Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

oral

There are no repeated dose studies with Na4EDTA available. 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/day.

 

inhalative

A NOAEC 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

Referenceopen allclose all

Endpoint:
sub-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
Qualifier:
no guideline available
Principles of method if other than guideline:
A 13 weeks 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 (10.0% ) 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 5.0% 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)
Sex:
male
Basis for effect level:
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 neither 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 Na2H2EDTA                                                                                            

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 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°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): 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.:
469 mg/kg bw/day (nominal)
Remarks:
original data: 3750 ppm; conversion factor according to EU risk assessment

Dose / conc.:
938 mg/kg bw/day (nominal)
Remarks:
original data: 7500 ppm; conversion factor 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.
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, 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.
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:
>= 938 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
histopathology: non-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 469 mg/kg bw/day 938 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 469 mg/kg bw/day 938 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.)

n.s. = not significant

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 study 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.:
248 mg/kg bw/day (nominal)
Remarks:
original data: 3750 ppm (conversion according to EU risk assessment)

Dose / conc.:
495 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: 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. Even though this effect was statistically significant, it is most likely a chance finding as treated animals did not exhibit any deviations from the control group in regard to clinical signs or pathology.
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:
>= 495 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
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.)

n.s. = not significant

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 (Read-Across)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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:
Batch Identification: Lot 64580436W0
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/m³ air (nominal)
Dose / conc.:
15 mg/m³ 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, non-treatment-related
Description (incidence and severity):
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 (see tables 1 and 2)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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 tobe treatment related and adverse.
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.
(see tables 3 and 4)
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Key result
Dose descriptor:
NOAEC
Effect level:
3 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEC
Effect level:
15 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEC
Effect level:
> 15 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified

Organ weights:

Table1: 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

Table 2: 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.

Table 3: 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

Table 4: Microscopic findings in lung and their grading

 

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

Histiocytosis,alveolar,d

0

0

6

10

0

0

5

10

·                  Grade1

 

 

6

4

 

 

5

6

·                  Grade2

 

 

 

6

 

 

 

4

Metaplasia mucous cells

0

0

0

4

0

0

0

5

·                  Grade1

 

 

 

4

 

 

 

5

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD TG 413 (Read-Across)

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 a NOAEL of 500 mg/kg bw/day (Wynn, 1970). Groups of 10 male Holzman rats received 1, 5 and 10% (respectively 500 mg/kg bw/day; 2500 mg/kg bw/day and 5000 mg/kg bw/day) 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 hematocrit and hemoglobin levels, livers appeared to be pale. Histological investigation failed to reveal any pathological alteration. From this investigation, a NOAEL of 500 mg/kg bw/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 x 3H2O) to rats revealed a NOAEL of 500 mg/kg bw/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 bw/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 bw/day) conducted with mice showed a NOAEL of 938 mg/kg bw/day.

A one month feeding study of Na2EDTA in rats revealed a NOAEL of 1125 mg/kg bw /day(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/day). 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 esophagus and forestomach by histopathology.

Inhalation

In a subacute repeated dose toxicity study (BASF, 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, 2014) according 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 90d study: although the number of exposures was factor 13 higher than in the 5d dose-range-finder study, the local effects at 15 mg/m3 in the subchronic inhalation study were comparably mild compared to the 5d dose-range-finder study that showed a more severe effect at 30 mg/m3.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available data are reliable and suitable for classification purposes under Regulation 1272/2008. It is justified to classify Na4EDTA for repeated dose effects due to its analogue substance Na2H2EDTA that induced local effects at low concentrations and the potential human relevance of the effects. However, due to the low severity and incidence of the effects at 15 mg/m3 in the subchronic inhalation toxicity study and the reversibility of the local effects, classification as STOT RE Cat.2 (H373:" May cause damage to organs through prolonged or repeated exposure”) under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179, is considered to be sufficient conservative.