Sodium 3-(p-anilinophenylazo)benzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction:
The Lowest observed effect concentration (LOAEL) value for the test substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate to female Charles Foster rats, after 20 and 30 exposure duration, was considered to be 250 mg/kg bw/day on the basis of observed effects on Clinical biochemistry, Estrous cycle, Reproductive performance, sexual maturity , gross pathology and histopathology.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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 peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Evaluation of the toxic effects of sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate on the female reproductive system.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material : sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
- Molecular formula : C18H14N3O3S.Na
- Molecular weight: 375.383 g/mol
- Smiles notation : c1(Nc2ccccc2)ccc(\N=N\c2cc(ccc2)S(=O)(=O)[O-])cc1.[Na+]
- InChl : 1S/C18H15N3O3S.Na/c22-25(23,24)18-8-4-7-17(13-18)21-20-16-11-9-15(10-12-16)19-14-5-2-1-3-6-14;/h1-13,19H,(H,22,23,24);/q;+1/p-1/b21-20+;
- Substance type: Organic
- Physical state: Solid

Species:

rat

Strain:

other: Charles Foster

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory-bred
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 90–120 days
- Weight at study initiation: between 110 and 120 g
- Fasting period before study: No
- Housing: Yes
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 258C628C
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12:12 h light and dark cycle
IN-LIFE DATES: From: To: not specified

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

No data

Details on mating procedure:

No data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

20 and 30 days

Frequency of treatment:

daily

Details on study schedule:

no data

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

750 mg/kg bw/day

No. of animals per sex per dose:

4 groups of 8 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

no data

Positive control:

Not specifed

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:

BODY WEIGHT: No
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OTHER:

Oestrous cyclicity (parental animals):

Yes

Sperm parameters (parental animals):

No

Litter observations:

No

Postmortem examinations (parental animals):

SACRIFICE
After completion of treatment period, the animals were sacrificed by cervical dislocation.

GROSS NECROPSY
Yes

HISTOPATHOLOGY / ORGAN WEIGHTS
Yes

Postmortem examinations (offspring):

No

Statistics:

Data obtained from the results were expressed as mean-6SEM. Statistical comparisons between the values obtained in control and in treated rats were carried out by using ANOVA followed by Student’s t test and Mann–Whitney U test for non-parametric data whichever is applicable. p<=0.05 was considered as significant.

Reproductive indices:

Diestrus index = number of day with clear diestrus smear / total duration of treatment x 100

Offspring viability indices:

No data

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The level of LH, FSH, and estradiol were significantly decreased in the MY-treated rats compared to control rats. The activities of some antioxidants enzymes in brain tissues of MY-treated rats were significantly decreased and the level of malondialdehyde (MDA), a marker of lipid peroxidn., in brain tissues of MY-treated rats was also significantly increased.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Light microscopic observations of brain tissue sections from treated rats showed mild structural changes in the brain tissues. Section studied from the cerebellum of control rats showed intact granule and purkinje cell layers. But in case of treated rats of higher doses in both treatment durations, a little dispersion was observed between granule cell layer and purkinje cell layer.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Treatment with higher doses of MY (500 and 750 mg/ kg bw/ day) caused a significant decrease in the number of estrous cycle and duration of proestrus, estrous, and metestrus phases with a concomitant significant increase in duration of diestrus phases in both 20 and 30 days treatment
duration. Diestrus index was also increased dose dependently following the administration of MY. In case of 250 mg/ kg bw/ day MY-treated rat, these alterations were the same but not as significant as higher doses. Significant changes in the cellular characteristics of vaginal smear in component phases of estrous cycle in treated group of rats compared to control rats.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Sexual maturity: The control rats exhibit an average GSI, while the treated group of rats showed less GSI according to the higher doses of treatment. We observed significant (p<0.05) decrease in the GSI in treated group of rats compared to control rats higher doses in both treatment durations, a little dispersion was observed between granule cell layer and purkinje cell layer.

Ovarian folliculogenesis in this study was also significantly impaired

Key result

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

gross pathology

neuropathology

reproductive function (oestrous cycle)

reproductive performance

Critical effects observed:

not specified

Remarks on result:

other: not specified

Critical effects observed:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The Lowest observed effect concentration (LOAEL) value for the test substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate to female Charles Foster rats, after 20 and 30 exposure duration, was considered to be 250 mg/kg bw/day on the basis of observed effects on Clinical biochemistry, Estrous cycle, Reproductive performance, sexual maturity , gross pathology and histopathology.

Executive summary:

The study was designed to examine the effect of test substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (Metanil yellow - MY) on estrous cycle rhythmicity and ovarian folliculogenesis in female rats. Rats have been exposed to MY at three doses of 250, 500, 750 mg/kg bw/day for two exposure durations, 20 and 30 days. Significant changes in the number and duration of estrous cycle along with prominent cytoarchitectural changes in the cellular characteristics of vaginal smear of component phases of estrous cycle in a dose and duration-dependent manner were observed in MY-treated rats compared to control rats. A significant decrease was also observed in serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol levels in MY-treated rats. Further, the activities of some antioxidants enzymes in brain tissues of MY-treated rats were significantly decreased and the level of malondialdehyde (MDA), a marker of lipid peroxidation, in brain tissues of MY-treated rats was also significantly increased. The ovarian folliculogenesis in this study was also significantly impaired in MY treated rats. In conclusion, MY impairs the estrous cycle and ovarian folliculogenesis in female rats by inhibiting the secretion of FSH and estradiol from the ovary, and inducing the oxidative stress in hypothalamic-pituitary-gonadal axis.

 

Hence, the Lowest observed effect concentration (LOAEL) value for the test substancesodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate to female Charles Foster rats, after 20 and 30 exposure duration, was considered to be250 mg/kg bw/day on the basis of observed effects on Clinical biochemistry, Estrous cycle, Reproductive performance, sexual maturity , gross pathology and histopathology.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is Klimisch 2 and from peer reviewed journal.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction:
Various experimental studies for target substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (CAS No. 587-98-4) has been investigated for potential of toxicity to reproduction and are presented below:

The study was designed by P. Nath, K. Sarkar, M. Mondal and G. Paul (Environmental Toxicology ,2016, 31(12), 2057-2067) to examine the effect of test substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (Metanil yellow - MY) on estrous cycle rhythmicity and ovarian folliculogenesis in female rats. Rats have been exposed to MY at three doses of 250, 500, 750 mg/kg bw/day for two exposure durations, 20 and 30 days. Significant changes in the number and duration of estrous cycle along with prominent cytoarchitectural changes in the cellular characteristics of vaginal smear of component phases of estrous cycle in a dose and duration-dependent manner were observed in MY-treated rats compared to control rats. A significant decrease was also observed in serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol levels in MY-treated rats. Further, the activities of some antioxidants enzymes in brain tissues of MY-treated rats were significantly decreased and the level of malondialdehyde (MDA), a marker of lipid peroxidation, in brain tissues of MY-treated rats was also significantly increased. The ovarian folliculogenesis in this study was also significantly impaired in MY treated rats. In conclusion, MY impairs the estrous cycle and ovarian folliculogenesis in female rats by inhibiting the secretion of FSH and estradiol from the ovary, and inducing the oxidative stress in hypothalamic-pituitary-gonadal axis.
Hence, the Lowest observed effect concentration (LOAEL) value for the test substancesodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate to female Charles Foster rats, after 20 and 30 exposure duration, was considered to be250 mg/kg bw/day on the basis of observed effects on Clinical biochemistry, Estrous cycle, Reproductive performance, sexual maturity , gross pathology and histopathology.

In a sub-chronic reproductive toxicity given by R. Sarkar and A. R. Ghosh (International Journal of Basic and Applied Medical Sciences, 2012 Vol. 2 (2) May-August, pp.40-42) for the substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate, Albino rats (R. norvegicus) in the age group of 2-3 months weighing 120-250 g were allowed to acclimatize to laboratory conditions for one week. Then the rats were divided into both control and two experimental groups. Rats of both control and treated groups were maintained on rat pellet diet. The rats of experimental groups were given 3.0 g/kg body weight of Metanil Yellow (MY) for 30 as well as 45 days. The rats of both control and treated were sacrificed under chloroform anesthesia and the testes were taken out and fixed in Bouin’s fixative for 24 hours for histopathological study. Paraffin sections were cut at 4-5 micron and stained in Delafield’s Haematoxylin and Eosin solution. After 30 days,some changes were observed like degeneration occurred in the somniferous tubules as well as spermatogonia , Vacuolations were found in the sertoli cells but maximum changes were found in the somniferous tubules were necrosed and pycnotic spermatocytes and highly vacuolated sertoli cells were also found after 45 days. Therefore, the Lowest observed adverse effect level (LOAEL) was considered to be 3000mg/kg bw/day for 30days, when male rats were treated with the test substancesodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate(CAS No. 587-98-4) for 30 and 45 days, on the basis of histopathological effects observed on somniferous tubules, spermatogonia, spermatocytes and Sertoli cells.

The reproductive toxicity of sodium 3 -[(4 -anilinophenyl)diazenyl] benzenesulfonate (CAS 587 -98 -4) was performed by A. Setyawati, R. R. Rahmawati, M. R. Hariri and A. Tenzer (Proceeding of ICGRC 2013) in Mice(Musmusculus) of strain Balb/C female 5 animals / group in concentration of 0.0, 0.435, 0.87, 1.305 and 1.74mg/20g bw/day (equivalents to 21.75, 43.5, 65.25 and 87 mg/kg bw/day respectively) by oral gavage route daily for from the 7th to 16th day of pregnancy. Every day, the pregnant female mice body weight was weighed and in the 18th day of pregnancy, the pregnant mice were dissected. Changes in body weight, Reproductive system,organ morphologywere observed. The Swelling on organs such as liver, kidney, and spleen were occurred starting from dose 0.87 mg/20g bw/day and weight loss also noted. There were also observed fetuses alive and resorption in control mice, but in treated mice all of dose caused fetal resorption, therefore the rate of fetal life was low.  Therefore, the Lowest observed adverse effect level (LOAEL) of the test substancesodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate(CAS 587-98-4) to female mice is considered to be 21.75 mg/kg bw/day on the basis of effects observed on Reproductive system (fetal resorption).

As per the data available for the target chemical sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate the LOAEL was found to be in a wide range of 21.75 – 3000 mg/kg bw/day affecting the reproductive organs of male and female rats as well as its reproductive system. Hence as per the available data the substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate is considered to be toxic to reproduction and hence can be classified as Reproductive toxic under category 2 as per CLP classification criteria.

Justification for classification or non-classification

As per the data available for the target chemical sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate the LOAEL was found to be in a wide range of 21.75 – 3000 mg/kg bw/day affecting the reproductive organs of male and female rats as well as its reproductive system. Hence as per the available data the substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate is considered to be toxic to reproduction and hence can be classified as Reproductive toxic under category 2 as per CLP classification criteria.