1-hydroxybenzotriazole

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Reproductive and developmental toxicity study of test chemical was performed on Sprague Dawley rats.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-hydroxybenzotriazole

EC Number:

219-989-7

EC Name:

1-hydroxybenzotriazole

Cas Number:

2592-95-2

Molecular formula:

C6H5N3O

IUPAC Name:

1H-1,2,3-benzotriazol-1-ol

Details on test material:

- Name of test material (as cited in study report): 1- Hydroxybenzotriazole hydrate
- Molecular formula: C6H5N3O
- Molecular weight: 135.126 g/mol
- Substance type: Organic
- Physical state: Solid, crystalline powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animals and env. conditions
TEST ANIMALS
- Source: Bioneeds India Private Limited
- Age at study initiation: 9 weeks
- Weight at study initiation: Males: 250.07 g to 299.20 g
Females: 204.08 g to 247.52 g
- Fasting period before study:
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum
- Water (e.g. ad libitum): Water was provided ad libitum
- Acclimation period: nineteen days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.6oC
- Humidity (%):40 to 69%,
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Details on exposure
PREPARATION OF DOSING SOLUTIONS:
The test chemical formulations were freshly prepared before dose administration on each day. The required quantity of test chemical was weighed in an aluminum foil, transferred in to a mortar and grind well using pestle by adding small quantity of vehicle until a clear suspension is formed. Thereafter, the entire quantity of the formulation was transferred into a measuring cylinder. Again, a small quantity of the vehicle was added to rinse the mortar and pestle and this was transferred into the measuring cylinder. The rinsing of mortar and pestle was repeated to ensure the complete transfer of the contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 25, 50 and 100 mg/mL of test chemical for low, mid and high dose groups respectively.

DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food)
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water):corn oil
- Concentration in vehicle: 0, 250,500,1000 mg/kg/day - Amount of vehicle (if gavage):10ml/kg

- Lot/batch no. (if required): A1712001
- Purity:

Details on mating procedure:

Details of mating
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Approx 64 days ( The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 30 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13)

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0,250, 500 , 1000mg/kg/day

No. of animals per sex per dose:

Total :96 (48 Males + 48 Females)
0 mg/kg bw/day: 12 male and 12 female
250mg/kg bw/day:12 male and 12 female
500mg/kg bw/day:12 male and 12 female
1000mg/kg bw/day:12 male and 12 female

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: Yes All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and preferably at the same time. Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity

BODY WEIGHT: Yes
Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period and on day 14 (fasting body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):Yes Feed consumption was measured for all animals once a week during premating and once for males during post mating period. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.

Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available

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

OTHER: Clinical Pathology- Hormone analysis
serum was pooled by litter for T4 analysis on both lactation day 4 and lactation day 13.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored for two weeks after five days of acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females.

Sperm parameters (parental animals):

Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells).

Litter observations:

The duration of gestation was recorded and calculated from day 0 of pregnancy. The day of littering was considered as lactation day 1. The number of pups born (dead and live) in a litter, sex, live births and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4) and the ratio of AGD to the cube root of pup body weight was calculated. All survived male pups were examined for appearance of nipples/areolae on postnatal day 13 (lactation day 13). The litter was observed daily in order to note the number of alive, dead and cannibalized pups. All the dead and sacrificed pups were examined and subjected to gross pathological examination. Fertility index for dams, sires and pup live birth index, mean litter size per group, survival index and sex ratio at birth were calculated.

Postmortem examinations (parental animals):

Postmortem examinations (Parent Animal)
SACRIFICE : The males were sacrificed after completion of 30 days of treatment, females were sacrificed on lactation day 14.The animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all the fasted animals was recorded prior to exsanguination. The vaginal smear of females on the day of necropsy (lactation day 14) was performed and stage of oestrus cycle was recorded. The animals were euthanized using deep CO2 followed by exsanguination .


GROSS NECROPSY

External and internal gross pathological examination. During necropsy, the males were randomized.
HISTOPATHOLOGY / ORGAN WEIGHTS

Detailed histopathological examination was performed on the ovaries, testes and epididymides of the animals from the control and the high dose group including all macroscopically abnormal tissues of all animals sacrificed at termination. The histopathological examination of epididymides was extended to the lower dose groups as treatment related effects observed at the high dose level to cover the gross lesions encountered.These tissues were embedded in paraffin wax, sectioned at 4 to 6 micrometers and stained with haematoxylin and eosin.

Postmortem examinations (offspring):

Postmortem examinations (offspring)
SACRIFICE
pups were sacrificed on lactation day 13.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data.After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests designated by the superscripts throughout the report as stated below:
*Statistically significant (P<0.05) change than the vehicle control group.
Note: Data of non-pregnant females and females mated but not littered was excluded from statistical analysis.
Type of Analysis
Parametric - One-way ANOVA with Dunnett’s post test
Non Parametric - Kruskal-Wallis Test
Cross Tabs -Chi-square test

Reproductive indices:

All the adult animals were observed for mating and fertility index.

Offspring viability indices:

Live birth index, pup survival index was observed.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs of toxicity observed at any of the tested dose group animals of either sex during the experimental period.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There was no mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reduction in mean body weight on day 14, 21 and 28 at G3 (500 mg/kg) and G4 (1000 mg/kg) group males; statistically significant reduction in mean body weight on day 21 at G2 (250 mg/kg) group males; statistically significant reduction in percent change in body weight during day 1 to 7, 1 to 14, 1 to 21 and 1 to 28 at all the tested dose group males when compared with vehicle control group was noted.
Statistically significant reduction in percent change in body weight during day 1 to 7 at G4 (1000 mg/kg) group females; statistically significant reduction in percent change in body weight during day 1 to 14 at G2 (250 mg/kg) and G3 (500 mg/kg) group females when compared with vehicle control group was noted.
These changes cannot considered as treatment related as the body weight and changer in body weight was increased when compared with day 1 within the group, no clinical signs of toxicity noted during the experimental period and no changes in feed consumption noted during this period.

There were no changes observed in the gestation body weight and percent change in gestation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals. However, statistical significant reduction in mean gestation body weight on GD0 and 7 was noted at G2(250 mg/kg) group animals when compared with vehicle control group. This change is considered as incidental due to lack of dose dependency and also no effects were noted in feed consumption during this period.
There were no changes observed in the lactation body weight and percent change in lactation body weight at any of the tested dose group animals when compared with vehicle control group animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a slight reduction in mean feed consumption observed at all the tested group animals of either sex during the pre-mating. These changes can be considered as treatment related as the reduction is consistent during the experimental period and also due to reduction in body weight and percent change in body weight with respect to day 1 during the experimental period. There were no changes observed in the feed consumption during gestation period at all the tested dose groups when compared with vehicle control group animals.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Sperm granulomas were detected in cauda of epididymides in three animals bilaterally and one animal unilaterally in G4 group. Two animals revealed lymphocytic inflammation and two animals revealed neutrophilic inflammation of cauda epididymides from G4 group. This change is considered as treatment related histopathological finding.
Dianne et al 2012, reported that sperm granulomas occur when immunologically competent cells gain access to sperm, which are antigenically foreign. This generally occurs following rupture of the luminal sperm intothe interstitium of the epididymis. Chemically induced sperm granulomasinclude caudal granulomas secondary to inflammation.
Lesions considered spontaneous and incidental was observed in one animal of control group. These lesions consisted of degeneration of seminiferous tubules of testes bilaterally and presence of cell debris in lumen of epididymides.
Epididymides evaluation from G3 (500 mg/kg) and G2 (250 mg/kg) group did not reveal any abnormalities.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in serum T4 levels in adult males and lactation day 13 pups at any of the tested dose groups when compared with vehicle control group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes observed in the oestrus cyclicity at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A total of twelve pairs were started for mating from each group, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups.
A total of 11 from vehicle control 10 from G2(250 mg/kg) group ,10 from G3(500 mg/kg) group and 2 from G4(1000 mg/kg) group females respectively, were found with a fertility rate of 91.7%, 83.3%, 83.3% and 16.7% respectively,
A total of 11 (91.7%), 10 (83.3%), 10 (83.3%) and 2 (16.7%) females from vehicle control, low dose, mid dose and high dose groups respectively, were littered and found to be pregnant; total of 1 (8.3%), 2 (16.7%), 2 (16.7%) and 7 (58.3%) females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations]; total of 0 (0.0%), 0 (0.0%), 0 (0.0%) and 3 (25.0%) females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-littered [evidence of implantations] during necropsy.
The females which were littered [11, 10, 10 and 2 females from vehicle control, low dose, mid dose and high dose groups respectively] only considered for mean calculations and statistical analysis for the following parameters.
There were no changes observed in the gestation length at any of the littered tested dose group animals when compared with vehicle control group animals.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

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

Remarks on result:

other: No toxic effects was observed at given dose level

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs and external anomalies observed in any of the pups of tested dose group animals during lactation period.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose groups when compared with vehicle control group dams. However, statistical significant reduction in mean male pup weight on lactation day 7 at G3 (500 mg/kg) and on lactation day 13 at G2 (250 mg/kg) was noted when compared with vehicle control group. This observation was not considered as treatment related as the occurrences were not dose dependent.

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):

There were no gross pathological lesions noticed at necropsy of pups (both live and dead).

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

There were no changes observed in ano-genital distance ratio of both male and female pups recorded on lactation day 4 at any of the tested dose group litters when compared with vehicle control group litters.
There were no occurrences of nipples in male pups of dams at any of the tested dose group litters and vehicle control group litters observed on lactation day 13.

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:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: No toxic effects were observed

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 500 mg/kg body weight, Based on the results obtained in the experiment, the fertility rate of 91.7%, 83.3%, 83.3% and 16.7% was noted from vehicle control, low dose, mid dose and high dose groups respectively. The reduction in fertility rate in both the sex at G4 (1000 mg/kg), the high dose group was considered as treatment related. The increase in epididymis and testes weight, occurrence of gross pathological findings (soft nodules on epididymis bilateral) and histopathological findings (sperm granulomas in cauda of epididymides bilateral and unilateral) at G4 (1000 mg/kg), the high dose group was considered as treatment related.

Executive summary:

The reproductive and developmental toxicity study of test chemical was performed on male and female Sprague Dawley rats in accordance with OECD Guideline for Testing of Chemicals, Number 421, “Reproduction/Developmental Toxicity Screening Test”. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle [corn oil], animals in G2, G3 and G4groups were administered with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively. The vehicle and test chemical formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 30 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).  Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results are found to be within the acceptable range of 85 to 115% the nominal concentration and the % RSD is less than 10.0%. All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter were screened for T4 levels. Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period. The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The anogenital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 31 for males and on lactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy. All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. No treatment related changes in body weight, percent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating period. There were no effects observed in absolute thyroid along with parathyroid weights and its relative weight with respect to terminal body weight at any of the tested dose group males. Statistical significant increase in absolute and relative testes weight at G4 (1000 mg/kg); statistical significant increase in relative testes weight at G2 (250 mg/kg) and G4 (500 mg/kg); statistical significant increase in relative epididymis weight at G3 (500 mg/kg) and G4 (1000 mg/kg) and statistical significant increase in relative prostate and seminal vesicles with coagulating glands weight at G2 (250 mg/kg) were noted when compared with vehicle control group. This changes at G4 (1000 mg/kg) group was considered as treatment related due to gross and histopathological findings and effect on fertility at this dose group. A total of 11, 10, 10 and 2 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 91.7%, 83.3%, 83.3% and 16.7%; total of 1, 2, 2 and 7 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a percent of 8.3%, 16.7%, 16.7% and 58.3%; total of 0, 0, 0 and 3 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-littered [evidence of implantations] with a percent of 0%, 0%, 0% and 25.0% during necropsy were noted. Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, and body weight and feed consumption during gestation and lactation at all the tested dose groups. A total of twelve pairs were started for mating from each group, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 11, 10, 10 and 2 females from vehicle control, low dose, mid dose and high dose groups respectively, were found with a fertility rate of 91.7%, 83.3%, 83.3% and 16.7%. A total of 11 (91.7%), 10 (83.3%), 10 (83.3%) and 2 (16.7%) females from vehicle control, low dose, mid dose and high dose groups respectively, were littered and found to be pregnant; total of 1 (8.3%), 2 (16.7%), 2 (16.7%) and 7 (58.3%) females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations]; total of 0 (0.0%), 0 (0.0%), 0 (0.0%) and 3 (25.0%) females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-littered [evidence of implantations] during necropsy. These observations are considered as treatment related. All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. During gross pathological examination, 25% of males from G4 group revealed white soft nodules on epididymis bilaterally which can be considered as treatment related due to adverse effects on fertility on this group when compared with other tested dose groups. There were no gross pathological lesions noticed at necropsy of pups (both live and dead). During histopathological examination, sperm granulomas were detected in cauda of epididymides in20% of males bilaterally and 8% of males unilaterally in G4 group. 20% of males revealed lymphocytic inflammation and 16.7% of males’ revealed neutrophilic inflammation of cauda epididymides from G4 group. This change is considered as treatment related histopathological finding. Hence The No Observed Adverse Effect  Level (NOAEL) of the test chemical was considered to be 500 mg/kg body weight, Based on the results obtained in the experiment, the fertility rate of 91.7%, 83.3%, 83.3% and 16.7% was noted from vehicle control, low dose, mid dose and high dose groups respectively. The reduction in fertility rate in both the sex at G4 (1000 mg/kg), the high dose group was considered as treatment related. The increase in epididymis and testes weight, occurrence of gross pathological findings (soft nodules on epididymis bilateral) and histopathological findings (sperm granulomas in cauda of epididymides bilateral and unilateral) at G4 (1000 mg/kg), the high dose group was considered as treatment related. Thus, based on all the observations and results, it was concluded that, The No Observed Adverse Effect Level (NOAEL) of the test chemical was considered to be 500 mg/kg body weight.