Registration Dossier

Administrative data

Description of key information

There are no repeated dose studies with the substance available. Studies on two structural analogues of the substance (Na3H-EDTA and Na2H2-EDTA) are available (for read-across justification refer to Section 13) which indicate no significant concerns for toxicity/carcinogenicity. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets generally accepted scientific principles For read-across justification refer to section 13.
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 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 7,500 ppm EDTA and 90.4 + / - 3.4% of the theoretical value at 3,750 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
Remarks:
Doses / Concentrations:
250; 500 mg/kg bw/day (original data: 3,750 ppm 7,500 ppm; conversion according to EU risk assessment)
Basis:
nominal in diet
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:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
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.

CLINICAL SIGNS
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.

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

GOSS PATHOLOGY
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.

HISTOPATHOLOGY: NEOPLASTIC
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).


Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Sex:
male/female
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.)
Conclusions:
The NOAEL following 103 weeks dietray exposure was 500 mg/kg/day
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Several studies available with the structurally related EDTA substances.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

A one month feeding study in rats of EDTA-Na2H2, a structural analogue of the substance, revealed a NOAEL of 2.25% in the diet, corresponding to 1,125 mg/kg/day (Kawamata, 1980). In this study the substance was incorporated at levels of 1, 2.25 and 5% in the diet, corresponding to 500, 1125 and 2500 mg/kg/day). Fifteen rats/sex/dose level were exposed over a period of one month. At the high dose level body weight was decreased, some mortalities occurred 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 reduced liver, spleen and thymus weights. Microscopic pathology revealed some parakeratosis in the oesophagus and forestomach. A 90 -day feeding study in rats with the same substance revealed a NOAEL of 500 mg/kg/day (Wynn, 1970). Groups of 10 male Holzman rats received 1, 5 and 10% (respectively 500, 2,500 and 5,000 mg/kg/day) EDTA-Na2H2 in the diet for 90 days. It should be noted that in this study detailed clinical chemistry investigations were not performed as required by OECD TG 408. 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 diarrhoea and were emaciated. Water consumption was increased. In the upper dose there was an intermittent decrease of haematocrit and haemoglobin levels and livers appeared to be pale. Histological investigation failed to reveal any pathological alteration.

A two-year feeding study in rats with another structural analogue (Na3H-EDTA x 3H2O) resulted in a NOAEL of 500 mg/kg/day (corresponding to 7,500 ppm in the diet) (NTIS, 1977). In this feeding study two dose levels were investigated - 3,750 ppm and 7,500 ppm (corresponding to approximately 250 and 500 mg/kg/day). No substance related toxic effects could be observed. This substance was also investigated in a study of the same duration in mice at the same dose levels (3,750 ppm and 7,500 ppm; corresponding to approximately 469 and 938 mg/kg/day). Again there were no treatment related changes and a NOAEL of 938 mg/kg/day was assigned.

When comparing the acute inhalation toxicity studies of HEDTA-Na3 and EDTA-Na2H2, the first one showed no lethality at the technically highest available concentration of almost 4000 mg/m3 for 4 h whereas the latter showed 30% mortality at 1000 mg/m3 for 6 h, the diffference being considered to be due to the higher binding capacity of EDTA compared to HEDTA. For EDTA-Na2H2 a 14 -day and a 90 -day inhalation toxicity study are available showing local effects in the respiratory tract mainly in the larynx. Based on the acute tests, much less local toxicity is expected for HEDTA upon repeated exposure. Moreover, in accordance with REACH Regulation 1907/2006 (Annex IX, 8.6.2 Column 2) testing by the inhalation route is appropriate if exposure of humans via inhalation is likely, taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size. As HEDTA-H3 is only produced as an aqueous solution there will be no exposure to dust particles, and also no exposure to the vapour because of the very low vapour pressure of HEDTA-H3.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Well documented study

Justification for classification or non-classification

According to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification and labelling is not indicated for repeat dose toxicity as clear functional disturbances or morphological changes were not apparent in chronic toxicity studies.