L-Aspartic acid, N,N'-1,2-ethanediylbis-, sodium salt (1:3)

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

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:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Safety evaluation studies of calcium EDTA

Author:

Oser BL, Oser M and Spencer HC

Year:

1963

Bibliographic source:

Toxicology and Applied Pharmacology 5, 142-162

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

In a multigeneration rat study, the parental (P) generation were fed the test substance from weaning and at sexual maturity underwent two successive matings. Exposure was continued throughout the study. Ten rats of each sex were selected from as many litters as possible from successive generations, and assigned to the F1, F2 and F3 generation, respectively, under the same exposure and mating regime as the P generation. The animals were observed for effects on fertility, gestation and lactation and for viability of the offspring. The P generation were exposed for 2 years, after which time the study was terminated (i.e. the F1, F2 and F3 generations were exposed for 18, 12 and 6 months, respectively). At study termination, the animals were examined for gross abnormalities and 15 organs and tissues were examined microscopically.

GLP compliance:

no

Remarks:

prior to GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Calcium disodium EDTA

IUPAC Name:

Calcium disodium EDTA

Constituent 2

Reference substance name:

Sodium calcium edetate

EC Number:

200-529-9

EC Name:

Sodium calcium edetate

Cas Number:

62-33-9

Molecular formula:

C10H12CaN2O8.2Na

IUPAC Name:

calcium disodium 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate

Constituent 3

Reference substance name:

calcium disodium 2-[2-[bis(2-oxido-2-oxoethyl)amino]ethyl-(2-oxido-2-oxoethyl)amino] acetate

IUPAC Name:

calcium disodium 2-[2-[bis(2-oxido-2-oxoethyl)amino]ethyl-(2-oxido-2-oxoethyl)amino] acetate

Details on test material:

- Name of test material (as cited in study report): calcium disodium EDTA dihydrate ("Calcium EDTA")
- Molecular formula (if other than submission substance): C10-H12-Ca-N2-O8.2Na
- Molecular weight (if other than submission substance): 374
- Smiles notation (if other than submission substance): [Na]OC(=O)CN1CCN(CC(=O)(O[Na]))CC(=O)O[Ca]OC(=O)C1
- InChl (if other than submission substance): 1S/C10H16N2O8.Ca.2Na/c13-7(14)3-11(4-8(15)16)1-2-12(5-9(17)18)6-10(19)20;;;/h1-6H2,(H,13,14)(H,15,16)(H,17,18)(H,19,20);;;/q;+2;2*+1/p-4
- Substance type: pure substance
- Physical state: solid; white flakes
- Analytical purity: 99%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable throughout period of the study
- Storage condition of test material: room temperature in polyethylene bottles

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Food and Drug Research Laboratories Inc., Maspeth 78, New York, USA
- Age at study initiation: at weaning (21 days)
- Weight at study initiation: no data given at start of exposure. At 12 weeks: (P) Males: 259-285 g; Females: 146-157 g; (F1) Males: 259-312 g; Females: 135-159 g; (F2) Males: 232-286 g; Females: 134-168 g; (F3) Males: 280-306 g; Females: 152-173 g
- Fasting period before study: no data
- Housing: individually (apart from during mating and lactation)
- Diet (e.g. ad libitum): "natural foods supplemented with inorganic salts and vitamins". Reported to "resemble the food consumption pattern of the US population with respect to the ratios of milk, meat and grain components"; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
- Concentration: 25% solution

Details on mating procedure:

- M/F ratio per cage: 1 male to 2 females
- Length of cohabitation: up to 3 weeks
- Proof of pregnancy: visually, by palpation or by weight increments. Not referred to by day of pregnancy
- After 21 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no, the female was regarded as infertile and matings discontinued
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

From weaning until 6, 12, 18, or 24 months after start of exposure for the F3, F2, F1 or P generations, respectively.

Frequency of treatment:

Daily in diet; ad libitum

Details on study schedule:

- F1, F2 and F3 parental animals not mated until 14 weeks after selection from the litters. A second mating took place one week after weaning of the offspring
- Selection of parents from F1, F2 and F3 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13 and 21 weeks (2 litters)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25/sex/dose in the P generation, then 10 rats/sex/dose in the F1, F2 and F3 generations

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on a range-finding study
- Rationale for animal assignment (if not random): random

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations for physical condition and behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no details; "for the 12-week postweaning period"

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/ day: food consumption for each of 10 rats/sex recorded as a representative sample of the group
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
10 rats/sex were selected at 21 days (at weaning) to provide the successive generations. The P generation were kept until they reached 2 years of age when the study was terminated (i.e. the F1, F2 and F3 generations were kept for 18, 12 and 6 months, respectively)

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F3 offspring:
viability and lactation index, postnatal mortality, weight gain, external and internal examinations, organ weights, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, the main organs, including gonads were examined

Postmortem examinations (parental animals):

SACRIFICE
- Male and maternal animals: 2 animals/sex/group were sacrificed at 12 weeks. All surviving animals in the 50 and 125 mg/kg bw/day groups not selected to provide the next generation were sacrificed after weaning of the second litters. Control animals and animals in the high-dose group continued on the diet for a total of 2 years.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively: in animals sacrified at 12 weeks; liver, kidneys, spleen, heart, adrenals, thyroid and gonads. At study termination, kidneys, pancreas, heart, spleen, lungs, bone marrow, stomach, small and large intestines, gonads, thyroid, parathyroids, lymph nodes, spinal cord, and tibia.

At 6 and 12 weeks from start of exposure in about one quarter of the animals/sex/group the following parameters were determined: blood haemoglobin levels, red and white blood cell counts, differential white cell counts, prothrombin time, blood sugar and non-protein nitrogen, serum calcium, urinary albumin and sugar.

Due to the sequestering action of calcium disodium EDTA which may interfere with mineral metabolism, the following additional examinations were made: ash content of tibias, examination for dental caries, xanthine oxidase in liver and carbonic anhydrase in serum.

Postmortem examinations (offspring):

SACRIFICE
The F1 and F2 offspring not selected as parental animals were sacrificed after weaning of the second litters. The F3 offspring were sacrificed at birth. The F1, F2 and F3 parental animals were sacrificed at 18, 12 and 6 months, respectively.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated were prepared for microscopic examination and weighed, respectively: kidney, pancreas, heart, spleen, lungs, bone marrow, stomach, small and large intestines, gonads, thyroid, parathyroid, lymph nodes, spinal cord, and tibia.

Statistics:

No data

Reproductive indices:

Fertility index, gestation index

Offspring viability indices:

Viability index (the proportion of offspring that survived 4 days or longer); lactation index (the proportion of offspring alive at 4 days that survived to weaning)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were evident

Mortality:

no mortality observed

Description (incidence):

No treatment-related deaths were seen. In the P generation, 3 deaths occurred in control animals at 10-12 weeks, 1 death occurred at 12 weeks at 50 mg/kg bw/day and 1 death occurred at 8 weeks at 125 mg/kg bw/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights were similar in all groups

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar in all groups

Dietry consumption, and therefore substance intake, were comparable between groups

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No significant differences were evident between the groups

Other effects:

no effects observed

Description (incidence and severity):

There were no significant differences in the levels of haemoglobin, haematocrit, blood sugar and non-protein nitrogen, urinary albumin and sugar, red and white blood cell counts, differential white cell counts and prothrombin times.significant differences were detected in the extent of dental caries in the high-dose and control groups.

The activity of the two metallo-enzymes, carbonic anhydrase and xanthine oxidase, were similar in all groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant differences in reproductive performance were evident between groups

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No effects were seen on clinical signs; mortality; body weight; food consumption and compound intake; food efficiency; water consumption and compound intake; gross pathology; organ weights; histopathology; mating index; fertility index; birth index; live birth index; pregnancy index; litter size; litter weight; pup weight; survival index; viability index; lactation index

Results: P1 (second parental generation)

Effect levels (P1)

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were evident and no deaths occurred in the F1, F2 or F3 generations

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant differences were seen in viability between groups in the F1, F2 or F3 generations

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and food consumption were similar in all groups in all generations

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Diet consumption, and therefore substance intake, was comparable between groups in all generations

Haematological findings:

no effects observed

Description (incidence and severity):

There were no significant differences in the levels of haemoglobin, haematocrit, blood sugar and non-protein nitrogen, urinary albumin and sugar, red and white blood cell counts, differential white cell counts and prothrombin times.

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights were similar in all groups

Gross pathological findings:

no effects observed

Description (incidence and severity):

No significant differences were evident between the groups

Histopathological findings:

no effects observed

Description (incidence and severity):

No significant differences were evident between the groups

Other effects:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE (OFFSPRING)
No significant differences in reproductive performance were evident between groups

No significant differences were detected in the extent of dental caries in the high-dose and control groups.

The activity of the two metallo-enzymes, carbonic anhydrase and xanthine oxidase, were similar in all groups.

Effect levels (F1)

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a good quality study (prior to GLP), no adverse effects were evident in a multigeneration reproduction study in rats fed calcium disodium EDTA at up to 250 mg/kg bw/day in the diet, considered the study NOAEL.

Executive summary:

In a good-quality study (prior to GLP), calcium disodium EDTA dihydrate was assessed for the potential to cause adverse effects in a multigeneration reproductive toxicity study in Wistar rats.

Groups of 25 rats of each sex (P generation) were fed 0, 50, 125 or 250 mg/kg bw/day in the diet from weaning (day 21) for up to 2 years. At 13 and 21 weeks of age, male rats were mated with females to provide two litters (F1 generation). At weaning of F1, 10 animals/sex were selected from as many litters as possible to provide the F2 generation. The F3 generation was similarly derived from F2 animals. All rats were fed the same diet as their parents throughout the study. The study was terminated 2 years after the first exposure of the P generation (i.e. the F1, F2 and F3 generations were terminated at 18, 12 and 6 months).

No treatment-related adverse effects were observed on the fertility, gestation or lactation index, or on viability of the offspring in any of the three generations of offspring. No treatment-related deaths, or changes in body weight gain or food consumption were observed across the generations. There were no treatment-related differences in the levels of haemoglobin, haematocrit, blood sugar and non-protein nitrogen, urinary albumin and sugar, red and white blood cell counts, differential white cell count or prothrombin times among any of the generations. Gross pathology and microscopic examination of 15 organs and tissues, including the gonads, at study termination was unremarkable. Therefore, the NOAEL was considered to be 250 mg calcium disodium EDTA/kg bw/day, the highest tested dose.

In conclusion, calcium disodium EDTA showed no evidence of reproductive or lactational effects when fed to rats at up to 250 mg/kg bw/day (highest tested dose) in a multigeneration study. Based on the structural similarity of the two ethylenediamines, it is expected that trisodium EDDS is also unlikely to induce reproductive toxicological effects under similar test conditions.