4'-aminoazobenzene-4-sulphonic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity Study:

A sub chronic experiment was performed to evaluate the effect of the test chemical on the reproductive parameters of the Swiss albino mice. The test chemical was administered to adult male mice in drinking water at doses of 0, 0.1, 1 and 2.5% for 13 weeks. After that period, the weights of testes, epididymides and seminal vesicles were determined. Sperm counts in the testis and epididymides, motility, morphology and testis histology were assessed. It was observed that body weight gain was significantly increased in 1% test chemical. This increased body weight gain is not evidence related dose. Food consumption values were significantly decreased at all experimental groups compared to control. However, liquid consumption was increased at all experimental groups. Liquid consumption was increased at all experimental groups. A decrease of relative testis and seminal vesicles weight were observed in all treated groups compared to control but not statistically significant. However, their absolute weight did not change. Seminiferous tubules were not identical with conjunctive tissue dystrophy in animals testes treated with 0.1% test chemical. Intercellular connections were reduced and imperfect and dilation in some seminiferous tubules of testis mice treated 1% test chemical. Significant damage was observed in testis mice treated with 2.5% test chemical extensive disruption in seminiferous tubules, widening of the interstitial spaces and loss leydig cells. Total number of spermatids count was reduced significantly in the mice administered 2.5% test chemical. Sperm concentration in epididymides was reduced in all treated groups but sperm epididymis reserves were reduced significantly only in mice treated with 2.5% test chemical. The percentage motility was reduced in 1 and 2.5% treated groups. Male mating index was decreased in the 2.5% treated groups compared to the control values. Weight and litter sizes were, however, decreased in comparison to litters sired from control males. Thus, based on all the above observations and results, it was observed that the NOAEL for the test chemical was found to be 50 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of 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:

data from handbook or collection of data

Justification for type of information:

Weight of evidence based on the data of the test chemical.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

The above experiments were performed to evaluate and assess the effects of the test chemicals on the reproductive parameters of the test animals.

GLP compliance:

not specified

Justification for study design:

No Data Available

Specific details on test material used for the study:

Details on test material:
- Molecular weight (if other than submission substance): 277.303 g/mol
- Substance type: Organic
- Physical state: No Data Available
- Impurities (identity and concentrations): No Data Available

Species:

other: Study 2: mouse; Study 3: Rat; Study 4: rat

Strain:

other: Study 2: Swiss Albino; Study 3: Sprague-Dawley; Study 4: Crl:CD(SD)IGS BR VAF/Plus

Details on species / strain selection:

No Data Available

Sex:

male/female

Details on test animals or test system and environmental conditions:

Study 2: Details on test animals and env. conditions
TEST ANIMALS
- Source: No Data Available
- Age at study initiation: (P) 4 weeks old
- Weight at study initiation: (P) 20±2.01 g
- Fasting period before study: No Data Available
- Housing: No Data
- Use of restrainers for preventing ingestion (if dermal): No
- Diet (e.g. ad libitum): Standard food pellets diet was given ad libitum
- Water (e.g. ad libitum): Water was provided ad libitum.
- Acclimation period: No Data Available

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

Study 3: TEST ANIMALS
- Source: Laboratory Animal Unit of the University of Hong Kong
- Weight at study initiation: Male rats: 300 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±1°C
- Humidity (%): 60-80%

Study 4: No details available

Route of administration:

other: Study 2: oral: drinking water; Study 3 and 4: oral: gavage

Vehicle:

other: Study 2: water; Study 3: corn oil; Study 4: 0.5% CMC (carboxymethyl cellulose)

Details on exposure:

Study 2: PREPARATION OF DOSING SOLUTIONS: The first group was given drinking water as a control, the second the drinking water containing 0.1% test chemical, the third the drinking water containing 1% test chemical and the fourth the drinking water containing 2.5% test chemical each for 13 weeks.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water was used as the vehicle
- Concentration in vehicle: Concentration of 0.1% (50 mg/kg bw), 1% (500 mg/kg bw) and 2.5% (1250 mg/kg bw) of test chemical for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): No data Available
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available

Study 3: PREPARATION OF DOSING SOLUTIONS: Test chemical was freshly suspended in corn oil

Study 4: PREPARATION OF DOSING SOLUTIONS: Dapsone was dissolved in 0.5% aq. CMC solution

Details on mating procedure:

Study 2: Details of mating
- M/F ratio per cage: Six males per dose group, were mated 1:1 with untreated females for 1 week.
- Length of cohabitation: 1 week
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: No Data Available
- 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): Individually
- Any other deviations from standard protocol: No Data Available

Study 4: Only males were treated. Untreated females were used to confirm male reproductive potential.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Study 2, 3 and 4: No Data Available

Duration of treatment / exposure:

Study 2: 13 weeks
Study 3: 6 weeks
Study 4: Approx. 100 consecutive days

Frequency of treatment:

Study 2: No Data Available
Study 3: Daily
Study 4: Daily

Details on study schedule:

Study 2: No Data Available
Study 3: The substance was administered via gastric intubation into a group of 7 male rats weighing about 300 g at the start of the treatment.
Study 4: F0 male rats were dosed with test material begining 63 days prior to cohabitation with untreated females and continuing through the day prior to sacrifice (doses administered for approx. 100 days).

Remarks:

Study 2: 0.1% (50 mg/kg bw), 1% (500 mg/kg bw) and 2.5% (1250 mg/kg bw) of test chemical for low, mid and high dose groups respectively.

Study 3: 0 or 50 mg/kg/day

Study 4: 0, 0.25, 0.5, 1 or 2 mg/kg/day

No. of animals per sex per dose:

Study 2: 6 males per dose group.
Study 3: Control: 6 males
50 mg/kg/day: 7 males
Study 4: 40 males per group were treated. 20 males per group were sacrificed following the last dose, 10 males per group were sacrificed following a 4 week recovery period and 10 males per group were sacrificed following a 10 week recovery period

Control animals:

yes, concurrent vehicle

Details on study design:

Study 2: No Data Available
Study 3: - Dose selection rationale: The dosage used was 10 times the human dose.
Study 4: Satellite groups: No, but 2 recovery groups were used.

Positive control:

Study 2, 3 and 4: No Data Available

Parental animals: Observations and examinations:

Study 2: Food and water consumption, body weights, Reproductive performance, Gross necropsy and histopathology were examined.
Study 3: Body weights, Mating procedure, percentage of pregnant females in each group, mean live embryo number per pregnant female, mean resorbed embryo number per pregnancy, mean number of corpora lutea of pregnancy, mean embryo crown-rump length were assessed.
Study 4: F0 male rats observed for viability and clinical signs twice daily. Body weight was recorded daily during the dosing period, then weekly for recovery animals. F0 females were monitored for body weight and clinical signs on gestation days 0, 7, 10 and 13.

Oestrous cyclicity (parental animals):

Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available

Sperm parameters (parental animals):

Study 2: Evaluation of sperm motility and morphology and Assessment of sperm production was examined.
Study 3: Sperm motility assessment and Determination of sperm concentration in the epididymis was conducted.
Study 4: Sperm concentration and motility were evaluated.

Litter observations:

Study 2: Litter size and weight after 7, 14 and 28 days of growth were examined.
Study 3: No Data available
Study 4: No Data Available

Postmortem examinations (parental animals):

Study 2: After mating, male mice were killed by cervical dislocation. Testes, epididymides were weighed immediately. The left epididymis was excised and placed in a Petri dish containing saline solution (NaCl 0.9%). The tail region tissue was minced with scalpels. Histologic examination of testis was performed. The left testis was fixed in formalin-buffer.

Study 3: On Day 21 of gestation, the females were killed to determine the following values:
(1) percentage of pregnant females in each group,
(2) mean live embryo number per pregnant female,
(3) mean resorbed embryo number per pregnancy,
(4) mean number of corpora lutea of pregnancy, and
(5) mean embryo crown-rump length.

Study 4: All F0 females were sacrificed on gestational day 13, cesarean-sectioned, gross necropsied and the number of corpora lutea , implantation sites, and viable and non-viable embryos were recorded. Following complettion of cohabitation, F1 males were subjected to gross necropsy, the reproductive organs (each testis, each epididymis, seminal vesicle and prostrate) were weighed, and sperm concentration and motility were evaluated.

Postmortem examinations (offspring):

Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available

Statistics:

Study 2: The data is expressed as mean ± SE. Statistical test one way ANOVA was applied to find significant difference between values of various parameters recorded for control and treated animals. p<0.05 was considered statistically significant.

Study 3: Differences between treated and control groups were compared by using an analysis of variance. All data are presented as mean ± s.e.m.

Study 4: No Data Available

Reproductive indices:

Study 2: Implantation Index, Litter Index, Male Mating Index
Study 3: The fecundity of each test group was represented by the 'Fecundity Index' which was calculated by: mean embryo number/mean corpus luteum number Day 21 embryo length % pregnant females.
Study 4: Fertility index (no. of rats pregnant/ No. of rats mated) was noted

Offspring viability indices:

Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available

Clinical signs:

no effects observed

Description (incidence and severity):

Study 3 and 4: no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: Body weight gain was significantly increased in 1% test chemical. This increased body weight gain is not evidence related dose.
Study 3: no effects observed
Study 4: no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Study 2: Food consumption values were significantly decreased at all experimental groups compared to control. However, liquid consumption was increased at all experimental groups.

Study 3: no effects observed.

Study 4: no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Study 2: Liquid consumption was increased at all experimental groups.

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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Study 2: Semniferous tubules were not identical with conjunctive tissue dystrophy in animals testes treated with 0.1% test chemical. Intercellular connections were reduced and imperfect and dilation in some semniferous tubules of testis mice treated 1% test chemical. Significant damage was observed in testis mice treated with 2.5% test chemical extensive disruption in semniferous tubules, widening of the interstitial spaces and loss leydig cells. Spermatogenic cells are affected and then depleted with absence of spermatozoa in the lumen.

Study 4: no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Study 4: no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Study 2: Total number of spermatids count was reduced significantly in the mice administered 2.5% test chemical. Sperm concentration in epididymides was reduced in all treated groups but sperm epididymis reserves were reduced significantly only in mice treated with 2.5% test chemical. The percentage motility was reduced in 1 and 2.5% treated groups.

Study 3: The capacity to initiate forward motility of the cauda epididymal spermatozoa was reduced by 30%.

Study 4: no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Study 2: Male mating index was decreased in the 2.5% treated groups compared to the control values.

Study 3: Reduced the fecundity of male rats by significantly reducing the embryo number over corpus luteum number per pregnant female. Dapsone reduced fecundity by more than 50% compared with normal rats.

Study 4: no effects observed

Study 3: The concentrations of dapsone in epididymal plasmas approached those in blood sera after 6 weeks treatment with the drugs

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: Not Specified

Critical effects observed:

not specified

System:

other: Not Specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Study 2: Study 2: Weight and litter sizes were, however, decreased in comparison to litters sired from control males.

Study 2: Weight and litter sizes were, however, decreased in comparison to litters sired from control males.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Weight and litter sizes were, however, decreased in comparison to litters sired from control males.

Remarks on result:

other: Not Specified

Critical effects observed:

not specified

System:

other: Not Specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Conclusions:

Study 2: Based on all the above observations and results, it was observed that the NOAEL for the test chemical was found to be 50 mg/kg bw.

Study 3: Based on all the observation and results, it was concluded that the LOAEL value of the test chemical, for effects on male fertility was observed to be 50 mg/kg/day based on the reduction of the fecundity of male rats.

Study 4: The NOAEL value of the test chemical for this study was found to be 2 mg/kg/day as no effect on fertility of male rats was observed at this dose level.

Executive summary:

Reproductive Toxicity Study:

The data from the reproductive toxicity studies are as follows:

Reproductive Toxicity Study 2:

A sub chronic experiment was performed to evaluate the effect of the test chemical on the reproductive parameters of the Swiss albino mice. The test chemical was administered to adult male mice in drinking water at doses of 0, 0.1, 1 and 2.5% for 13 weeks. After that period, the weights of testes, epididymides and seminal vesicles were determined. Sperm counts in the testis and epididymides, motility, morphology and testis histology were assessed. It was observed that body weight gain was significantly increased in 1% test chemical. This increased body weight gain is not evidence related dose. Food consumption values were significantly decreased at all experimental groups compared to control. However, liquid consumption was increased at all experimental groups. Liquid consumption was increased at all experimental groups. A decrease of relative testis and seminal vesicles weight were observed in all treated groups compared to control but not statistically significant. However, their absolute weight did not change. Seminiferous tubules were not identical with conjunctive tissue dystrophy in animals testes treated with 0.1% test chemical. Intercellular connections were reduced and imperfect and dilation in some seminiferous tubules of testis mice treated 1% test chemical. Significant damage was observed in testis mice treated with 2.5% test chemical extensive disruption in seminiferous tubules, widening of the interstitial spaces and loss leydig cells. Total number of spermatids count was reduced significantly in the mice administered 2.5% test chemical. Sperm concentration in epididymides was reduced in all treated groups but sperm epididymis reserves were reduced significantly only in mice treated with 2.5% test chemical. The percentage motility was reduced in 1 and 2.5% treated groups. Male mating index was decreased in the 2.5% treated groups compared to the control values. Weight and litter sizes were, however, decreased in comparison to litters sired from control males. Thus, based on all the above observations and results, it was observed that the NOAEL for the test chemical was found to be 50 mg/kg bw.

Reproductive Toxicity Study 3:

The study was performed to evaluate the effects of the test chemical on the fertility and fecundity of male rats. Its effects on the viability of epididymal spermatozoa and their penetration into the epididymis were also studied. The test animals Sprague-Dawley rats were obtained from the Laboratory Animal Unit of the University of Hong Kong. They were reared and maintained under SPF conditions at an ambient temperature of 20+/- 1°C and a relative humidity of 60-80%. The test chemical was freshly suspended in corn oil and was fed via gastric intubation into a group of 6-12 male rats weighing about 300g at the start of the treatment. The dosage used was 10 times the human dose and treatment was for 6 weeks. Each trial also contained a group of 6-12 control rats to which corn oil only was fed. The body weight of each rat was taken before each feeding. In the study, body weights, Mating procedure, percentage of pregnant females in each group, mean live embryo number per pregnant female, mean resorbed embryo number per pregnancy, mean number of corpora lutea of pregnancy, mean embryo crown-rump length were assessed. Sperm motility assessment and Determination of sperm concentration in the epididymis was conducted. The fecundity of each test group was represented by the 'Fecundity Index' which was calculated by: mean embryo number/mean corpus luteum number Day 21 embryo length % pregnant females. The test chemical reduced the fecundity of male rats by significantly reducing the embryo number over corpus luteum number per pregnant female. The numbers of female rats impregnated by the males treated with the test chemical. The capacity to initiate forward motility of the caudal epididymal

spermatozoa were reduced by the test chemical by 30%. No effects on the body weights of the test animals were observed due to the treatment with the test chemicals. Thus, based on all the observation and results, it was concluded that the LOAEL value of the test chemical, for effects on male fertility was observed to be 50 mg/kg/day based on the reduction of the fecundity of male rats.

Reproductive Toxicity Study 4:

The above experiment was performed to evaluate and assess the effects of the test chemical on the fertility and general reproduction toxicity of male rats.In this study, male rats were treated for 63 days prior cohabitation with untreated females. The animals were treated with doses of 0, 0.25, 0.5, 1 or 2 mg/kg/day. Only males were treated. Untreated females were used to confirm male reproductive potential.No significant differences in fertility parameters and sperm parameters of F0 males were observed at any of the doses.No significant differences in the number of implantations and viable embryos in the females that did become pregnant were observed.The NOAEL value of the test chemical for this study was found to be 2 mg/kg/day as no effect on fertility of male rats was observed at the exposure levels examined in this study.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

The data is from a Klimisch 2 database.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive Toxicity Study:

The data from the reproductive toxicity studies are as follows:

Reproductive Toxicity Study 2:

A sub chronic experiment was performed to evaluate the effect of the test chemical on the reproductive parameters of the Swiss albino mice. The test chemical was administered to adult male mice in drinking water at doses of 0, 0.1, 1 and 2.5% for 13 weeks. After that period, the weights of testes, epididymides and seminal vesicles were determined. Sperm counts in the testis and epididymides, motility, morphology and testis histology were assessed. It was observed that body weight gain was significantly increased in 1% test chemical. This increased body weight gain is not evidence related dose. Food consumption values were significantly decreased at all experimental groups compared to control. However, liquid consumption was increased at all experimental groups. Liquid consumption was increased at all experimental groups. A decrease of relative testis and seminal vesicles weight were observed in all treated groups compared to control but not statistically significant. However, their absolute weight did not change. Seminiferous tubules were not identical with conjunctive tissue dystrophy in animals testes treated with 0.1% test chemical. Intercellular connections were reduced and imperfect and dilation in some seminiferous tubules of testis mice treated 1% test chemical. Significant damage was observed in testis mice treated with 2.5% test chemical extensive disruption in seminiferous tubules, widening of the interstitial spaces and loss leydig cells. Total number of spermatids count was reduced significantly in the mice administered 2.5% test chemical. Sperm concentration in epididymides was reduced in all treated groups but sperm epididymis reserves were reduced significantly only in mice treated with 2.5% test chemical. The percentage motility was reduced in 1 and 2.5% treated groups. Male mating index was decreased in the 2.5% treated groups compared to the control values. Weight and litter sizes were, however, decreased in comparison to litters sired from control males. Thus, based on all the above observations and results, it was observed that the NOAEL for the test chemical was found to be 50 mg/kg bw.

Reproductive Toxicity Study 3:

The study was performed to evaluate the effects of the test chemical on the fertility and fecundity of male rats. Its effects on the viability of epididymal spermatozoa and their penetration into the epididymis were also studied. The test animals Sprague-Dawley rats were obtained from the Laboratory Animal Unit of the University of Hong Kong. They were reared and maintained under SPF conditions at an ambient temperature of 20+/- 1°C and a relative humidity of 60-80%. The test chemical was freshly suspended in corn oil and was fed via gastric intubation into a group of 6-12 male rats weighing about 300g at the start of the treatment. The dosage used was 10 times the human dose and treatment was for 6 weeks. Each trial also contained a group of 6-12 control rats to which corn oil only was fed. The body weight of each rat was taken before each feeding. In the study, body weights, Mating procedure, percentage of pregnant females in each group, mean live embryo number per pregnant female, mean resorbed embryo number per pregnancy, mean number of corpora lutea of pregnancy, mean embryo crown-rump length were assessed. Sperm motility assessment and Determination of sperm concentration in the epididymis was conducted. The fecundity of each test group was represented by the 'Fecundity Index' which was calculated by: mean embryo number/mean corpus luteum number Day 21 embryo length % pregnant females. The test chemical reduced the fecundity of male rats by significantly reducing the embryo number over corpus luteum number per pregnant female. The numbers of female rats impregnated by the males treated with the test chemical. The capacity to initiate forward motility of the caudal epididymal

spermatozoa were reduced by the test chemical by 30%. No effects on the body weights of the test animals were observed due to the treatment with the test chemicals. Thus, based on all the observation and results, it was concluded that the LOAEL value of the test chemical, for effects on male fertility was observed to be 50 mg/kg/day based on the reduction of the fecundity of male rats.

Reproductive Toxicity Study 4:

The above experiment was performed to evaluate and assess the effects of the test chemical on the fertility and general reproduction toxicity of male rats.In this study, male rats were treated for 63 days prior cohabitation with untreated females. The animals were treated with doses of 0, 0.25, 0.5, 1 or 2 mg/kg/day. Only males were treated. Untreated females were used to confirm male reproductive potential.No significant differences in fertility parameters and sperm parameters of F0 males were observed at any of the doses.No significant differences in the number of implantations and viable embryos in the females that did become pregnant were observed.The NOAEL value of the test chemical for this study was found to be 2 mg/kg/day as no effect on fertility of male rats was observed at the exposure levels examined in this study.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study :

Developmental studies were performed on SPF-derived Wistar female rats. Study was conducted in two stages as the preliminary study four groups of 15 pregnant rats was administered by gavage from day 0-19 of pregnancy. In second study, 30 pregnant rats were used with same protocol. On 21 days the animals were killed and ovaries and uterus removed. The number of corpora lutea in each ovary was recorded and the foetuses examined. Live foetuses, embryonic and foetal resorptions and dead foetuses were counted and the number and position of implantation sites were recorded. In the second study, half the foetuses in the control and top dose groups were examined for skeletal malformations and half for visceral defects. No abnormalities in condition or behaviour of the dams were observed in either study. At autopsy, no signs of embryo-toxicity or teratogenicity were observed. Thus, based on all the observations and results it was concluded that, the NOAEL for the test chemical was found to be 2500 mg/kg bw for SPF-derived Wistar rats.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of 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:

data from handbook or collection of data

Justification for type of information:

Weight of evidence based on the studies of different test chemical.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

The above experiments were performed to evaluate and assess the effects of the test chemical on the developmental parameters of the test animals.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on test material:
- Molecular weight (if other than submission substance): 277.303 g/mol
- Substance type: Organic
- Physical state: No Data Available
- Impurities (identity and concentrations): No Data Available

Species:

other: Study 2: Rat; Study 3: Mice Study 4: Rat

Strain:

other: Study 2: Wistar; Study 3: Swiss; Study 4: Crl:CD(SD)IGS BR VAF/Plus

Details on test animals or test system and environmental conditions:

Study 2: No Data Available

Study 3: TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Sex: Female
- Housing: solid-bottom polycarbonate cages with stainless steel wire lids and certified hardwood cage litter
- Diet (e.g. ad libitum): NIH-07 Certified Mouse/Rat Diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67-77°F
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12:12 light:dark cycle

Study 4: Sex: female
No other details available

Route of administration:

other: Study 2: oral: unspecified; Study 3 and 4 : oral:gavage

Vehicle:

other: Study 2: Unspecified; Study 3: 0.5% aqueous methylcellulose; Study 4: CMC (carboxymethyl cellulose)

Details on exposure:

Study 2: No Data Available

Study 3: Total daily dose volume was 20 ml/kg

Study 4: PREPARATION OF DOSING SOLUTIONS: 0.5% carboxymethyl cellulose in purified deionised water was used as the vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study 2: No Data Available

Study 3: Analytical verification done by HPLC

Study 4: No Data Available

Details on mating procedure:

Study 2: No Data Available

Study 3: - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Overnight
- Proof of pregnancy: The morning on which a copulation plug was found in the vagina was designated as gd 0.

Study 4: - Impregnation procedure: cohoused
- If cohoused: Virgin females were paired with males
- Proof of pregnancy: Day on which mating was confirmed by presence of vaginal plug or sperm in vaginal smear was referred to as day 0 of pregnancy

Duration of treatment / exposure:

Study 2: For first and second study : from day 0-19 of pregnancy

Study 3: 10 days (Gestation day 6 to 15)

Study 4: 15 days prior to pairing upto day 17 of gestation

Frequency of treatment:

Study 2: Exact frequency was not mentioned

Study 3: Twice daily

Study 4: Daily

Duration of test:

Study 2: From F1 to F3 generation

Study 3: 17 days

Study 4: Upto day 21 of pregnancy

Remarks:

Study 2: 0, 0.1, 1.0 or 3% (0, 50, 500,1500 mg/kg bw per day)

Study 3: 0, 50, 100 or 200 mg/kg/day

Study 4: 0, 12, 30 and 75 mg/kg/day

No. of animals per sex per dose:

Study 2: First study:15 animals
Second study: 30 animals

Study 3: 20-21 mated females per group

Study 4: 25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

Study 2: No Data Available

Study 3: - Dose selection rationale: Dose selection was based on previous studies in which CD-1 mice were treated by gavage with dapsone from gd 6 through 15

Study 4: No Data Available

Maternal examinations:

Study 2: Body weight of the maternal animals was examined.

Study 3: Body weight (g) was recorded on the mornings of gd 0, 6-15, and 17, and immediately following sacrifice on gd 17.
Females were observed for clinical condition at least once/day on gd 0 to 5 (prior to dosing). From gd 6 through 15, females were observed for clinical condition twice daily at dosing. On gd 17, females were observed for clinical condition at weighing and at scheduled termination.
Feed consumption was monitored during the study, with measurements on the mornings of gd 0, 6, 9, 12, 15 and 17.
The body, and uterus of each timed-mated female were weighed. Thoracic and abdominal cavities were examined.

Study 4: Maternal survival and body weight

Ovaries and uterine content:

Study 2: The number of corpora lutea in each ovary was recorded and the foetuses examined.

Study 3: Ovarian corpora lutea were counted. Pregnancy status was confirmed by uterine examination. Uterine contents were examined to determine the number of implantation sites, resorptions, dead fetuses, and live fetuses.

Study 4: Number of live, dead and resorbed fetuses were determined

Fetal examinations:

Study 2: Live foetuses, embryonic and foetal resorptions and dead foetuses were counted and the number and position of implantation sites were recorded.

Study 3: Dead fetuses were counted, weighed, and discarded. All live fetuses were counted, weighed, sexed (external), and examined for external morphological abnormalities, including cleft palate.

Study 4: Live fetuses were weighed and examined for external, visceral and skeletal abnormalities.

Statistics:

Study 2: No data available

Study 3: No Data Available

Study 4: No Data Available

Indices:

Study 2: No data available

Study 3: Resorption Index, Fetal Viability Index, Implantation Index

Study 4: Resorption Index, Fetal Viability Index.

Historical control data:

No Data Available

Clinical signs:

no effects observed

Description (incidence and severity):

Study 2: No Data Available

Study 3: no effects observed

Study 4: Increased licking, sniffing, chewing and salivation in animals were observed at 75 mg/kg/day.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Study 2: No Data Available

Study 3: 10% of the maternal mortality was observed after the administration of the test chemical.

Study 4: no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study 2: No data available

Study 3: Maternal body weight change were each reduced at the highest dose of the test chemical (200 mg/kg/day).

Study 4: Significantly reduced body weight gain at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Study 2: No data available

Study 3: There was a tendency for the test chemical consuming groups to consume more feed than the controls. Maternal feed consumption was usually at or above controls for subsequent measurement periods.

Study 4: Slightly reduced at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment).

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

Study 2: No data available

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:

effects observed, treatment-related

Description (incidence and severity):

Study 2: No data available
Study 3: Gravid uterine weight were each reduced at the highest dose of the test chemical (200 mg/kg/day).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Study 2: No data available
Study 3: At scheduled necropsy (gd 17), gross findings included enlarged spleens in 1, 12, 13 or 17 dams in the control through high dose groups, respectively.

Neuropathological findings:

not specified

Description (incidence and severity):

Study 2: No data available
Study 3: No data available
Study 4: No data available

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Not Specified
Study 4: Not Specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Implantation losses were observed in the highest dose group.
Study 4: no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 4: no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Indices of prenatal mortality appeared to be elevated at the highest dose of the test chemical.
Study 4: Number. of early resorptions were significantly increased at 75 mg/kg/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Indices of prenatal mortality appeared to be elevated at the highest dose of the test chemical.
Study 4: Not Specified

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Indices of prenatal mortality appeared to be elevated at the highest dose of the test chemical.
Study 4: Not Specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 4: Not Specified

Other effects:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Not Specified
Study 4: Not Specified

Details on maternal toxic effects:

Study 2: Maternal toxic effects:no effects
Details on maternal toxic effects:
At autopsy, no signs of embryo-toxicity or teratogenicity were observed.

Study 3: Maternal toxic effects:yes
Details on maternal toxic effects:
Maternal mortality (10%) was noted at the high dose, but otherwise clinical observations were unremarkable.
At scheduled necropsy (gd 17), gross findings included enlarged spleens in 1, 12, 13 or 17 dams in the control through high dose groups,
respectively. Maternal body weight, weight change, and gravid uterine weight were each reduced at the highest dose of the test chemical.

Study 4: Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality: None
Clinical signs: Increased licking, sniffing, chewing and salivation at 75 mg/kg/day.
Body weight: significantly reduced body weight gain at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment)
Food consumption: slightly reduced at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment)
No effects on latency to mate or fertility index

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Developmental Toxicity

Remarks on result:

other: Not Specified

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Fetal body weights (male, female or both) were significantly reduced by 25% at the highest dose of the test chemical.
Study 4: no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Indices of prenatal mortality appeared to be elevated at the highest dose of the test chemical (e.g., 16% resorbed implantation sites vs. 2% for controls).
Study 4: Number of live fetuses were slightly reduced at 30 mg/kg/day; significantly reduced at 75 mg/kg/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Not Specified
Study 4: Not Specified

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Not Specified
Study 4: Not Specified

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Study 2: No effects observed
Study 3: Not Specified
Study 4: Not Specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: Not Specified
Study 3: Unremarkable effects on the dosing of the test chemical were observed.
Study 4: Not Specified

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: Not Specified
Study 3: Unremarkable effects on the dosing of the test chemical were observed.
Study 4: Not Specified

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: Not Specified
Study 3: Unremarkable effects on the dosing of the test chemical were observed.
Study 4: Not Specified

Other effects:

not specified

Description (incidence and severity):

Study 2: Not Specified
Study 4: Not Specified

Details on embryotoxic / teratogenic effects:

Study 2: Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
No abnormalities in condition or behaviour of the dams were observed in either study

Study 3: Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Indices of prenatal mortality appeared to be elevated at the highest dose of dapsone (e.g., 16% resorbed implantation sites vs. 2% for controls).
Fetal body weights (male, female or both) were significantly reduced by 25% at the highest dose of dapsone. Altered incidences of fetal morphological anomalies (malformations or variations) were noted, but patterns of effects varied across replicates.

Study 4: Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Mean implantations: reduced at 75 mg/kg/day
Body weight of live fetuses: no remarkable observations
No. of live fetuses at C-section: Slightly reduced at 30 mg/kg/day; significantly reduced at 75 mg/kg/day
No. of early resorptions: significantly increased at 75 mg/kg/day only

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

Remarks on result:

other: Not Specified

Abnormalities:

not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Conclusions:

Study 2: From all the observations and results it was concluded that, the NOAEL for the test chemical was found to be 2500 mg/kg bw for SPF-derived Wistar rats.

Study 3: Based on all the observations and results, it was concluded that the NOAEL value for the test chemical was found to be 100 mg/kg/day for parental as well as F1 generation.

Study 4: The NOAEL value of the test chemical for the F1 generation was 12 mg/kg/day based on the reduction in number of implantations, increase in early resorption rate and reduction in mean litter size.

Executive summary:

Developmental Toxicity Study:

The data from the developmental toxicity studies are as follows:

Developmental Toxicity Study 2:

Developmental studies were performed on SPF-derived Wistar female rats. Study was conducted in two stages as the preliminary study four groups of 15 pregnant rats was administered by gavage from day 0-19 of pregnancy. In second study, 30 pregnant rats were used with same protocol. On 21 days the animals were killed and ovaries and uterus removed. The number of corpora lutea in each ovary was recorded and the foetuses examined. Live foetuses, embryonic and foetal resorptions and dead foetuses were counted and the number and position of implantation sites were recorded. In the second study, half the foetuses in the control and top dose groups were examined for skeletal malformations and half for visceral defects. No abnormalities in condition or behaviour of the dams were observed in either study. At autopsy, no signs of embryo-toxicity or teratogenicity were observed. Thus, based on all the observations and results it was concluded that, the NOAEL for the test chemical was found to be 2500 mg/kg bw for SPF-derived Wistar rats.

Developmental Toxicity Study 3:

The above experiment was performed to evaluate and assess the effect of the test chemical on the developmental parameters of the test animals. Timed-mated Swiss albino (CD-1®) mice were dosed by gavage with the test chemical 0, 50, 100 or 200 mg/kg/day. on gestational days (gd) 6 through 15. The vehicle was 0.5% aqueous methylcellulose. Total daily doses were administered in two divided doses (morning and afternoon), each with a volume of 10 ml/kg body weight. The oral route corresponds to one of the commonly used routes in human patients. Timed-mated mice (20-21 per group) were monitored at regular intervals for clinical signs of toxicity, feed consumption, and body weight. At necropsy (17), the following observations were made: clinical condition; maternal body, and gravid uterine weights; pregnancy status; and number of corpora lutea. In the gravid uterus, the numbers of prenatal deaths (resorptions and/or dead fetuses) and live fetuses were recorded. Live fetuses were weighed, sexed, and examined for morphological anomalies (external, visceral, and skeletal). For the test chemical, maternal mortality (10%) was noted at the high dose, but otherwise clinical observations were unremarkable. At scheduled necropsy (17), gross findings included enlarged spleens in 1, 12, 13 or 17 dams in the control through high dose groups, respectively. Maternal body weight, weight change, and gravid uterine weight were each reduced at the highest dose of the test chemical (200 mg/kg/day). Maternal feed consumption was usually at or above controls for subsequent measurement periods. Indices of prenatal mortality appeared to be elevated at the highest dose of the test chemical (e.g., 16% resorbed implantation sites vs. 2% for controls). Fetal body weights (male, female or both) were significantly reduced by 25% at the highest dose of the test chemical. Thus, based on all the observations and results, it was concluded that the NOAEL value for the test chemical was found to be 100 mg/kg/day for parental as well as F1 generation.

Developmental Toxicity Study 4:

The above experiment was performed to evaluate and assess the effect of the test chemical on the developmental parameters of the test animals. 25 females per group were selected in this study. The animals were dosed with the test chemical in concentration of 0, 12, 30 and 75 mg/kg/day. The maternal animals were dosed from 15 days prior to pairing upto day 17 of gestation. The maternal animals were observed for Maternal survival and body weight and Number of live, dead and resorbed fetuses were also determined. Live fetuses were weighed and examined for external, visceral and skeletal abnormalities. It was observed that no mortality was present in any of the maternal animals at any dose groups. However, increased licking, sniffing, chewing and salivation in animals were observed at 75 mg/kg/day. Significantly reduced body weight gain at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment). Slightly reduced at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment). Also, no effects on latency to mate or fertility index was observed at any dose group. Number of early resorptions were significantly increased at 75 mg/kg/day. In fetal parameters, no remarkable effects were observed on fetal body weights. Although, number of live fetuses were slightly reduced at 30 mg/kg/day; significantly reduced at 75 mg/kg/day. Thus, based on all the observations and results, it was concluded that the NOAEL value of the test chemical for the F1 generation was 12 mg/kg/day based on the reduction in number of implantations, increase in early resorption rate and reduction in mean litter size.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The data is from a Klimisch 2 source.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental Toxicity Study:

The data from the developmental toxicity studies are as follows:

Developmental Toxicity Study 2:

Developmental studies were performed on SPF-derived Wistar female rats. Study was conducted in two stages as the preliminary study four groups of 15 pregnant rats was administered by gavage from day 0-19 of pregnancy. In second study, 30 pregnant rats were used with same protocol. On 21 days the animals were killed and ovaries and uterus removed. The number of corpora lutea in each ovary was recorded and the foetuses examined. Live foetuses, embryonic and foetal resorptions and dead foetuses were counted and the number and position of implantation sites were recorded. In the second study, half the foetuses in the control and top dose groups were examined for skeletal malformations and half for visceral defects. No abnormalities in condition or behaviour of the dams were observed in either study. At autopsy, no signs of embryo-toxicity or teratogenicity were observed. Thus, based on all the observations and results it was concluded that, the NOAEL for the test chemical was found to be 2500 mg/kg bw for SPF-derived Wistar rats.

Developmental Toxicity Study 3:

The above experiment was performed to evaluate and assess the effect of the test chemical on the developmental parameters of the test animals. Timed-mated Swiss albino (CD-1®) mice were dosed by gavage with the test chemical 0, 50, 100 or 200 mg/kg/day. on gestational days (gd) 6 through 15. The vehicle was 0.5% aqueous methylcellulose. Total daily doses were administered in two divided doses (morning and afternoon), each with a volume of 10 ml/kg body weight. The oral route corresponds to one of the commonly used routes in human patients. Timed-mated mice (20-21 per group) were monitored at regular intervals for clinical signs of toxicity, feed consumption, and body weight. At necropsy (17), the following observations were made: clinical condition; maternal body, and gravid uterine weights; pregnancy status; and number of corpora lutea. In the gravid uterus, the numbers of prenatal deaths (resorptions and/or dead fetuses) and live fetuses were recorded. Live fetuses were weighed, sexed, and examined for morphological anomalies (external, visceral, and skeletal). For the test chemical, maternal mortality (10%) was noted at the high dose, but otherwise clinical observations were unremarkable. At scheduled necropsy (17), gross findings included enlarged spleens in 1, 12, 13 or 17 dams in the control through high dose groups, respectively. Maternal body weight, weight change, and gravid uterine weight were each reduced at the highest dose of the test chemical (200 mg/kg/day). Maternal feed consumption was usually at or above controls for subsequent measurement periods. Indices of prenatal mortality appeared to be elevated at the highest dose of the test chemical (e.g., 16% resorbed implantation sites vs. 2% for controls). Fetal body weights (male, female or both) were significantly reduced by 25% at the highest dose of the test chemical. Thus, based on all the observations and results, it was concluded that the NOAEL value for the test chemical was found to be 100 mg/kg/day for parental as well as F1 generation.

Developmental Toxicity Study 4:

The above experiment was performed to evaluate and assess the effect of the test chemical on the developmental parameters of the test animals. 25 females per group were selected in this study. The animals were dosed with the test chemical in concentration of 0, 12, 30 and 75 mg/kg/day. The maternal animals were dosed from 15 days prior to pairing upto day 17 of gestation. The maternal animals were observed for Maternal survival and body weight and Number of live, dead and resorbed fetuses were also determined. Live fetuses were weighed and examined for external, visceral and skeletal abnormalities. It was observed that no mortality was present in any of the maternal animals at any dose groups. However, increased licking, sniffing, chewing and salivation in animals were observed at 75 mg/kg/day. Significantly reduced body weight gain at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment). Slightly reduced at 30 mg/kg/day and above during treatment period. Increased at 30 mg/kg/day and above on days 18-21 (after stopping of treatment). Also, no effects on latency to mate or fertility index was observed at any dose group. Number of early resorptions were significantly increased at 75 mg/kg/day. In fetal parameters, no remarkable effects were observed on fetal body weights. Although, number of live fetuses were slightly reduced at 30 mg/kg/day; significantly reduced at 75 mg/kg/day. Thus, based on all the observations and results, it was concluded that the NOAEL value of the test chemical for the F1 generation was 12 mg/kg/day based on the reduction in number of implantations, increase in early resorption rate and reduction in mean litter size.

Justification for classification or non-classification

On the basis of the information and data of the available studies, it is concluded that the test chemical might not be classified as a reproductive and developmental toxicant as per CLP regulation.