9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and hexyl and octyl derivs.

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity Summary:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test chemical on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition, and early neonatal development. There was no test chemical related adverse effects that were observed during the study on parental as well as developmental parameters of the pups. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Weight of evidence based on the data of the read-across chemicals.

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:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: Guidelines for 28-Day Repeat Dose Toxicity Test of Chemicals (Japan's own inspection work)

Principles of method if other than guideline:

The above studies were used to evaluate the effect of the test chemical on the reproductive parameters and also on the reproductive organs of the animals.

GLP compliance:

yes

Species:

rat

Strain:

other: Study 2: Sprague-Dawley; Study 3 and 4: Crj: CD(SD)

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: In-house bred animals
- Age at study initiation: (P) x 9 weeks
- Weight at study initiation: (P) Males: 236.64 g to 296.64 g; Females: 204.64 g to 260.61 g
- Fasting period before study: No Data Available
- 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.
i. Pre mating: Two animals of same sex and group per cage were housed.
ii. Mating: During mating, two animals (one male and one female) of same group were housed.
iii. Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Use of restrainers for preventing ingestion (if dermal): 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 to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Healthy and young adult animals were acclimatized for nineteen days (including fourteen days of oestrus cycle evaluation) to experimental room conditions and females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment and were observed for clinical signs once daily.
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.

IN-LIFE DATES: From: 30 May 2018
To: 20 July 2018

Study 4: TEST ANIMALS
- Source: Charles River Co., Ltd. (Atsugi city, Kanagawa Prefecture)
- Age at study initiation: 6 weeks
- Weight at study initiation: 131 to 146 g for males and 112 to 129 g for females
- Fasting period before study: No data
- Housing: The animals were raised in the rearing room (W 9.8 × D 8.2 × H 2.5 m, 200.9 m ^ 3) of the barrier system. Using a washing type rearing machine (W 674.2 × D 80.0 × H 175.5 cm) of Tokyo Giken service (Fuchu-shi, Tokyo), a metallic front and bed network breeding cage (W 20.0 × D 28.2 × H 18.0 cm, one animal was housed in a breeding cage space of 10, 152 cm ^ 3). The breeding cages were replaced every other biweekly, and the feeders were changed once a week.
- Diet (e.g. ad libitum): Radiation sterilized improved NIH release rat / mouse feed produced by Oriental Yeast Co., Ltd. (Chuo-ku, Tokyo) was ingested ad libitum
- Water (e.g. ad libitum): No data
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1 ° C
- Humidity (%): 55 ± 5%
- Air changes (per hr): 150 ~ 300 lux, 12 hours (7 am lights a day, 7 pm off)
- Photoperiod (hrs dark / hrs light): 20 times / Hour

IN-LIFE DATES: From: To: No data

Route of administration:

oral: gavage

Vehicle:

other: Study 2: corn oil; Study 3: 0.5%w/v Methylcellulose aqueous solution（suspended); Study 4: CMC (carboxymethyl cellulose)

Details on exposure:

Study 2: Details on exposure
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This rinsing procedure was repeated until complete transfer of test item formulation into 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 item for low, mid and high dose groups respectively.
Chromatographic Conditions:
Column: BDS Hypersil C18 100 × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min.
Injection volume: 10 µL
Wavelength: 254 nm
Run time: 16 minutes
Column temperature: 40°C

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): The test item was not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL. Hence corn oil was used as vehicle for test item formulation preparation. Also, corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: Concentration of 0, 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): 100 ml
- Lot/batch no. (if required): A1712001
- Purity: No Data Available

Study 3: Details on exposure
PREPARATION OF DOSING SOLUTIONS: No Data Available

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): 0.5 % Methylcellulose solution was used because the test chemical was insoluble in water.
- Concentration in vehicle: 5 mL/kg
- Amount of vehicle (if gavage): No Data Available
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available

Study 4: PREPARATION OF DOSING SOLUTIONS: A predetermined amount of the test substance was precisely weighed every specified dose (100, 300 and 1,000 mg / kg), suspended in a 0.5% sodium carboxymethyl cellulose aqueous solution using an agate mortar, and 0.1% (w / v) Tween 80 was added and sufficiently mixed to prepare the dose

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% carboxymethylcellulose sodium / water solution containing 0.1% Tween 80
- Concentration in vehicle: 0,100, 300, 1000 mg/kg/day
- Amount of vehicle (if gavage): 0.5 % CMC Na solution with 0.1% Tween 80

Details on mating procedure:

Study 2: 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: No Data Available
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. The female was placed with the same male until pregnancy occurs by evidence of sperm in vaginal smear until two weeks have elapsed. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear.
- Further matings after two unsuccessful attempts: [no / yes (explain)]: No
- After successful mating each pregnant female was caged (how): No Data Available
- Any other deviations from standard protocol: No Data Available

Study 4: Mating was not performed for this study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study 2: The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This rinsing procedure was repeated until complete transfer of test item formulation into 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 item for low, mid and high dose groups respectively.
Chromatographic Conditions:
Column: BDS Hypersil C18 100 × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min.
Injection volume: 10 µL
Wavelength: 254 nm
Run time: 16 minutes
Column temperature: 40°C

Study 4: Although it was confirmed that the administration solution was stable for 2 weeks after storage in the refrigerator, preparation was carried out once a week in this test. Concentration analysis of the administration solution was performed on the preparations of administration 1 and 4 weeks for all groups, and it was in the range of 99.0 to 104% of the prescribed concentration and was appropriately prepared.

Duration of treatment / exposure:

Study 2: 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.
Study 3: Males: 42 Days
Females: 41-45 days(from 14 days before mating to day 4 of lactation）
Study 4: 28 days

Frequency of treatment:

Study 2: The test item or vehicle was administered to animals through oral (gavage) route once daily.
Study 3: Daily
Study 4: Daily

Details on study schedule:

No Data Available

Remarks:

Study 2: 0, 250, 500 and 1000 mg/kg bw
Study 3: 0, 40, 200 and 1000 mg /kg bw
Study 4: 0,100, 300, 1000 mg/kg bw

No. of animals per sex per dose:

Study 2: 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.
Study 3:
Males: 12 animals/group/sex (5 animals for recovery group)
Females: 12 animals/group/sex (5 animals for recovery group)
Study 4: Total no of animals: 60
Test group
0 mg/kg/day-5 male and 5 female
100 mg/kg/day-5 male and 5 female
300 mg/kg/day-5 male and 5 female
1000 mg/kg/day-5 male and 5 female

Recovery group:
0 mg/kg/day-5 male and 5 female
1000 mg/kg/day-5 male and 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

Study 4: - Dose selection rationale:
Preliminary studies with 2 weeks dosing for dose setting were performed at 4 doses of 0, 200, 600 and 1,800 mg / kg / day.
As a result, dead animals were not found in any of the administration groups, and no changes that could be attributed to administration of the test substance were observed in body weight, feed intake, pathological autopsy findings and organ weight. Therefore, the maximum dose in the 28-day repeated dose test was set to the limit dose of 1,000 mg / kg, divided by the common ratio 3 below, and the medium dose was set to 300 mg / kg and the low dose to 100 mg / kg. A control group to which only carrier (0.5% carboxymethylcellulose sodium aqueous solution containing 0.1% Tween 80) was administered was provided.
- Rationale for animal assignment (if not random):
The animals were grouped by the random extraction method
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
Yes, recovery group was included in the 0 and 1000 mg/Kg/day group
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): No data
- Other: No data

Positive control:

No Data Available

Parental animals: Observations and examinations:

Study 2: Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

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
- Time Schedule: 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.

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

Study 3: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No Data Available

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No Data Available

BODY WEIGHT: Yes
- Time schedule for examinations: No Data Available

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

OTHER: No Data Available

Study 4: CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes
:

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: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: No data
OTHER: Organ weight; Yes
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals were observed every morning, afternoon twice (once on holidays) every day
- Cage side observations checked in table [No.?] were included.
the presence or absence of intoxication symptoms, behavior abnormalities, the presence of dead animals and the presence or absence of dead animals, etc. were recorded.

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule:
No data

BODY WEIGHT: Yes
- Time schedule for examinations:
once a week from the start of administration until the end of recovery test.

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, Feed intake (g / week) was calculated by measuring the remaining amount fed once a week.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

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

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
No data
- Dose groups that were examined:
No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
At the end of administration and at the end of the recovery period
- Anaesthetic used for blood collection: Yes, ether anaesthesia was given to animals
- Animals fasted: Yes, for 16 hrs
- How many animals:
All animals
- Parameters checked in table [No.?] were examined.
The hematocrit value (HCT: corrected from the volume of total erythrocyte), the average red blood cell volume (WBC: dark field plate method), red blood cell count (RBC: dark field plate method), hemoglobin amount (HGB: cyanomethemoglobin method) (Calculated from MCV: RBC, HCT), mean red blood cell hemoglobin amount (calculated from MCH: HGB, RBC), mean red blood cell hemoglobin concentration (calculated from MCHC: HGB, HCT), platelet count (PLT: dark field plate method) (Flow cytochemistry method) was measured using a blood automatic analyzer THMS. For calculating the reticulocyte (RC) ratio, whole blood was stained with Capilot, and a blood smear sample was prepared and stored. In addition, the prothrombin time (guick 1-step method) and the activated partial thromboplastin time (clotting method) were measured for blood plasma of blood citrate-added blood using an automatic blood coagulation measuring apparatus KC-40.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
At the end of administration and at the end of the recovery period
- Animals fasted: Yes, for 16 hrs
- How many animals:
All animals
- Parameters checked in table [No.?] were examined.
By using serum, total protein (Buret method), albumin (BCG method), A / G ratio (calculated value), blood sugar (hexokinase method), neutral fat (enzymatic method), total cholesterol (enzymatic method), urea EKTACHEM 700N (Kodak Company, USA) is used for nitrogen (BUN: urease modification method), calcium (alizarin method), inorganic phosphorus (molybdenum blue method), sodium (electrode method), potassium (electrode method) and chlorine Glutamic acid oxaloacetate transaminase (GOT: Karmen modification method), glutamic acid pyruvic acid transaminase (GPT: Wroblewski and LaDue improved method), γ-glutamyl transpeptidase (γ-GTP: enzymatic method) and alkaline phosphatase (ALP : Bessey-Lowry-Brock Improvement Method) was measured with Centrifi Chem ENCORE II

URINALYSIS: Yes
- Time schedule for collection of urine:
Prior to hemotological examination, urine was collected using a urine collector for 24 hours (from 10 am until the next morning at 10 a.m.)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table [No.?] were examined.
Urine volume, color tone and turbidity were inspected, and then urine density meter UR - S , Itabashi-ku, Tokyo) was used to measure the urine specific gravity, the urine was centrifuged, the sediment was stained by the Sternheimer method, and examined by a microscope. The pH, occult blood, ketone bodies, sugar, protein, bilirubin and urobilinogen were measured using N-Martistic SG test paper and CLINITEK 200

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
No data
- Dose groups that were examined:
No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
No data

IMMUNOLOGY: No data
- Time schedule for examinations:
No data
- How many animals:
No data
- Dose groups that were examined:
No data No data
- Parameters checked in table [No.?] were examined.
No data

OTHER: No data

Oestrous cyclicity (parental animals):

Study 2: 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.

Study 3: Yes, Oestrus cycle was examined.

Sperm parameters (parental animals):

No Data Available

Litter observations:

Study 2: 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.

Study 3: Yes, The effect of the test chemical was observed on the litter parameters.

Study 4:

Postmortem examinations (parental animals):

Study 2: Postmortem examinations (Parent Animal):
SACRIFICE:
- Male animals: The males were sacrificed after completion of 30 days of treatment, females were sacrificed on lactation day 14 and pups were sacrificed on lactation day 13. 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. During necropsy, the males were randomized.
- Maternal animals: 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 Asphyxiation and subjected to gross necropsy, external and internal gross pathological examination.

GROSS NECROPSY
- Gross necropsy consisted of Epididymides, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands, Glans penis, ovaries, Prostate and Seminal vesicles and coagulating glands, Testes, Uterus with cervix, Thyroid along with parathyroid glands and other gross lesions in other organs of respiratory, digestive and nervous system were observed.

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 was extended to lower dose groups for gross findings like, tail and non-glandular stomach to cover all gross lesions encountered. The tissues were embedded in paraffin wax, sectioned at 4 to 6 micrometers and stained with haematoxylin and eosin. The testes were sectioned at 3 to 4 micrometers and stained with Hematoxylin and Eosin stain and also with Periodic Acid-Schiff (PAS) and Hematoxylin stain. PAS stain aided spermatogenesis evaluation.

Study 3: SACRIFICE
- Male animals: All surviving animals.
- Maternal animals: All surviving animals

GROSS NECROPSY
- Yes, gross necropsy was performed on all the organs in abdomen, viscera and reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes, Organs weights and Histopathology was performed.

Study 4: GROSS PATHOLOGY: Yes, Pathologic dissection was conducted by ether anesthetizing the animals at the end of the administration and at the end of the recovery period and killing them to exsanguination. Macroscopic abnormalities were recorded on pathological autopsy findings recording sheets. Weight was measured for brain, liver, kidney, spleen, adrenal gland, testis and ovary, and the organ weight / body weight ratio was calculated.

Organs / tissues that showed a change in the above weighted organ and pituitary, eyeball, thyroid (including parathyroid gland), heart, lung, stomach, bladder, bone marrow (femur) and macroscopic findings were 10% And fixed with formalin solution

HISTOPATHOLOGY: Yes, Histopathological examination was performed on the heart, liver, spleen, kidney and adrenal glands of the control group and high dose group among the fixed organs / tissues. Thin cut specimens were prepared according to a conventional method, and stained with hematoxylin and eosin, followed by a microscopic examination.

Postmortem examinations (offspring):

Study 2: The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups were examined for gross abnormalities and the findings were recorded. The thyroid along with parathyroid was collected from one male and one female pup per litter on lactation day 13. The thyroid along with parathyroid from adults and pups were preserved in 10% v/v Neutral Buffered Formalin. The thyroid along with parathyroid from adults was weighed post fixation. The histopathological examination of thyroid from pups and adults was not conducted as there were no treatment related effects noted in T4 levels of adult males and lactation day 13 pups.

Statistics:

Study 2: The data was subjected to various statistical analyses using SPSS software version 22.
For Body weight, Percent change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ weights, Anogenital distance ratio, Mean pup weight, Live birth Index, Pup survival index, One-way ANOVA with Dunnett’s post test was used. For, Pre/post implantation loss, Pre/post natal loss, No. of resorptions per dam, Corpora lutea per dam, Implantations per dam, No. of live/dead pups/dam, Sex ratio and Litter size, Kruskal-Wallis Test was used. For, Pregnancy rate, No. of litters with/without resorptions, No. of dams with/without live young born, No. of dams with/without dead pups , Chi-square test was used.

Study 4: The body weight, feed intake, hematology test value, biochemical test value, urine test value (only urine volume and urine specific gravity), organ weight and organ weight / body weight ratio of each test group are determined according to the automatic discrimination method shown below , Bartlett's equidistance test was first carried out. In the case of equal variance, a one-way ANOVA is performed, and if the variance is significant, Dunnett's multiple comparison test if the number of samples in each group is the same, and Duncan's multiple range test if the number of samples in each group is different, And significant differences between each dose group were tested. In Bartlett's equality variance test, Kruskal-Wallis rank test was carried out in case of unequal variance, and significant difference between control group and each dosing group was tested by nonparametric Dunnett's multiple comparison test in case of significance. For dose correlation, significant differences were tested using Jonckheere's trend test. The significance level was carried out with a one-sided test of 5 and 1%

Reproductive indices:

Study 2: Male Mating Index, Male Fertility Index, Female Mating Index, Female Fertility Index, Precoital Interval (Days), Mean Gestational Length, Gestation Index (%), Mean number of corpora lutea, Mean number of Implantations, Pre-Implantation Loss (%), Pre-natal Loss/Post-Implantation Loss (%), Pre-natal Loss (No.), Post-natal Loss on lactation day 4/13, Post-natal Loss on lactation day 4/13 (%)

Study 3: Gestational Index, estrous cycle examination, Copulation Index, Copulation interval, fertility index, gestation period, no of corpora lutea, number of implantations, implantation index, delivery index, nursing behaviour.

Offspring viability indices:

Study 2: Mean Litter Size per Group, Live Birth Index (%) per dam, Precoital Interval (Days), Pup Survival index (%) on lactation day 4, Sex ratio, Percentage of male/female offspring (%), Ano-genital Distance to cube root of the body weight Ratio (AGD ratio).
Study 3: Pup Viability Index

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: The detailed clinical examination of animals did not reveal any changes at any of the tested dose group animals of either sex during the experimental period. There were no treatment related clinical signs of toxicity at any of the tested dose group animals of either sex during the experimental period. However, dark green colored fecal matter was observed at all the tested dose group animals. This coloration is due to dark green color of the test item but not any adverse effects of test chemical.

Study 3: Test item coloured fecal matter in both male and female animals was observed in both the dose groups.

Study 4: No significant effect were observed at the dose level of 100, 300, 1000 mg/kg/day of treated group and recovery group as compared to control.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

Study 2: There were no treatment related mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.

Study 3: No Mortality was observed in all animals in either of the dose groups.

Study 4: No mortality was observed at the dose level of 100, 300, 1000 mg/kg/day of treated and recovery group as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Study 2: There were no changes in mean body weight and percent change in body weight with respect to day 1 at all the tested doe groups of either sex during pre-mating and mating periods when compared with vehicle control group was noted.

Study 3: No abnormal effects were observed in body weight changes in males or females due to the test chemical administration.

Study 4: There was no difference between the groups during the administration period and the recovery period in both males and females, and there was no difference in body weight gain during the administration period (0 to 4 weeks) and during the recovery period (4 to 6 weeks).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: There were no changes in feed consumption at all the tested doe groups of either sex during pre-mating and mating periods when compared with vehicle control group was noted. However, statistical significant reduction in average feed consumption during week 2 of pre-mating period at all the tested dose group females and statistical significant reduction in average feed consumption during week 2 of pre-mating period at G2 (250mg/kg) and G3 (500 mg/kg) males was noted when compared with vehicle concurrent control group. These changes may not to be considered as treatment related as the reduction is not consistent during the experimental period.

Study 3: No abnormal effects were observed in feed consumption patterns in males or females due to the test chemical administration.

Study 4:In males, there was no difference between the groups during the administration period and recovery period, and no difference was observed in total intake between 0 and 4 weeks.

In females, food intake decreased in 1 week of administration in 300 and 1,000 mg / kg group compared with control group, but in both groups after 2 weeks of administration there was no difference from control group. Regarding the total food intake for 0 to 4 weeks, there was no difference between the 1,000 mg / kg group and the control group, but the 300 mg / kg group decreased in comparison with the control group.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

Study 2: no effects observed

Study 3: No abnormal effects were observed in hematological parameters in males or females due to the test chemical administration.

Study 4: a) Results at the end of administration
In males, white blood cell count decreased in 100 mg / kg group compared with control group.
In females, the ratio of neutrophils decreased and the proportion of lymphocytes increased in the 100 mg / kg group.

b) Result at end of recovery period
There was no difference in males between the control group and 1,000 mg / kg group.
In females, MCHC decreased in the 1,000 mg / kg group.

In the test results at the end of the administration period and at the end of the recovery test, there was no difference between the groups in the prothrombin time and activated partial thromboplastin time in both males and females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: 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. However, statistical significant increase in serum T4 levels in adult males was noted at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group males. This change is not considered as treatment related as the change is not dose dependant.

Study 3: Significant decrease in Albumin content, Total Protein, and significant increase in α2-Globulin (%) in males of 1000 mg/kg bw was observed. No effects in females were observed. However, these effects were considered to be toxicolgically insignificant since, therse effects were observed to be reversed in the recovery group.

Study 4: a) Results at the end of administration
In males, A / G increased in all test substance-administered groups, and GPT and γ-GTP increased in the 1,000 mg / kg group as compared with the control group. As another change, blood glucose decreased in the 100 mg / kg group.
In females, γ-GTP decreased in all test substance-administered groups, and potassium decreased in the 300 mg / kg group.

b) Results at the end of the recovery period
Chlorine increased in the 1,000 mg / kg group of males and calcium decreased in the female 1,000 mg / kg group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Study 3: Abnormal coloration in the urine was observed in males and females at 200 mg/kg bw and 1000 mg/kg bw dose groups. This coloration was attributed to the test chemical or a metabolite of the test chemical.

Study 4: a) Results at the end of administration
There was no difference between the groups in all the test items for both males and females.

b) Results at the end of the recovery period
There were no difference in test item between 1,000 mg / kg group and control group in both males and females.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Study 2: No effect of the test chemical was observed on the grip strength, locomotor activity and other functional observation battery parameters in either sex at all the dose groups.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: No treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs. Neutrophils infiltration at submucosa of non-glandular stomach was observed in one male from G4 (1000 mg/kg) group. This lesion considered as spontaneous, incidental because of lack of consistency. One male from vehicle control group revealed cytoplasmic vacuolation in Sertoli cells of testes. This lesion considered as spontaneous and incidental. A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings. The histopathological examination was extended to lower dose groups for gross findings like, tail and non-glandular stomach. There were no treatment related changes noted in these tissues at all the dose groups during microscopic evaluation. Reproductive organs (Testes, Epididymides and Ovaries) did not show any pathological findings in G4 (1000 mg/kg) group animals.

Study 3: No histopathological abnormalities were observed in any animals of either sex and at all the dose levels, including the high dose recovery groups.

Study 4: Histopathology was performed on the control group dissected at the end of the treatment and on the sexes of the 1,000 mg / kg group. As a result, no difference was observed between both groups in both males and females. The main findings including the control group were small basolateral and granular nests in liver cells, basophilization and calcification of tubular epithelial cells of the kidneys, vacuolation of bundle bands of the adrenal gland, etc

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Study 2: 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.

Study 3: No effects on the estrous cyclicity due to the test chemical was observed at any dose groups.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Study 2: 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 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. There were no treatment related changes observed in the copulatory interval and number of conceiving days at any of the tested dose group animals when compared with vehicle control group animals.

Study 3: No effects on the reproductive performance due to the test chemical was observed at any dose groups.

Based on all the data of observations and results, the test chemical was not considered as toxic to reproductive parameters.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

other: Not Specified

Critical effects observed:

not specified

System:

other: Not Specified

Clinical signs:

no effects observed

Description (incidence and severity):

Study 2: There were no clinical signs and external anomalies observed in any of the pups of tested dose group animals during lactation period.

Study 3: no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

Study 2: There were no treatment related changes observed in pup survival index of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.

Study 3: no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: 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 all the tested dose groups; statistical significant reduction in mean female pup weight on lactation day 7 and 13 at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group. This observation was not considered as treatment related as the occurrences were not consistent, no changes were noted in pup observations during this period and also the changes are not dose dependent.

Study 3: no effects observed

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:

no effects observed

Description (incidence and severity):

Study 2: no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: 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. However, statistical significant increase in serum T4 levels in adult males was noted at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group males. This change is not considered as treatment related as the change is not dose dependant and no changes were noted in absolute and relative weight of thyroid in these groups.

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Description (incidence and severity):

Study 2: no effects observed

Study 3: no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Study 2: No treatment related changes in ano-genital distance ratio were noted.

Study 3: no effects observed

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Study 2: No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

Study 3: no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

Study 2: 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.

Study 3: no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

Study 2: no effects observed

Study 3: no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Study 2: No effects were observed on number of pups delivered, number of live pups, live birth index, viability index and sex ratio on day 0 and day 4 at any dose groups.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Study 3: Based on the observation and results of the test chemical, no effects were observed on the developmental parameters of the pups at any dose levels.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

haematology

clinical biochemistry

gross pathology

histopathology: non-neoplastic

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:

Based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw and the test chemical is likely to be non hazardous as a reproductive toxicant.

Executive summary:

Reproductive Toxicity Study:

The data from all the reproductive toxicity studies are as follows:

Reproductive Toxicity Study 2:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test chemical on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition, and early neonatal development.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 G4 groups 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 item 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). 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. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating period. No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose groups of either sex. 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 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy were noted.Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weight and feed consumption during gestation and lactation at all the tested dose groups. 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, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Reproductive Toxicity Study 3:

In a combined repeated and reproductive toxicity studies according to OECD 422 guideline, the test chemical was administered to Crl:CD (SD) male and female rats, aged 10 weeks old at the initiation of dosing. The rats were dosed at the dose levels of 0, 40, 200, 1000 mg/kg/day with two revesal groups of 0 and 1000 mg/kg bw/day as control and high dose group recovery group, respectively. The vehicle used in the study was 0.5%w/v Methylcellulose aqueous suspended solution. Male animals were dosed for 42 Days, while females were dosed for 41 -45 days. After the administration ofthe test chemical, test item coloured fecal matter in both male and female animals was observed in both the dose groups. However, no Mortality was observed in all animals in either of the dose groups. No abnormal effects were observed in body weight changes and feed consumption in males or females due to the test chemical administration. In hematological parameters, No abnormal effects were observed in hematological parameters in males or females due to the test chemical administration. In blood chemistry parameters, significant decrease in Albumin content, Total Protein, and significant increase in α2-Globulin (%) in males of 1000 mg/kg bw was observed. No effects in females were observed. However, these effects were considered to be toxicolgically insignificant since, therse effects were observed to be reversed in the recovery group. Also, abnormal coloration in the urine was observed in males and females at 200 mg/kg bw and 1000 mg/kg bw dose groups. This coloration was attributed to the test chemical or a metabolite of the test chemical. No effect of the test chemical was observed on the grip strength, locomotor activity and other functional observation battery parameters in either sex at all the dose groups. No effect of the test chemical was observed on the grip strength, locomotor activity and other functional observation battery parameters in either sex at all the dose groups. No adverse effects were observed on any abdominal, visceral or reproductive organs / 100 grams of body weight due to the test chemical in either of the sex at any dose groups. In gross necropsy, Colored aqueous content in the alimentary tract was observed in males, and in males and females both 40 mg/kg bw dose group and 200 mg/kg bw dose group, respectively. Also, mucosal discoloration of alimentary tract in males of 200 mg/kg bw was also observed. Colored aqueous content in the alimentary tract and  mucosal discoloration of alimentary tract in both males and females were observed in 1000 mg/kg bw dose group. However, these effects were reversed in the recovery group doses and thus were not considered to be of toxicological significance. No histopathological abnormalities were observed in any animals of either sex and at all the dose levels, including the high dose recovery groups. No effects on the estrous cyclicity due to the test chemical was observed at any dose groups. No effects on the reproductive performance due to the test chemical was observed at any dose groups. No effects were observed on number of pups delivered, number of live pups, live birth index, viability index and sex ratio on day 0 and day 4 at any dose groups. Also, no test chemical changes wele noted in the number of pupsdelivered, number of live pups, live birth index,viability index or sex ratio on day 0 and 4, clinical signs, body weights, external examinations, body weights or necropsy. Thus, based on all the observations and results it was concluded that the NOAEL for the test chemical was found to be 1000 mg/kg bw.

Reproductive Toxicity Study 4:

A 28 days repeated dose toxicity study for the test chemical was assessed for its possible toxic nature. For this purpose subacute study was performed according to guideline for 28-Day Repeat Dose Toxicity Test of Chemicals (Japan). The test substance was exposed to Crj:CD (SD) male and female rats by oral gavage at the concentration of 0,100, 300, 1000 mg/kg/day for 28 days . Recovery group animals were also included in the study from 0 and 1000 mg/Kg/day. Animals were observed for clinical signs, changes in body weight, food consumption, hematology, clinical chemistry, urine analysis, organ weight, gross and histopathology. As a result of clinical observation, no abnormal animals were observed in either group of males and females during the administration period and recovery period, and no death cases were observed. Body weights did not differ between groups throughout the administration period and recovery period in both males and females. In the males, no difference was observed in the consumption of food during the administration period and the recovery period, and in females, in the 300 and 1,000 mg / kg group, the feeding amount decreased in one week after administration compared to the control group, but after 2 weeks, no significant changes were noted. The food intake of 0 to 4 weeks were almost unchanged between the 1,000 mg / kg group and the control group, so it was not thought that it was a change to the effect of the test substance. As a result of the blood morphological examination, a decrease in white blood cell count observed in the 100 mg / kg group of males at the end of administration, a decrease in the ratio of neutrophils observed in the female 100 mg / kg group, an increase in lymphocyte ratio were all dose - correlated changes. Also, with regard to the reduction of MCHC observed in the female 1,000 mg / kg group at the end of the recovery period, it was a slight change not observed at the end of administration, and the hematocrit and hemoglobin levels were not different from the control group It was not considered a meaningful change from. As a result of the blood coagulation test, no difference was observed between the control group of male and female and the test substance administered group at any examination time, and it was considered that there was no influence of administration of the test substance. As a result of biochemical examination, the increase in A / G observed in all male test substance-administered groups tended to be rather low in the control group (background value 1.38 ± 0.15, n = 35), 100 mg / kg group was minor and there was no dose correlation, GPT and γ-GPT in the 1,000 mg / kg group were extremely minor changes, both considered to be toxicologically meaningless change It was done. For females, γ-GPT decreased in all test substance-administered groups, potassium decreased in the 300 mg / kg group, but the former was a minor change and the latter was a dose-uncorrelated change. In addition, the examination at the end of the recovery period showed an increase in chlorine in the male 1,000 mg / kg group, a decrease in calcium in the female same dose group, but a slight change. As a result of the urine test, there was no item for which differences between groups were observed in both males and females, and there was no effect of administration of the test substance. As a result of organ weight measurement, in the measurement at the end of administration, there was no difference between the groups for both the actual weight and the relative weight for all weighing organs for both males and females. At the end of the recovery period, the increase in brain weight and adrenal relative weight was observed in the male 1,000 mg / kg group, and the increase in splenic phase versus weight was observed in the female 1,000 mg / kg group, but both were slight Since it was a change and it was not observed at the end of administration, it was not considered to be a toxicologically important change. As a result of pathological examination, lesions suggested by administration of the test substance, neither macroscopically nor histologically, were observed in both males and females. No significant effects were observed in treated animals compared to control. Therefore the no observed adverse effect level (NOAEL) for the test chemical was considered to be 1000mg/kg/day when Crj:CD (SD) male and female rats were exposed by oral gavage route of exposure for 28 days .

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is from Klimisch 1 source.

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 all the reproductive toxicity studies are as follows:

Reproductive Toxicity Study 2:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test chemical on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition, and early neonatal development.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 G4 groups 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 item 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). 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. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating period. No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose groups of either sex. 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 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy were noted.Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weight and feed consumption during gestation and lactation at all the tested dose groups. 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, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Reproductive Toxicity Study 3:

In a combined repeated and reproductive toxicity studies according to OECD 422 guideline, the test chemical was administered to Crl:CD (SD) male and female rats, aged 10 weeks old at the initiation of dosing. The rats were dosed at the dose levels of 0, 40, 200, 1000 mg/kg/day with two revesal groups of 0 and 1000 mg/kg bw/day as control and high dose group recovery group, respectively. The vehicle used in the study was 0.5%w/v Methylcellulose aqueous suspended solution. Male animals were dosed for 42 Days, while females were dosed for 41 -45 days. After the administration ofthe test chemical, test item coloured fecal matter in both male and female animals was observed in both the dose groups. However, no Mortality was observed in all animals in either of the dose groups. No abnormal effects were observed in body weight changes and feed consumption in males or females due to the test chemical administration. In hematological parameters, No abnormal effects were observed in hematological parameters in males or females due to the test chemical administration. In blood chemistry parameters, significant decrease in Albumin content, Total Protein, and significant increase in α2-Globulin (%) in males of 1000 mg/kg bw was observed. No effects in females were observed. However, these effects were considered to be toxicolgically insignificant since, therse effects were observed to be reversed in the recovery group. Also, abnormal coloration in the urine was observed in males and females at 200 mg/kg bw and 1000 mg/kg bw dose groups. This coloration was attributed to the test chemical or a metabolite of the test chemical. No effect of the test chemical was observed on the grip strength, locomotor activity and other functional observation battery parameters in either sex at all the dose groups. No effect of the test chemical was observed on the grip strength, locomotor activity and other functional observation battery parameters in either sex at all the dose groups. No adverse effects were observed on any abdominal, visceral or reproductive organs / 100 grams of body weight due to the test chemical in either of the sex at any dose groups. In gross necropsy, Colored aqueous content in the alimentary tract was observed in males, and in males and females both 40 mg/kg bw dose group and 200 mg/kg bw dose group, respectively. Also, mucosal discoloration of alimentary tract in males of 200 mg/kg bw was also observed. Colored aqueous content in the alimentary tract and  mucosal discoloration of alimentary tract in both males and females were observed in 1000 mg/kg bw dose group. However, these effects were reversed in the recovery group doses and thus were not considered to be of toxicological significance. No histopathological abnormalities were observed in any animals of either sex and at all the dose levels, including the high dose recovery groups. No effects on the estrous cyclicity due to the test chemical was observed at any dose groups. No effects on the reproductive performance due to the test chemical was observed at any dose groups. No effects were observed on number of pups delivered, number of live pups, live birth index, viability index and sex ratio on day 0 and day 4 at any dose groups. Also, no test chemical changes wele noted in the number of pupsdelivered, number of live pups, live birth index,viability index or sex ratio on day 0 and 4, clinical signs, body weights, external examinations, body weights or necropsy. Thus, based on all the observations and results it was concluded that the NOAEL for the test chemical was found to be 1000 mg/kg bw.

Reproductive Toxicity Study 4:

A 28 days repeated dose toxicity study for the test chemical was assessed for its possible toxic nature. For this purpose subacute study was performed according to guideline for 28-Day Repeat Dose Toxicity Test of Chemicals (Japan). The test substance was exposed to Crj:CD (SD) male and female rats by oral gavage at the concentration of 0,100, 300, 1000 mg/kg/day for 28 days . Recovery group animals were also included in the study from 0 and 1000 mg/Kg/day. Animals were observed for clinical signs, changes in body weight, food consumption, hematology, clinical chemistry, urine analysis, organ weight, gross and histopathology. As a result of clinical observation, no abnormal animals were observed in either group of males and females during the administration period and recovery period, and no death cases were observed. Body weights did not differ between groups throughout the administration period and recovery period in both males and females. In the males, no difference was observed in the consumption of food during the administration period and the recovery period, and in females, in the 300 and 1,000 mg / kg group, the feeding amount decreased in one week after administration compared to the control group, but after 2 weeks, no significant changes were noted. The food intake of 0 to 4 weeks were almost unchanged between the 1,000 mg / kg group and the control group, so it was not thought that it was a change to the effect of the test substance. As a result of the blood morphological examination, a decrease in white blood cell count observed in the 100 mg / kg group of males at the end of administration, a decrease in the ratio of neutrophils observed in the female 100 mg / kg group, an increase in lymphocyte ratio were all dose - correlated changes. Also, with regard to the reduction of MCHC observed in the female 1,000 mg / kg group at the end of the recovery period, it was a slight change not observed at the end of administration, and the hematocrit and hemoglobin levels were not different from the control group It was not considered a meaningful change from. As a result of the blood coagulation test, no difference was observed between the control group of male and female and the test substance administered group at any examination time, and it was considered that there was no influence of administration of the test substance. As a result of biochemical examination, the increase in A / G observed in all male test substance-administered groups tended to be rather low in the control group (background value 1.38 ± 0.15, n = 35), 100 mg / kg group was minor and there was no dose correlation, GPT and γ-GPT in the 1,000 mg / kg group were extremely minor changes, both considered to be toxicologically meaningless change It was done. For females, γ-GPT decreased in all test substance-administered groups, potassium decreased in the 300 mg / kg group, but the former was a minor change and the latter was a dose-uncorrelated change. In addition, the examination at the end of the recovery period showed an increase in chlorine in the male 1,000 mg / kg group, a decrease in calcium in the female same dose group, but a slight change. As a result of the urine test, there was no item for which differences between groups were observed in both males and females, and there was no effect of administration of the test substance. As a result of organ weight measurement, in the measurement at the end of administration, there was no difference between the groups for both the actual weight and the relative weight for all weighing organs for both males and females. At the end of the recovery period, the increase in brain weight and adrenal relative weight was observed in the male 1,000 mg / kg group, and the increase in splenic phase versus weight was observed in the female 1,000 mg / kg group, but both were slight Since it was a change and it was not observed at the end of administration, it was not considered to be a toxicologically important change. As a result of pathological examination, lesions suggested by administration of the test substance, neither macroscopically nor histologically, were observed in both males and females. No significant effects were observed in treated animals compared to control. Therefore the no observed adverse effect level (NOAEL) for the test chemical was considered to be 1000mg/kg/day when Crj:CD (SD) male and female rats were exposed by oral gavage route of exposure for 28 days .

Justification for classification or non-classification

Based on the above available data for test chemical it is likely to be non hazardous as a reproductive toxicant.