Disodium dihydrogen ethylenediaminetetraacetate

Endpoint summary

Description of key information

Several repeated dose studies using Na2EDTA are available. Additionally toxicological information on other Na EDTA salts has been considered as under physiological conditions (pH 7-9) any EDTA salt as well as edetic acid will dissociate into the sodium cations and the respective anionic species of edetic acid depending on the dissociation equilibria of edetic acid. Under the assumption of this equivalence it is likely that all EDTA salt chelate ions in vivo. A 90-day study with Na2H2EDTA as well as 2 years feeding studies with Na3EDTA on rats and mice provide reliable toxicological information for an overall NOAEL of about 500 mg/kg bw. A NOAEC value of 3 mg/m³ air was established in a subchronic toxicity study with Na2H2EDTA.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

A 13-week feeding study on rats was performed using 3 different dose groups and one control group. After 13 weeks 50 % of the animals of each group were sacrificed and tissues examined for gross and histopathologic changes. The remaining animals were placed on control diet for 4 weeks. Thereafter animals were sacrificed and examined for gross and histopathologic changes.

GLP compliance:

no

Species:

rat

Strain:

other: Holtzman

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 120 ± 6 g
- Diet: Ground Purina Laboratory Chow

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The top level (10 %) diet was prepared by adding the appropriate amount of Na2H2EDTA to the basic diet. This was then diluted with the basic diet to prepare the 5 % diet. Lower concentrations were similarly prepared by dilution of the next highest concentration.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

original data: 1 % ; conversion factor: 20

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

original data: 5 % ; conversion factor: 20

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

original data: 10 % ; conversion factor: 20

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Mean food consumption g/animal

WATER CONSUMPTION
- not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 4th and 13th week
- Parameters checked: hematocrit, hemoglobin, total and differential white blood cell counts, prothrombin times

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at the end of the 4th and 13th week
- Parameters checked: calcium level

URINALYSIS: Yes
- Time schedule for collection of urine: not specified
- Parameters checked: Calcium

OPHTHALMOSCOPIC EXAMINATION: No
CLINICAL CHEMISTRY: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: liver, kidney, spleen, lung, heart, urogenital system, digestive organs, and muscle
HISTOPATHOLOGY: Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day dose group: nothing abnormal detected
2500 mg/kg bw/day dose group: diarrhea starting at the third day
5000 mg/kg bw/day dose group: animals were emaciated, diarrhea starting at the third day

Mortality:

mortality observed, treatment-related

Description (incidence):

2500 mg/kg bw/day dose group: 2/10 animals died
5000 mg/kg bw/day dose group: 6/10 animals died

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day dose group: no difference to the control group
2500; 5000 mg/kg bw/day dose group: statistically significant reduced body weight gain (control: 326 g; 2500 mg/kg bw/day dose group: 185 g; 5000 mg/kg bw/day dose group: 4 g)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day dose group: normal food consumption
2500; 5000 mg/kg bw/day dose group: statistically significant lower food consumption than the control

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

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day dose group: nothing abnormal detected
2500 mg/kg bw/day dose group: twice the water consumption of the control
5000 mg/kg bw/day dose group: nothing abnormal detected

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

after 4 weeks: hematocrits and hemoglobins of the 5000 mg/kg bw/day dose group slightly depressed
after 13 weeks: nothing abnormal detected in any of the groups

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

serum calcium level did not differ from the control

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

total calcium:
500 mg/kg bw/day dose group: no difference to the control
2500; 5000 mg/kg bw/day dose group: ca. twice as much as in the control

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

500; 2500 mg/kg bw/day dose group: nothing abnormal detected
5000 mg/kg bw/day dose group: pale livers

Histopathological findings: non-neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Critical effects observed:

not specified

RECOVERY

Once the survivors were placed on a control diet the diarrhea, when present, subsided the within 24 hours. The animal that had previously received 5000 mg/kg bw/day Na2H2EDTA still had low food consumption and died within l week. All other animals survived this 4-week period . The animals that had received 2500 mg/kg bw/ day Na2H2EDTA gained weight more slowly than did the other animals and weighed significantly less than controls at the end of the 4-week withdrawal period. The chelating agent was not detectable in the urine after 2-3 days, nor in the feces after 7 days. The autopsies revealed no significant findings.

- EGTA was better tolerated in the diet than EDTA

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles

Principles of method if other than guideline:

A bioassay for possible carcinogenicity was conducted by administering the test material in feed to B6C3F1 mice. The chemical was administered to 50 males and 50 females at low and high concentrations, for 103 weeks. Matched-control groups were composed of 20 males and 20 females. Animals were analysed for mortality, clinical signs, histopathological as well as gross pathological changes.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, USA
- Weight at study initiation: 19-22 g
- Age at study initiation: 28 days
- Housing: five per cage in solid polycarbonate cages
- Diet: prepared from Wayne Lab Blox Meal (Allied Mills, Inc.) ad libitum
- Water: acidulated water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 45-55
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 16/8

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): 2 times/week
- Mixing appropriate amounts with (Type of food): Wayne Lab Blox Meal (Allied Mills, Inc.)
- Storage temperature of food: 4°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed, using FDA methods to determine the efficiency of the mixing procedure and the stability of the test chemical in feed. Recoveries were found to be 90.3 ± 1.4% of the theoretical value at 7500 ppm EDTA and 90.4 ± 3.4 % of the theoretical value at 3750 ppm. It was concluded from these results that the preparations contained reasonably accurate concentrations of EDTA and were mixed homogeneously, and that the chemical was stable in feed for at least a week.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

daily

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

original data: 3750 ppm; conversion according to EU risk assessment

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

original data: 7500 ppm; conversion according to EU risk assessment

No. of animals per sex per dose:

50 (except for the control, which consisted of only 20 animals)

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: approximately once a month

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; all major organs, not specified but presumably all organs used for histopathology: skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.
HISTOPATHOLOGY: Yes; skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.

Statistics:

- Statistical tests of differences in survival between groups are compared using the method of Cox (1972) for two groups and an extension of this method by Tarone (1975) for more than two groups.
- Statistical analysis of the incidence of tumors was made using the Fisher exact test (Cox, 1970) to compare a control group to a group of treated animals at each dose. In addition, the Armitage and Cochran test for linear trend in proportions, with continuity correction (Armitage, 1971), was used.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Ataxia occurred in a low-dose male at 8 months, and ascites was noted in mice of both sexes during the second year of the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No statistically significant difference in survival between the different groups in both sexes. (males: 5/50 (10 %) of the low-dose group, 2/50 (4 %) of the high-dose group, and 1/20 (5 %) of the control; females: 0/50 low-dose female mice, 1/20 (5 %) of the control group and 3/50 (6 %) of the high-dose group)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In male mice only the high-dose group showed throughout most of the test period a decrease in average body weight compared to the controls. In female mice average body weights of the treatment groups were depressed in a dose-related manner during the test period, although the effect was small. No individual data given. No information whether this effect is statistically relevant is available.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Nothing reported.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A variety of neoplasms was observed in both treated and control animals. Each type observed has been encountered previously as a spontaneous lesion in the mouse. However, the incidence of neoplasms in all groups was high in the hematopoietic, endocrine, digestive, and respiratory systems. The incidence of neoplasms in other systems was variable. (See table 1 and 2).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: neoplastic

mortality

Critical effects observed:

not specified

Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Mice Fed EDTA Trisodium Salt in the Diet

Morphology (p-value)	Control	625 mg/kg bw/day	1250 mg/kg bw/day
Hematopoietic System:  Malignant Lymphoma	2/20 (n.s.)	7/46 (n.s.)	7/48 (n.s.)
Thyroid: C-cell Adenoma	0/10 (n.s.)	1/29 (n.s.)	1/33 (n.s.)
Pituitary: Chromophobe Adenoma	1/13 (n.s.)	0/19 (n.s.)	1/26 (n.s.)
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma	2/18 (0.096)	2/50 (n.s.)	3/49 (n.s.)
Liver: Hepatocellular Adenoma and Neoplastic Nodule	3/19 (n.s.)	10/44 (n.s.)	10/47 (n.s.)

Table 2: Analyses of the Incidence of Primary Tumors at Specific Sites in Female Mice Fed EDTA Trisodium Salt in the Diet

Morphology (p-value)	Control	625 mg/kg bw/day	1250 mg/kg bw/day
Hematopoietic System:  Malignant Lymphoma	5/19 (n.s.)	11/49 (n.s.)	12/47 (n.s.)
Thyroid: C-cell Adenoma	1/12 (n.s.)	3/33 (n.s.)	1/34 (n.s.)
Pituitary: Chromophobe Adenoma	2/12 (n.s.)	6/34 (n.s.)	4/29 (n.s.)
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma	0/19 (n.s.)	3/47 (n.s.)	3/49 (n.s.)
Liver: Hepatocellular Adenoma and Neoplastic Nodule	0/19 (n.s.)	1/46 (n.s.)	1/47 (n.s.)

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

A bioassay for possible carcinogenicity was conducted by administering the test material in feed to Fischer 344 rats. The chemical was administered to 50 males and 50 females at low and high concentrations, for 103 weeks. Matched-control groups were composed of 20 males and 20 females. Animals were analysed for mortality, clinical signs, histopathological as well as gross pathological changes.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Schmidt, Madison, Wisconsin, USA
- Weight at study initiation: 85-110 g
- Age at study initiation: 28 days
- Housing: four per cage in solid polycarbonate cages
- Diet: prepared from Wayne Lab Blox Meal (Allied Mills, Inc.) ad libitum
- Water: acidulated water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 45-55%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 16/8

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): 3 times/week
- Mixing appropriate amounts with (Type of food): Wayne Lab Blox Meal (Allied Mills, Inc.)
- Storage temperature of food: 4°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed, using FDA methods to determine the efficiency of the mixing procedure and the stability of the test chemical in feed. Recoveries were found to be 90.3 ± 1.4% of the theoretical value at 7500 ppm EDTA and 90.4 ± 3.4% of the theoretical value at 3750 ppm. It was concluded from these results that the preparations contained reasonably accurate concentrations of EDTA and were mixed homogeneously, and that the chemical was stable in feed for at least a week.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

daily

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

original data: 3750 ppm; conversion according to EU risk assessment

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

original data: 7500 ppm; conversion according to EU risk assessment

No. of animals per sex per dose:

50 (except for the control, which consisted of only 20 animals)

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: approximately once a month

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; all major organs, not specified but presumably all organs used for histopathology: skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.
HISTOPATHOLOGY: Yes;
skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.

Statistics:

- Statistical tests of differences in survival between groups are compared using the method of Cox (1972) for two groups and an extension of this method by Tarone (1975) for more than two groups.
- Statistical analysis of the incidence of tumors was made using the Fisher exact test (Cox, 1970) to compare a control group to a group of treated animals at each dose. In addition, the Armitage and Cochran test for linear trend in proportions, with continuity correction (Armitage, 1971), was used.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No significant signs were observed among test animals during the first year of the study. In the 6 months preceding termination of the test, corneal opacities, ascites, and urine stains, occurred in both treatment and control groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The male rats exhibited a negative dose-related trend in survival with the probability level of P = 0.103; the treated and control groups of male rats can thus be considered as comparable to each other in survival. The female rats also exhibited a negative dose-related trend in survival, but in this case the effect was statistically significant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Average body weights of treated male and female rats were comparable to those of the matched controls throughout the study.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Inflammatory and degenerative changes were observed in about the same frequency in all groups. These lesions appeared to be related to age and no to the administration of the chemical.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of neoplasms was high in the reproductive and endocrine systems and lower in the hematopoietic, respiratory, integumentary, and digestive systems. No neoplasms were observed in the nervous, musculoskeletal, or urinary systems or in organs of special sense. A number of tumors occurred in other organ systems of both sexes, controls as well as treated animals. In some instances the incidence of tumors in the controls exceeded that of the treated animals. With the possible exception of endocrine tumors in the males, no clear association between the incidence of tumors, treatment, or sex could be established (see table 1 and 2).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: neoplastic

mortality

Critical effects observed:

not specified

Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Rats Fed EDTA Trisodium Salt in the Diet

Morphology (p-value)	Control	250 mg/kg bw/day	500 mg/kg bw/day
Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia	3/20 (n.s.)	4/50 (n.s.)	4/50 (n.s.)
Adrenal: Pheochromocytoma	2/20 (n.s.)	5/49 (n.s.)	4/50 (n.s.)
Thyroid: C-cell Adenoma	0/17 (n.s.)	6/45 (p = 0 0.08)	3/38 (n.s.)
Pituitary: Chromophobe Adenoma	0/18 (p = 0.089)	3/47 (n.s.)	5/44 (n.s.)
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma	1/18 (n.s.)	2/50 (n.s.)	3/49 (n.s.)
Liver: Hepatocellular Adenoma and Neoplastic Nodule	0/20 (n.s.)	1/48 (n.s.)	1/50 (n.s.)
Testis: Interstitial-cell Tumor	19/20 (n.s.)	43/50 (n.s.)	44/50 (n.s.)

Table 2 Analyses of the Incidence of Primary Tumors at Specific Sites in Female Rats Fed EDTA Trisodium Salt in the Diet

Morphology (p-value)	Control	250 mg/kg bw/day	500 mg/kg bw/day
Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia	1/20 (n.s.)	8/50 (n.s.)	0/50 (n.s.)
Adrenal: Pheochromocytoma	1/20 (n.s.)	1/49 (n.s.)	1/48 (n.s.)
Thyroid: C-cell Adenoma	0/11 (n.s.)	0/36 (n.s.)	1/37 (n.s.)
Pituitary: Chromophobe Adenoma	6/19 (n.s.)	10/48 (n.s.)	11/50 (n.s.)
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma	0/20 (n.s.)	3/48 (n.s.)	2/48 (n.s.)
Liver: Neoplastic Nodule	0/20 (n.s.)	1/48 (n.s.)	0/48 (n.s.)
Uterus: Endometrial Stromal Polyp	5/20 (n.s.)	6/50 (n.s.)	7/50 (n.s.)
Mammary Gland: Fibroadenoma	4/20 (n.s.)	3/50 (n.s.)	3/50 (n.s.)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

103-week feeding study

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:

2009-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 412 (Repeated Dose Inhalation Toxicity: 28/14-Day)

Deviations:

yes

Remarks:

Dosing until Day 5 only

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.: of test material: 06088797V0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Expiry date: 1 September 2011

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Age: 7 weeks (approx)
Identification: Tattooing of ears
All animals free of disease and clinical signs
Rats housed together (5 animals per cage) in Polysulfon cages
Bedding: Type Lignocel fibres, dust free bedding
Woodne gnawing blocks for enrichment
Rooms: Fully ariconditioned, temperature range 20 to 24 degrees celcius, 30 to 70% humidity
Light/dark cycle of 12 hours (6 am to 6pm light, 6pm to 6 am dark)
Food, drinking water and bedding/enrichment materials were analysed for chemical and microbiological contaminants.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: MMAD : 2.0 - 2.7 µm
GSD: 2.0 - 2.22

Details on inhalation exposure:

A dust aerosol was generated using a dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed into the inhalation system. The test substance was mixed with Aerosil R972 prior to facilitate aerosol generation.

The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets. The rats were restrained in glass exposure tubes with their snouts projecting into the inhalation chamber.
The animals did not ave access to feed or water during the exposure period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of the inhalation atmospheres were analyzed using gravimetry. Daily means were calculated based on 2 measured samples per concentration and exposure. From the Daily mean values of each concentration, mean concentrations and standard deviations were derived.
Consistency of concentrations in each inhalation system was continuously monitored using scattered light photometry.
Particle size analysis was conducted using a cascade impactor.

Duration of treatment / exposure:

Exposures: 6 hours per day
High dose group (1000 mg/m³) was exposued for 1 day only
All other groups were exposed for 5 consecutive days

Frequency of treatment:

Daily

Dose / conc.:

30 mg/m³ air (nominal)

Remarks:

actual: 33.3 (± 2.3) mg/m³

Dose / conc.:

300 mg/m³ air (nominal)

Remarks:

actual: 320 (± 27) mg/m³

Dose / conc.:

1 000 mg/m³ air (nominal)

Remarks:

actual: 1103 (± 52) mg/m³

No. of animals per sex per dose:

10 animals per dose group
An additional 10 animals for the high dose group and control

Control animals:

yes, sham-exposed

Details on study design:

The animals were exposed to a respirable dust aerosol for 6 hours per day for 5 consecutive days. The exception was the high dose group (1000mg/m³) where exposure was for one day only due to mortality observed.
In the control, low and mid dose groups, 5 animals were sacrificed on the day after the last exposure period and 5 were sacrificed 17 days after the last exposure.
10 additional control animals and the 14 surviving high dose group animals were sacrificed on day 14 of the study (14 days after first exposure).

Observations and examinations performed and frequency:

Mortality:
- observations of toxicity or mortality were made twice daily (weekdays) and once daily (weekends and public holidays)

Clinical Observations:
- Once during the pre-flow period and on post-exposure days. At least three times (before, during and after exposure) on exposure days.

Bodyweight:
- Start of pre-flowm start of exposuer period, once per week prior to necropsy

Food consumption:
- Determined weekly and calculated as mean food consumption in grams per animal per day (no statistical evaluation possible due to grouphousing conditions)

Clinical Pathology:
- On day of sacrifice, blood aken from retro-orbital venous plexus fromfasted animals.

Hematology:
- Leucocyte count, erthyrocyte count, hemoglobin, hematocrit, MCV, MCH, MCHC, Platelet count, differential blood count, reticulocytes count, clotting anlyses (prothrombin time)

Clinical Chemistry:
- ALT, AST, ALP, GGT

Blood chemistry:
- sodium, potassium, chloride, inorg. phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

Sacrifice and pathology:

5 animals per dose group were sacrificed and underwent gross macroscopic examination the day after the last exposure.
5 animals from each dose group (control, 30, and 300 mg/m³) were sacrificed at the end of the recovery period.
All animals dying prematurely were examined histologically.
Surviving animals in the 1000 mg/m³ group and 10 control animals were sacrificed and examined on day 14.

Necropsy:
- all animals were necropsied and assessed for gross pathology. Animals dying intercurrently or were killed in a moribund state wee necropsied as soon as possible after their death.

Organ Weights:
- Adrenals, brain, epididymides, heart, liver, kidneys, lungs, spleem, testes, thymus thyroid glands

Organ/tissue fixation:
- All gross lesions, adrenal glands, brain with olfactory bulb, bone marrow (femur), eyes with optic nerve, heart, kidneys, liver, larynx/pharynx, lymph nodes, nose, oesophagus, seminal vesicle, spinal cord, stomach (forestomach and glandular), spleen, testes, thyroid, thymus, trachea, urinary bladder
- One lobe of the liver from the lobus Dexter medialis and the lobus sinster lateralis was fixed in Carnoy's solution and embedded in paraplast. All other tissues were fixed in formulin.

Histology (All animals per group):
- All gross lesions, nasal cavity (4 levels), larynx (3 levels), trachea, lungs (5 lobes), lymph nodes (tracheobronchial and mediastinal), kidneys and liver

Statistics:

Body weight/body weight change, food consumption - comparison of each group with control using DUNNETTS test (two-sided) for the hypothesis of equal means
Clinical pathology, urine volumes, urine specific gravity, Weight parameters - Non-parametric one-way analysis using Kruskal-wallis test (two sided). If resulting p-value was less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two sided) for equal means.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloerection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration sounds, piloerection, reduced fur care

Mortality:

mortality observed, treatment-related

Description (incidence):

6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloerection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration sounds, piloerection, reduced fur care

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased bodyweight change in mid and high dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decreased food consumption between days 0 and 1 in mid and high dose group

Food efficiency:

not examined

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:

effects observed, treatment-related

Description (incidence and severity):

Lung weight increase in low and mid dose group

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Congestion, edema and multifocal hemorraghes in lungs of high dose group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Histopathology results:
High dose: Multifocal hemorraghes in the lungs; Inflammatory cell infiltrates

Mid dose:
Larynx: laryngeal, epithelial necrosis, multifocal, in various levels of the larynx
Inflammatory cell infiltrates in various levels of the larynx
laryngeal squamous metaplasia, multifocal, in various levels of the larynx
Regenerative hyperplasia of the laryngeal epithelium, multifocal, in various levels of the larynx
Lungs: Regenerative hyperplasia of bronchiolar epithelium (predominantly: medium bronchi, terminal bronchioles)
Mucous cell hyperplasia in large bronchi
interstitial infiltration of eosinophylic granulocytic cells

Low dose:
Larynx: Laryngeal, epithelial necrosis, multifocal, at the base of the epiglittis (level 1)
Inflammatory cell infiltrates at the base of the epiglottis (level 1)
Lungs: Regenerative hyperplasia of the bronchiolar epithelium (predominantly medium bronchi and terminal bronchioles)
Mucous cell hyperplasia in large bronchi
interstitial infiltration of eosinophylic granulocytic cells.

There were no histopathological findings in any of the recovery group animals. Thus all pathology was reversible within the recovery period.

Dose descriptor:

LOAEC

Effect level:

30 mg/m³ air (nominal)

Based on:

act. ingr.

Remarks:

Na2H2EDTA

Sex:

male

Basis for effect level:

other: histopathology of the respiratory tract and lung weights

Critical effects observed:

not specified

no NOAEC identified

Conclusions:

Inhalation exposure to 1000 mg/m³ disodium EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorraghes and inflammatory cell infiltrates. Inhalation exposure of rats to disodium EDTA for 6 hours per day, 5 consecutive days cause concentration dependant lesions inthe larynx and lungs that were fully reversible within 14 days. Due to histopahological changes in the low dosegroup a no observed effect level could not be determined.

Executive summary:

In a subacute repeated dose toxicity study (BASF SE, 2009) 10 male Wistar rats per dose were exposed to a respirable dust aerosol of Na2H2EDTA for 6 hours per day for 5 consecutive days at concentrations of 0, 30, 300, 1000 mg/m³ air.

Exposure in the high dose group (1000 mg/m³) was for one day only due to mortality observed. Inhalation exposure to 1000 mg/m³ disodium EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorraghes and inflammatory cell infiltrates.

Inhalation exposure of rats to disodium EDTA for 6 hours per day, 5 consecutive days cause concentration dependant lesions in the larynx and lungs that were fully reversible within 14 days. Due to histopahological changes in the low dose group a no observed effect level could not be determined.

The LOAEC was considered to be 30 mg/m³ air.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.: of test material: Lot 64580436W0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: Homogeneous
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 7 weeks
- Housing: up to 5 animals per cage in Polysulfon cages (H-Temp [PSU])
- Diet (e.g. ad libitum): 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): A light/dark rhythm of 12 hours was maintained: 06.00 a.m.; 06.00 p.m. light; 06.00 p.m.; 06.00 a.m. dark

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Test group 1 (0.5 mg/m³): MMAD (µm) 2.3-2.8; Geometric standard deviation 1.7-2.2
Test group 2 (3 mg/m³): MMAD (µm) 2.0-2.4; Geometric standard deviation 1.8-2.0
Test group 3 (15 mg(m³): MMAD (µm) 2.3-2.5; Geometric standard deviation 1.8-2.1

Details on inhalation exposure:

For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned dilution air and passed into the inhalation system. To achieve stable concentration in the atmosphere, a part of generated atmosphere was replaced by fresh conditioned air.
The inhalation atmosphere was maintained inside aerodynamic exposure systems, consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room. All test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures. The animals did not have access to water or feed during the exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analyzed by gravimetry in all test groups including. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 2 measured samples in test group 1 and 3 measured samples per concentration and exposure in test groups 2 and 3. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.
Scattered light photometry was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices.
The particle size analysis was carried out with a cascade impactor.

Duration of treatment / exposure:

Exposures: 6 hours per day

Frequency of treatment:

daily (5 consecutive days/week), 13 weeks, 65 exposures in total

Dose / conc.:

0.5 mg/m³ air (nominal)

Dose / conc.:

3 mg/L air (nominal)

Dose / conc.:

15 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined for evident signs of toxicity or mortality twice a day on working days and once a day on Saturdays, Sundays and public holidays

CLINICAL OBSERVATIONS: Yes
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 42, 84

BODY WEIGHT: Yes
- Time schedule for examinations: at start of the pre-exposure, at start of the exposure, then twice a week (Monday, Friday), and prior to gross necropsy

FOOD CONSUMPTION:
- Food consumption was determined cage-wise weekly (Monday-Friday) and calculated as g food/animal/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the exposure period (day -1/ -2) the eyes of all animals, and at the end of the study (day 82/83) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY: Yes
All animals per test group and sex at the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
Parameters: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, DBC, RET

CLINICAL CHEMISTRY: Yes
All animals per test group and sex at the end of the administration period
- Animals fasted: Yes
Parameters. ALT, AST, ALP, GGT, NA, K, CL, INP, CA, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB, TRIG, CHOL

URINALYSIS: Yes
All animals per test group and sex at the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity / Open filed observations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals assigned for light microscopic examination were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: all animals sacrificed on schedule
Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus

Organ/Tissue Fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal gland, Eyes with optic nerve and eyelid (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus

HISTOPATHOLOGY: Yes
List of organs and tissues of histological examinations: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Colon, Duodenum, Esophagus, Femur with knee joint, Heart, Ileum, Jejunum, Kidneys, Larynx (3 levels), Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric), Mammary gland (female), Nasal cavity (4 levels), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus

Statistics:

Body weight, body weight change - comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the
hypothesis of equal means
Feces, rearing, grip, strength length, forelimbs, grip, strength length, hindlimbs, footsplay, test, motor activity - Non-parametric one-way analysis
using KRUSKAL-WALLIS test (twosided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
Blood parameters - For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians
Weight parameters - Non-parametric one-way analysis using KRUSKAL-WALLIS test (twosided).If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Clinical signs:

no effects observed

Description (incidence and severity):

One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.

Mortality:

no mortality observed

Description (incidence):

One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Dose descriptor:

LOAEC

Effect level:

15 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: histopathology

Dose descriptor:

NOAEC

Effect level:

> 15 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: histopathology

Dose descriptor:

NOAEC

Effect level:

3 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathology

Critical effects observed:

not specified

Organ weights:

Relative changes of absolute liver and lung weights in comparison to the control

	Male animals			Female animals
Test group (mg/m³)	1 (0.5)	2 (3)	3 (15)	1 (0.5)	2 (3)	3 (15)
Liver				107%	102%	109%*
Lungs	99%	93%	108%*	107%*	112%**	124%**

* : p <= 0.05, **: p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when

compared to the control group 0.

Relative organ weights

When compared with control group 0 (=100%), the following mean relative organ weights

were significantly changed (statistically significant changes printed in bold):

	Female animals
Test group (mg/m³)	1 (0.5)	2 (3)	3 (15)
Lungs	104%	112%**	120%**

* : p <= 0.05, **: p <= 0.01

 

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The increased lung weights in males of test group 3 (15 mg/m³) and females of all test groups might be related to the treatment. No histopathologic finding could explain the weight increase. In males, the relative lung weight was not significantly changed. Furthermore was the lung weight of female animals within the range of historical control data. Therefore the lung weight increase was regarded to be not adverse.

Treatment-related findings were observed in male and females with incidences and grading according to the table below

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (0.5)	2 (3)	3 (15)	0 (0)	1 (0.5)	2 (3)	3 (15)
No.of animals	10	10	10	10	10	10	10	10
Hyperplasia (m)focal	0	0	0	0	0	0	0	1
Metaplasia, squamous	0	0	1	6	0	0	1	3
·                  Grade1			1	2			1	1
·                  Grade2				4				2
Epithelial alteration	2	4	6	4	1	7	8	6
·                  Grade1	2	4	5	1	1	7	8	2
·                  Grade2			1	3				4
Infiltrates, granulocytic								2
·                 Grade2								2

Whenever no grading was given the finding was recorded to be present

Animals of all test groups as well as single control animals revealed minimal to slight epithelial alteration at the base of the epiglottis. The term was used, if at the base of the epiglottis a small focal area was covered by flattened epithelium, which differed from the normal cuboidal to columnar laryngeal epithelium. Secondary, some animals of test group 3 (15 mg/m³) and 2 (3 mg/m³) showed a minimal to slight focal squamous metaplasia in this region. These findings were regarded to be test substance related and adaptive. One female of test group 3 (15 mg/m³) revealed a small focal hyperplasia of the laryngeal epithelium at the base of the epiglottis and in addition slight granulocytic cell infiltrates. These infiltrates were also observed in a second female of this test group. These findings were regarded to be treatment related and adverse.

Microscopic findings in lung and their grading

	Maleanimals				Femaleanimals
Test group (mg/m³)	0 (0)	1 (0.5)	2 (3)	3 (15)	0 (0)	1 (0.5)	2 (3)	3 (15)
No.ofanimals	10	10	10	10	10	10	10	10
Histiocytosisalveolar,d	0	0	6	10	0	0	5	10
·                  Grade1			6	4			5	6
·                  Grade2				6				4
Metaplasiamucouscells	0	0	0	4	0	0	0	5
·                  Grade1				4				5

Animals of test group 2 and 3 (3 and 15 mg/m³) revealed minimal to slight increased numbers of alveolar histiocytes. These histiocytes showed an eosinophilic cytoplasm, occasionally with clear vacuoles and were located as single cells within the alveoli all over the lung lobes. When compared to the control animals, males and females of test group 3 (15 mg/m³) showed a minimal to slight increase in number of mucous cells in the large bronchi. These findings were regarded to be treatment related effects and adaptive.

Testes

Tubular degeneration (up to moderate) was observed in control (8 animals affected) and test group 3 animals (7 animals affected) in similar incidences. This finding was characterized by randomly affected (not stage specific) tubules with sloughed spermatogenic cells, vacuolation of the spermatogenic epithelium or missing germ cell layers. This effect in the testis is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in similar incidences in control animals.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Executive summary:

Inhalation exposure of rats to Trilon BD for 90 day (65 exposures) did not lead to any substance-related clinical signs of toxicity. Nor were there any effect in clinical chemistry, hematology. Histological examination revealed some effects in larynx at the highest tested concentration of 15 mg/m³ in female animals. No signs of systemic toxicity were observed up to a concentration of 15 mg/m³. Signs of local toxicity were observed only at the high concentration of 15 mg/m³ in female animals.

Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effects in larynx was 3 mg/m3, the NOEC for systemic effects is 15 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Sufficient quality due to guideline study (OECD TG 413)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

ORAL ROUTE

A 90 days feeding study of Na2EDTA in rats revealed an NOAEL of 500 mg/kg bw (Wynn, 1970). Groups of 10 male Holzman rats received 1, 5 and 10 % (respectively 500 mg/kg bw; 2500 mg/kg bw and 5000 mg/kg bw) Na2EDTA in the diet for 90 days. The mid and high dose animals expressed a significant decrease of body weights and food consumption. Dose dependent mortality was evident by 20 % in the 5 % and 60 % in the 10 % group. In these groups animals exhibited diarrhea and were emaciated. Water consumption was increased. In the upper dose there was an intermittent decrease of hematocrite and hemoglobin levels, livers appeared to be pale. Histological investigation failed to reveal any pathological alteration. From this investigation, an NOAEL of 500 mg/kg/day equivalent to 1 % in diet can be deduced for male rats. It should be noted that in this study no complete clinical biochemistry has been performed as required by OECD 408 guideline.

A two year feeding study with (Na3EDTA × 3 H2O) to rats revealed a NOAEL of 500 mg/kg/day (corresponding to 7500 ppm in the diet) (NTIS, 1977). In this feeding study with two dose levels 3750 ppm and 7500 ppm (corresponding to approximately 250 and 500 mg/kg/day) no substance related toxic effects could be observed.

The same study at the same dose levels (3750 ppm and 7500 ppm; corresponding to approximately 469 and 938 mg/kg/day) conducted with mice showed a NOAEL of 938 mg/kg bw/day. There were also no treatment related changes.

A one month feeding study of Na2EDTA in rats revealed a NOAEL of 1125 mg/kg bw (this corresponds to 2.25 % in the diet) (Kawamata, 1980). In this study test substance was incorporated at levels of 1, 2.25 and 5 % in the diet (this corresponds to 500, 1125 and 2500 mg/kg bw). 15 rats per sex and dose level were exposed over a period of one month. At the upper dose level body weight decrease, some mortalities and a reduction of total leucocytes and lymphocytes as well as an increase of bound urine nitrogen (BUN) and a decrease Ca serum levels were found. Pathological investigation at this dose level revealed a decrease of liver, spleen and thymus weight. Some parakeratosis was detected in the oesophagus and forestomach by histopathology.

INHALATION

In a subacute repeated dose toxicity study (BASF SE, 2009), 10 male Wistar rats per concentration were exposed to a respirable dust aerosol of Na2H2EDTA for 6 hours per day for 5 consecutive days at concentrations of 0, 30, 300, 1000 mg/m³ air.

Exposure in the high dose group (1000 mg/m³) was for one day only due to mortality observed. Inhalation exposure to 1000 mg/m³ disodium EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorrhages and inflammatory cell infiltrates.

Inhalation exposure of rats to Na2H2EDTA for 6 hours per day, 5 consecutive days caused concentration-dependent lesions in the larynx and lungs that were fully reversible within 14 days. Due to histopathological changes in the low dose group, a No-Observed-Adverse-Effect-Concentration could not be determined. The LOAEC was considered to be 30 mg/m³ air.

The study was planned as range finder study and conducted according to OECD Guideline 412.

In a subchronic repeated dose toxicity study (BASF SE, 2014) according to to OECD guideline No. 413, Wistar rats were exposed to a respirable dust aerosol of Na2H2EDTA for 6h/d on 5 consecutive d/w for 13 weeks (65 exposures in total) at concentrations of 0, 0.5, 3, 15 mg/m³ air.

Inhalation exposure of rats to Na2H2EDTA did not lead to any substance-related clinical signs of toxicity. Nor were there any effect in clinical chemistry, hematology. Histological examination revealed some effects in larynx at the highest tested concentration of 15 mg/m³. No signs of systemic toxicity were observed up to a concentration of 15 mg/m³.

Signs of local toxicity were observed only at the high concentration of 15 mg/m³. Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effect in larynx was 3 mg/m³, the NOEC for systemic effect is 15 mg/m³.

The local key effect of respirable Na2H2EDTA in the respiratory tract (larynx) is assumed to be mainly concentration-related; hence, the impact of exposure time should be low at subcritical concentrations, which was confirmed by the 90 -day study: although the number of exposures was factor 13 higher than in the 5-day dose-range-finder study, the local effects at 15 mg/m³ in the subchronic inhalation study were comparably mild compared to the 5-day dose-range-finder study that showed a more severe effect at 30 mg/m³.

Justification for classification or non-classification

The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008 as amended for the tenth time by Regulation (EU) 2017/776. It is justified to classify Na2H2EDTA for repeated dose effects due to the low concentrations of the substance that induces the local effects with a potential human relevance of the effects. However, due to the low severity and incidence of the effects at 15 mg/m³ in the subchronic toxicity study and the reversibility of the local effects, a STOT RE Cat.2 (H373: May cause damage to organs through prolonged or repeated exposure) is considered sufficiently conservative.