Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-12-23 to 2016-04-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 2015-07-28
Deviations:
yes
Remarks:
Some additional organs (i.e. kidneys, adrenals, heart, thymus), blood samples for hematology /clinical chemistry were gathered from all adult rats of all dose groups. Food and water consumption was measured.
Principles of method if other than guideline:
The study design followed OECD Guideline 421 “Reproduction/ Developmental Toxicity Screening Test” adopted on 28th July 2015. Additional to this guideline, organs (i.e. kidneys, adrenals, heart, thymus) and blood samples for hematology /clinical chemistry were gathered from all adult male and female rats of all dose groups for a more detailed analysis at termination. Food and water consumption was measured. Food and water consumption was not measured during cohabitation period.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): (Benzothiazol-2ylthio) Acetic Acid
- Physical state: light yellow white powder
- Analytical purity: 99.63 %
- Purity test date: 2015-11-10
- Lot/batch No.: 41273
- Expiration date of the lot/batch: 2017-01-23
- Storage condition of test material: Cool and dry (+2°C to + 8°C)
Specific details on test material used for the study:
TEST MATERIAL
- lot/batch No.of test material: 41273
- Expiration date of the lot/batch: 2017-01-23
- Purity: 99.63%
- Purity test date: 2015-11-10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool and dry (+2°C to +8°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was observed to form homogenous suspension in the vehicle (i.e. 5% (w/v) Na-CMC in Milli-Q water) and founds to be stable and resuspendable in the vehicle at 1 mg/mL and 100 mg/mL up to 8 days when stored at room temperature and refrigerated condition.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Sufficient quantities of test item were weighed in a glass breaker and transferred to a mortar and grounded to a fine powder. Small volume of the vehicle was added in a stepwise manner until a uniform suspension was obtained. Further, a small volume of vehicle was added to mortar to rinse with vehicle to ensure the test item was completely transferred. The final volume was made up with 5% (w/v) Na-CMC in Milli-Q water to obtain a desired concentration.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily prior to start of treatment. After weighing the required quantity and transfer to a mortar the test item was grounded to a fine powder. A small volume of vehicle was added in stepwise manner to mortar with continuous trituration till a uniform suspension was obtained. The mixture was transferred into the measuring cylinder. Further, a small volume of vehicle was added to the mortar and rinsed with vehicle to ensure the test item is completely transferred. The final volume was made up with 0.5% (w/v) Na CMC in Milli-Q water to attain the desired concentration.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the solubility and suspensibility test carried out at the same testing facilitythe test item was soluble in Na CMC (medium viscosity) in Milli-Q water..
- Concentration in vehicle: 0.5% (w/v)

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: The pre-coital time was calculated for each female.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After successful mating each pregnant female was housed individually until lactation day 14.
Analytical verification of doses or concentrations:
yes
Remarks:
The analysis was done as per the method validated under Advinus Study No. G9831.
Details on analytical verification of doses or concentrations:
The test item (Benzothiazol-2-ylthio) acetic acid in the dose formulations was determined using High Performance Liquid Chromatograph (HPLC) with UV Detector.

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during week 4 of the treatment period. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation. In case of vehicle control, duplicate samples were drawn only from middle layer. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient conditions for reanalysis purpose as a backup

The analysis results were within ± 15 % of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %
Duration of treatment / exposure:
Males: The dose formulations were administered orally by gavage to specific group of male rats at approximately the same time each day (varied by ± 3 hours) for 43 days in total. Treatment started two consecutive weeks prior to mating and the treatment was continued during the mating period for approximately 2 weeks post mating, after which the males were sacrificed.
Females: The dose formulations were administered orally by gavage to the specific group of rats at approximately the same time each day (varied by ± 3 hours), two weeks prior to the mating period and was continued through mating, pregnancy and up to LD 13 (lactation day).
The animals in the vehicle control group were handled in an identical manner to the treatment group and administered vehicle only.
The dose volume administered was constant, at 10 mL/kg body weight. The actual volume was calculated for individual animals using the most recent body weight.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
31.25 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 /sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose concentrations are based on the results of Dose Range Finding Study for Reproduction / Developmental Toxicity Screening Test of (Benzothiazol-2-ylthio) acetic acid in Wistar Rats by Oral Route (Advinus Study No.: N2597) conducted at Advinus Therapeutics Limited.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Blood samples were collected for hematology, coagulation and clinical chemistry analysis for males prior to necropsy and for females on lactation day 4.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to treatment on Day 1 and at weekly intervals thereafter during treatment period. On the days of detailed clinical examination, general clinical signs were not performed.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards)

BODY WEIGHT: Yes
- Time schedule for examinations:
i. Individual body weights were recorded at the beginning (on Day 1) of the treatment, weekly thereafter and at termination.
ii. All dams were weighed on GD 0, 7, 14 and 20 and on LD 0, 4 and 13.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
To determine the food intake/rat/day, food consumption per cage was calculated by using the food consumed per week per cage divided by the number of rats per cage and the number of days in the intervening period to determine the food intake g/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams of the treated groups was recorded during GD 0-7, 7-14 and 14-20 and for Day 0-4, 4-7, 7-11 and 11-13 of lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
The cage wise water consumption was calculated twice weekly by using the water consumed at each measuring interval per cage and divided by the number of animals per cage and the number of days in the intervening period to determine the water intake (g/rat/day).
Water consumption was not measured during the cohabitation period.
Water consumption of pregnant dams of the treated groups was recorded during GD 0-4, 4-7, 7-11, 11-14, 14-18 and 18-20 and for Days 0-4, 4-7, 7-11 and 11-13 of lactation period.
Oestrous cyclicity (parental animals):
Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. Oestrous cycle length and normality was evaluated by vaginal smears for two weeks during the treatment period (prior to cohabitation) and during mating. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. The overall pattern of each female was characterized as regularly cycling (having recurring 4–5 day cycles).
At the time of necropsy, a vaginal smear was examined to determine the stage of the oestrous cycle.
Sperm parameters (parental animals):
Parameters examined in all P0 male parental generations:
Testis weight, epididymis weight, sperm count in testes, sperm motility, sperm morphology.
All stages of spermatogenesis are normal.
Litter observations:
GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.
The litters were observed daily in order to note the number of alive, dead and cannibalized pups.

GROSS EXAMINATION OF DEAD PUPS:
Yes, All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination for external and internal abnormalities; Possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After six weeks of treatment all surviving males of P0 generation were sacrificed. After six weeks of treatment period, all male rats were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis.
- Maternal animals: All surviving female rats (dams) were sacrificed at lactation day 14. Female rats (dams) were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis.

GROSS NECROPSY
- Gross necropsy was performed at lactation day 14 after overnight fasting and consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- All the surviving F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic).

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the cervical, thoracic, and abdominal viscera. At lactation day 13 thyroid gland from one male and one female pup from each litter was collected and preserved in 10% NBF for histopathological examination. The thyroid weight was determined after fixation.
Statistics:
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0.

All quantitative variables like body weight, food consumption, water consumption, oestrous cycle length, hormone levels, ano-genital distance, terminal fasting body weights (excluding the dams with body weights greater than the day before without fasting and dams with all the pups either found dead or cannibalized before lactation day 14), clinical pathology (haematology clinical chemistry and coagulation) parameters, thyroid hormone levels, organ weights and organ weight ratios (only in males) were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroscedastic) data were transformed, before using ANOVA. Means between treatment groups and vehicle control group were compared using Dunnett’s test.

Pre-implantation loss (%), post-implantation loss (%), no. of corpora lutea, implantations, pre-coital interval and gestation length (days) were analysed after suitable transformation (√ x + ½) of the data. One-way ANOVA was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done for the significant group differences.

Z test was performed for testing the differences in proportions for mating and fertility indices.

Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated throughout the report as stated below:

+/-: Significantly higher (+)/lower (-) than the vehicle control group.
Reproductive indices:
Reproductive Performance Data of Parents
a. Male mating index (%)

Number of males with evidence of mating
= --------------------------------------------- x 100
Number of males cohabited

b. Male fertility index (%)

Number of males siring a litter
= --------------------------------- x 100
Number of males cohabited

c. Female mating index (%)

Number of females mated
= -------------------------------- x 100
Number of females cohabited

d. Fecundity index (%)

Number of pregnant females (confirmed at necropsy)
= ----------------------------------------------------------- x 100
Number of females with confirmed mating

e. Female fertility index (%)

Number of pregnant females (confirmed at necropsy)
=------------------------------------------------------------ x 100
Number of females used for mating

f. Mean number of corpora lutea (CL)/group

Total number of CL
=-------------------------------------
Total number of pregnant animals

g. Mean number of implantations/group

Total number of implantations
= ---------------------------------------
Total number of pregnant animals

h. Implantation index

No. of implantation sites
= ------------------------- x 100
No. of corpora lutea

i. Pre-implantation loss (%)

Number of CL - Number of implantation sites
= -------------------------------------------------------- x 100
Number of CL

j. Post implantation loss (%)
Number of implantation sites - Number of live pups
= --------------------------------------------------------- x100
Number of implantations

k. Gestation index

Number of females with live born pups
= ------------------------------------------------------- x 100
Number of females with evidence of pregnancy
(confirmed at necropsy)
Offspring viability indices:
Litter Data
a. Mean litter size per group

Total Number of pups
= --------------------------------------------------
Total Number of pregnant /littered animals

b. Live birth index (%)

Number of viable pups born (at first observation)
= ------------------------------------------------------ x 100
Total Number of pups born (at first observation)

c. Day 4 survival index (%)

Number of viable pups on LD 4
= ----------------------------------- x 100
Number of viable pups born

d. Ano-genital Distance to cube root of the boy weight Ratio (AGD ratio):

Ano-genital Distance
= --------------------------------------------
Cube root of the body weight (BW1/3)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg Bwt/day, the mean body weights in males were significantly lower on Days 36 and 43 with reduction of up to 8.4%, when compared to vehicle control group.
Mean body weight of dams was not affected by treatment during gestation and lactation period at all dose groups tested.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- In males the average food consumption was signifcant lower at 125 mg/kg bw/day dose in week one of premating period and stays lower compared to control group throughout the treatment period. Food consumption was unaffected at 31.25 and 62.5 mg/kg bw/day dose gorups.
- In dams food consumption were unaffected by the treatment during gestation and lactation period at all the doses tested.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- Water consumption was significantly higher in males at 125 mg/kg Bwt/day on weeks 2, 5 and 6 with increase up to 61.3%, when compared to vehicle control group. The change in the water intake was considered test item-related.
- In dams significantly higher total and per day water intake were observed at 125 mg/kg Bwt/day during Gestation Days 11-14 and 0-20, when compared to vehicle control. These changes were considered test item-related.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological parameters were unaffected by the treatment of females at all dose groups tested.
A decrease in absolute (30%) and relative (32%) reticulocytes count in males at 125mg/kg bw/day was considered test item related. In males of the 31.25, 62.5 mg/kg bw/day dose groups, a decrease in absolute reticulocytes (about 22 and 21 %) was observed, which was considered toxicological insignificant, as there was no dose correlation as the magnitude of decrease was higher at low dose group when compared with mid dose group.
There were no test item-related changes in coagulation parameters in all the doses tested in adult animals.
There were no significant changes in thyroid stimulation hormone (TSH) and thyroxin hormone levels (T4) in adult animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males of the 125 mg/kg bw/day dose group a decreased globulin concentration (8%) was considered test item related. The decreased food intake could have contributed to reduction in globuline concentration.
A few variations in other clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
All other clinical chemistry parameter were not affected by treatment in males at 31.25, 62.5 and 125 mg/kg Bwt/day dose groups.
Clinical chemistry parameter were not affected by treatment in females at 31.25, 62.5 and 125 mg/kg Bwt/day dose groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related histopathological changes were observed in the reproductive organs in both male and females. The staging of spermatogenesis did not reveal any stage specific changes. The spermatogenic cycle observed in the different seminiferous tubules of testes were complete and none of the stages of spermatogenesis were arrested in all the males examined.
Test item related histopathological changes were observed only in non-reproductive organs of males of the high dose group at 125 mg/kg bw/day. The incidence of these changes are described as follows: In adrenals cortical hypertrophy of minimal severity was observed in zona fasciculata of 3 males. In thymus lymphoid depletion of minimal severity was observed in 4 males of the 125 mg/kg bw /day dose group. These changes were considered as secundary change resulting from decreased body weight and reduced food consumption.
All other single or low incidences of microscopic findings observed in different groups were considered incidental and not related to test item as they were randomly distributed in different groups of rats.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
test mat. (dissolved fraction)
Remarks:
dissolved in CMC 0.5% (w/v),
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat. (dissolved fraction)
Remarks:
dissolved in CMC 0.5% (w/v),
Sex:
female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
reproductive function (oestrous cycle)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no

Results: P1 (second parental generation)

General toxicity (P1)

Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mean litter size was reduced for the 125 mg/kg Bwt/day dose group, associated with lower mean number of implantations. The mean viable litter size was lower at 62.5 and 125 mg/kg Bwt/day doses, statistically significant at 62.5 mg/kg Bwt/day associated with higher post-implantation losses at these doses. Day 4 survival index was significantly lower at all the doses tested, due to the total death/cannibalism of pups observed in dams from Day 1 and 2-4. The number of pups dead/ cannibalised from Day 5-13 was higher at 125 mg/kg Bwt/day dose group, when compared to control group.
Other reproductive parameters were not affected.
Body weight and weight changes:
no effects observed
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:
no effects observed
Description (incidence and severity):
There were no significant changes in Thyroid Stimulating Hormone (TSH) and Thyroxin (T4). Decreased thyroxin hormone (T4) in pups on lactation day 13 in all treated groups was considered toxicologically insignificant as it was not associated with any changes in thyroid stimulating hormone and also because there were no related microscopic changes.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No changes attributable to test item were detected in the ano-genital distance (AGD) and ratio of AGD to the cube root of body weight of either sex.
However, in female pups, reduction in the ano-genital distance was observed at 31.25 mg/kg Bwt/day when compared to the vehicle control with the reduction of 8.6%. In male pups, the reduction in the ano-genital distance ratio was observed at 31.25 and 62.5 mg/kg Bwt/day with the reduction of 4.8%. In female pups, the reduction in the ano-genital distance ratio was observed at 31.25 mg/kg Bwt/day with the reduction of 8.8%.
These changes were considered to be incidental because of the lack of any dose relationship.
The male pups did not exhibit areola/nipple retention on PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant changes in thyroid weight of pups recorded on lactation day 13.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
No test item related microscopic changes in thyroid gland of pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

- The mean number of male, female and total pups per litter at all tested doses were unaffected by treatment.
- The mean weight of male, female and total pups per litter at all doses tested were unaffected by treatment.
- No changes attributable to test item were detected in the Ano-genital Distance (AGD) and ratio of AGD to the cube root of body weight of either sex. However, In male pups, the reduction in the ano-genital distance ratio was observed at 31.25 and 62.5 mg/kg Bwt/day with the reduction of 4.8%. In female pups, the reduction in the ano-genital distance ratio was observed at 31.25 mg/kg Bwt/day with the reduction of 8.8%.
- The male pups did not exhibit areola/nipple retention on PND 13.
- Test item had no treatment-related effects on the number of pregnancies, number littered. There were no external abnormalities in live or dead pups in any of the groups.
- The mean litter size was reduced for the 125 mg/kg Bwt/day dose group, associated with lower mean number of implantations. The mean viable litter size was lower at 62.5 and 125 mg/kg Bwt/day doses, statistically significant at 62.5 mg/kg Bwt/day, associated with higher post-implantation losses at these doses.
Day 4 survival index was significantly lower at all the doses tested, due to the total death/cannibalism of pups observed in dams from Day 1 and 2-4.
The number of pups found dead/ cannibalised from Day 5-13 was higher at 125 mg/kg Bwt/day dose group, when compared to control group.
Other reproductive parameters were not affected.

Effect levels (F1)

Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
other: Due to significantly reduced postnatal survival rates of pups at 62.5 and 125 mg/kg Bwt/day dose group,

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
62.5 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Daily oral (gavage) administration of (Benzothiazol-2-ylthio) acetic acid to male and female Wistar rats at dose levels of 31.25, 62.5 and 125 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) resulted in:
• Changes observed in males were restricted to the highest dose level tested (i.e. 125 mg/kg Bwt/day) and included: decreased body weight, food consumption, reticulocytes count, globulin concentration; increased kidney weight, decreased thymus weights and increased adrenal weight and microscopic changes in lymphoid depletion observed in thymus and cortical hypertrophy of zona fasciculata in adrenals. Based on these observations, the no observed adverse effect level (NOAEL) for toxicity in males is considered to be 62.5 mg/kg Bwt/day.
• No treatment-related adverse changes were observed in females up to the highest dose tested. The no observed adverse effect level (NOAEL) for maternal toxicity is considered 125 mg/kg Bwt/day.
• Adverse effects on fertility and reproductive performance were observed at the 125 mg/kg Bwt/day group. Due to reduced implantation index and mean litter size at 125 mg/kg Bwt/day dose group, the no observed adverse effect level (NOAEL) for reproductive toxicity of (Benzothiazol-2-ylthio) acetic acid is considered to be 62.5 mg/kg Bwt/day.
• Due to significantly reduced postnatal survival rates of pups at 62.5 and 125 mg/kg Bwt/day dose group, the no observed adverse effect level (NOAEL) for developmental toxicity is considered 31.25 mg/kg Bwt/day.
Executive summary:

The purpose of this study in Wistar rats was to generate information concerning the effects of (Benzothiazol-2-ylthio) acetic acid on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item (Benzothiazol-2-ylthio) acetic acid was suspended in vehicle [0.5% (w/v) Carboxymethyl cellulose (CMC) sodium salt in Milli-Q®water] and administered by oral gavage at the dose levels of 31.25 (G2), 62.5 (G3) and 125 (G4) mg/kg Bwt/day to male and female Wistar rats, at the dose volume of 10 mL/kg Bwt/day. Similarly, vehicle was administered to rats in the vehicle control group. Each group consisted of 10 male and 10 female rats. The prepared dose formulations were administered once daily to specific group of rats prior to mating, during mating and post mating periods (for males), during pregnancy and up to lactation Day 13 (for females).

The identity of (Benzothiazol-2-ylthio) acetic acid was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No.G9831. Based on the results, the test item was found to be stable for up to 8 days at 1 and 100 mg/mL concentrations when stored at room temperature and refrigeration condition. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during Week 4 of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the claimed concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food and water consumption were recorded weekly except during the cohabitation period.Female rats were also weighed onGestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7, 11 and 13. Water consumption was recorded on GD 4, 7, 11, 14, 18 and 20 and on LD 4, 7, 11 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 4. All the survived male pups were examined for the appearance nipples/areolae on PND 13. After six weeks of treatment period, all male rats were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis. The animals were subjected to detailed necropsy at sacrifice after overnight fasting and study plan specified tissues were collected. Female rats (dams) were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis. A gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination.

Tissues collected from all animals were examined microscopically for histopathological changes. Histopathological examination of the testes was also included a qualitative assessment of stages of spermatogenesis. All gross lesions were examined in all the groups.

Under the experimental conditions employed, the following results were obtained:

Clinical Signs and Mortality:There were no treatment related clinical signs or mortality observed at any of the doses tested.

Body Weights, Food and Water Consumption:At 125 mg/kg Bwt/day, the mean body weights were significantly lower on Days 36 and 43 in males with reduction of up to 8.4%, when compared to vehicle control group. The food consumption was unaffected in males at all the doses tested; however, the water consumption was significantly higher at 125 mg/kg Bwt/day dose.

In females, the body weights and food consumption were unaffected by the treatment during pre-mating, gestation and lactation period at all the doses tested. During gestation period, total and per day water intake was significantly increased at all the doses tested. The increased water intake observed in both the sexes was considered test item-related non-adverse, as all animals appeared normal during routine clinical observations.

Fertility Parameters:Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

Litter Parameters:The mean litter size was reduced for the 125 mg/kg Bwt/day dose group, associated with lower mean number of implantations. The mean viable litter size was lower at 62.5 and 125 mg/kg Bwt/day doses, statistically significant at 62.5 mg/kg Bwt/day,associated with higher post-implantation losses at these doses.Day 4 survival index was significantly lower at all the doses tested, due to the total death/cannibalism of pups observed in dams from Day 1 and 2-4. The number of pups dead/cannibalised from Day 5-13 was higher at 125 mg/kg Bwt/day dose group, when compared to control group.

At 125 mg/kg Bwt/day dose, the higher pre-implantation loss was observed, which in-turn resulted in reduced implantation index. 

No treatment-related changes in the ano-genital distance and ano-genital distance ratio were observed at any of the doses tested when compared to the control group. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested. There were no significant changes in thyroid stimulating hormone (TSH) and thyroxin hormone levels in adult animals and pups.

Clinical Pathology:At 125 mg/kg Bwt/day dose, the test item-related changes includes, decreased reticulocytes count and globulin concentration in males. There were no test item-related changes in coagulation in both sexes at all dose groups tested. There were no other changes in haematology or clinical chemistry observed in adult males and females than outlined above.

Fasting Body Weights, Organ Weights and its Ratios: At 125 mg/kg Bwt/day dose, the treatment-related changes observed in males include: decrease in terminal fasting body weight, increased kidney and adrenal weights and decreased thymus weights. There were no test item-related changes in thyroid weights of pups.

Gross Pathology:There were no test-related gross pathological changes in both parents and pups 

Histopathology:Microscopically, lymphoid depletion observed in thymus and cortical hypertrophy of zona fasciculata in adrenals of males was considered as secondary change resulting from decreased body weights. There were no test item-related changes in male and female reproductive organs.

There were no test item-related microscopic changes in thyroid gland of pups.

Conclusions

Daily oral (gavage) administration of (Benzothiazol-2-ylthio) acetic acid to male and female Wistar rats at dose levels of 31.25, 62.5 and 125 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) resulted in:

·       Changes observed in males were restricted to the highest dose level tested (i.e. 125 mg/kg Bwt/day) and included: decreased body weight, food consumption, reticulocytes count, globulin concentration; increased kidney weight, decreased thymus weights and increased adrenal weight and microscopic changes in lymphoid depletion observed in thymus and cortical hypertrophy of zona fasciculata in adrenals. Based on these observations, the no observed adverse effect level (NOAEL) for toxicity in males is considered to be 62.5 mg/kg Bwt/day.

·      No treatment-related adverse changes were observed in females up to the highest dose tested. Theno observed adverse effect level (NOAEL) for maternal toxicityis considered 125 mg/kg Bwt/day.

·       Adverse effects on fertility and reproductive performance were observed at the 125 mg/kg Bwt/day group. Due to reduced implantation index and mean litter size at 125 mg/kg Bwt/day dose group, theno observed adverse effect level (NOAEL) for reproductive toxicityof (Benzothiazol-2-ylthio) acetic acid is considered to be 62.5 mg/kg Bwt/day.

·       Due to significantly reduced postnatal survival rates of pups at 62.5 and 125 mg/kg Bwt/day dose group, the no observed adverse effect level (NOAEL) for developmental toxicity is considered 31.25 mg/kg Bwt/day.