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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from J-Check

Data source

Reference
Reference Type:
other: Authorized database
Title:
Preliminary Reproduction Toxicity Screening Test of 4,4'-Diamino-2,2'-stilbenedisulfonic acid by Oral Administration in Rats
Author:
J-Check
Year:
2010
Bibliographic source:
Ministry of Health, Labour and Welfare", "Ministry of the Environment" and "National Institute of Technology and Evaluation, J-Check, 2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Preliminary Reproduction Toxicity Screening Test
Principles of method if other than guideline:
Preliminary Reproduction Toxicity Screening Test of 4,4'-Diamino-2,2'-stilbenedisulfonic acid by Oral Administration in Rats
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4,4'-Diamino-2,2'-stilbenedisulfonic acid
- Molecular formula (if other than submission substance): C14H14N2O6S2
- Molecular weight (if other than submission substance): 370.42 g/mole
- Substance type: Organic
- Physical state: Light yellow powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4,4'-Diamino-2,2'-stilbenedisulfonic acid
- Molecular formula (if other than submission substance): C14H14N2O6S2
- Molecular weight (if other than submission substance): 370.42 g/mole
- Substance type: Organic
- Physical state: Light yellow powder

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Japan Co., Ltd
- Age at study initiation: (P) x wks; (F1) x wks: 8 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: 313-350 g for male and 183 - 222 g for female.
- Fasting period before study:
- Housing: Animals were housed in a polycarbonate cage with an experimental animal floor covering (petit chips: Charles River Japan), one after starting administration per cage, one each for male and female during the mating period and one litter during the feeding period.
- Diet (e.g. ad libitum): Autoclave-sterilized solid feed filtering by 5 μm, ad libitum
- Water (e.g. ad libitum): Tap water irradiated with ultraviolet rays, ad libitum
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 25 ° C
- Humidity (%): 40 to 70%
- Air changes (per hr): 12 times / hour
- Photoperiod (hrs dark / hrs light): lighting 12 hours / day (7: 00 to 19: 00).

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
other: Sodium Carboxymethyl Cellulose
Remarks:
0.5%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 4,4'-Diamino-2,2'-stilbene disulfonic acid suspended in 0.5% CMC-Na aqueous solution

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% Sodium Carboxymethyl Cellulose
- Concentration in vehicle: 0 (vehicle), 40, 200, 1000 mg/kg/day
- Amount of vehicle (if gavage): 10 ml / kg
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 7 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Female vaginal plaques were collected every morning,
stained with Giemsa and examined in a microscopic examination. In cases where vaginal plug formation or
sperm was found in vaginal plaque specimens, the mating was established, and that day was taken as the 0th day
of pregnancy.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)] No data available
- After successful mating each pregnant female was caged (how): No data available
- Any other deviations from standard protocol: For one female in the 200 mg / kg group, males that are scheduled to live together died, so they were living together
with the males already confirmed in the same group. From these results, the number of days of mating (the number of days required for mating after the mating), the number of estrus periods missed until the mating was established, the mating rate ([number of copulatory animals / number of living animals] × 100) Conception rate ([number of pregnant animals / number of copulatory animals] × 100) was calculated.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males, 41 days
Females, from 14 days before mating to Day 3 of lactation
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Total: 80
0 mg/kg/day: 10 male, 10 female
40 mg/kg/day: 10 male, 10 female
200 mg/kg/day: 10 male, 10 female
1000 mg/kg/day: 10 male, 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In repeated 8-day preliminary test (dose: 0, 100, 300, 1000 mg / kg) using SD rats of both sexes, even in the 1000 mg / kg technical limit dose amount group, both weight and autopsy No change was noticed. Therefore, in this study, the high dose was 1000 mg / kg, the median dose was 200 mg / kg and the low dose was 40 mg / kg at the common ratio 5.
- Rationale for animal assignment (if not random):
- Other:

Examinations

Parental animals: Observations and examinations:
Survival, clinical signs, body weight and food consumption were examined.
Estrous cyclicity (parental animals):
Estrous cyclicity and Conception rate in females was evaluated.

Corpus luteums, number of landings and presence or absence of implantation were examined.
Sperm parameters (parental animals):
not specified
Litter observations:
General condition, number of births, the number of births born, the number of stillborn infants, the sex and Body weight were examined.
Postmortem examinations (parental animals):
Oragn weight, gross pathology and histopathology were examined.
Postmortem examinations (offspring):
gross pathology were examined.
Statistics:
Weighing data were tested for equal variance by the Bartlett method, and one-way ANOVA was performed when the variance was uniform, and Kruska 1-Wallis test was performed when the variance was not uniform. When a significant difference was observed between the groups, the Dunnett method or Dunnett method was used if the number of cases in each group was constant, and the Scheff method or Scheff type multiple comparison test was performed if not constan. However, the items marked with * below are from the Kruskal-Wallis test. The counting data was tested by Fisher's direct stochastic method. The level of significance was set at 5% or less. For the data on newborn babies, the average value calculated for each mother animals was used as the statistical unit. Body weight, food intake, organ weight, number of days required for mating *, number of estrous cycles missed by mating establishment, gestation period *, number of corpus luteum, implantation number, implantation rate *, delivery rate *, neonatal number, fertility rate *, Neonatal survival rate *, neonatal weight by multiple comparison test and Copulation rate, conception rate, birth rate, sex ratio (male / female) Fisher's direct stochastic method.
Reproductive indices:
Lactation, nesting, presence or absence of feeding, gestation, birth rate, implantation rate, delivery rate were observed.
Offspring viability indices:
Yes, viability index were examined.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No change was observed in general condition of male and female rats as compared to control.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
At 200 mg / kg, one male died after the start of administration on the fourth day.
At necropsy, perforation was found in the lung, so it was judged to be death due to administration error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect on body weight and weight gain were observed in treated rats as compared to control.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In male rat at 1000 mg / kg, from the start of administration to the 14th day a significant increase in food consumption were observed, but after that, the values were similar to those of the control group, and no significant difference was observed. In females, no significant change was observed throughout the entire period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant change in both the actual weight and the body weight ratio in testis and epididymis were observed in treated rats as compared to control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Miniaturization of bilateral testes was observed in each case of 40 and 1000 mg / kg, but it was judged as
random change from the expression frequency did.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histological changes were found in the epididymal, non-mating and non-pregnant animal ovaries.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
No effect was observed on estrous cycle of treated female rats as compared to contorl.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant effect on copulation rate and conception rate, labor and nursing behaviors, gestation period, luteinum count, implantation number, implantation rate, birth rate and
delivery rate of treated rats were observed as compared to control.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (estrous cycle)
reproductive performance
other: No effect observed

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
At 200 mg / kg, trace tail was found in one pup.
No abnormality due to the test substance was observed in the general state.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant difference in neonatal survival rate were observed in treated pups as compared to control.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant change in body weight and body weight gain were observed in treated pups as compared to control.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were observed in both surviving animals and dead animals as compared to control.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
other: No effect observed

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Crj:CD (SD) male and female rats treated with 4,4'-Diamino-2,2'-stilbenedisulfonic acid orally by gavage.
Executive summary:

In a Preliminary Reproduction Toxicity Screening Test, Crj:CD (SD) male and female rats were treated with 4,4'-Diamino-2,2'-stilbenedisulfonic acid in the concentration of 0 (vehicle), 40, 200, 1000 mg/kg/day orally by gavage. At 200 mg / kg, one male died after the start of administration on the fourth day. At necropsy, perforation was found in the lung, so it was judged to be death due to administration error. No change was observed in general condition and body weight and weight gain were observed in treated rats as compared to control. In male rat at 1000 mg / kg, from the start of administration to the 14th day a significant increase in food consumption were observed, but after that, the values were similar to those of the control group, and no significant difference was observed. In females, no significant change was observed throughout the entire period. Similarly, No effect was observed on estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, delivery index, parturition or maternal behavior were observed as compared to control of P generation. No significant change in both the actual weight and the body weight ratio in testis and epididymis were observed. Miniaturization of bilateral testes was observed in each case of 40 and 1000 mg / kg, but it was judged as random change from the expression frequency did. No histological changes were found in the epididymal, non-mating and non-pregnant animal ovaries of p generation rats were observed as compared to control. In addition, No significant differences in numbers of offspring or live offspring, sex ratio, live birth index, viability index and body weight of pups were observed as compared to control. No abnormalities were observed in both surviving animals and dead animals as compared to control. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Crj:CD (SD) male and female rats treated with 4,4'-Diamino-2,2'-stilbenedisulfonic acid orally by gavage.