Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
14 September 1998 to 20 January 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, to GLP; on related material

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trisodium EDDS (under code)
- Molecular formula (if other than submission substance): C10-H13-N2-O8. 3Na
- Molecular weight (if other than submission substance): 358
- Smiles notation (if other than submission substance): [Na+].[Na+].[Na+].OC(=O)[C@H](CC([O-])=O)NCCNC(CC([O-])=O)C([O-])=O
- Substance type: technical product
- Physical state: colourless liquid
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: refrigerated, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P) Males: 106-132 g; Females: 105-132 g
- Fasting period before study: no data
- Housing: individually, except during mating and postpartum period, in stainless steel, wire-bottomed cages
- Diet (e.g. ad libitum): conventional with mineral supplement containing twice the National Research Council recommended daily intake of copper, iron and zinc (as EDDS acid has been shown to chelate these minerals in previous studies), ad libitum
- Water (e.g. ad libitum): chlorinated, reverse osmosis membrane processed, deionised water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared weekly with continuous stirring at concentrations of 9, 25 and 70 mg/mL

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility for a further 7 days
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually with nesting material
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data. [Duplicate samples were taken from the top, middle and bottom of each concentration on first day of dosing and then monthly and sent away for analysis.]
Duration of treatment / exposure:
From 70 days before mating until postpartum day 21
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 18 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 90, 250 or 700 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses determined by the sponsor
- Rationale for animal assignment (if not random): computer-generated (weight-ordered) randomization
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: twice daily
- Cage side observations included: deaths, and in females behaviour, abortions and premature deliveries.

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: daily

BODY WEIGHT: yes
- Time schedule for examinations: males - on day 1 of dosing, then weekly. Females - on day 1 of dosing, then weekly until gestation, then on gestation days 0, 6, 10, 15 and 25 (if necessary) and on lactation days 1,4, 7, 10, 14 and 21.

FOOD CONSUMPTION: yes
- Time schedule: males - weekly during dosing period. Females - weekly until gestation, then on gestation days 0, 6, 10, 15 and 25 (if necessary) and on lactation days 1,4, 7, 10 and 14.

Oestrous cyclicity (parental animals):
Estrous cycle observed for 14 days prior to cohabitation
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were sacrificed and not examined further.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals were evidently sacrificed after weaning (although the methods section of the report states males were sacrificed once pregnancy was confirmed)
- Maternal animals: all surviving animals were sacrificed after their litter was weaned. Females that did not deliver a litter were sacrificed on gestation day 25.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for possible future microscopic examination and weighed: prostate, pituitary and brain; uterus and ovaries (of females); epididymides, seminal vesicles and testes (of males). All gross lesions were retained and those from dams were examined histologically. Histopathology was performed on the testes of all control and high dose males, and any males that failed to induce pregnancy. Micropscopic examination of the ovaries of all females that were not pregnant was also carried out.

OTHER EXAMINATIONS
Baseline serum levels of copper, iron and zinc in 5 animals/sex were taken prior to treatment. Blood was collected from females at 40 days of age and terminal sacrifice and from males on day of sacrifice and the serum analysed for copper, iron and zinc levels.
Postmortem examinations (offspring):
SACRIFICE of pups kept until weaned
- The F1 offspring were sacrificed at 21 days of age
- These animals were not subjected to postmortem examinations.

GROSS NECROPSY
None

HISTOPATHOLOGY / ORGAN WEIGTHS
None

SACRIFICE of pups culled due to standardization
- The F1 offspring were sacrificed at 4 days of age
- These animals were subjected to postmortem examinations.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Cross-section of the brain at the level of the frontal-parietal suture for hydrocephaly.
Statistics:
Bartlett's test of homogeneity of variances. If the Bartlett's test was not significant, then the analysis of variance was applied and if this was significant, Dunnett's test was used to identify the statistical significance of the individual groups. If the Bartlett's test was significant, the Kruskal-Wallis test or Fisher's exact test were applied as appropriate.
Discrete natural delivery data were evaluated using the Kruskal-Wallis test.
Reproductive indices:
Precoital index (days in cohabitation before mating), mating index, fertility index, gestation index
Offspring viability indices:
Number and sex of offspring/litter, number of implantation sites, condition of pups, litter size and viability, number of pups surviving to lactation days 1 and 4, lactation index (number of pups surviving to weaning), percentage survival and sex ratio.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no treatment-related deaths or clinical signs reported

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A slight (about 5%), but not statistically significant, reduction in body weight gain was observed in the high-dose males (throughout the study) and females (prior to mating only), compared to the controls. Food consumption was slightly reduced (not statistically significant) in males at the top-dose level throughout the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effects were seen at any stage of the estrous cycle.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No differences were seen in sperm number, motility, cauda epididymal sperm count or density.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The precoital, mating and fertility indices were similar in all groups. The number of implantations and litters, lactation index and sex ratios of the pups were similar between groups. A slight, non dose-related non-significant, reduction in the number of pregnant dams was seen; 23 (95.8%), 22 (88.0%), 22 (91.7%) and 20 (80.0%) pregnant females in the 0, 90, 250 and 700 mg/kg bw/day groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No differences were seen in the weights of the epididymis, cauda epididymis, testes, seminal vesicles, pituitary or brain weights in any groups. The absolute prostate weights were slightly lower (p¿0.05) in the 250 mg/kg bw/day males. In females, the weights of the pituitary, brain, ovaries and uterus were comparable between all groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No significant differences were observed in gross pathology between groups.

OTHER FINDINGS (PARENTAL ANIMALS)
The zinc levels in serum were elevated in males in all treatment groups and in females only at the top dose. There were no changes in the serum levels of copper and iron.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
ca. 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
ca. 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Viability was similar in all groups.

CLINICAL SIGNS (OFFSPRING)
No effects were attributed to the test substance. In all groups, persistent clinical effects, each occurring in 1 pup/group consisted of tip of tail missing, microphthalmia, bent tail and umbilical hernia.

BODY WEIGHT (OFFSPRING)
No significant differences were seen between groups.

ORGAN WEIGHTS (OFFSPRING)
No data.

GROSS PATHOLOGY (OFFSPRING)
No significant differences were seen between the groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
ca. 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a GLP study conducted according to OECD Guideline 415, no adverse affects on measures of fertility and development (or systemic toxicity) were seen in groups of rats administered trisodium EDDS by stomach tube at up to 700 mg/kg bw/day (considered the study NOAEL) in a one-generation reproduction study.
Executive summary:

In a GLP study conducted according to OECD Guideline 415, trisodium EDDS was assessed for its ability to induce reproductive and developmental toxicity in rats. Groups of 25 Spague-Dawley rats of each sex were given 0, 90, 250 or 700 mg/kg bw/day of the test substance (in water) by oral gavage for 70 days before mating, and throughout mating, pregnancy and weaning. Culling of the offspring occurred on day 4 after birth to standardize the litters, and the remaining offspring were killed on day 21 after delivery. The animals were observed for mortality, clinical signs of toxicity, body weight gain, feed consumption, changes in the estrus cycle, precoital index, mating index, fertility, gestation index, number and sex of offspring, litter size and viability, and lactation index. At sacrifice, gross necropsy of the thoracic, abdominal and pelvic viscera was carried out, the reproductive organs, brain and pituitary were weighed and sperm evaluations were performed. The females were examined for the number and distribution of implantation sites. Necropsies were performed on the pups culled at 4 days for external and internal abnormalities and the brains were sectioned to detect hydrocephaly.

No significant systemic adverse effects were observed on the parental animals, and the reproductive and developmental parameters examined were unremarkable. Therefore the study NOAEL was considered to be 700 mg/kg bw/day.

In conclusion, trisodium EDDS did not affect fertility or development in rats orally administered up to 700 mg/kg bw/day in a one-generation reproductive toxicity study. Therefore, according to EU CLP and DSD regulations, trisodium EDDS would not be classified as a reproductive or developmental toxicant under the test conditions.

[Data on trisodium EDDS is considered relevant to use for understanding the potential reproductive toxicity of EDDS acid, and is acceptable for using as read-across information.]