4-[(5-amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)azo]-2,5-dichlorobenzenesulphonic acid

Toxicity to reproduction

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

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

Test material information

Constituent 1

Reference substance name:

4,4'-diamino-2,2'-stilbenedisulfonic acid

Cas Number:

81-11-8

Molecular formula:

C14H14N2O6S2

IUPAC Name:

4,4'-diamino-2,2'-stilbenedisulfonic acid

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 or test system 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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 4

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.

Oestrous 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 generation)

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

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: oestrous 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 (oestrous 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±1.1^c)	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	10^g)	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±0.3^f)	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.