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Toxicological information

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: Autharized database
Title:
Simple reproductive toxicity test for oral administration using rat of 4,4'-diamino-2,2'-stilbenedisulfonic acid
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental 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 :C14H14N2O6S2
- Molecular weight :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 :C14H14N2O6S2
- Molecular weight :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 males, and 183 - 222 g for females.
- Fasting period before study:
- Housing: Animals were housed in a polycarbonate cage with an experimental animal bedding (Petitip: Charles River Japan)
- Diet (e.g. ad libitum): Autoclave-sterilized solid feed for laboratory animals (CRF-1: Oriental Yeast Co., Ltd.) and 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):
- Photoperiod (hrs dark / hrs light): 12 times / hour, 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: 0.5% Sodium Carboxymethyl Cellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test chemical 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 solution
- 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 Vaginal plug formation or sperm was found in vaginal plaque specimens referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
homogeneity, stability and concentration were analysied
Duration of treatment / exposure:
Males, 41 days
Females, from 14 days before mating to Day 3 of lactation
Frequency of treatment:
Daily
Details on study schedule:
not specified
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
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: Dose were selected baed on result of repeated 8-day preliminary test (dose: 0, 100, 300, 1000 mg / kg) using SD rats. 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): Animals were sorted by randomized stratified random extraction method.
- Other:
Positive control:
not specified

Examinations

Parental animals: Observations and examinations:
Mortality, General condition, breastfeeding, nesting, presence or absence of feeding, clinical signs, body weight and food consumption were examined.
Estrous cyclicity (parental animals):
Numbers of estrus periods were examined.
Sperm parameters (parental animals):
not specified
Litter observations:
Numbers of offspring or live offspring, sex ratio, the live birth index and body weight were examined.
Postmortem examinations (parental animals):
Organ weight, Gross pathology and histopathology were examined.
Postmortem examinations (offspring):
Gross pathology was 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 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. Items to be tested by multiple comparison test for 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 and Fisher's direct stochastic method for Copulation rate, conception rate, birth rate, sex ratio (male / female)
Reproductive indices:
Copulation index, fertility index, implantation index, gestation index, delivery index and live birth index were examined.
Offspring viability indices:
Viability index on day 0 and 4 were examined.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical sign that could be attributed to the treatment were observed.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
When treated with 200 mg/kg bw, one male rat died after the start of administration on the fourth day, and as a result of 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 main body weight were observed in treated male and female rats as compared to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
When treated with 1000 mg/kg bw, significant increase in food consumption of male rats were observed from the start of administration to the 14th day, 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 efffect on absolute and relative testis and epididymis weight of male rats were observed as compared to control.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When treated wtih 40 and 1000 mg / kg, Miniaturization of bilateral testes were observed in each case of male rats.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Atrophy of seminiferous tubules in bilateral testes were observed in 1 male control and testis miniaturization of 2 males at 1000 mg/kg bw, 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 animals 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 on number of estrous cycle were observed n treated female rats as compared to control.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No effects on reproductive parameters including the copulation index, fertility index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, delivery index, parturition or maternal behavior, numbers of offspring or live offspring, sex ratio and the live birth index 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
Remarks on result:
other: overall no reproductive toxicity 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):
No clinical sign in pups were observed as compared to control.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant effect on survival rat of pups were observed as compared to control.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant effect on body weight at day 0 and day 4 and weight gain of 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 and dead pups.
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
Remarks on result:
other: overall no developmental toxicity 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

Any other information on results incl. tables

Table 1. Absolute and relative organ weights in male rats treated orally with 4,4'-diamino-2,2'-stilbenedisulfonic acid in the preliminary reproduction toxicity screening test

Dose (mg/kg/day)

0

40

200

1000

Number of rats examined

10

10

9

10

Body weight (g)

514±29.3

518±25.9

503±21.6

516±44.7

Absolute organ weight

Testes (g)

3.36±0.19

3.30±0.31

3.38±0.20

3.27±0.36

Epididymides (g)

1.22±0.09

1.10±0.10

1.18±0.10

1.11±0.14

Relative organ weight

Testes (g)

0.65±0.03

0.64±0.07

0.67±0.04

0.64±0.06

Epididymides (g)

0.24±0.02

0.21±0.03

0.24±0.02

0.22±0.02

 Values are expressed as Mean±S.D.

Table 2. Fertility and pregnancy data in rats treated orally with 4,4'-diamino-2,2'-stilbenedisulfonic acid in the preliminary reproduction toxicity screening test

Dose (mg/kg/day)

0

40

200

1000

Number of pairs examined

10

10

10

10

Number of pairs with successful mating

9

10

8

10

Mating Index (%)a)

90.0

100.0

80.0

100.0

Number of pregnant females

9

10

8

9

Fertility index (%)b)

100.0

100.0

100.0

90.0

Pairing days until mating

2.3±1.1c)

2.9±1.3

2.9±1.3

2.7±1.2

Number of estrous stages without mating

0.1±0.3

0.0±0.0

0.0±0.0

0.0±0.0

 a) Mating index (%) = (number of pairs with successful mating/number of pairs examined) x100

b) Fertility index (%) = (Number of pregnant animals/ number of pairs with successful mating) x 100

c) Values are expressed as Mean ± S.D.

Table 3. Delivery and litter data in rats treated orally with 4,4'-diamino-2,2'-stilbenedisulfonic acid in the preliminary reproduction toxicity screening test

Dose (mg/kg/day)

0

40

200

1000

Number of females examined

9

10g)

8

9

Number of females with live pups

9

9

7

9

Gestation index (%)a)

100.0

100.0

87.5

100.0

Gestation length (Days)

22.9±0.3f)

22.7±0.5

22.5±0.8

22.6±0.5

Number of corpora lutea

18.8±4.7

18.7±2.7

17.1±5.4

17.1±4.0

Number of implantation sites

15.3±4.8

15.6±4.5

15.4±5.6

14.8±5.0

Implantation index (%)b)

81.2

82.9

85.9

83.0

Delivery index (%)c)

93.7

94.8

80.0

90.3

Number of pups delivered

14.2±4.3

14.7±4.2

13.0±6.0

14.0±5.1

Number of live pups on day 0

14.1±4.3

14.7±4.2

12.9±6.2

13.9±5.1

Live birth index (%)d)

99.1

100.0

87.5

99.4

Sex ratio (male/female)

0.73 (54/74)

1.20 (72/60)

1.08(54/50)

1.03(64/62)

Number of live pups on day 4

14.1±4.3

14.7±4.2

14.7±3.7

13.6±5.3

Viability index on day 4 (%)e)

100.0

98.6

100.0

87.5

Body weight of pups on day 0 (Male)

                                                    (Female)

                                     On day 4 (male)

                                                    (female)      

7.0±0.8

6.7±0.8

11.6±1.9

11.1±2.3

7.1±0.9

6.7±0.7

11.3±1.7

10.7±1.4

6.5±0.4

6.1±0.5

10.6±1.2

9.9±1.3

6.9±0.5

6.5±0.7

10.4±1.2

9.8±1.1

Body weight gain of pups from day 0 to day 4

                                                        (male)

                                                    (Female)

4.5±1.2

4.4±1.5

4.2±0.9

4.0±0.8

4.0±1.0

3.6±1.0

3.5±0.8

3.4±0.7

a) Gestation index (%) = (Number of females with live pups/Number of pregnant females) x 100

b) Implantation index (%) = (Number of implantation sites /Number of corpora lutea) x 100

c) Delivery index (%) = (Number             of pups delivered / Number of implantation sites) x 100

d) Live birth index (%) = (Number of live pups on day 0/Number of pups delivered) x 100

e) Viability index (%) = (Number of live pups on day 4/ Number of live pups on day 0) x 100

f) Values are expressed as Mean ± S.D.

g) One female was not examined because the pregnancy was noticed at the time of necropsy after the mating period.

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 rat 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 in 0.5% Sodium Carboxymethyl Cellulose solution for 41 days in male and from 14 days before mating to Day 3 of lactation in female. one male rat died after the start of administration on the fourth day at 200 mg/kg bw, and as a result of necropsy, perforation was found in the lung, so it was judged to be death due to administration error. No clinical sign and body weight change were observed. Significant increase in food consumption of male rats was observed at 1000 mg/kg bw from the start of administration to the 14th day, 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 effects on reproductive parameters including the number of estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, delivery index, parturition or maternal behavior, numbers of offspring or live offspring, sex ratio and the live birth index were observed as compared to control. No significant effect on survival rat, clinical sign and body weight at day 0 and day 4 and weight gain of pups as compared to control. In addition, No effect on absolute and relative testis and epididymis weight of male rats were observed as compared to control. Miniaturization of bilateral testes was observed in each case of 40 and 1000 mg / kg male rats. Atrophy of seminiferous tubules in bilateral testes were observed in 1 male control and testis miniaturization of 2 males at 1000 mg/kg bw, 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 animals ovaries. No abnormalities were observed in both surviving animals and dead pups. 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.