1,1,1"((methylsilanetriyl)tris)(oxy))tris(2-methylpropan-2-amine)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical  through oral gavage.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across substance

Justification for type of information:

Data for the target chemical is summarized based on the read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

WoE report is based on two toxicity to screening of repeated and reproductive tocicity studies as-
WoE 2 and WoE 3.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

WoE 2
TEST ANIMALS
- Source: In-House Bred at sa-FORD, Animal Facility (CPCSEA Registration No. 1256/bc/09/CPCSEA)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation.
- Weight at study initiation: Male: Minimum: 240 g Maximum: 315 g
Female: Minimum: 210 g Maximum: 260 g
- Fasting period before study: No data
- Housing: A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20[cm]). Cage rotation was carried out weekly during study period except during mating for males and females both and during gestation and lactation for females. Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet from approved supplier was offered ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum.
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.30 to 22.70 °C
- Humidity (%): 43.90 to 67.60%
- Air changes (per hr): 12 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: To:

WoE 3
No data available

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: WoE 2 : Corn oil; WoE 3; water

Details on exposure:

WoE 2
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

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): Corn oil. The test chemical was soluble in corn oil
- Concentration in vehicle:
- Amount of vehicle (if gavage): 0.5 ml/100g body weight
- Lot/batch no. (if required): MR301015, MR161215
- Purity: No data

WoE 3
PREPARATION OF DOSING SOLUTIONS:Test chemical was dissolved in water to give a dose of 0, 250, 500 and 1000 mg/Kg bwDIET PREPARATION- Rate of preparation of diet (frequency): No data available - Mixing appropriate amounts with (Type of food): No data available - Storage temperature of food: No data available VEHICLE- Justification for use and choice of vehicle (if other than water): Water- Concentration in vehicle: 0, 250, 500, or 1000 mg/kg/day - Amount of vehicle (if gavage): No data available - Lot/batch no. (if required): No data available - Purity: No data available

Details on mating procedure:

WoE 2
- M/F ratio per cage: One male and one female (1:1)
- Length of cohabitation: Female rats were housed with same male until pregnancy occurs or two weeks elapsed.
- Proof of pregnancy: Mating was confirmed by observation of sperm positive vaginal smear. The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data
- Further matings after two unsuccessful attempts: Yes, Re-mating of unsuccessfully paired female was done with proven male of the same group.
- After successful mating each pregnant female was caged (how): No data
- Any other deviations from standard protocol: No data

WoE 3
- Length of cohabitation: 2 weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

WoE 2
The analytical method was validated with respect to the following parameters.

Specificity:
The specificity will be evaluated by analysing the solvent used, standard solution, and sample solution.

Linearity:
The linearity was carried out by preparing and analyzing the standard solutions of at least 6 concentrations (covering the target analyte concentration i.e. 5 ppm,10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm ). A plot was drawn between the concentration and the response. The correlation coefficient, slope and intercept was calculated.

Assay accuracy and precision:
Assay accuracy and precision was carried out by fortifying the standard in vehicle at two levels (covering the target analyte concentration i.e., 10 ppm & 100 ppm). Five preparations were carried out at each concentration level selected. Two controls along with the assay accuracy samples were analysed. The mean, SD, % RSD was calculated. Assay accuracy was reported as the mean % recovery whereas the precision was reported as % RSD.

Homogeneity:
The homogeneity of the dose formulation prepared was determined by sampling and analyzing the formulation at top, middle and bottom layers. Sampling was done in two replicates from each layer.

Stability:
The stability of the prepared dose formulation was determined by analysing the sample at different time points (Stability was determined by sampling and analyzing the aliquots from the sample stored at 25 ± 2°C at the time points of 0, 2 and 6 hours).Two replications was analyzed at each time point.

WoE 3
No data available

Duration of treatment / exposure:

WoE 2
64 days

WoE 3
Total:110
Premating period -71 days Mating period-14 daysGestation period-21 days Postpartum day -4.

Frequency of treatment:

WoE 2
Total days: 64
All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Males were further dosed till 47th day . Females were dosed during pregnancy and upto day 4 post partum.

WoE 3
Daily

Details on study schedule:

No data available

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1 group (WoE 2)

Dose / conc.:

308 mg/kg bw/day

Remarks:

G2 group (WoE 2)

Dose / conc.:

556 mg/kg bw/day

Remarks:

G3 group (WoE 2)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G4 group (WoE 2)

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1R group (recovery) (WoE 2)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G4R group (recovery) (WoE 2)

Remarks:

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

No. of animals per sex per dose:

WoE 2
Total: 124 ( 104 Test animals + 20 recovery animals)
Test animals:
0 mg/Kg bw: 13 males and 13 females
308 mg/Kg bw: 13 males and 13 females
556 mg/Kg bw: 13 males and 13 females
1000 mg/Kg bw: 13 males and 13 females

Recovery animals:
0 mg/Kg bw: 5 males and 5 females
1000 mg/Kg bw: 5 males and 5 females

WoE 3
Total no of animals-96
0 mg/kg/day - 12 male and 12 female
250 mg/kg/day- 12 male and 12 female
500 mg/kg/day- 12 male and 12 female
1000 mg/kg/day- 12 male and 12 female

Control animals:

yes, concurrent vehicle

Details on study design:

WoE 2
- Dose selection rationale: The dose levels were selected based on the information provided by Sponsor.
- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. The animals were allocated to the different test groups using validated software or the ‘Group Allocation’ function in the MS Excel Add-in “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights will be considered within ± 20% of the groups mean.
- Other: No data

WoE 3
No data available

Positive control:

No data available

Parental animals: Observations and examinations:

WoE 2
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the acclimatization and study period
- Cage side observations checked in table [No.?] were included. Mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations of animals of all groups were made once a day. Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.

Observations included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic
or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards)

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, During pre-mating, pregnancy and lactation, feed consumption were measured at least weekly. Feed consumption was not measured during mating period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT), Activated Partial Thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb) , Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN) – Calculated, Globulin (Glob) - Calculated, Alb/ Glb (A:G) – Calculated, Bile acids

URINALYSIS: Not specified
- Time schedule for collection of urine: Not specified
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined. Not specified

NEUROBEHAVIOURAL EXAMINATION:Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not specified

IMMUNOLOGY: Not specified
- Time schedule for examinations: Not specified
- How many animals: Not specified
- Dose groups that were examined: Not specified
- Parameters checked in table [No.?] were examined. Not specified

OTHER:
Functional Battery Observations: Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for five males and five females from control and treatment groups, during the last week of treatment and that of recovery groups, in the last week of recovery period.

Animals were subjected to examinations of various functional parameters which included; motor activity measurements using OPTO–VARIMEX 4, an automated animal activity measuring system; fore limb and hind limb grip strength, using grip strength meter; hind limb foot splay record and sensory reactivity measurements.

WoE 3
Body weight and food consumption was observed.

Oestrous cyclicity (parental animals):

WoE 2
Estrous cycle was monitored for its regularity during treatment period and in cohabitation for confirmation of pregnancy.

WoE 3
Any irregularity in estrous cycle were observed. Implantation sites and ovarian corpora lutea also examined.

Sperm parameters (parental animals):

No data available

Litter observations:

WoE 2
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No data
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. No data

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. Live pups were counted and sexed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum. Live pups were weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum.

GROSS EXAMINATION OF DEAD PUPS:
Terminally sacrificed pups and pups died during the course of study were subjected to gross pathology

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No data

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No data

WoE 3
Pup viability, individual pup weights, litter sizes.and clinical signs were observed at birth and on lactation day 4.

Postmortem examinations (parental animals):

WoE 2
GROSS PATHOLOGY: Yes, At scheduled sacrifice date, all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents. Simultaneously gross lesions examination was performed in accordance with the Standard Operating Procedure (SOP) of the Laboratory. Number of implatation sites in uterus and number of corpora lutea of all pregnant females were counted during necropsy examination.

Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted and gross pathological observations were recorded.

Following organs from randomly selected 5 male and 5 female rats were collected and preserved : Adrenals, Aorta, Bone (femur) with joint, Brain (cerebrum, cerebellum, mid brain), Cecum, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mammary glands, Mesenteric and Mandibular lymph node, Oesophagus, Ovaries with oviduct, Pancreas, Peyer's Patches, Pituitary, Prostate and Seminal vesicle with coagulating glands, Rectum, Salivary glands, Sciatic Nerve, Skeletal muscle, Skin, Spleen, Spinal Cord (cervical, mid-thoracic and lumbar), Sternum with marrow, Stomach, Testes, Thymus, Thyroid with Parathyroids, Trachea, Urinary Bladder, Uterus, Cervix with Vagina

Testes, epididymides, prostate and seminal vesicle with coagulating glands of all male rats and ovaries, uterus and cervix with vagina of all female rats were collected and preserved. Thyroid gland of one male and one female pup from each litter was collected and preserved. Collected Organs/tissues were fixed and preserved in 10 % Neutral buffered formalin. Testes and epididymis were initially fixed in Bouin’s fluid for approximately 24 hr, then 4 changes were given in 70% alcohol and transferred to 10 % neutral buffered formalin for preservation. Eyes were initially fixed in Modified Davidson‘s fluid for approximately 24 hr and then transferred to 10 % neutral buffered formalin for preservation.

Organ Weight: Weighing of brain, adrenals, ovaries with oviduct, testes, epididymides, heart, liver, kidneys, thymus and spleen was performed for randomly selected 5 male and 5 female rats. Testes and epididymides of all male rats were weighed. Before weighing adherent tissue/fat from organs were trimmed off and were kept in normal saline.

HISTOPATHOLOGY: Yes, All the preserved organs (Testes, epididymides, prostate and seminal vesicle with coagulating glands, ovaries, uterus and cervix with vagina) of all the rats, all the preserved tissues of randomly selected five male and five female rats of groups G1 and G4 and preserved thyroid of one male and one female pup of each litter were subjected to histopathological examination. All the tissues were trimmed, processed, embedded in paraffin wax. Sections were cut at a thickness of 3-5 micron and stained with hematoxylin and eosin stain. Processed tissues were subjected to histopathological examination. The prepared slides were examined under microscope by the Pathologist to note histopathological lesions, if any in different organs. Special attention was paid to observe effect of test item on reproductive system and spermatogenesis. The observed abnormalities were described according to morphological character, distribution, severity.

WoE 3
Gross pathology and histopathology were examined. A histological examination of all tissues saved was conducted for all animals in the control and 1000 mg/kg/day group. Tissue examination in the other groups was limited to relevant gross lesions and tissues demonstrating treatment-related histological effects at 1000 mg/kg/day

Postmortem examinations (offspring):

WoE 2
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age. No data
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Gross pathology- Pups: Terminally sacrificed pups and pups died during the course of study were subjected to gross pathology

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. No data

WoE 3
No data available

Statistics:

WoE 2
Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks

WoE 3
No data available

Reproductive indices:

WoE 2
Pregnancy index/fertility index ,Mean Post-Implantation Loss (%), Post-natal Loss (%) were determined.

WoE 3
Corpora Luta Counts and Mean Number of Pups/Litter were examined.

Offspring viability indices:

WoE 2
Pups Survival Index (%) was determined.

WoE 3
Offspring viability indices was observed on day 0 and 4 of lactation.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

WoE 2
No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period.

Statistically significant decrease was observed in number of rears of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on pre-treatment as compared to control G1 (0 mg/kg body weight). The statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G3 (556 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G4 (1000 mg/kg body weight) male at week 4 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) male at week 6 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G2 (308 mg/kg body weight), G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) female at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G4 (1000 mg/kg body weight) female at week 5 as compared to control G1 (0 mg/kg body weight).

The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration.

WoE 3
no data available

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

WoE 2
No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period

WoE 3
no data available

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

WoE 2
A statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) male on day 30 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) female on day 20 of gestation as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4-R (1000 mg/kg body weight) male on day 29, 36, 41 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on day 1-8, 1-14 whereas statistically significant decrease was observed in percent body weight change of G4 (1000 mg/kg body weight) male on day 1-21, 1-28, 1-30, 1-37, 1-44, 1-46 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change during gestation period of G4 (1000 mg/kg body weight) female on day 0-14, 0-20 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G4-R (1000 mg/kg body weight) male on day 1-8, 1-15, 1-22, 1-29 as compared to control G1-R (0 mg/kg body weight).

Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.

WoE 3
Significant reductions in body weight in animals were observed at 1000 mg/kg/day. Weight gain of pregnant females was less at 1000 mg /kg/day from gestational day 1 to day 7 (87.5% of control) but similar to the other groups at intervals after gestational day 7.
These changes observed were inconsistent, hence not considered as effect of the test item administration.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in feed consumption was observed in G4 (1000 mg/kg body weight) female on gestation day 14-20 as compared to the control group G1. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.

WoE 3
Significant reductions in food consumption were observed in P male and female rats.

Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration

Food efficiency:

no effects observed

Description (incidence and severity):

WoE 2
Formulations were found to be homogeneous and stable upto 6 hour in vehicle corn oil. The mean active ingredient content at 61.6, 111.2 and 200 mg/ml concentration of Methyl Phenyl acetate (CAS No.:101-41-7) was 61.770, 110.321 and 200.007 mg/ml on day 1; 62.045, 110.902 and 198.199 mg/ml on day 21 and 60.726, 111.912 and 201.231 mg/ml on day 40, respectively

WoE 3
No data available

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All hematological parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant decrease observed for MCHC, WBC in males of G4 (1000 mg/kg body weight) as compared to G1, statistically significant increase observed
for aPTT in males of G4 (1000 mg/kg body weight) and G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for RBC, HCT, HGB, WBC in males of G4-R (1000 mg/kg body weight) as compared to G1-R. Statistically significant decrease observed for PT in females of G3 (
556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for MCHC and statistically significant increase observed for RBC, HCT, HGB in females of G4-R (1000 mg/kg body weight) as compared to G1-R.

The above changes were inconsistent, not related to the test item and may be due to the preanalytical and analytical variables

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant increase observed for ALT and statistical ly significant decrease observed for Sodium (Na) in males of G4 (1000 mg/kg Body weight) as compared
to G1. Statistically significant increase observed for Creatinine in males of G2 (308 mg/kg Body weight) as compared to G1. Statistically significant decrease observed for Total Protein and statistically significant increase observed for A/G ratio in females of G3 (556 mg/kg Body weight) as compared to G1.

The above changes were inconsistent, not dose dependent hence considered as incidental in nature.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.

Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups except a statistically significant decrease was observed in hindlimb foot splay in G4-R (1000 mg/kg body weight) male as compared to the
repective control group G1-R.

The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence, considered as incidental and not attributed to the effect of test item administration.

Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group except statistically significant decrease was observed in ST=Stereotypic time in G2, G3 and G4 male as compared to control group G1 and G4-
R in female as compared to G1-R.

The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination of control group and rats treated at 308, 556 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes Liver: focal to multifocal minimal lymphocytic infiltration (Male: G1:1/5, G4:2/5; Female: G1: 1/5; G4: 2/5); focal minimal necrosis (Male: G1:1/5; Female: G1: 1/5); Kidneys: focal minimal lymphocytic infiltration (Male: G1:2/5; Female: G4:1/5); focal mild mineralization (Female: G1:1/5); Lungs: multifocal minimal lymphocytic infiltration (Male:G1:1/5, G4:1/5; Female: G1: 2/5, G4: 3/5); focal minimal histiocyte infiltration (Female: G1: 1/5, G4: 1/5); Heart: focal minimal lymphocytic infiltration (Male: G1:1/5, G4:1/5); Aorta: focal minimal aneurysm (Male:G1:1/5, G4:1/5); Mandibular Lymph Node: focal moderate cystic dilation of cortex (Female: G4:1/5); Stomach: focal mild squamous epithelium hyperplasia (Female: G1: 1/5); Mesenteric lymph node: focal moderate cystic dilation of cortex (Female:G1:1/5); Spleen: focal to diffuse minimal to mild extramedullary hematopoesis (Female: G1: 2/5, G4: 3/5); Thymus: mild to moderate atrophy (Female: G1:3/5, G4:4/5); focal mild
cystic epithelial dilation (Male: G4:1/5; Female: G1: 1/5, G4:1/5); Trachea: focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration (Male: G1:3/5, G4:3/5; Female: G1: 2/5, G4:1/5); Adrenal s: unilateral accessory adrenocortical tissue (Male: G1:1/5, G4:1/5); Testes: focal to multifocal minimal to mild retention of mature sperm (Male: G1:4/13, G2:8/13, G3:8/13, G4:8/13); focal minimal to mild degeneration of seminiferous tubules (Male: G1:2/13, G2:1/13, G3:1/13, G4:1/13); focal to multifocal minimal sloughing of Pachytene Spermatocyte (Male: G1:2/13, G2:2/13, G3:2/13, G4:2/13); focal minimal sloughing of round spermatid (Male: G1:1/13, G2:1/13, G3:1/13, G4:1/13); focal mild infiltration of multinucleated giant cells (Male: G1:1/13); Seminal Vesicles: multifocal mild neutrophilic /lymphocytic infiltration (Male: G1:1/13); Prostate: focal moderate necrotic debris in lumen (Male: G2:1/13); Uterus: multifocal to diffuse mild reduction of stromal cells (Female: G1:1/13; G4:2/13); focal moderate necrosis (Female: G3:1/13); multifocal mild to moderate nodular hyperplasia (Female: G1:1/13; G2:1/13; G4:1/13); Cervix: focal minimal lymphocytic infiltration (Female: G2:1/13). Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance.

From the patho-morphological results presented, it is concluded that, the treatment of Methyl Phenyl acetate at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the Test Item.

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item related changes in estrous cyclicity and precoital interval were observed. In control group G1 and treatment group G2 all the females showed regular cyclicity i.e. 3-5 days estrous cycle; while in group G3, 3 females and in group G4, 4 females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. Precoital Interval was calculated, all females showed precoital interval less than 5 days, except 1, 1 and 4 females from G1, G3 and G4, respectively which showed precoital interval more than 5 days.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

WoE 2
Pregnancy index was found to be 92.31, 84.62, 84.62 and 61.54 in G1, G2, G3 and G4 respectively. Marked decrease in Pregnancy index / Fertility index in G4(1000 mg/kg body weight) was considered to be treatment related.

WoE 3
Significant decrease in corpora lutea counts and subsequently lower implantation site counts and litter sizes were observed at 1000 mg/kg/day treated female rat. No effect on Number of Pups/Litter were observed in treated female rats.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

ophthalmological examination

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

other: Not Specified

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

reproductive performance

Remarks on result:

other: no toxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

WoE 2
There was no statistically significant difference between the control (G1) and treatment groups for mortality, no. of live births and survival index.

WoE 3
No mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no statistically significant difference between the control (G1) and treatment groups for pups weight at birth and PND4 and weight gain at PND4

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Pups died during course of study revealed various lesions among the control and treated groups viz., external examination emaciated carcass (Male: G1:2/55, G2:1/44, G3:5/35; Female: G1:3/56, G2:1/30, G3:6/54); Cannibalism (Male: G1:3/55, G3:2/35; Female: G1:2/56, G3:3/54); Tearing of Neck Muscle (Female: G3:1/54; G4:1/18) and internal examination absence of milk in stomach (Male: G1: 6/55, G2: 6/44, G3: 12/35, G4: 3/16; Female: G1: 8/56, G3: 14/54, G4: 2/18); blood clot in thoracic cavity (Male: G1: 2/55, G2: 3/44, G3: 1/35; Female: G1: 1/56, G3: 1/54, G4: 1/18); reddish discoloration of brain (Male: G1: 1/55, G2: 1/44, G3: 1/35; Female: G1: 1/56, G3: 3/54, G4: 1/18); reddish discoloration of lungs (Male: G1: 5/55, G2: 5/44, G3: 7/35, G4: 1/16; Female: G2: 1/30, G3: 10/54, G4: 2/18); paleness of liver (Male: G1: 1/55, G2: 2/44, G3: 1/35; Female: G3: 4/54, G4: 2/18); congested intestine (Female: G1: 1/56, G3: 1/54); autolytic changes (Female: G2: 1/30, G3: 2/54, G4: 1/18)

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Pups sex ratio (Male/Female) was found to be 55/57, 44/30, 43/58, and 21/26 in G1, G2, G3 and G4 respectively.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

WoE 3
Viability: No effect on viability of treated pups were observed till day 4 of lactation as compared to control.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

556 mg/kg bw (total dose)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

other: Fertility index/pregnancy index was decreased at dose level of 1000 mg/Kg bw in dams which was correlated to the viability of offsprings

Remarks on result:

other: No developmental toxic effects observed

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

other: No adverse effect

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw (total dose)

Treatment related:

no

Conclusions:

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical through oral gavage.

Executive summary:

Data available of the read across chemicals has been reviewed to determine the effect of the test chemical on Screening of repeated and reproductive toxicity. The studies are as mentioned below:

A Combined repeated dose repro-developmental toxicity study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test chemical in Wistar rats. The animals were randomly allocated to the four main groups (13/sex/group) and two recovery groups (5/sex/group). The doses selected for main groups were; 0 (G1-control),308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4) daily for 64 days. The recovery groups G1-R and G4-R were dosed with similar doses of respective main groups.Vehicle corn oil to G1 and G1-R and test item to G2, G3, G4 and G4-R animals were administered by oral gavage route each day during the dosing period. No mortality and morbidity were observed any of the groups of animals throughout the study period. Animals of all dose groups were observed for Clinical signs/ symptoms daily once during the experimental period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements were comparable and no statistically significant changes were revealed in animals of treatment groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the repective control groups. Motor activity measurements were comparable and no changes were revealed in any of the animals of all treated groups of both the sexes as compared to control group. Estrous cycle was evaluated for checking the regularity during treatment period and in cohabitation for confirmation of pregnancy. No test chemical related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. All hematological and clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups. No treatment related changes were observed in any of the treatment groups.  At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to the respective control group rats. External and visceral examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. From the patho-morphological results presented, it is concluded that, the treatment of Methyl Phenyl acetate at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs. Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the test chemical.Based on all the observations and results, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical  through oral gavage.

In a repeated dose reproduction –development toxicity study, male and female rats were treated with test chemical in the concentration of 0, 250, 500 and 1000 mg/kg/day orally by gavage in water. Test was conducted as per OECD 422. Significant decrease in body weight of P1 animals were observed at 1000 mg/kg/day. Weight gain of pregnant females was less at 1000 mg /kg/day from gestational day 1 to day 7 (87.5% of control) but similar to the other groups at intervals after gestational day 7. Significant reductions in food consumption were observed in P1 male and female rats. Similarly, reproductive toxicity was observed in treated female rats at 1000 mg/kg bw such as significant decrease in corpora lutea counts and subsequently lower implantation site counts and litter sizes were observed at 1000 mg/kg/day treated female rat. But, No effect on Number of Pups/Litter were observed in treated female rats. In addition, No effect on viability of treated pups were observed till day 4 of lactation as compared to control at 250, 500 and 1000 mg/kg bw/day. Therefore, NOAEL was considered to be 500 mg/kg bw/day for P generation and 1000 mg/kg bw/day for F1 generation when male and female rats were treated with test chemical orally by gavage for 110 days.

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical  through oral gavage.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

556 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

K2

Additional information

Data available of the read across chemicals has been reviewed to determine the effect of the test chemical on Screening of repeated and reproductive toxicity. The studies are as mentioned below:

A Combined repeated dose repro-developmental toxicity study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test chemical in Wistar rats. The animals were randomly allocated to the four main groups (13/sex/group) and two recovery groups (5/sex/group). The doses selected for main groups were; 0 (G1-control),308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4) daily for 64 days. The recovery groups G1-R and G4-R were dosed with similar doses of respective main groups.Vehicle corn oil to G1 and G1-R and test item to G2, G3, G4 and G4-R animals were administered by oral gavage route each day during the dosing period. No mortality and morbidity were observed any of the groups of animals throughout the study period. Animals of all dose groups were observed for Clinical signs/ symptoms daily once during the experimental period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements were comparable and no statistically significant changes were revealed in animals of treatment groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the repective control groups. Motor activity measurements were comparable and no changes were revealed in any of the animals of all treated groups of both the sexes as compared to control group. Estrous cycle was evaluated for checking the regularity during treatment period and in cohabitation for confirmation of pregnancy. No test chemical related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. All hematological and clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups. No treatment related changes were observed in any of the treatment groups.  At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to the respective control group rats. External and visceral examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. From the patho-morphological results presented, it is concluded that, the treatment of Methyl Phenyl acetate at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs. Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the test chemical.Based on all the observations and results, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical  through oral gavage.

In a repeated dose reproduction –development toxicity study, male and female rats were treated with test chemical in the concentration of 0, 250, 500 and 1000 mg/kg/day orally by gavage in water. Test was conducted as per OECD 422. Significant decrease in body weight of P1 animals were observed at 1000 mg/kg/day. Weight gain of pregnant females was less at 1000 mg /kg/day from gestational day 1 to day 7 (87.5% of control) but similar to the other groups at intervals after gestational day 7. Significant reductions in food consumption were observed in P1 male and female rats. Similarly, reproductive toxicity was observed in treated female rats at 1000 mg/kg bw such as significant decrease in corpora lutea counts and subsequently lower implantation site counts and litter sizes were observed at 1000 mg/kg/day treated female rat. But, No effect on Number of Pups/Litter were observed in treated female rats. In addition, No effect on viability of treated pups were observed till day 4 of lactation as compared to control at 250, 500 and 1000 mg/kg bw/day. Therefore, NOAEL was considered to be 500 mg/kg bw/day for P generation and 1000 mg/kg bw/day for F1 generation when male and female rats were treated with test chemical orally by gavage for 110 days.

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical  through oral gavage.

Justification for classification or non-classification

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical through oral gavage and was not classified.