Reaction mass of 2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide] and 2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2-methylphenyl)-3-oxobutyramide] and 2-[[3,3'-dichloro-4'-[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-4-yl]azo]-3-oxo-N-(o-tolyl)butyramide

Endpoint summary

Description of key information

Subacute toxicity of the test item was investigated in an 21 days aerosol inhalation study in rats, which were exposed to 0, 52, 151, 401 mg/m³ test item (6 h/d, 5 d/w).

A slight but significant (p < 0.01) decrease in the bodyweight of the male rats an the highest concentration at termination of the exposure period was observed when compared with those of the controls and the other treated animals.

A slight change was found to occur in the differential leucocyte count of male and female rats at the highest exposure level, where a higher percentage of polymorphonuclear neutrophils and a lower percentage of lymphocytes was observed. This change persisted upon cessation of treatment.

The lungs of all rats from the highest concentration group were slightly enlarged and yellowish in colour. The yellowish dis­coloration of the lungs was also noted in rats from the group which was sacrificed after an additional recovery period of 21 days. The lung weights, the lung to bodyweight and lung to brain weight ratios of these animals were significantly increased. The weight increase persisted in rats of this group after anadditional recovery period of 21 days.

Upon histopathological examination the lungs of all rats from the highest concentration group showed focal accumulation of slightly basophilic material and of numerous foamy cells in the alveoli. In frozen sections large amount of minute yellow-brownforeign particles (1 - 3 µm) in the lumen of the alveoli, in the cytoplasm of some foamy cells, in the lumen of occasional small bronchi and in the macrophages in the interstitium were observed. There was also slight focal lymphohistiocytic infiltration in the interstitium. Focal pneumoconiosis showing brown-yellow foreign particles (dissolved by the procedure used for tissue embedding) was also observed in the treated rats from the top concentration level after the withdrawal period of 3 weeks. Small accumulation of brown-yellow particles was occasionally found in the intra­pulmonal lymphoid tissue of these animals.

In the rats treated with the intermediate concentration level the lungs showed at autopsy slight yellowish discoloration. Histo­pathology revealed brown-yellow, fat soluble particles in the alveoli and in the interstitium of the lungs. In 8 out of 20 rats from this group focal accumulation of foamy cells in several alveoli was observed as well.

The yellowish discoloration of the lungs was not observed in rats from the lowest concentration group. Neither accumulation of foamy cells in the alveoli, nor interstitial inflammatory infiltration was seen upon histopathology in these animals. Minute brown­yellow fat soluble foreign particles were however found in frozen sections in the interstitium of the lungs and in occasional alveoli.

No other gross or microscopical changes which could be related to the inhalation of the test item were found in the treated animals.

It can be inferred from the observations made during the above study that the "no observable effect level" for rats is below 52 mg/m³ of air. The effects observed at the lowest test concentration were only due to the deposition of the test material but did not cause adverse effects like inflammation etc. Therefore, the lowest test concentration can be regarded as "no observable adverse effect level".

In oral studies (short term as well as chronic) with the close analogue substance (PY12) no adverse effects were detected up to the limit dose or the highest dose tested.

In a short term repeated dose inhalation study with another close analogue substance (PY13) no adverse effects were found.

No study with dermal application is available

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 JUL 2000 to 02 OCT 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study (OECD TG 422)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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
- strain: Wistar Crl:(WI) BR (outbred, SPF)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: group means males: 489-501 g; group means females: 267-271 g
- Fasting period before study: no
- Housing:
acclimatisation period: 5 animals per sex per cage, stainless steel suspended cages with wire mesh floors
mating period: 1 female together with 1 male, stainless steel suspended cages with wire mesh floors
after mating: animals were housed individually, Macrolon cages (Type III)
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 26 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: polyethylene glycol 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- formulations (w/w) were prepared daily within 4 hours prior to dosing
- storage at ambient temperature

VEHICLE
- vehicle: polyethylene glycol 400, specific gravity: 1.125
- Justification for use and choice of vehicle: based on trial fromulations
- Concentration in vehicle: 0 mg/g; 4.45 mg/g; 17.8 mg/g; 89.9 mg/g
- Amount of vehicle: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical study was performed to check accuracy of preparation and to determine stability and homogeneity of test substance preparation.
Test samples were dissolved in chloroform by sonication and analysed spectrophotometrically.

Duration of treatment / exposure:

F0-males:
- for 2 weeks prior to mating, throughout mating and after mating at least until the minimum total dosing period of 28 days had been completed
F0-females:
2 weeks prior to mating, throughout mating, and pregnancy and at least up to, and including the day before sacrifice

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a dose range finding study with the test item (5 days oral exposure of male and female rats to 50, 200 and 1000 mg/kg bw/day did not cause changes in clinical appearance, body weights, food consumption, macroscopic examination and organ weights)

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily in the home cage and twice during the complete study in a standard arena


BODY WEIGHT: Yes
- Time schedule for examinations:
F0-males and F0-females: on the first day of dosing and weekly thereafter
mated females were weighed daily from day 0 until day 20 of gestation inclusive
F0-females were weighed daily during lactation
F1-pubs were weighed on day 1 and 4 post partum


FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly, but not during the mating period

WATER CONSUMPTION: Yes
- Time schedule for examinations: subjective appraisal during the study, no quantitative investigation


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to killing
- Anaesthetic used for blood collection: Yes (ether anaesthesia)
- Animals fasted: Yes (overnight with a maximum of 20 hours)
- How many animals: 5 males and 5 females randomly selected from each group
- Parameters: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, red cell distribution width, total leucocyte count, differential leucocyte count, prothrombin time, partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just prior to killing
- Animals fasted: Yes (overnight with a maximum of 20 hours)
- How many animals: 5 males and 5 females randomly selected from each group
- Parameters: alanine aminotransferase, aspartate aminotransferase, total bilirubin, total cholesterol, triglycerides, creatinine, glucose, urea, total protein, albumin, globulin, albumin globulin ratio, alkaline phosphatase, sodium, potassium, chloride, calcium, phosphorus


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once, males during week 4 of treatment, femals during lactation (all before blood sampling)
- Dose groups that were examined: all dose groups
- Battery of functions tested: hearing ability / pupillary reflex / static righting reflex / grip strength / motor activity


OTHER:
Observations on females and litters, calculation of reproduction parameters are reported in section 7.8.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (cranial, thoracic, abdominal tissues and organs with special attention to the reproductive organs)
HISTOPATHOLOGY: Yes
from all animals:
accessory sex organs
epididymides (fixed in Bouin's fixative)
ovaries
testes (fixed in Bouin's fixative)
all macroscopic lesions

from 5 animals/sex/group:
adrenal glands
bone marrow
brain
cervix
heart
kidneys
liver
lung, infused with formalin
lymph nodes - mandibular, mesenteric
oesophagus
sciatic nerve
small and large intestines (including Peyer's patches)
spinal cord -cervical, midthoracic, lumbar
spleen
stomach
thymus
thyroid including parathyroid
trachea
urinary bladder
uterus
vagina
all gross lesions

Other examinations:

ORGAN WEIGHTS
- from all males: epididymides, testes
- from 5 animals/sex/group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus

Statistics:

- Dunnett-test
- Steel-test
- exact Fisher-test

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:

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment related clinical signs were observed.

Almost all animals of the control and treated groups showed scabbing, red/brown staining of various body parts, and diarrhoea. Erythema of the anus was seen in all males during the last three weeks of the study period, and piloerection was observed in almost all females. The vehicle (polyethylene glycol) that was used, is known to cause these effects. Therefore, these findings were considered to be of no toxicological significance.
Almost all animals showed alopecia of various parts of the body. Alopecia is commonly seen in group housed rats and therefore no toxicological significance was attached to this finding. Greenish/yellowish faeces and yellow staining of various body parts were observed in almost all animals of the treated groups. This was probably due to the staining properties of the test substance.
Incidental findings included lethargy, rales, hunched posture, laboured respiration, piloerection and reddish faeces in males, chromodacryorrhoea, salivation, and emaciation and are commonly.

One male receiving 200 mg/kg was killed in extremis after showing severe clinical signs. Two males (one of the control and one of the 50 mg/kg dose group) died spontaneously. These three deaths were considered to be unrelated to treatment, because there were no macroscopic findings suggesting a relation with treatment and there were no other death among the animals. No unscheduled deaths occurred among the females.


BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls.

Except on day 8 of the pre-mating period, when the males of the treatment groups showed a statistical significant reduction in body weight gain when compared to the control group. And on days 2 and 3 of lactation, when females of the 50 mg/kg dose group showed a statistical significant reduced body weight gain. These incidental reductions were considered to have arisen by chance and not to represent a change of toxicological significance.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no differences in food consumption (absolute and relative) between treated and control animals.

Except on days 2-3 of lactation, when females of the 50 mg/kg dose group showed a statistical significant reduced food consumption (absolute and relative) when compared to the control group. This incidental difference showed no dose relationship and was considered to has arisen by chance and to be of no toxicological relevance.

HAEMATOLOGY
The haematological profile was not disturbed by treatment.

Statistically significant differences for Erythrocyte counts (RBC) of males and females in group 2 (50 mg/kg) did not form a dose response relationship. Therefore, this finding was considered to be of no toxicological relevance.
Monocyte counts were statistically significant decreased in males of group 3 (200 mg/kg). Haemoglobin (HB), haematocrit (HCT), and mean corpuscular haernagiobin (MCHC) values of females in group 2 were statistically significant increased when compared to that of the controls. However, similar findings in the opposite sex were absent and no dose relationship was observed. Therefore, the toxicological significance of these findings was considered to be doubted.


CLINICAL CHEMISTRY
The clinical blochemistry parameters were not changed by treatment.

Alanine aminotransferase (ALAT) activity, Aspartate aminotransferase (ASAT) activity, and inorganic phosphate values were statistically significant decreased and total protein values were statistically significant increased in the males of group 3 (200 mg/kg) when compared of the control values. Alanine aminotransferase (ALAT) activity, Aspartate aminotransferase (ASAT) activity, bilirubin, and triglyceride values were increased among high dose females, being mainly attributable to one high individual value (female no. 77). On exclusion of this value, average values were slightly decreased for ALAT and ASAT, and bilirubin and triglyceride were similar when compared to the controls.
Creatine values were statistically significant decreased in females of group 2 (50 mg/kg), and glucose values were statistically significant increased in females of group 3 (200 mg/kg) when cempared to the control values. However, similar findings in the opposite sex were absent and no dose relationship was observed. Therefore, the toxicological significance of these findings were considered to be irrelevant.
Chloride values of males of group 4 (1000 mg/kg) were statistical significant decreased, and total globuline values were statistical significant increased in males of groups 3 and 4. This was considered to be due to slightly high or low control values respectively.


NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the treated animals, when compared to control animals.

Extremely increased motor activity was noted by the high sensors for the 200 mg/kg treated males (no. 23, 25 and 28). Since an increase in motor activity recorded by the high sensors is unlikely to occur without an concurrent change recorded by the low sensors, the increased motor activity was considered to have occurred by chance and of no toxicological significance.
Increased motor activity was noted by the low sensors for the 50 mg/kg (no. 14) and 1000 mg/kg treated males (no. 32 and 33). Since no corroborative findings were noted during clinical observations of the animals, this was considered to have occurred by chance without any toxicological relevance.
Decreased motor activity was noted by the low sensors for one control female (no. 41) and one 1000 mg/kg treated female (no. 71). Since the counts per sample period were often very low or even zero, these recordings were excluded from interpretation.


ORGAN WEIGHTS
Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY
Macroscopic observations of the remaining animals at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Six males of the 1000 mg/kg and 2 males of the 200 mg/kg dose group showed a greenish contents in the gastro-intestinal tract. In the absence of correlated microscopic findings these findings were considered to be due to the presence of undigested pigment, and although treatment-related, of limited toxicological significance.
At the 1000 mg/kg dose group, one male showed yellow foci on the lungs, and another male showed yellow foci on the lungs, a yellowish discolouration of the trachea and oesophagus. These findings were considered to be due to gavage errors, and without toxicological significance.
One male of the control group which died spontaneously showed a thoracic cavity containing cloudy gray/white fluid. One male of 200 mg/kg dose group was killed in extremis and showed a gastro-intestinal tract which contained fluid and was distended with gas, a dark red discolouration of the glandular mucosa of the stomach, and a spleen reduced in size.
Incidental findings included an enlarged caecum or adrenal glands, the testes, epididymis or seminal vesicles reduced in size, and red foci on the thymus.
Three females in the 200 mg/kg dose group and one female in the 50 mg/kg dose group showed a dark red discolouration of the mandibular lymph node. Incidental findings included a caecum with a greenish contents, pelvic dilatation of the kidney, a red-brown discolouration of the clitoral gland, irreguter surface of the spleen, and a thymus reduced in size.


HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance.

All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

OTHER:
There were no treatment related findings on reproduction. Details are presented in section 7.8.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

no

Conclusions:

There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, reproduction, litter observation, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment.
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the test item administered by oral gavage in Wistar rats (10/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 50, 200 and 1000 mg/kg/day. Oral dosing of male and fernale Wistar rats (10 per sex and group) with the test item at dose levels of 50, 200 or 1000 mg/kg b.w./day for at least 28 days, revealed no treatment-related findings on parental animals, on fertility, on embryo-foetal development, or on pup development. From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See read across document in chapter 13

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Route of administration:

oral: gavage

Vehicle:

other: polyethylene glycol 400

Frequency of treatment:

once daily

No. of animals per sex per dose:

10

Positive control:

none

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:

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment related clinical signs were observed.

Almost all animals of the control and treated groups showed scabbing, red/brown staining of various body parts, and diarrhoea. Erythema of the anus was seen in all males during the last three weeks of the study period, and piloerection was observed in almost all females. The vehicle (polyethylene glycol) that was used, is known to cause these effects. Therefore, these findings were considered to be of no toxicological significance.
Almost all animals showed alopecia of various parts of the body. Alopecia is commonly seen in group housed rats and therefore no toxicological significance was attached to this finding. Greenish/yellowish faeces and yellow staining of various body parts were observed in almost all animals of the treated groups. This was probably due to the staining properties of the test substance.
Incidental findings included lethargy, rales, hunched posture, laboured respiration, piloerection and reddish faeces in males, chromodacryorrhoea, salivation, and emaciation and are commonly.

One male receiving 200 mg/kg was killed in extremis after showing severe clinical signs. Two males (one of the control and one of the 50 mg/kg dose group) died spontaneously. These three deaths were considered to be unrelated to treatment, because there were no macroscopic findings suggesting a relation with treatment and there were no other death among the animals. No unscheduled deaths occurred among the females.


BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls.

Except on day 8 of the pre-mating period, when the males of the treatment groups showed a statistical significant reduction in body weight gain when compared to the control group. And on days 2 and 3 of lactation, when females of the 50 mg/kg dose group showed a statistical significant reduced body weight gain. These incidental reductions were considered to have arisen by chance and not to represent a change of toxicological significance.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no differences in food consumption (absolute and relative) between treated and control animals.

Except on days 2-3 of lactation, when females of the 50 mg/kg dose group showed a statistical significant reduced food consumption (absolute and relative) when compared to the control group. This incidental difference showed no dose relationship and was considered to has arisen by chance and to be of no toxicological relevance.

HAEMATOLOGY
The haematological profile was not disturbed by treatment.

Statistically significant differences for Erythrocyte counts (RBC) of males and females in group 2 (50 mg/kg) did not form a dose response relationship. Therefore, this finding was considered to be of no toxicological relevance.
Monocyte counts were statistically significant decreased in males of group 3 (200 mg/kg). Haemoglobin (HB), haematocrit (HCT), and mean corpuscular haernagiobin (MCHC) values of females in group 2 were statistically significant increased when compared to that of the controls. However, similar findings in the opposite sex were absent and no dose relationship was observed. Therefore, the toxicological significance of these findings was considered to be doubted.


CLINICAL CHEMISTRY
The clinical blochemistry parameters were not changed by treatment.

Alanine aminotransferase (ALAT) activity, Aspartate aminotransferase (ASAT) activity, and inorganic phosphate values were statistically significant decreased and total protein values were statistically significant increased in the males of group 3 (200 mg/kg) when compared of the control values. Alanine aminotransferase (ALAT) activity, Aspartate aminotransferase (ASAT) activity, bilirubin, and triglyceride values were increased among high dose females, being mainly attributable to one high individual value (female no. 77). On exclusion of this value, average values were slightly decreased for ALAT and ASAT, and bilirubin and triglyceride were similar when compared to the controls.
Creatine values were statistically significant decreased in females of group 2 (50 mg/kg), and glucose values were statistically significant increased in females of group 3 (200 mg/kg) when cempared to the control values. However, similar findings in the opposite sex were absent and no dose relationship was observed. Therefore, the toxicological significance of these findings were considered to be irrelevant.
Chloride values of males of group 4 (1000 mg/kg) were statistical significant decreased, and total globuline values were statistical significant increased in males of groups 3 and 4. This was considered to be due to slightly high or low control values respectively.


NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the treated animals, when compared to control animals.

Extremely increased motor activity was noted by the high sensors for the 200 mg/kg treated males (no. 23, 25 and 28). Since an increase in motor activity recorded by the high sensors is unlikely to occur without an concurrent change recorded by the low sensors, the increased motor activity was considered to have occurred by chance and of no toxicological significance.
Increased motor activity was noted by the low sensors for the 50 mg/kg (no. 14) and 1000 mg/kg treated males (no. 32 and 33). Since no corroborative findings were noted during clinical observations of the animals, this was considered to have occurred by chance without any toxicological relevance.
Decreased motor activity was noted by the low sensors for one control female (no. 41) and one 1000 mg/kg treated female (no. 71). Since the counts per sample period were often very low or even zero, these recordings were excluded from interpretation.


ORGAN WEIGHTS
Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY
Macroscopic observations of the remaining animals at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Six males of the 1000 mg/kg and 2 males of the 200 mg/kg dose group showed a greenish contents in the gastro-intestinal tract. In the absence of correlated microscopic findings these findings were considered to be due to the presence of undigested pigment, and although treatment-related, of limited toxicological significance.
At the 1000 mg/kg dose group, one male showed yellow foci on the lungs, and another male showed yellow foci on the lungs, a yellowish discolouration of the trachea and oesophagus. These findings were considered to be due to gavage errors, and without toxicological significance.
One male of the control group which died spontaneously showed a thoracic cavity containing cloudy gray/white fluid. One male of 200 mg/kg dose group was killed in extremis and showed a gastro-intestinal tract which contained fluid and was distended with gas, a dark red discolouration of the glandular mucosa of the stomach, and a spleen reduced in size.
Incidental findings included an enlarged caecum or adrenal glands, the testes, epididymis or seminal vesicles reduced in size, and red foci on the thymus.
Three females in the 200 mg/kg dose group and one female in the 50 mg/kg dose group showed a dark red discolouration of the mandibular lymph node. Incidental findings included a caecum with a greenish contents, pelvic dilatation of the kidney, a red-brown discolouration of the clitoral gland, irreguter surface of the spleen, and a thymus reduced in size.


HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance.

All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

OTHER:
There were no treatment related findings on reproduction. Details are presented in section 7.8.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

no

Conclusions:

There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, reproduction, litter observation, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment.
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the test item administered by oral gavage in Wistar rats (10/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 50, 200 and 1000 mg/kg/day. Oral dosing of male and fernale Wistar rats (10 per sex and group) with the test item at dose levels of 50, 200 or 1000 mg/kg b.w./day for at least 28 days, revealed no treatment-related findings on parental animals, on fertility, on embryo-foetal development, or on pup development. From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From JAN 1974 to APR 1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Basic data given, comparable to standards; restrictions: e.g. no data on clinical biochemistry, haematology, urine analysis

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not applicable

Remarks:

The study was performed to test carcinogenicity of the test item. That's the reason why some details usually requested in a repeated dose toxicity study (e.g. data on clinical biochemistry, haematology) are missing.

GLP compliance:

no

Remarks:

study performed before implementation of GLP

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S. IVANOVAS GmbH & Co, Med. Versuchstierzucht KG (Kissleg, Germany)
- Age at study initiation: 38 (males) -42 (females) days
- Weight at study initiation: 100 - 108 g
- Housing: in groups of 2 or 3 animals in Macrolon cages (Type III)
- Diet: Altromin 1321 (Altromin, Lage), ad libitum
- Water: tap water, ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 0.5
- Humidity (%): 60 +/- 3
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily

Dose / conc.:

0 ppm

Remarks:

in diet

Dose / conc.:

1 000 ppm

Remarks:

in diet

Dose / conc.:

3 000 ppm

Remarks:

in diet

Dose / conc.:

9 000 ppm

Remarks:

in diet

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


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 AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once, immediately before sacrifice
- Dose groups that were examined: all animals


HAEMATOLOGY: No


CLINICAL CHEMISTRY: No

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
at the end of the exposure period the following investigations were performed:
- audiometry (using a simple sound test)
- inspection of denture
- organ weights of 7-8 organs (heart, liver, lungs, spleen, kidney, thymus, brain, testis)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (animals of the control and highest dose group; paraffin sections, Haematoxylin-Eosin staining):
heart, lung, liver (additionally: frozen sections with Sudan staining), kidney, spleen, adrenal, thymus, pituitary, brain, gonads, thyroid, prostate/uterus, seminal vesicle/mammary gland, stomach, duodenum, colon, salivary gland, lymph nodes, eye and optic nerve, urinary bladder, bone marrow, neoplastic lesions, bones
- histopathological investigations of animals of the lower dose groups were performed, if they had died prematurely or were sacrificed in the meantime and revealed macroscopic findings

Statistics:

- variance analysis according to Peto
- Student's t-test

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:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

9 000 ppm

Based on:

other: ppm in diet

Sex:

male/female

Basis for effect level:

other: no substance related toxicity or carcinogenicity was observed; 9000 mg/kg diet (ppm) correspond to 555.3 mg/kg bw/day and 579.4 mg/kg bw/day in male and female rats, respectively (calculated in the study report on basis of food consumption)

Key result

Critical effects observed:

no

- no effects on behaviour, appearance, faeces, feed and drinking water uptake, eyes, hearing, dentition, mortality, body weight development

- no substance induced macroscopic or histological changes

- no substance related effects on the tumour incidence

- 1000, 3000, 9000 ppm test item in diet correspond to 62, 186, 555 mg/kg bw/day in male and 64, 194, 579 mg/kg bw/day in female rats, respectively

- no 3,3'-dichlorobenzidine was detected in the urine samples (limit of detection: 3 µg/10 ml urine; 0.3 ppm)

Conclusions:

Chronic feeding of Sprague Dawley rats with up to 9000 ppm test item did not cause any adverse effect. The NOAEL in this study was 9000 ppm in diet (corresponding to 555.3 mg/kg bw/da and 579.4 mg/kg bw/day in male and female rats, respectively).

Executive summary:

Sprague Dawley rats (50 per sex per dose) were exposed to 1000, 3000, 9000 ppm test item in diet (corresponding to 62, 186, 555 mg/kg bw/day and 64, 194, 579 mg/kg bw/day in male and female rats, respectively) for 104 weeks. The test item did neither induce toxicity nor tumorigenicity. The NOAEL in this study was 9000 ppm in diet.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because a reliable chronic toxicity study is available, conducted with an appropriate species and route of administration

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

reliabe with restriction

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 NOV 1978 to 12 DEC 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no GLP)

Principles of method if other than guideline:

21-days repeated dose inhalation toxicity study

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: RAI f SPF (RA25)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: mean body weights males: 172-177g; mean body weights females: 165-171g
- Housing: groups of 5; Macrolon cages type 3
- Diet: pelleted standard diet, Nafag No. 890 (NAFAG, Gossau SG, Switzerland)
- Water: tap water, ad libitum
- Acclimatisation to exposure conditions: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 15

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: 70-80% < 7µm in diameter

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: in PVC-tubes which were positioned radially around the exposure chamber
- System of generating particulates/aerosols: by injecting three different amounts of the solid test material with the help of a Grafix Exaktomat Injector (Cerutti AG, Bern, Switzerland) into an airstream which was discharged into the exposure chamber
- Temperature, humidity, pressure in air chamber (group means): 23 °C, 50-56%, 2 atm
- Air flow rate: 20 l/min
- Method of particle size determination: gravimetrically with a 4 stage cascade impactor on selectron filters, pore size 0.2 µm and 25 mm diameter



TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter
- Samples taken from breathing zone: yes
- Frequency: 5 times a day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter

Duration of treatment / exposure:

21 days (15 exposure days), 6 hours/day

Frequency of treatment:

5 days per week

Remarks:

Doses / Concentrations:
0, 52, 151, 401 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: daily, except weekends, during exposure; weekly during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: twice weekly during exposure; weekly during 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: Yes


WATER CONSUMPTION: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all dose groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haemoglobin, methaemoglobin, carboxyhaemoglobin, erythrocytes, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, reticulocytes, inclusion bodies, thrombocytes, prothrombin time, activated partial thromboplastin time, leucocytes (total count and differential count)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Animals fasted: Yes
- How many animals: all
- Parameters examined: glucose, urea, total protein, protein electrophoresis, electrolytes, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transpeptidase


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
- organ weights: yes (at the end of the exposure and recovery period; brain, heart, liver, kidney,adrenals, thymus, gonades, lung)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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
No death occurred during the exposure and recovery period. No toxic symptoms were observed in the animals of the control and test groups.

BODY WEIGHT AND WEIGHT GAIN
There was a slight but significant (p>/= 0.01) decrease in the male animals of the highest concentration group at day 21 and females at days 3, 7 and 10. During the recovery period the bodyweight gain was again comparabel to that of the control.

FOOD CONSUMPTION
No significant differences between treated animals and controls.

FOOD EFFICIENCY
No significant differences between treated animals and controls.

WATER CONSUMPTION
No effects.

OPHTHALMOSCOPIC EXAMINATION
No occular changes were observed.

HAEMATOLOGY
The observed haematological findings between treated rats and controls were generally unremarkable.
A slight change with statistical significance (p
CLINICAL CHEMISTRY
No significant differences between treated animals and controls.


ORGAN WEIGHTS
Absolute and relative lung weigths (lung to body weight and lung to brain weight) of males and females of the highest dose group were significantly elevated in comparison to the control group at the end of the exposure and at the end of the recovery period. Lung to body weights (but not the absolute lung weights) of males of the mid dose group were also elevated at the end of the exposure period (not measured at the end of the recovery period). The organ weights of the other organs were comparable in the control and treated groups.

GROSS PATHOLOGY
The lungs of all rats from the highest concentration group were slightly enlarged, with yellowish tinge. The lungs of all rats from the intermediate concentration group showed slightly yellowish tinge. The lungs of the lowest concentration group showed no compound related gross anatomical changes. The yellwoish discoloration of lungs was noted also in rats from the highest concentration group which were sacrificed after an additional recovery period of 21 days.

HISTOPATHOLOGY: NON-NEOPLASTIC
The eyes of all animals showed posttraumatic peribulbar lesions which were results of blood collection performed before sacrifice of the animals and were of no toxicologic relevance.
The lungs of all rats from the highest concentration group showed focal accumulation of slighly basophilic material and foamy cells in numerous alveoli. In Sudan stained frozen sections minute yellow-brown particles in the lumen of alveoli, in the cytoplasm of some foamy cells, in the lumen of occasional small bronchi and in the macrophages in the interstitium were seen. There was only minimal focal lymphohistiocytic infiltration in the interstitium.
The lungs of all rats from the intermediate concentration group showed , in the frozen section, moderate focal accumulation of brown-yellow minute particles in the macrophages in the interstitium and in very occasional alveoli. In the lungs of 4/10 male and 4/10 female rats from this group minimal focal accumulation of foamy cells in very occasional alveoli was observed.
The lungs of the rats from the lowest concentration goup showed no accumulation of foamy cells in the alveoli. In the frozen sections however minimal focal accumulation of brown-yellow particles in the macrophages in the interstitium and in very occasional alveoli was seen.
At the end of the recovery period the lungs of all animals from the highest concentration group showed focal accumulation of minute brown-yellow foreign particles in the interstitium. Few minute foreign particles in the alveoli and occasional ones in the intrapulmonal lymphatic tissue were observed as well.

Key result

Dose descriptor:

NOAEC

Effect level:

52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathologic investigation revealed minimal deposition of test material in the lung, but this was not accompanied by an inflammatory or any other adverse response

Key result

Critical effects observed:

no

Analysed test item concentrations were:

0 mg/m3

52 +/- 4 mg/m3

151 +/- 9 mg/m3

401 +/- 15 mg/m3

Conclusions:

The test item did not elicit relevant toxicological effects after subacute inhalation exposure except for some local effects in the lungs of the treated animals probably due to the particulate matter of the test item and not due to inherent toxicity. Therefore, no NOEL can be derived from this study. Whereas inflammatory effects up to pneumoconiosis occurred in the mid and high concententration groups, the effects observed at the lowest test concentration were only due to the deposition of the test material, but not adverse. Therefore the lowest test concentration can be regarded as NOAEC.

Executive summary:

Subacute toxicity of the test item was investigated in an 21 days aerosol inhalation study in rats, which were exposed to 0, 52, 151, 401 mg/m3 test item (6 h/d, 5 d/w).

A slight but significant (p < 0.01) decrease in the bodyweight of the male rats an the highest concentration at termination of the exposure period was observed when compared with those of the controls and the other treated animals.

A slight change was found to occur in the differential leucocyte count of male and female rats at the highest exposure level, where a higher percentage of polymorphonuclear neutrophils and a lower percentage of lymphocytes was observed. This change persisted upon cessation of treatment.

The lungs of all rats from the highest concentration group were slightly enlarged and yellowish in colour. The yellowish dis­coloration of the lungs was also noted in rats from the group which was sacrificed after an additional recovery period of

21 days. The lung weights, the lung to bodyweight and lung to brain weight ratios of these animals were significantly increased. The weight increase persisted in rats of this group after anadditional recovery period of 21 days.

Upon histopathological examination the lungs of all rats from the highest concentration group showed focal accumulation of slightly basophilic material and of numerous foamy cells in the alveoli. In frozen sections large amount of minute yellow-brownforeign particles (1 - 3 um) in the lumen of the alveoli, in the cytoplasm of some foamy cells, in the lumen of occasional small bronchi and in the macrophages in the interstitium were observed. There was also slight focal lymphohistiocytic infiltration in the interstitium. Focal pneumoconiosis showing brown-yellow foreign particles (dissolved by the procedure used for tissue embedding) was also observed in the treated rats from the top concentration level after the withdrawal period of 3 weeks. Small accumulation of brown-yellow particles was occasionally found in the intra­pulmonal lymphoid tissue of these animals.

In the rats treated with the intermediate concentration level the lungs showed at autopsy slight yellowish discoloration. Histo­pathology revealed brown-yellow, fat soluble particles in the alveoli and in the interstitium of the lungs. In 8 out of 20 rats from this group focal accumulation of foamy cells in several alveoli was observed as well.

The yellowish discoloration of the lungs was not observed in rats from the lowest concentration group. Neither accumulation of foamy cells in the alveoli, nor interstitial inflammatory infiltration was seen upon histopathology in these animals. Minute brown­yellow fat soluble foreign particles were however found in frozen sections in the interstitium of the lungs and in occasional alveoli.

No other gross or microscopical changes which could be related to the inhalation of the test item were found in the treated animals.

It can be inferred from the observations made during the above study that the "no observable effect level" for rats is below 52 mg/m³of air. The effects observed at the lowest test concentration were only due to the deposition of the test material but did not cause adverse effects like inflammation etc. Therefore, the lowest test concentration can be regarded as "no observable adverse effect level".

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

see chapter 13

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Slight deposits of test material in the lungs were not considered as adverse

Key result

Critical effects observed:

no

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 27 NOV 1978 to 9 JAN 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no GLP)

Principles of method if other than guideline:

21-days repeated dose inhalation toxicity study

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: RAI f SPF (RA25)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: mean body weights males: 174-177g; mean body weights females: 166-168g
- Housing: groups of 5; Macrolon cages type 3
- Diet: pelleted standard diet, Nafag No. 890 (NAFAG, Gossau SG, Switzerland)
- Water: tap water, ad libitum
- Acclimatisation to exposure conditions: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 15

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: > 80% < 7µm in diameter

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: in PVC-tubes which were positioned radially around the exposure chamber
- System of generating particulates/aerosols: by injecting three different amounts of the solid test material with the help of a Grafix Exaktomat Injector (Cerutti AG, Bern, Switzerland) into an airstream which was discharged into the exposure chamber
- Temperature, humidity, pressure in air chamber (group means): 22.5-22.9 °C, 41.7-44.9%, 2 atm
- Air flow rate: 20 l/min
- Method of particle size determination: gravimetrically with a 4 stage cascade impactor on selectron filters, pore size 0.2 µm and 25 mm diameter



TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter
- Samples taken from breathing zone: yes
- Frequency: 5 times a day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter

Duration of treatment / exposure:

15 exposure days, 6 hours/day

Frequency of treatment:

5 days per week

Remarks:

Doses / Concentrations:
0, 54, 157, 410 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: daily, except weekends, during exposure; weekly during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: twice weekly during exposure; weekly during 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: Yes


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all dose groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haemoglobin, methaemoglobin, erythrocytes, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, reticulocytes, inclusion bodies, thrombocytes, prothrombin time, activated partial thromboplastin time, leucocytes (total count and differential count)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Animals fasted: Yes
- How many animals: all
- Parameters examined: glucose, urea, total protein, protein electrophoresis, electrolytes, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transpeptidase


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
- organ weights: yes (at the end of the exposure and recovery period; brain, heart, liver, kidney, adrenals, thymus, gonades, lung)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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:

no effects observed

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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
No death occurred during the exposure and recovery period. No toxic symptoms were observe in the animals of the control and test groups.

BODY WEIGHT AND WEIGHT GAIN
There was a slight but significant (p>/= 0.01) decrease in the body weigth gain of the male rats of the highest concentration group during the exposure period, when compared with that of the recovery period of these animals and the other treated and control group.

FOOD CONSUMPTION
No significant differences between treated animals and controls.

FOOD EFFICIENCY
No significant differences between treated animals and controls.

WATER CONSUMPTION
No data.

OPHTHALMOSCOPIC EXAMINATION
No occular changes were observed.

HAEMATOLOGY
The observed haematological findings between treated rats and controls were generally unremarkable.


CLINICAL CHEMISTRY
No significant differences between treated animals and controls.


ORGAN WEIGHTS
With the exception of the absolute and relative lung weigths, which were significantly increased in male and female rats of the highest dose group after the exposure as well as the recovery period, the organ weights, organ to bodyweight and organ to brainweight ratios were generally comparabel between the control and treated rats.

GROSS PATHOLOGY
In the treated rats from all concentration level groups yellowish or yellow discoloration of the lungs was seen at autopsy. No other compound related anatomical changes were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the lungs of the animals from the highest concentration group (410 mg/m3) histological signs of a slight pneumoconiosis were seen. There were small brown-yellow birefringent particles in the lumen of numerous alveoli, small bronchi, in the numerous histiocytes in the interstitium and in the peribronchial lymphatic tissue. The size of these particles varied between 2-4 µm in diameter. In numerous alveoli focal accumulation of foamy cells containing usually birefringent particles in their cytoplasm was observed. There was slight focal lymphohistiocytic infiltration in the vicinity of the foreign particles. After a recovery period of 21 days only minimal or questionable regression of the pneumoconiosis was seen in the animals from the highest concentration group.
In the lungs of the animals from the intermediate concentration group (157 mg/m3) focal accumulation of small brown-yellow birefringent particles in the cytoplasm of histiocytic elements in the interstitium, in occasional alveoli, in the lumen of a few bronchi and occasionally in the peribronchial lymphatic tissue was found.
Similar only slightly less pronounced finding in the lung was observed in the rats from the lowest concentration group (54 mg/m3).
Neither accumulation of the foamy cells in the lungs nor lymphohistiocytic inflammatory infiltration in the interstitium were noted in rats from the lowest and intermediate concentration groups.
All other minor changes seen in some control and treated rats were only incidental in nature and not related to the inhalation of the test item.

Dose descriptor:

NOAEC

Effect level:

157 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: histopathologic investigation revealed minimal deposition of test material in the lung, but this was not accompanied by an inflammatory or any other adverse response

Critical effects observed:

not specified

Analysed test item concentrations were:

0 mg/m3

54 +/- 6 mg/m3

157 +/- 9 mg/m3

410 +/- 12 mg/m3

Conclusions:

The test item did not elicit relevant toxicological effects after subacute inhalation exposure except for some local effects in the lungs of the treated animals probably due to the particulate matter of the test item and not due to inherent toxicity. Therefore, no NOEL can be derived from this study. Whereas inflammatory effects up to pneumoconiosis occurred in the high concententration group, the effects observed at the mid and lowest test concentration groups were mainly due to the deposition of the test material, but not adverse. Therefore the 157 mg/m3 can be regarded as NOAEC.

Executive summary:

Subacute toxicity of the test item was investigated in an 21 days aerosol inhalation study in rats, which were exposed to 0, 54, 157, 410 mg/m3 test item (6 h/d, 5 d/w).

The bodyweights of the male and female rats of the highest treatment group were slightly decreased during the exposure period when compared with those of the control and other treated groups. In the treated rats from all concentration level groups yellowish or yellow discoloration of the lungs was seen at autopsy. Slight but significant increase of both absolute and relative weights of the lungs was noted in rats from the top concentration group. Histopathology revealed in the lungs of these rats pneumoconiosis showing numerous brown-yellow, birefringent particles about 2 - 4 um in diameter in the lumen of numerous alveoli, in small bronchi, in numerous histiocytes in the interstitium and in peribronchial lymphatic tissue, associated with focal accumulation of foamy pneumocytes in the alveoli and focal lymphohistiocytic infiltration.

After a recovery period of 21 days, to which a further group of rats exposed to the 410 mg/m³concentration of the tested compound was subjected, practically no regression of the pulmonal changes was observed.

In treated rats from the 157 mg/m³and 54 mg/m³concentration groups focal accumulation of minute brown-yellow, birefringent particles was observed in the cytoplasm of histiocytic elements in the interstitium, in occasional alveoli, in the lumen of a few small bronchi and very occasionally also in the peribronchial lymphatic tissue, however without obvious accumulation of foamy cells in the alveoli and without inflammatory infiltration.

Other minor microscopical findings obtained in some treated and control animals were only incidental in nature and not related to the inhalation of the test item.

It can be inferred from the observations made during the above study that the "no observable effect level" for rats is below 54 mg/m³air.

The effects observed at the mid and low test concentration were only due to the deposition of the test material but did not cause adverse effects like inflammation etc. Therefore, the mid test concentration of 157 mg/m3 can be regarded as "no observable adverse effect level".

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because a reliable chronic toxicity study is available, conducted with an appropriate species and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 NOV 1978 to 12 DEC 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no GLP)

Principles of method if other than guideline:

21-days repeated dose inhalation toxicity study

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: RAI f SPF (RA25)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: mean body weights males: 172-177g; mean body weights females: 165-171g
- Housing: groups of 5; Macrolon cages type 3
- Diet: pelleted standard diet, Nafag No. 890 (NAFAG, Gossau SG, Switzerland)
- Water: tap water, ad libitum
- Acclimatisation to exposure conditions: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 15

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: 70-80% < 7µm in diameter

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: in PVC-tubes which were positioned radially around the exposure chamber
- System of generating particulates/aerosols: by injecting three different amounts of the solid test material with the help of a Grafix Exaktomat Injector (Cerutti AG, Bern, Switzerland) into an airstream which was discharged into the exposure chamber
- Temperature, humidity, pressure in air chamber (group means): 23 °C, 50-56%, 2 atm
- Air flow rate: 20 l/min
- Method of particle size determination: gravimetrically with a 4 stage cascade impactor on selectron filters, pore size 0.2 µm and 25 mm diameter



TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter
- Samples taken from breathing zone: yes
- Frequency: 5 times a day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

gravimetrically on selectron filters, pore size 0.2 µm and 50 mm in diameter

Duration of treatment / exposure:

21 days (15 exposure days), 6 hours/day

Frequency of treatment:

5 days per week

Remarks:

Doses / Concentrations:
0, 52, 151, 401 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: daily, except weekends, during exposure; weekly during recovery


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: twice weekly during exposure; weekly during 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: Yes


WATER CONSUMPTION: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all dose groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haemoglobin, methaemoglobin, carboxyhaemoglobin, erythrocytes, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, reticulocytes, inclusion bodies, thrombocytes, prothrombin time, activated partial thromboplastin time, leucocytes (total count and differential count)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at the end of the exposure
- Animals fasted: Yes
- How many animals: all
- Parameters examined: glucose, urea, total protein, protein electrophoresis, electrolytes, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transpeptidase


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
- organ weights: yes (at the end of the exposure and recovery period; brain, heart, liver, kidney,adrenals, thymus, gonades, lung)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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
No death occurred during the exposure and recovery period. No toxic symptoms were observed in the animals of the control and test groups.

BODY WEIGHT AND WEIGHT GAIN
There was a slight but significant (p>/= 0.01) decrease in the male animals of the highest concentration group at day 21 and females at days 3, 7 and 10. During the recovery period the bodyweight gain was again comparabel to that of the control.

FOOD CONSUMPTION
No significant differences between treated animals and controls.

FOOD EFFICIENCY
No significant differences between treated animals and controls.

WATER CONSUMPTION
No effects.

OPHTHALMOSCOPIC EXAMINATION
No occular changes were observed.

HAEMATOLOGY
The observed haematological findings between treated rats and controls were generally unremarkable.
A slight change with statistical significance (p
CLINICAL CHEMISTRY
No significant differences between treated animals and controls.


ORGAN WEIGHTS
Absolute and relative lung weigths (lung to body weight and lung to brain weight) of males and females of the highest dose group were significantly elevated in comparison to the control group at the end of the exposure and at the end of the recovery period. Lung to body weights (but not the absolute lung weights) of males of the mid dose group were also elevated at the end of the exposure period (not measured at the end of the recovery period). The organ weights of the other organs were comparable in the control and treated groups.

GROSS PATHOLOGY
The lungs of all rats from the highest concentration group were slightly enlarged, with yellowish tinge. The lungs of all rats from the intermediate concentration group showed slightly yellowish tinge. The lungs of the lowest concentration group showed no compound related gross anatomical changes. The yellwoish discoloration of lungs was noted also in rats from the highest concentration group which were sacrificed after an additional recovery period of 21 days.

HISTOPATHOLOGY: NON-NEOPLASTIC
The eyes of all animals showed posttraumatic peribulbar lesions which were results of blood collection performed before sacrifice of the animals and were of no toxicologic relevance.
The lungs of all rats from the highest concentration group showed focal accumulation of slighly basophilic material and foamy cells in numerous alveoli. In Sudan stained frozen sections minute yellow-brown particles in the lumen of alveoli, in the cytoplasm of some foamy cells, in the lumen of occasional small bronchi and in the macrophages in the interstitium were seen. There was only minimal focal lymphohistiocytic infiltration in the interstitium.
The lungs of all rats from the intermediate concentration group showed , in the frozen section, moderate focal accumulation of brown-yellow minute particles in the macrophages in the interstitium and in very occasional alveoli. In the lungs of 4/10 male and 4/10 female rats from this group minimal focal accumulation of foamy cells in very occasional alveoli was observed.
The lungs of the rats from the lowest concentration goup showed no accumulation of foamy cells in the alveoli. In the frozen sections however minimal focal accumulation of brown-yellow particles in the macrophages in the interstitium and in very occasional alveoli was seen.
At the end of the recovery period the lungs of all animals from the highest concentration group showed focal accumulation of minute brown-yellow foreign particles in the interstitium. Few minute foreign particles in the alveoli and occasional ones in the intrapulmonal lymphatic tissue were observed as well.

Key result

Dose descriptor:

NOAEC

Effect level:

52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathologic investigation revealed minimal deposition of test material in the lung, but this was not accompanied by an inflammatory or any other adverse response

Key result

Critical effects observed:

no

Analysed test item concentrations were:

0 mg/m3

52 +/- 4 mg/m3

151 +/- 9 mg/m3

401 +/- 15 mg/m3

Conclusions:

The test item did not elicit relevant toxicological effects after subacute inhalation exposure except for some local effects in the lungs of the treated animals probably due to the particulate matter of the test item and not due to inherent toxicity. Therefore, no NOEL can be derived from this study. Whereas inflammatory effects up to pneumoconiosis occurred in the mid and high concententration groups, the effects observed at the lowest test concentration were only due to the deposition of the test material, but not adverse. Therefore the lowest test concentration can be regarded as NOAEC.

Executive summary:

Subacute toxicity of the test item was investigated in an 21 days aerosol inhalation study in rats, which were exposed to 0, 52, 151, 401 mg/m3 test item (6 h/d, 5 d/w).

A slight but significant (p < 0.01) decrease in the bodyweight of the male rats an the highest concentration at termination of the exposure period was observed when compared with those of the controls and the other treated animals.

A slight change was found to occur in the differential leucocyte count of male and female rats at the highest exposure level, where a higher percentage of polymorphonuclear neutrophils and a lower percentage of lymphocytes was observed. This change persisted upon cessation of treatment.

The lungs of all rats from the highest concentration group were slightly enlarged and yellowish in colour. The yellowish dis­coloration of the lungs was also noted in rats from the group which was sacrificed after an additional recovery period of

21 days. The lung weights, the lung to bodyweight and lung to brain weight ratios of these animals were significantly increased. The weight increase persisted in rats of this group after anadditional recovery period of 21 days.

Upon histopathological examination the lungs of all rats from the highest concentration group showed focal accumulation of slightly basophilic material and of numerous foamy cells in the alveoli. In frozen sections large amount of minute yellow-brownforeign particles (1 - 3 um) in the lumen of the alveoli, in the cytoplasm of some foamy cells, in the lumen of occasional small bronchi and in the macrophages in the interstitium were observed. There was also slight focal lymphohistiocytic infiltration in the interstitium. Focal pneumoconiosis showing brown-yellow foreign particles (dissolved by the procedure used for tissue embedding) was also observed in the treated rats from the top concentration level after the withdrawal period of 3 weeks. Small accumulation of brown-yellow particles was occasionally found in the intra­pulmonal lymphoid tissue of these animals.

In the rats treated with the intermediate concentration level the lungs showed at autopsy slight yellowish discoloration. Histo­pathology revealed brown-yellow, fat soluble particles in the alveoli and in the interstitium of the lungs. In 8 out of 20 rats from this group focal accumulation of foamy cells in several alveoli was observed as well.

The yellowish discoloration of the lungs was not observed in rats from the lowest concentration group. Neither accumulation of foamy cells in the alveoli, nor interstitial inflammatory infiltration was seen upon histopathology in these animals. Minute brown­yellow fat soluble foreign particles were however found in frozen sections in the interstitium of the lungs and in occasional alveoli.

No other gross or microscopical changes which could be related to the inhalation of the test item were found in the treated animals.

It can be inferred from the observations made during the above study that the "no observable effect level" for rats is below 52 mg/m³of air. The effects observed at the lowest test concentration were only due to the deposition of the test material but did not cause adverse effects like inflammation etc. Therefore, the lowest test concentration can be regarded as "no observable adverse effect level".

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

see chapter 13

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Slight deposits of test material in the lungs were not considered as adverse

Key result

Critical effects observed:

no

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because a reliable chronic toxicity study is available, conducted with an appropriate species and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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:

other:

Critical effects observed:

not specified