 +/- 3.0*
Week 13	16.9 +/- 2.2*	42.6 +/- 2.9*	55.3 +/- 2.2*	2.1 +/- 1.2	94.2 +/- 1.4	1.59 +/- 0.08*	1808.0 +/- 631.6*	29.2 +/- 2.5*
Recovery
Week 12	2.7 +/- 0.82	88.8 +/- 0.9	9.3 +/- 0.9*	1.9 +/- 0.4	93.8 +/- 1.5	0.386 +/- 0.06	192.1 +/- 57.6*	1.1 +/- 0.19
Week 32	2.2 +/- 0.08	94.9 +/- 1.1	2.6 +/- 1.3	2.5 +/- 0.5	94.2 +/- 2.4	0.339 +/- 0.02	152.0 +/- 68.8	0.46 +/- 0.06

* Significantly different from age-matched control group; p # 0.05.

 

Table 2.

Lung Burdens after Subchronic Exposure of Rats to Crystalline and Amorphous Silicas (µg SiO₂/lung)

		Weeks of Exposure				Weeks of Exposure
Treatment Group		6.5		13		12		32
Control		55.9 +/- 40.4		42.5 +/- 16.9		28.1 +/- 13		39.8 +/- 8.7
Crystalline, 3 mg/m^3		335.6 +/- 28.3*		819.0 +/- 83.3*		657.6 +/- 28*		743.0 +/- 14.5*
Amorphous, 50 mg/m^3		755.9 +/- 22.9*		882.7 +/- 83.1*		156.0 +/- 38.6*		92.6 +/- 38.6

 

Note. Values represent the ⴟ +/- SD; n = 4 rats/treatment.

* Significantly different from age-matched control group; p # 0.05.

Reference 7

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The initial sample used in this study contained 45% of cristobalite and approximately 55% of amorphous silica. The respirable fraction was 3.8% including 1.8% crystalline silica. In order to be compliant with the OECD TG 412 (which recommends the generation of aerosols with mass median aerodynamic diameters ranging from 1 to 3 μm), the test material was micronized to allow that the majority of material/particles could reach the deep lung (alveoli). The test method of processing of the material resulted in the content of respirable crystalline silica being increased from 1.8% to approximately 45%, which is approximately double the worse-case level found in commercially produced material. Under these conditions, the toxicity observed in the study material reflects and confirms the health hazards of extremely high concentrations of the crystalline silica. However, as stated above, the study material is not reflective of the toxicity of, or risk factor associated with, the much lower concentration of crystalline silica contained in commercially available product, the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: males: 8 weeks; females 9 weeks (at delivery)
- Weight at study initiation: males: 216.7-294.3 g; females 169.1-209.1 g (at acclimatization)
- Fasting period before study: n/a
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment.
- Diet: Pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations..
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except during the periods when the animals were restrained in the
exposure tubes.
- Acclimation period: Eight or nine days under test conditions after clinical health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2 and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The the group means of the chemically determined mass median aerodynamic diameters (MMAD) were within the target range of 1 to 3 μm. Single measurements slightly above the upper limit were considered to be acceptable. The corresponding geometric standard deviations (GSD) were also within the target range of 1.5 to 3. The results of the gravimetric determinations were considered to be comparable.
Overall, deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. Hence, the particle size distribution and MMADs obtained were considered to be appropriate.
The values for gravimetrically and chemically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) for Soda-ash flux-calcined kieselguhr are shown in Tables 1 & 2 below.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system. Ports for animal exposure are positioned radially around the nose-only, flow-past exposure chamber on several different levels.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR 3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. The generated test aerosol was diluted as necessary with compressed air to achieve the concentrations required for this study.
- Temperature in air chamber: 22.6/-0.2 - 23.2 +/- 0.2 deg C
- Relative humidity: 1.2 +/- 0.9 - 5.0 +/- 1.7 %
- Air flow rate: The flow of air at each tube was 1.0 L/min
- Method of particle size determination: The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. The airflow rate through the impactor was 1 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentrations were determined gravimetrically and chemically (please refer to detailed description below)

VEHICLE
Compressed air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric determination of the aerosol concentration was performed six times during each exposure for groups 2 to 4. Test aerosol samples were collected onto Millipore®durapore filters, Type HVLP using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port. The duration of sampling was sufficient to ensure reliable results. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
To determine the concentration of the test item based on the Silicon in the generated aerosol chemical analyses of test aerosol samples were performed twice per week and on two occasions of these selected exposures for groups 2 to 4. Filters sampled for gravimetric determination were used for chemical determination. An appropriate number of samples collected were transferred into labeled appropriate vials, forwarded at ambient temperature to the scientist responsible for formulation analysis and stored at room temperature (range of 20 ± 5 °C) until analysis. They were analyzed for Silicon by Harlan Laboratories Ltd. using an AAS method developed by Harlan Laboratories Ltd.

Duration of treatment / exposure:

6 hours/exposure

Frequency of treatment:

5 days/week at approximately 24 hour intervals for four consecutive weeks.
Following the treatment period there was a 9 week recovery period.

Remarks:

Doses / Concentrations:
0.044 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0.207 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0.700 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

20 animals/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale:Target Dose Levels were selected based on data from the 5 day dose range finding inhalation toxicity study. In the 5-day inhalation toxicity study, rats were dosed with target concentrations of 0, 0.20, 0.60 and 2.0 mg/L of the test item. A statistical significant increase in absolute lungs weights was observed at the mid dose in females and at the high dose in males and females. At the other doses, males and females already showed an increase in lung weight although not statistically significant compared to controls. According to the results of this preliminary study,
the dose-levels of 0.050, 0.20 and 0.80 mg/L were selected for the current study.
- Rationale for animal assignment: random

Positive control:

No positive control was used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily during acclimatization and recovery and twice dily during treatment.
- Cage side observations: mortality, viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly during acclimatization and recovery. On exposure days, once before, during and after the daily treatment, and once daily on non-exposure days of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatization and recovery, and twice weekly during treatment.

FOOD CONSUMPTION:
- Food consumption: Recorded (per cage) weekly during acclimatization, treatment and recovery.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatization (main study and recovery animals) and week 4 of treatment (main study animals).
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 18 hours before sampling
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a
- Battery of functions tested: sensory activity / grip strength / motor activity / other: n/a

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Other examinations:

BRONCHO-ALVEOLAR LAVAGE:

5 animals/sex/group were sacrificed approximately 24 h after the last exposure and a further 5 animals/sex/group were sacrificed approximately 24 h after 9-week recovery. The lungs of each dose group were washed six times with approximately 4 mL physiological saline at room temperature by slow instillation and withdrawal of fluid, with thoracic massage.
The following parameters were determined:
- Enzymatic activities of lactate dehydrogenase and alkaline phosphatase which were used as indicators of possible plasma membrane damage and/or cell lysis.
- Total protein which was indicative of inflammatory processes and damage to the alveolar capillary barrier.
- Phospholipids which was indicative of disturbances in the metabolic activity of type II epithelial cells.

Statistics:

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios, ophthalmoscopic examination as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for
the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied to the ophthalmoscopic and macroscopic findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled treatment and recovery periods. No clinical signs were noted during the course of this study.

BODY WEIGHT AND WEIGHT GAIN
Marginal mean body weight loss was noted between day 1 and day 4 of treatment in high dose group males attaining statistical significance for the body weight gain.
These animals gained weight thereafter and a dose relation was no longer observed from week 3 onwards. Accordingly, further statistical significant differences for the body weight development for low and high dose group males were considered to be incidental. Body weight gain in females was
similar across all groups.

FOOD CONSUMPTION
There were no effects on the food consumption that were considered to be related to treatment. Statistically significant decreased food intake on some occasions for males of the low and mid dose groups during the treatment period were considered to be incidental in the absence of a dose relationship. A statistically significantly increased food intake on same occasions for males of the mid and high dose groups was also considered to be fortuitous.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
No ophthalmoscopic findings were observed which were considered to be related to the treatment.
Corneal opacity observed at the end of the treatment period in some animals across all groups was already present before exposure start.

HAEMATOLOGY
At the end of the treatment period the absolute and relative neutrophil levels were increased in males and females of all three groups treated with the test substance. Statistical significance was attained in the mid and high dose groups for the absolute or relative levels in females and males, respectively.
At the end of the recovery period the effects on the neutrophils increased and a clear dose relation ship was recorded for the absolute levels. Statistical significant differences to controls were recorded for the relative levels in males in all groups treated with the test substance and for the absolute levels in males
and females in all groups treated with the test substance (except for low dose males). In addition, the absolute monocyte levels were statistically significantly increased in mid dose females and in both sexes at the high dose. Accordingly, the total leukocyte count was also increased in males and females in all groups treated with the test substance, attaining statistical significance in the high dose group.

CLINICAL CHEMISTRY
Aspartate aminotransferase was increased in both sexes in the high dose group at the end of the recovery period. Statistical significance was attained for males. Statistically significant differences in cholesterol, triglycerides and phospholipids at either the end of the treatment period or the 9 week recovery period were within the range of historical control data and therefore not considered to be related to treatment.

URINALYSIS
There were no findings in urinalysis that were considered to be test item-related.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Absolute and related lung weights were dose-dependently increased in both sexes in all groups treated with the test substance at the end of treatment. Statistical significance was obtained for the mid and high dose groups.
After the end of recovery, the there was a further dose-dependent increase in absolute and relative lungs weights and statistical significance was obtained for both sexes in all groups treated with the test substance (except for the absolute lung weight and the lung weight to body ratio for low dose females). In addition, the absolute and relative spleen weights were increased in both sexes in the high dose group, attaining statistical significance for males. Absolute and relative adrenal weights were increased in high dose males, attaining statistical significance for the absolute weight and the adrenal to brain weight ratio. Absolute and relative liver weights were increased in high dose females, attaining statistical significance for the liver to body weight ratio.
There were no further changes in organ weights that were considered to be test item-related. Single statistically significant differences between treated groups and controls were considered to be incidental as there was no dose-relationship and/or alterations were not consistently present in both sexes.

GROSS PATHOLOGY
The tracheobronchial/mediastinal lymph nodes were recorded as enlarged in two mid dose females and one male and three females in the high dose group at the end of the treatment period and in all animals treated with the test substance after the end of the recovery period.
All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
After the end of treatment, diffuse alveolar histiocytosis in the lungs was noted at a dose dependently increased severity in all treated animals. Amorphous material (considered to be the test item) was noted at a dose-dependently increased incidence and severity in mid and high dose males and females. In addition, perivascular/peribronchial lymphoid hyperplasia was noted some high dose animals.
In the tracheobronchial lymph nodes, histiocytic granulomas were noted in most treated animals at a dose-dependent severity. This finding was generally accompanied by lymphoid hyperplasia.
After the end of recovery period diffuse alveolar histiocytosis, the amorphous material and the perivascular/peribronchial lymphoid hyperplasia were still present in the lungs. However, the severity grade for the alveolar histiocytosis increased in all treatment groups and the incidences and the severity for the amorphous material were similar but this finding was additionally noted in two low dose females. In addition, the severity grade incidence for the lymphoid hyperplasia increased in the high dose group and this finding was noted in one low dose male and some mid dose animals. This hyperplasia was accompanied by histiocyte granulomas, partly with dose-dependent fibrosis. Finally, microgranulomas at the bronchiolar-alveolar junction at a dose-dependent severity were
noted in most treated animals.
In the tracheobronchial lymph nodes, histiocytic granulomas and lymphoid hyperplasia were still present but the severity increased as well as the incidence to all animals. In addition, there was fibrosis present in all these lymph nodes at a generally dose-dependently increased severity.
The other findings noted were within the range of incidental background lesions commonly recorded in animals of this strain and age.

HISTORICAL CONTROL DATA
Available for Haematology, Clinical chemistry and urinalysis. attached as file historical data.pdf

BRONCHO-ALVEOLAR LAVAGE
Lactate dehydrogenase was increased in the broncho-alveolar lavage fluid at the end of treatment in both sexes of all treated groups, attaining statistical significance in high dose males and in low and mid dose females. In addition, phospholipids and alkaline phosphatase were also increased in males and females but only attaining statistical significance on some occasions in mid and high dose groups. Finally, total protein was increased in the high dose group attaining statistical significance in females.
In addition, the number of cells in the broncho-alveolar lavage fluid increased dose-dependently in all reated groups attaining statistical significance in both sexes of the mid and high dose groups. This increase resulted mainly from macrophages and neutrophils but the absolute number of lymphocytes were also increased. The statistical significant increase of the cell viability in mid and high dose males was considered to be a consequence of these increased neutrophil levels.
After the end of the recovery period, completed recovery was noted for alkaline phosphatase and partial recovery was noted for phospholipids and lactate dehydrogenase: phospholipids were increased at the end of treatment in mid dose males and in both sexes at the high dose, attaining
statistical significance in the high dose group; lactate dehydrogenase remained increased in low and mid dose males and both sexes at the high dose, attaining statistical significance in this group.
A progression of the effects was noted for total protein and cell counts in the broncho-alveolar lavage fluid: Total protein was dose-dependently increased in males in all treated groups and in mid and high dose females attaining statistical significance in high dose males and in mid and high dose females. The number of cells increased in a dose-dependent manner and reached statistical significance in all reatment groups with a similar differential cell count as at the end of treatment.

Some further inter-group variations in hematology, clinical biochemistry, urinalysis, and broncho-alveolar lavage parameters occasionally achieved statistical significance reflecting changes that were considered to be in the normal range, displayed no dose-relationship and/or were not consistently present in both sexes. These changes were considered not to be test item related.
In addition, statistical significant decreases for differential cell counts were a consequence of the increase of neutrophils.

Dose descriptor:

NOAEC

Basis for effect level:

other: On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Summary tables for the results of examinations are attached as Summary tables Part 1.pdf and Summary tables Part 2.pdf

Executive summary:

In this 28-day repeat dose inhalation study performed according to OECD TG 412 under GLP, kieselguhr soda ash flux-calcined was administered to Wistar rats (20 animals/sex/dose) by nose-only, flow-past inhalation for a period of 5 days/week (6 hours/day) for 4 consecutive weeks at aerosol concentrations of 0.044, 0.207 and 0.700 mg/L air.

 

The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 9-week treatment-free recovery period following the treatment period in selected animals of all groups. Sub-groups of male and female animals served for determination of broncho-alveolar lavage fluid (BALF) parameters on day 28 of treatment or were kept for a 9-week off-dose period. Mortality, clinical signs, body weight, food consumption, organ weights, macroscopic and microscopic findings were recorded and ophthalmoscopic examinations and clinical laboratory investigations were performed. BALF samples were collected after the end of treatment and after recovery.

 

No premature deaths and no clinical signs were observed except for a slight and transient effect on body weight gain at the high dose. A dose-dependent increase in lung weights was recorded from the low dose to the high dose at the end of the treatment. A further increase in lung weights and lymph nodes had increased in size at the end of the recovery period. An increase in spleen, adrenals and liver weights at the high-dose at the end of the recovery period was observed but the relevance of these findings was not certain. In the alveoli, histiocytosis was observed increasing in incidence and severity from the low dose to the high dose at the end of the treatment. A progression in incidence and severity was also observed to occur after the recovery period. The presence of the test item was detected in the alveoli of the animals of the mid and high dose groups at the end of the treatment period. This was observed to persist at least until the end of the recovery period and additionally it was observed in 2/10 animals of the low dose recovery group.

 

In the observations on the peribronchial/perivascular zone, lymphoid hyperplasia was observed in the high dose group animals at the end of the treatment period. There was a progression in severity after the recovery period and additionally it was observed in some mid-dosed animals of the recovery group. The occurrence of microgranuloma and fibrosis was observed at the end of the treatment-free period. For the tracheobronchial lymph nodes, histiocytic granuloma was observed increasing in severity from the low dose to the high dose at the end of the treatment. There was a progression in incidence and severity as shown by the occurrence of fibrosis after the recovery period.

There were no adverse effects detected in organs other than the lungs. Some adaptive responses may have been induced in the liver, adrenals and spleen in response to the irritation and damage to the lung tissue.

 

On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Reference 8

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The initial sample used in this study contained 45% of cristobalite and approximately 55% of amorphous silica. The respirable fraction was 3.8% including 1.8% crystalline silica. In order to be compliant with the OECD TG 412 (which recommends the generation of aerosols with mass median aerodynamic diameters ranging from 1 to 3 μm), the test material was micronized to allow that the majority of material/particles could reach the deep lung (alveoli). The test method of processing of the material resulted in the content of respirable crystalline silica being increased from 1.8% to approximately 45%, which is approximately double the worse-case level found in commercially produced material. Under these conditions, the toxicity observed in the study material reflects and confirms the health hazards of extremely high concentrations of the crystalline silica. However, as stated above, the study material is not reflective of the toxicity of, or risk factor associated with, the much lower concentration of crystalline silica contained in commercially available product, the registered substance.

Justification for type of information:

The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: males: 8 weeks; females 9 weeks (at delivery)
- Weight at study initiation: males: 216.7-294.3 g; females 169.1-209.1 g (at acclimatization)
- Fasting period before study: n/a
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment.
- Diet: Pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations..
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except during the periods when the animals were restrained in the
exposure tubes.
- Acclimation period: Eight or nine days under test conditions after clinical health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2 and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The the group means of the chemically determined mass median aerodynamic diameters (MMAD) were within the target range of 1 to 3 μm. Single measurements slightly above the upper limit were considered to be acceptable. The corresponding geometric standard deviations (GSD) were also within the target range of 1.5 to 3. The results of the gravimetric determinations were considered to be comparable.
Overall, deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. Hence, the particle size distribution and MMADs obtained were considered to be appropriate.
The values for gravimetrically and chemically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) for Soda-ash flux-calcined kieselguhr are shown in Tables 1 & 2 below.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system. Ports for animal exposure are positioned radially around the nose-only, flow-past exposure chamber on several different levels.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR 3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. The generated test aerosol was diluted as necessary with compressed air to achieve the concentrations required for this study.
- Temperature in air chamber: 22.6/-0.2 - 23.2 +/- 0.2 deg C
- Relative humidity: 1.2 +/- 0.9 - 5.0 +/- 1.7 %
- Air flow rate: The flow of air at each tube was 1.0 L/min
- Method of particle size determination: The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. The airflow rate through the impactor was 1 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentrations were determined gravimetrically and chemically (please refer to detailed description below)

VEHICLE
Compressed air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric determination of the aerosol concentration was performed six times during each exposure for groups 2 to 4. Test aerosol samples were collected onto Millipore®durapore filters, Type HVLP using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port. The duration of sampling was sufficient to ensure reliable results. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
To determine the concentration of the test item based on the Silicon in the generated aerosol chemical analyses of test aerosol samples were performed twice per week and on two occasions of these selected exposures for groups 2 to 4. Filters sampled for gravimetric determination were used for chemical determination. An appropriate number of samples collected were transferred into labeled appropriate vials, forwarded at ambient temperature to the scientist responsible for formulation analysis and stored at room temperature (range of 20 ± 5 °C) until analysis. They were analyzed for Silicon by Harlan Laboratories Ltd. using an AAS method developed by Harlan Laboratories Ltd.

Duration of treatment / exposure:

6 hours/exposure

Frequency of treatment:

5 days/week at approximately 24 hour intervals for four consecutive weeks.
Following the treatment period there was a 9 week recovery period.

Remarks:

Doses / Concentrations:
0.044 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0.207 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

20 animals/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale:Target Dose Levels were selected based on data from the 5 day dose range finding inhalation toxicity study. In the 5-day inhalation toxicity study, rats were dosed with target concentrations of 0, 0.20, 0.60 and 2.0 mg/L of the test item. A statistical significant increase in absolute lungs weights was observed at the mid dose in females and at the high dose in males and females. At the other doses, males and females already showed an increase in lung weight although not statistically significant compared to controls. According to the results of this preliminary study,
the dose-levels of 0.050, 0.20 and 0.80 mg/L were selected for the current study.
- Rationale for animal assignment: random

Positive control:

No positive control was used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily during acclimatization and recovery and twice dily during treatment.
- Cage side observations: mortality, viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly during acclimatization and recovery. On exposure days, once before, during and after the daily treatment, and once daily on non-exposure days of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatization and recovery, and twice weekly during treatment.

FOOD CONSUMPTION:
- Food consumption: Recorded (per cage) weekly during acclimatization, treatment and recovery.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatization (main study and recovery animals) and week 4 of treatment (main study animals).
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 18 hours before sampling
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 weeks (main study animals) and after 9 weeks of recovery (recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a
- Battery of functions tested: sensory activity / grip strength / motor activity / other: n/a

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Other examinations:

BRONCHO-ALVEOLAR LAVAGE:

5 animals/sex/group were sacrificed approximately 24 h after the last exposure and a further 5 animals/sex/group were sacrificed approximately 24 h after 9-week recovery. The lungs of each dose group were washed six times with approximately 4 mL physiological saline at room temperature by slow instillation and withdrawal of fluid, with thoracic massage.
The following parameters were determined:
- Enzymatic activities of lactate dehydrogenase and alkaline phosphatase which were used as indicators of possible plasma membrane damage and/or cell lysis.
- Total protein which was indicative of inflammatory processes and damage to the alveolar capillary barrier.
- Phospholipids which was indicative of disturbances in the metabolic activity of type II epithelial cells.

Statistics:

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios, ophthalmoscopic examination as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for
the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied to the ophthalmoscopic and macroscopic findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled treatment and recovery periods. No clinical signs were noted during the course of this study.

BODY WEIGHT AND WEIGHT GAIN
Marginal mean body weight loss was noted between day 1 and day 4 of treatment in high dose group males attaining statistical significance for the body weight gain.
These animals gained weight thereafter and a dose relation was no longer observed from week 3 onwards. Accordingly, further statistical significant differences for the body weight development for low and high dose group males were considered to be incidental. Body weight gain in females was
similar across all groups.

FOOD CONSUMPTION
There were no effects on the food consumption that were considered to be related to treatment. Statistically significant decreased food intake on some occasions for males of the low and mid dose groups during the treatment period were considered to be incidental in the absence of a dose relationship. A statistically significantly increased food intake on same occasions for males of the mid and high dose groups was also considered to be fortuitous.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
No ophthalmoscopic findings were observed which were considered to be related to the treatment.
Corneal opacity observed at the end of the treatment period in some animals across all groups was already present before exposure start.

HAEMATOLOGY
At the end of the treatment period the absolute and relative neutrophil levels were increased in males and females of all three groups treated with the test substance. Statistical significance was attained in the mid and high dose groups for the absolute or relative levels in females and males, respectively.
At the end of the recovery period the effects on the neutrophils increased and a clear dose relation ship was recorded for the absolute levels. Statistical significant differences to controls were recorded for the relative levels in males in all groups treated with the test substance and for the absolute levels in males
and females in all groups treated with the test substance (except for low dose males). In addition, the absolute monocyte levels were statistically significantly increased in mid dose females and in both sexes at the high dose. Accordingly, the total leukocyte count was also increased in males and females in all groups treated with the test substance, attaining statistical significance in the high dose group.

CLINICAL CHEMISTRY
Aspartate aminotransferase was increased in both sexes in the high dose group at the end of the recovery period. Statistical significance was attained for males. Statistically significant differences in cholesterol, triglycerides and phospholipids at either the end of the treatment period or the 9 week recovery period were within the range of historical control data and therefore not considered to be related to treatment.

URINALYSIS
There were no findings in urinalysis that were considered to be test item-related.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Absolute and related lung weights were dose-dependently increased in both sexes in all groups treated with the test substance at the end of treatment. Statistical significance was obtained for the mid and high dose groups.
After the end of recovery, the there was a further dose-dependent increase in absolute and relative lungs weights and statistical significance was obtained for both sexes in all groups treated with the test substance (except for the absolute lung weight and the lung weight to body ratio for low dose females). In addition, the absolute and relative spleen weights were increased in both sexes in the high dose group, attaining statistical significance for males. Absolute and relative adrenal weights were increased in high dose males, attaining statistical significance for the absolute weight and the adrenal to brain weight ratio. Absolute and relative liver weights were increased in high dose females, attaining statistical significance for the liver to body weight ratio.
There were no further changes in organ weights that were considered to be test item-related. Single statistically significant differences between treated groups and controls were considered to be incidental as there was no dose-relationship and/or alterations were not consistently present in both sexes.

GROSS PATHOLOGY
The tracheobronchial/mediastinal lymph nodes were recorded as enlarged in two mid dose females and one male and three females in the high dose group at the end of the treatment period and in all animals treated with the test substance after the end of the recovery period.
All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
After the end of treatment, diffuse alveolar histiocytosis in the lungs was noted at a dose dependently increased severity in all treated animals. Amorphous material (considered to be the test item) was noted at a dose-dependently increased incidence and severity in mid and high dose males and females. In addition, perivascular/peribronchial lymphoid hyperplasia was noted some high dose animals.
In the tracheobronchial lymph nodes, histiocytic granulomas were noted in most treated animals at a dose-dependent severity. This finding was generally accompanied by lymphoid hyperplasia.
After the end of recovery period diffuse alveolar histiocytosis, the amorphous material and the perivascular/peribronchial lymphoid hyperplasia were still present in the lungs. However, the severity grade for the alveolar histiocytosis increased in all treatment groups and the incidences and the severity for the amorphous material were similar but this finding was additionally noted in two low dose females. In addition, the severity grade incidence for the lymphoid hyperplasia increased in the high dose group and this finding was noted in one low dose male and some mid dose animals. This hyperplasia was accompanied by histiocyte granulomas, partly with dose-dependent fibrosis. Finally, microgranulomas at the bronchiolar-alveolar junction at a dose-dependent severity were
noted in most treated animals.
In the tracheobronchial lymph nodes, histiocytic granulomas and lymphoid hyperplasia were still present but the severity increased as well as the incidence to all animals. In addition, there was fibrosis present in all these lymph nodes at a generally dose-dependently increased severity.
The other findings noted were within the range of incidental background lesions commonly recorded in animals of this strain and age.

HISTORICAL CONTROL DATA
Available for Haematology, Clinical chemistry and urinalysis. attached as file historical data.pdf

BRONCHO-ALVEOLAR LAVAGE
Lactate dehydrogenase was increased in the broncho-alveolar lavage fluid at the end of treatment in both sexes of all treated groups, attaining statistical significance in high dose males and in low and mid dose females. In addition, phospholipids and alkaline phosphatase were also increased in males and females but only attaining statistical significance on some occasions in mid and high dose groups. Finally, total protein was increased in the high dose group attaining statistical significance in females.
In addition, the number of cells in the broncho-alveolar lavage fluid increased dose-dependently in all reated groups attaining statistical significance in both sexes of the mid and high dose groups. This increase resulted mainly from macrophages and neutrophils but the absolute number of lymphocytes were also increased. The statistical significant increase of the cell viability in mid and high dose males was considered to be a consequence of these increased neutrophil levels.
After the end of the recovery period, completed recovery was noted for alkaline phosphatase and partial recovery was noted for phospholipids and lactate dehydrogenase: phospholipids were increased at the end of treatment in mid dose males and in both sexes at the high dose, attaining
statistical significance in the high dose group; lactate dehydrogenase remained increased in low and mid dose males and both sexes at the high dose, attaining statistical significance in this group.
A progression of the effects was noted for total protein and cell counts in the broncho-alveolar lavage fluid: Total protein was dose-dependently increased in males in all treated groups and in mid and high dose females attaining statistical significance in high dose males and in mid and high dose females. The number of cells increased in a dose-dependent manner and reached statistical significance in all reatment groups with a similar differential cell count as at the end of treatment.

Some further inter-group variations in hematology, clinical biochemistry, urinalysis, and broncho-alveolar lavage parameters occasionally achieved statistical significance reflecting changes that were considered to be in the normal range, displayed no dose-relationship and/or were not consistently present in both sexes. These changes were considered not to be test item related.
In addition, statistical significant decreases for differential cell counts were a consequence of the increase of neutrophils.

Dose descriptor:

NOAEC

Basis for effect level:

other: On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Summary tables for the results of examinations are attached as Summary tables Part 1.pdf and Summary tables Part 2.pdf

Executive summary:

In this 28-day repeat dose inhalation study performed according to OECD TG 412 under GLP, kieselguhr soda ash flux-calcined was administered to Wistar rats (20 animals/sex/dose) by nose-only, flow-past inhalation for a period of 5 days/week (6 hours/day) for 4 consecutive weeks at aerosol concentrations of 0.044, 0.207 and 0.700 mg/L air.

 

The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 9-week treatment-free recovery period following the treatment period in selected animals of all groups. Sub-groups of male and female animals served for determination of broncho-alveolar lavage fluid (BALF) parameters on day 28 of treatment or were kept for a 9-week off-dose period. Mortality, clinical signs, body weight, food consumption, organ weights, macroscopic and microscopic findings were recorded and ophthalmoscopic examinations and clinical laboratory investigations were performed. BALF samples were collected after the end of treatment and after recovery.

 

No premature deaths and no clinical signs were observed except for a slight and transient effect on body weight gain at the high dose. A dose-dependent increase in lung weights was recorded from the low dose to the high dose at the end of the treatment. A further increase in lung weights and lymph nodes had increased in size at the end of the recovery period. An increase in spleen, adrenals and liver weights at the high-dose at the end of the recovery period was observed but the relevance of these findings was not certain. In the alveoli, histiocytosis was observed increasing in incidence and severity from the low dose to the high dose at the end of the treatment. A progression in incidence and severity was also observed to occur after the recovery period. The presence of the test item was detected in the alveoli of the animals of the mid and high dose groups at the end of the treatment period. This was observed to persist at least until the end of the recovery period and additionally it was observed in 2/10 animals of the low dose recovery group.

 

In the observations on the peribronchial/perivascular zone, lymphoid hyperplasia was observed in the high dose group animals at the end of the treatment period. There was a progression in severity after the recovery period and additionally it was observed in some mid-dosed animals of the recovery group. The occurrence of microgranuloma and fibrosis was observed at the end of the treatment-free period. For the tracheobronchial lymph nodes, histiocytic granuloma was observed increasing in severity from the low dose to the high dose at the end of the treatment. There was a progression in incidence and severity as shown by the occurrence of fibrosis after the recovery period.

There were no adverse effects detected in organs other than the lungs. Some adaptive responses may have been induced in the liver, adrenals and spleen in response to the irritation and damage to the lung tissue.

 

On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

Reference 9

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: 5 day dose range finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Principles of method if other than guideline:

The purpose of this 5-day dose range finding inhalation study was to obtain preliminary information about the toxicity of Soda-ash flux-calcined kieselguhr when administered by nose only, flow-past inhalation exposure for a period of 5 days (6 hours/day). The results of this study were used to select the dose levels for further toxicity studies.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at delivery): Males: 8 weeks, Females: 9 weeks
- Weight (at acclimatization): Males: 209.7 to 236.9 g (± 6%), Females: 163.1 to 181.9 g (± 6%)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 82/09) rat maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: Eight days under test conditions after clinical health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2 and 4 hours, respectively.



ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: relative humidity range: (30 – 70%)
- Air changes: 10 – 15 air changes per hour
- Photoperiod: There was a 12-hour fluorescent light/12- hour dark cycle with music during the light period

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Remarks on MMAD:

MMAD / GSD: The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the chemical results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA). The target ranges were 1 to 3 μm for the MMAD and 1.5 to 3 for the GSD.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR 3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer
- Temperature, humidity, pressure in air chamber:
- Air flow rate: The flow of air at each tube was 1.0 L/min
- Method of particle size determination: The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor.


TEST ATMOSPHERE
- Brief description of analytical method used: To determine the concentration of the test item based on the Silicon content in the generated aerosol, chemical analyses of test aerosol samples were performed twice daily for groups 2 to 4. The filters taken for gravimetric determination were used for chemical analysis.They were analyzed for Silicon by Harlan Laboratories Ltd. using an AAS method developed by Harlan Laboratories Ltd.

Analytical verification of doses or concentrations:

yes

Frequency of treatment:

6-hours daily, at approximately 24-hour intervals.

Remarks:

Doses / Concentrations:
0.18, 0.58 and 1.57 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

5

Details on study design:

- Dose selection rationale: Target Concentrations were selected by the Sponsor based on data from the acute inhalation toxicity study performed at Harlan Laboratories Ltd. (study C88248). In addition, the target concentration for group 4 was considered to be the technical limit based on a 6 hour exposure).

Observations and examinations performed and frequency:

Viability / Mortality: Twice daily during treatment, once daily during acclimatization.

Clinical Signs: Were recorded once daily during treatment (after exposure) and on days 1 and 5 of acclimatization

Food Consumption: Was recorded (per cage) on days 1 and 5 of acclimatization and on days 1, 3 and 5 during the treatment period.

Body Weights: Were recorded on days 1 and 5 (each individual animal) of acclimatization and on days 1, 3 and 5 during treatment.

Sacrifice and pathology:

All animals were weighed and necropsied (samples taken are detailed in table 1)

Statistics:

laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test [(many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: All animals survived the scheduled treatment period. No clinical signs were noted during the course of the study.

BODY WEIGHT AND WEIGHT GAIN: Mean body weight loss was recorded in both male and female animals of group 4 over the five day treatment period. In male animals a mean of 11.9 g was lost between day 1 and 5. Over the same time period the female animals lost a mean of 2.7 g. Mean group body weights of all groups including control did not return to before treatment values. Statistically significant reduced body weight gain was observed in male rats of group 4 only when compared with the control group.

FOOD CONSUMPTION: A reduced mean food consumption was recorded on treatment days 1 to 3 and 3 to 5 for groups 4 animals when compared to days 5 to 10 for acclimatization.


ORGAN WEIGHTS: Absolute lung weights of group 4 males and group 3 and 4 females were significantly increased. Furthermore, significant increases were recorded for lung/body weight and lung/brain weight ratios of male and female animals of groups 3 and 4 and males of groups 3 and 4 and females of group 4, respectively.


MACROSCOPIC FINDINGS: No macroscopic findings were recorded which were considered to be related to treatment with the test item


MICROSCOPIC FINDINGS: There was a dose dependent increase in the incidence of alveolar histiocytosis. This finding was seen in one male of group 1, two males and females of group 2 and all animals of groups 3 and 4. In addition, alveolitis was observed in one male of group 3 and in all males and females of group 4. Furthermore, microgranulomas were noted in one male and female of group 3 and all males and females of group 4 and amorphous material in alveoli was found in four males and three females of group 4. These findings in the lungs are considered to represent an irritant effect of the test item. All other findings noted were considered to be incidental findings commonly noted in animals of this strain and age.

Critical effects observed:

not specified

The chemically determined mean aerosol concentrations were close to target for groups 2 and 3 and below the target for group 4. lower concentrations for group 4 were considered to be acceptable in view of the significant effects on body weight. Details on aerosol concentrations are summarized below:

Group	Achieved gravimetric aerosol concentration [mg/L]	Achieved chemical aerosol concentration [mg/L]	Target chemical aerosol concentration [mg/L]	Chemical aerosol concentration relative to target [%]
2	0.21 ± 0.00 (n=5, CV=2.4%)	0.18 ± 0.03 (n=5, CV=14.0%)	0.20	91.4%
3	0.61 ± 0.02 (n=5, CV=3.1%)	0.58 ± 0.05 (n=5, CV=9.4%)	0.60	96.2%
4	1.91 ± 0.11 (n=5, CV=5.7%)	1.57 ± 0.18 (n=5, CV=11.5%)	2.0	78.5%

The values for gravimetrically and chemically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) were as follows:

	Gravimetric determination
Group	MMAD [μm] and (mean GSD)	Mean percentage of particles < 3 μm
2	2.46 (2.93)	57.3%
3	2.47 (2.65)	58.0%
4	2.81 (1.91)	54.1%

The gravimetric determined Mass Median Aerodynamic Diameters (MMAD) were within the target range of 1 to 3 μm. Overall, deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. Hence, the particle size distribution and MMADs obtained were considered to be appropriate. The results of the chemical determined Mass Median Aerodynamic Diameters (MMAD) obtained by atomic absorption spectrometry (AAS) were considered to be unreliable, due to dissolving impactor stages which contained silicon.

The group mean values for temperature, relative humidity and oxygen concentration measured during the exposures were as follows:

Group	Temperature (°C)	Relative humidity (%)	Oxygen concentration (%)
1	22.8 ± 0.1 (n=5)	1.8 ± 0.2 (n=5)	20.6 ± 0.0 (n=5)
2	23.1 ± 0.1 (n=5)	1.6 ± 1.0 (n=5)	20.3 ± 0.0 (n=5)
3	23.0 ± 0.1 (n=5)	2.8 ± 0.2 (n=5)	20.7 ± 0.0 (n=5)
4	22.5 ± 0.1 (n=5)	4.9 ± 1.2 (n=5)	20.6 ± 0.1 (n=5)

All parameters were very consistent during the treatment period and similar for all groups. Therefore, they were considered to be satisfactory for this type of study.

Conclusions:

Exposure to Soda-ash flux-calcined kieselguhr at chemically determined aerosol concentrations of 0.18, 0.58 and 1.57 mg/L air resulted in effects on food consumption, body weight and lung weight as well as microscopic findings in the lung.

Reduced food consumption as well as reduced body weight gain and body weight loss were observed in animals of group 4.

Dose-related alveolar histiocytosis was observed in animals of all groups. Furthermore, alveolitis was seen in one male of group 3 and all animals of group 4 as well as increased absolute and relative lung weights was observed in the test item-treated groups 3 and 4. Additionally, microgranulomas were found in one male and female of group 3 and in all animals of group 4 and amorphous material in the alveoli was observed in most animals of group 4. These findings in the lungs were considered to represent an irritant effect of the test item. On the basis of the microscopic findings in the lung, a NOEL could not be established. Based on the results of this study, aerosol concentrations of 0.050, 0.20 and 0.80 mg/L air were considered to be suitable target concentrations for a 4-week inhalation study.

Reference 10

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study does not fulfill the requirements of a current OECD guideline study and has not been conducted according to GLP.

Principles of method if other than guideline:

Dogs, guinea pigs and rats were exposed daily, 6 hours per day, 5 days per week for periods up to 2.5 years to flux-calcined diatomaceous earth of 61% cristobalite content and 0.7 µ mass medium particle size. The concentrations were 2, 5 and 50 million particles per cubic foot of air (mppcf). Dogs were given an additional 10 month period of observation to study possible progression of tissue reaction.

GLP compliance:

no

Remarks:

Study predates GLP

Species:

other: Dog, guinea pig and rat

Strain:

other: Dogs: mongrels, guinea pigs: not stated, rats: wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dogs were obtained froma local supplier, guinea pigs were obtained from Rockland Farms (New City, New York); and the rats were from Hamilton Laboratory Animals (Cincinnati, Ohio)
- Fasting period before study: No food or water was available to animals during the daily six hour exposures.
- Diet: While in their housing vages th dogs were fed one daily ration of Bordens Dog Meal moistened with a mixture of equal amounts of water and powdered skim milk; guinea pigs and rats had available food and tap water ad libitum. Guinea pigs had Purina Rabbit Pellets supplemented twice weekly with greens, the rats had Rockland Rat Diet.
- Acclimation period: All animals, both those to be exposed and controls were pre-exposed to the control chamber to normal atmospheres of filtered and temperature and humidity controlled chambers for 6 hours, five days per week for 3 weeks.

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
In an effort to simulate the stress of work associated with industrial environment on the possible toxicity of low level, flux-calcined diatomaceous earth, separate groups of 10 control and 10, 5 mppcf exposed rats were placed in large motor driven rotating cages located in their respective exposure chambers. The cages were rotated at 7 rpm, 5 minutes each hour, starting after the first hour during a continuous 6 hour exposure. The rats were exposed daily in this manner for one year.

The dynamic type exposure chmabers were cubical units of 145 cubic feet capacity with conical galvanized-iron tops and aluminium double wall and floor construction. Control animals were exposed in a similar manner but recieved only filtered temperature controlled air.

TEST ATMOSPHERE
Chamber air samples were collected by two methods; the midget impringer and a semi continuous sampling analysis and recording method utilizing the Sinclair-Phoenix Aerosol and Smoke Photometer. A summary of the tabulated monthly cumulative chamber dust concentrations for each level for the 30 months of operation is shown in table 2.

Details on analytical verification of doses or concentrations:

Refer to table 2

Duration of treatment / exposure:

Up to 2.5 years

Frequency of treatment:

Daily, six hours per day

Remarks:

Doses / Concentrations:
2 mppcf
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
5 mppcf
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
50 mppcf
Basis:
nominal conc.

No. of animals per sex per dose:

Refer to table 1 for total number of animals

Control animals:

yes, concurrent no treatment

Details on study design:

Separate groups of animals were exposed at 2 and 5 million particles of flux-calcined diatomaceous earth per cubic foot of air 6 hours per day, 5 days per week on a 2 shift basis. The dual shift was necessitated by a shortage of exposure chambers, but was discontinued after 8 weeks when additional chamber became available. Table 1 shows that in addition to the groups of dogs, guinea pigs and rats exposed daily at the 2 and 5 mppcf levels, separate groups of rats and guinea pigs were exposed to an intermittent peak concentration of 50 mppcf for one hour, 3 times per week; and other group of rats and guinea pigs were exposed intermittently at the same peak 50 mppcf exposure.

Sacrifice and pathology:

Lungs, hilar lymph nodes, heart, liver, kidney, spleen, adrenal, small intestine (duodenum) and hepatic lymph nodes were preapred for histologic examination. In addition, sections of the trachea, pancreas and urinary bladder of the dogs were saved.

In an effort to determine the extent of retention of cristobalite in the lung tissue from each scheduled, serially sacrificed control, 2 mppcf and 5 mppcf dog was submitted for x-ray diffraction analysis.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

BODY WEIGHTS
All groups of dogs gained as much and sometimes more than controls. Guinea pigs showed no depressed growth at any level of exposure; the final body weights at the end of 12 and 18 months compared favourably with the controls. The terminal average body weights of rats exposed at the 5+50 mppcf level, however were considerably below those of the corresponding controls, as were those exposed at the 5 mppcf level.

HEMATOLOGY
There were no changes in the hematologic varaibles, hematocrit, hemoglobin and red and white cell counts that were determined on dogs trimonthly throughout the 2.5 year exposure nor in dogs that were retained another 10 months for observation.

X RAY DIFFRACTION OF THE LUNG
Spectrometric x-ray diffraction analyses of lungs of dogs exposed at dust levels of 2 and 5 mppcf indicated a cumulative deposition of cristobalite that plateaued after about 1800 hours at the lower level and after 3000 hours at the higher. The average value of the cristobalite content in dry lung tissue for the 2 mppcf level was 0.18% (range 0.14% - 0.27%) and 0.40% (range 0.37%-0.43%) for those exposed at 5 mppcf. No amount of cristobalite was detected in control animals.

PATHOLOGY
Detailed histologic evaluation of tissues of serially sacrificed animals showed that no fibrosis of the pulmonary parencgyma resulted in any of the species under any condition of exposure. However, there was no dust level which did not exhibit a histiocytic and giant cell infiltration in the pulmonary parenchyma, and moderate numbers of hyalinized fibrotic nodules developed in hilar lymph nodes in dogs at the 5 mppcf level and to a lesser degree at the 2 mppcf level. No recognizable pathologic abnormalities were observed in any other organs.

Dose descriptor:

NOAEL

Effect level:

5 other: mppcf

Sex:

male/female

Basis for effect level:

other: Based on no observed fibrotic changes in dogs

Critical effects observed:

not specified

Table 2: Thirty month average cumulative chamber dust concentration data

Nominal conc.	Actual average conc.	Deviation		Average range	Exposure days	Exposure hours	Calendar days	Gtvalue (mppcf-hr)
mppcf	mppcf	mppcf	%	mppcf				Actual	nominal
2	2.17	+0.45	20.7	1.6 – 3.1	630	3770	927	7,917	7540
5	5.27	+0.81	13.3	3.7 – 7.1	632	3766	927	19,734	18,830
50	46.8	-9.9		33.5 – 65.2	372	422	920	19,800	21,100

Conclusions:

The NOAEL from the study is 5 mppcf (equivalent to 0.14 mg/kg) based on no observed fibrotic changes while a NOEL was not found in the study as effects were already present at 2 mppcf

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1.3 mg/m³

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified