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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1977

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium hydrogen ethylenediaminetetraacetate
EC Number:
205-758-8
EC Name:
Trisodium hydrogen ethylenediaminetetraacetate
Cas Number:
150-38-9
Molecular formula:
C10H16N2O8.3Na
IUPAC Name:
trisodium hydrogen 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate
Test material form:
solid

Test animals

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

Administration / exposure

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 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
Doses / concentrationsopen allclose all
Dose / conc.:
3 750 ppm
Remarks:
469 mg/kg bw/day
(conversion factor according to EU risk assessment)
Dose / conc.:
7 500 ppm
Remarks:
938 mg/kg bw/day
(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

Examinations

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.

Results and discussion

Results of examinations

Details on results:
MORTALITY
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)

CLINICAL SIGNS
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.

BODY WEIGHT AND WEIGHT GAIN
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 PATHOLOGY
Nothing reported.

HISTOPATHOLOGY: NEOPLASTIC
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 below)


Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 938 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: non-neoplastic
mortality

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.)

Applicant's summary and conclusion