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Carcinogenicity

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Description of key information

Carcinogenicity Study (no guideline followed, NTP study design), rat:

NOAEL (carcinogenicity) >= 7500 ppm (equivalent to 495 mg/kg bw)

Carcinogenicity Study (no guideline followed, NTP study design), mouse:

NOAEL (carcinogenicity) >= 7500 ppm (equivalent to 938 mg/kg bw/day)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: 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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
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
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
Details on 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 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 were noted 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)
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 were found in both treated and control animals that were well known from historical controls of the same strain. There was a high incidence of tumors in the hematopoictic, endocrine, digestive, and respiratory systems. The incidence of neoplasms in other systems was variable. For all tumor types observed no statistical significance were seen between incidences in dose groups and control groups (see table 1 and 2).
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
>= 938 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no indication of increase tumour incidence up to and including the highest dose tested
Dose descriptor:
NOAEL
Remarks:
general toxicity
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: neoplastic
mortality

Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Mice

Morphology (p-value) Control Low dose High dose
Hematopoietic System:  Malignant Lymphoma  2/20 (n.s.) 7/46 (n.s.) 7/48 (n.s.)
Weeks to first observed tumor: 91 73 87
Thyroid: C-cell Adenoma 0/10 (n.s.) 1/29 (n.s.) 1/33 (n.s.)
Weeks to first observed tumor: - 104 105
Pituitary: Chromophobe Adenoma 1/13 (n.s.) 0/19 (n.s.) 1/26 (n.s.)
Weeks to first observed tumor: 105 - 105
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma 2/18 (0.096) 8/44 (n.s.) 12/45 (n.s.)
Weeks to first observed tumor: 105 99 96
Liver: Hepatocellular Adenoma and Neoplastic Nodule 3/19 (n.s.) 10/44 (n.s.) 10/47 (n.s.)
Weeks to first observed tumor: 103 84 105

Table 2: Analyses of the Incidence of Primary Tumors at Specific Sites in Female Mice

Morphology (p-value) Control  Low dose  High dose
Hematopoietic System:  Malignant Lymphoma  5/19 (n.s.) 11/49 (n.s.) 12/47 (n.s.)
Weeks to first observed tumor: 85 99 93
Thyroid: C-cell Adenoma 1/12 (n.s.) 3/33 (n.s.) 1/34 (n.s.)
Weeks to first observed tumor: 105 99 105
Pituitary: Chromophobe Adenoma 2/12 (n.s.) 6/34 (n.s.) 4/29 (n.s.)
Weeks to first observed tumor: 105 105 105
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma 0/19 (n.s.) 3/47 (n.s.) 3/49 (n.s.)
Weeks to first observed tumor: - 105 102
Liver: Hepatocellular Adenoma and Neoplastic Nodule 0/19 (n.s.) 1/46 (n.s.) 1/47 (n.s.)
Weeks to first observed tumor: - 99 105

n.s. = not significant

Conclusions:
No test item-related carcinogenic potentail could be detected in the study. Under the conditions of the test the NOAEL for carcinogenicity in male and female mice is >= 938 mg/kg bw/day.
Endpoint:
carcinogenicity: 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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
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
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 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
Post exposure period:
none
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 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):
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 statitsically significant (p =0.029).
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 not to the administration of the chemical.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A high incidence of neoplasms occurred 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.
No tumor appeared in a statistically significant positive trend in either dose groups or sexes. A variety of endocrine tumors were found, some types occurring only in treated animals. However, these tumors occurred in low numbers and have frequently been seen in untreated animals in other studies. Therefore, they are probably unrelated to treatment .

Males:
Interstitial-cell tumors of the testes were observed in nearly all male rats in each feeding group. This high incidence of interstitial-cell tumors in both treated and control animals reflects this commonly occurring age-related lesion in the male Fischer 344 rat.

Females:
The distribution of neoplasms in the reproductive system among control and treated rats was random, the tumors occurred mainly in the uterus . The majority of these were endometrial stromal polyps. However, one adenocarcinoma and one leiomyosarcoma occurred at 495 ppm. An ovarian cystadenoma was detected in a single 248 ppm-dose rat
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
>= 495 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no indication of increase tumour incidence up to and including the highest dose tested
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
>= 495 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

Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Rats

Morphology (p-value) Control Low dose High dose
Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia 3/20 (n.s.) 4/50 (n.s.) 4/50 (n.s.)
Weeks to first observed tumor:  76 104 102
Adrenal: Pheochromocytoma 2/20 (n.s.) 5/49 (n.s.) 4/50 (n.s.)
Weeks to first observed tumor:  104 104 67
Thyroid: C-cell Adenoma 0/17 (n.s.) 6/35 (p = 0 0.08) 3/38 (n.s.)
Weeks to first observed tumor:  - 104 67
Pituitary: Chromophobe Adenoma 0/18 3/47 (n.s.) 5/44 (n.s.)
Weeks to first observed tumor:  - 88 104
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma 1/18 (n.s.) 2/50 (n.s.) 3/49 (n.s.)
Weeks to first observed tumor:  104 95 67
Liver: hepatocellular Adenoma and Neoplastic Nodule 0/20 (n.s.) 1/48 (n.s.) 1/50 (n.s.)
Weeks to first observed tumor:  - 104 104
Testis: Interstitial-cell Tumor 19/20 (n.s.) 43/50 (n.s.) 44/50 (n.s.)
Weeks to first observed tumor:  88 85 95

Table 2: Analyses of the Incidence of Primary Tumors at Specific Sites in Female Rats

Morphology (p-value) Control Low dose High dose
Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia 1/20 (n.s.) 8/50 (n.s.) 0/50 (n.s.)
Weeks to first observed tumor:  104 80 -
Adrenal: Pheochromocytoma 1/20 (n.s.) 1/49 (n.s.) 1/48 (n.s.)
Weeks to first observed tumor:  98 104 104
Thyroid: C-cell Adenoma 0/11 (n.s.) 0/36 (n.s.) 1/37 (n.s.)
Weeks to first observed tumor:  - - 104
Pituitary: Chromophobe Adenoma 6/19 (n.s.) 10/48 (n.s.) 11/50 (n.s.)
Weeks to first observed tumor:  95 104 104
Lung: Alveolar/Bronchiolar Adenoma and Carcinoma 0/20 (n.s.) 3/48 (n.s.) 2/48 (n.s.)
Weeks to first observed tumor:  - 104 104
Liver: Neoplastic Nodule 0/20 (n.s.) 1/48 (n.s.) 0/48 (n.s.)
Weeks to first observed tumor:  - 104 -
Uterus: Endometrial Stromal Polyp 5/20 (n.s.) 6/50 (n.s.) 7/50 (n.s.)
Weeks to first observed tumor:  104 96 85
Mammary Gland: Fibroadenoma 4/20 (n.s.) 3/50 (n.s.) 3/50 (n.s.)
Weeks to first observed tumor:  85 96 97

- Not all animals were examined histopathologically, due to cannibalism or advanced state of autolysis.

Conclusions:
No test item-related carcinogenic potentail could be detected in the study. Under the conditions of the test the NOAEL for carcinogenicity in male and female rats is >= 495 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
495 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable and consistent studies (Klimisch score 2).

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on the carcinogenic potential of trisodium hydrogen EDTA (CAS 150-38-9) do not indicate adverse effects up to and including the highest doses tested. Therefore, the available data are conclusive but not sufficient for classification according to Regulation (EC) No 1272/2008.

Additional information

Standard carcinogenicity studies in mice and rats using trisodium hydrogen EDTA (CAS 150-38-9) did not demonstrate that the test substance is carcinogenic in experimental animals (NTIS, 1977).

50 male and 50 female Fischer 344 rats were administered with 3750 and 7500 ppm (equivalent to about 248 and 495 mg/kg bw/day) daily in the diet for 103 weeks. Matched control groups were composed of 20 male and 20 female animals. The average body weights of treated males and females were similar to those of the matched controls throughout the study. No treatment-related clinical signs were observed and no statistically significant differences in survival noted. Inflammatory and degenerative changes were observed in about the same frequency in all groups. The lesions appeared to be related to age and not to the administration of the test substance. A high incidence of tumors has been observed in the reproductive and endocrine systems and low incidences occurred in the hematopoietic, respiratory, integumentary, and digestive systems. No neoplasms were observed in the nervous, musculoskeletal, or urinary systems. No tumor appeared in a statistically significant positive trend in either dose groups or sexes. A variety of endocrine tumors were found, some types occurring only in treated animals. However, these tumors occurred in low numbers and have frequently been seen in untreated animals in other studies. Therefore, they are probably unrelated to treatment.

In a similar study in mice, 50 male and 50 female B6C3Fl mice were administered with the same dose concentrations (3750 and 7500 ppm, equivalent to about 469 and 938 mg/kg bw/day) daily in the feed for 103 weeks. Matched control groups were composed of 20 male and 20 female animals. In male mice only the high-dose group showed a decrease in average body weight compared to the controls throughout most of the study period. In female mice average body weights of the treatment groups were depressed in a dose-related manner during the study period, although the effect was small. No treatment-related clinical signs were observed and no statistically significant differences in survival noted. Inflammatory and degenerative changes were observed in about the same frequency in all groups. The lesions appeared to be related to age and not to the administration of the test substance. A variety of neoplasms were found in both treated and control animals that were well known from historical controls of the same strain. There was a high incidence of tumors in the hematopoictic, endocrine, digestive, and respiratory systems. The incidence of neoplasms in other systems was variable. For all tumor types observed no statistical significance were seen between incidences in dose groups and control groups. With the exception of a splenic hemangioma in a control female and a 3750 ppm-male, all of the tumors of the hematopoietic system were malignant lymphomas or leukemias. The distribution of endocrine tumors varied little between treated and control mice. For all groups, the incidences of hepatic neoplasms were considerably higher in males than in females. Liver tumors occurred in the 3750 ppm-dose (10/44, 22 %) and 7500 ppm-dose (10/47, 21 %) male groups. Percentage was approximately the same as in the male controls (3/19, 16 %). Primary neoplasms of the respiratory system were observed in both treated and control groups. The highest incidence of pulmonary neoplasms was found in the 7500 ppm-dose male mice (control: 2/18, 11 %; 3750 ppm: 8/44, 18 %; 7500 ppm: 12/45, 26 %). This may suggest a treatment related effect. Lung tumors were frequently seen in mice of this strain and age, and therefore, the increase of incidence in this mouse study is probably not related to treatment. In conclusion, the non-significantly increased incidence of some tumor types observed in the study provides no clear evidence of carcinogenic effects in the mice.