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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity study

Based on the data available from different studies for target chemical 1-phenylethanol (98-85-1)and its structuraly similar read across substance 2-phenylethan-1-ol CAS 60-12-8 / EC 200-456-2)andMethyl phenyl acetate; methyl phenylacetate (CAS 101-41-7) via oral and dermal route;LOAEL for 1-phenylethanol (98-85-1)was considered to be in range of 4.3 -556 mg/kg bw/day for F0, F1 generation but as adverse effects like markedly decreased pregnancy index / fertility index, In addition, morphological change was observed in fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals at different dose group. Thus, comparing this value with the criteria of CLP regulation 1-phenylethanol (98-85-1)is likely to classify as reproductive and developmental toxicant.

 

Link to relevant study records

Referenceopen allclose all

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 from various test chemicals
Justification for type of information:
Weight of evidence approach based on the available information from various test 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
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 reproductive toxicity studies on rats
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: 1.Wistar 2.Crl:COBSCD(SD)BR 3.Long- Evans rats
Details on species / strain selection:
The rat is the commonly used species for toxicity studies and also recommended by the regulatory guidelines specified in the study plan.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Study 1.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. Bedding material of batch No. SPAR-30/2015 (Sparconn Life Sciences Bangalore) was used in this study and a copy of report of microbial and chemical contaminants analysed periodically by manufacturer of bedding material are incorporated in the raw data.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet of batch no. 004915, 041215 and 041015 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: A conventional laboratory pelleted diet of batch no. 004915, 041215 and 041015 from approved supplier (Nutrivet Life Sciences, Pune) was offered. Aqua guard filtered drinking water in bottles was offered.

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: November 16, 2015 to March 26, 2016
Route of administration:
other: 1,3& 4.oral: gavage 2.oral: feed
Vehicle:
other: 1.corn oil 4.sunflower oil
Details on exposure:
Study1.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
Study 2.Details on exposure
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet
- Storage temperature of food:
Study 3.Details on exposure
PREPARATION OF DOSING SOLUTIONS: test material was suspended in distilled water

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:4.3, 43 and 432 mg/kg
- Amount of vehicle (if gavage):20 mL/kg
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
Study1.- M/F ratio per cage: One male and one female
- 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.
- 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
Study 3.Pregnant female rats were used
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study1.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.
Study2.High performance liquid chromatographic (HPLC) analyses were used to validate concentration, homogeneity and stability of test material throughout the study
Duration of treatment / exposure:
Study1.
64 days
Study2.& 3.
10 days ( from day 6 through day 15)
Study4.
4 days ( GD 4 (preimplantation) or GDs 10 through 12 (during embryogenesis)
Frequency of treatment:
Daily
Details on study schedule:
No data
Remarks:
Study1.Test animals:
0 (G1-control), 308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4)
Recovery animals: 0 (G1-R) or 1000 (G4-R) mg/Kg bw
Study2.
0, 1000, 3000 or 10,000 ppm (calculated doses were 83, 266 and 799 mg/kg for the respective exposure groups).
Study3
.0,4.3, 43 and 432 mg/kg
Study4.
0,508 mg/kg
No. of animals per sex per dose:
Study1.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
Study2.Total:112
0mg/kg bw/day:28 female
83mg/kg bw/day:28 female
266mg/kg bw/day:28 female
799mg/kg bw/day:28 female
Study3.Total:38
0mg/kg bw/day:19female
4.3mg/kg bw/day:7female
43mg/kg bw/day:7female
432mg/kg bw/day:5 female
Study4.Total:20
0mg/kg bw/day:10female
508mg/kg bw/day:10female

Control animals:
yes, concurrent vehicle
Details on study design:
1.- 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
3.Doses were selected to be 0.24, 2.4 and 24% of the LD50 value published by Owston et al. (1981)
Positive control:
No data
Parental animals: Observations and examinations:
Study1.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.
Oestrous cyclicity (parental animals):
1.Estrous cycle was monitored for its regularity during treatment period and in cohabitation for confirmation of pregnancy.
Sperm parameters (parental animals):
No data
Litter observations:
Study1.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
Postmortem examinations (parental animals):
Study 1.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.
2,3&4Postmortem examinations (Parent Animal)
SACRIFICE: The animalswere euthanized on GD 20 and in utero parameters were assessed on the basis of litter values.

GROSS NECROPSY: yes
HISTOPATHOLOGY / ORGAN WEIGHTS: yes
Postmortem examinations (offspring):
Study1.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
2,3.&4
All live fetuses were examined for examination of the fetuses for external, soft tissue and skeletal alterations (malformations and variations).
Statistics:
Study1.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
Reproductive indices:
Study1.Pregnancy index/fertility index was determined
Offspring viability indices:
No data
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Study1.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.
Study2.No obvious clinical signs of toxicity was noted.
Study3.Severe maternal intoxication was observed at the 432 mg/kg dose group immediately after administration and persisted overnight on each day of treatment. No clinical effects were observed at the 4.3 and 43 mg/kg doses.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
Study1.No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period.
Study2.No deaths occurred during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Study1.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.

These changes observed were inconsistent, hence not considered as effect of the test item administration.
Study2.No effects were observed at 83mg/kg and 266mg/kg dose group . At 799mg/kg dose group , reduced maternal feed consumption and slight weight loss occurred on treatment days 1 and 2 (GDs 6–7, 7–8).
Study3.reduced weight gain was evident at the 43 mg/kg dose
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.
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):
Study1Formulations 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
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):
Study1.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):
Study1.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):
Study1.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):
Study1.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):
Study1.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):
Study1.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):
Study1.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.
Study.2.mean values for embryo-fetal loss, litter sizes, sex ratios were equivalent in control and test group
Study4.No adverse effect on fetal ossification occurred when test material was administered preimplantation
Dose descriptor:
LOAEL
Effect level:
> 4.3 - <= 799 mg/kg bw (total dose)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Significant changes in the pregnancy index were found at 1000 mg/Kg bw.
Remarks on result:
other: No toxic effects observed
Critical effects observed:
yes
Lowest effective dose / conc.:
4.3 mg/kg bw/day (nominal)
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Study1.There was no statistically significant difference between the control (G1) and treatment groups for mortality, no. of live births and survival index
Study3.Mean litter size was reduced at the 43 and 430 mg/kg dose levels
Study4.Fetal viability were unaffected.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Study1.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.
Study2.The mean fetal body weights, was equivalent in control and test groups.
Study3.Fetal growth retardation (reduced body weight and crown-rump length) was observed at the 4.3 and 432 mg/kg doses, with runting (body weight <2.7 g) occurring in 5 of 30, 0 of 61 and 32 of 51 fetuses at the 0.43, 43 and 432 mg/ kg dose levels, respectively. This absence of dose-dependency also was probably related to the relatively few litters evaluated.
4.Fetal body weight were unaffected.
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):
Study1.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).
2.The number, type and distribution of fetal malformations, anomalies and skeletal variations also were unaffected by maternal exposure to test material at concentrations as high as 10,000 ppm (799 mg/kg/day), although at 799mg/kg dose group there was a possible marginal delay in fetal ossification (a slightly higher incidence of fetuses with incomplete ossification of some sites, such as the sacro-caudal vertebral arches)
Study3.The percentage of dead or malformed fetuses per total implants was 55%, 97% and 100% at 4.3, 43 and 432 mg/kg, respectively. A dose–related increase in malformations was reported, although how the number of fetuses affected was determined is unclear, because the number of fetuses evaluated by each methodology and the total number of fetuses available for evaluation were not provided. Reduced cranial- bone ossification, a variant, was observed at a low incidence (0 of 224, 7 of 60, 2 of 65 and 6 of 51 fetuses in the 0, 0.43, 43
and 432 mg/kg dose groups, respectively). Additional retardations of ossification of the limbs, ribs and tail were evident at the 43 and 432 mg/kg dose levels. Microphthalmia (small eyes) and hydronephrosis (enlarged renal pelvis) occurred at 0.43, 4.3 and 432 mg/kg doses. Additional malformations included open eyes, micromelia (small paw) and a low incidence of heart defects at the 43 and 432 mg/kg doses. The 432 mg/kg dose was also associated with spina bifida (open spinal column), phocomelia (short limb), sirenomelia (joined limbs), micrognathia (short jaw) and webfoot/club foot.
Study4.Small delays in some bone ossification sites occurred in treated fetuses, as compared with controls, when the test material was administered during the period of organogenesis, observations compatible with the small but biologically unimportant reduction in fetal body that also occurred in these fetuses. No other effects were observed

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):
Study1.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
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
> 4.3 - <= 799 mg/kg bw (total dose)
Based on:
test mat.
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
Critical effects observed:
yes
Lowest effective dose / conc.:
4.3 mg/kg bw/day (nominal)
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Reproductive effects observed:
yes
Lowest effective dose / conc.:
4.3 mg/kg bw/day (nominal)
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
Low Observed Adverse Effect Level (LOAEL) is considered to be in range of 4.3-799mg/kg bw. When male and female wistar rats were treated with 1-phenylethanol (98-85-1) orally.
Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of 1-phenylethanol (98-85-1).The studies are as mentioned below:

Study1.

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 material 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 item 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 Item.

Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material in Wistar Rats with 14 days recovery, where in 0, 308, 556 and 1000 mg/kg body weight, doses were tested; No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with test material orally.

Study2.

Reproductive and developmental toxicity study of test materialwas performed onCrl:COBSCD(SD)BR female rats. The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet in dose concentration 0, 1000, 3000 or 10,000 ppm (calculated doses were 83, 266 and 799 mg/kg for the respective exposure groups). The concentrations tested were based on a pilot study in which dietary inclusion of up to 9000 ppm test material was well tolerated in pregnant rats and did not result in unacceptable loss of diet palatability. High performance liquid chromatographic (HPLC) analyses were used to validate concentration, homogeneity and stability of test material throughout the study. 28 female rats per dose group were exposed to test material on GDs 6 through 15, In all the animals Feed consumption was measured daily, and body weights were taken on GDs 1, 3, 6 and then on alternate days until necropsy on GD 20, when in utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for external, soft tissue and skeletal alterations (malformations and variations).

No deaths occurred during the study.No obvious clinical signs of toxicity was noted. No effects were observed at 83mg/kg and 266mg/kg dose group . At 799mg/kg dose group , reduced maternal feed consumption and slight weight loss occurred on treatment days 1 and 2 (GDs 6–7, 7–8). Calculated mean group intake of test material was 0, 83, 266 and 799 mg/kg/day for the pregnant rats in the 0, 1000, 3000 and 10,000 ppm groups, respectively. mean values for embryo-fetal loss, litter sizes, sex ratios were equivalent in control and test group also the mean fetal body weights, was equivalent in control and test groups.The number, type and distribution of fetal malformations, anomalies and skeletal variations also were unaffected by maternal exposure to test material at concentrations

as high as 10,000 ppm (799 mg/kg/day), although at799mg/kg dose group there was a possible marginal delay in fetal ossification(a slightly higher incidence of fetuses with incomplete ossification of some sites, such as the sacro-caudal vertebral arches).HenceNo Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 799mg/kg. Whenfemalerats were treated withtest material by orally for GDs 6 through 15.

Study 3.

The reproductive and developmental toxicity study of test material was performed onpregnant Long- Evans rats. The aqueous suspension of test material was administered by gavage at dose concentration 4.3, 43 and 432 mg/kg to rats on GDs 6 through 15. All doses were administered at a volume of 20 mL/kg,Group sizeswere 19, 7, 7 and 5 pregnant rats in the control (distilled water),4.3, 43 and 432 mg/kg dose groups, respectively. Doses wereselected to be 0.24, 2.4 and 24% of the LD50 value published byOwston et al. (1981).The dams were observed daily for signs of toxicity and lethality. Body weights were recorded on GDs 0 through 15 and 20. All dams were overdosed with ether on GD 20, and their fetuses were delivered by Cesarean-section. Observations were made for uterine weight, litter weight, individual pup weight, and numbers of live and dead pups, resorptions, implantations, sex distribution, crown-rump length and corpora lutea. All live pups were examined for gross external malformations. As report that 224, 51, 65 and 60 fetuses in the four respective dose groups were evaluated for both soft tissue and skeletal examinations. It is unclear how this number was attained, because soft tissue evaluation was by sectioning (Wilson’s method), precluding skeletal evaluation of the same specimen (alcian blue-alizarin red S-staining).

 

Severe maternal intoxication was observed at the 432 mg/kg dose group immediately after administration and persisted overnight on each day of treatment. No clinical effects were observed at the 4.3 and 43 mg/kg doses but reduced weight gain was evident at the 43 mg/kg dose. Mean litter size was reduced at the 43 and 430 mg/kg dose levels .Fetal growth retardation (reduced body weight and crown-rump length) was observed at the 4.3 and 432 mg/kg doses, with runting (body weight <2.7 g) occurring in 5 of 30, 0 of 61 and 32 of 51 fetuses at the 0.43, 43 and 432 mg/ kg dose levels, respectively. This absence of dose-dependency also was probably related to the relatively few litters evaluated and thepattern of postimplantation loss (resorption)

The percentage of dead or malformed fetuses per total implants was 55%, 97% and 100% at 4.3, 43 and 432 mg/kg, respectively. A dose–related increase in malformations was reported, although how the number of fetuses affected was determined is unclear, because the number of fetuses evaluated by each methodology and the total number of fetuses available for evaluation were not provided. Reduced cranial- bone ossification, a variant, was observed at a low incidence (0 of 224, 7 of 60, 2 of 65 and 6 of 51 fetuses in the 0, 0.43, 43 and 432 mg/kg dose groups, respectively). Additional retardations of ossification of the limbs, ribs and tail were evident at the 43 and 432 mg/kg dose levels. Microphthalmia (small eyes) and hydronephrosis (enlarged renal pelvis) occurred at 0.43, 4.3 and 432 mg/kg doses. Additional malformations included open eyes, micromelia (small paw) and a low incidence of heart defects at the 43 and 432 mg/kg doses. The 432 mg/kg dose was also associated with spina bifida (open spinal column), phocomelia (short limb), sirenomelia (joined limbs), micrognathia (short jaw) and webfoot/club foot. As there were effects at all three dose levels, a NOAEL could not be determined from this study and is presumed to be < 4.3 mg/kg

Study4.

The reproductive and developmental toxicity study of test material was performed onpregnant rats.Test material in sunflower oil was administered to pregnant rats (10 per group) by gavage on GD 4 (preimplantation) or GDs 10 through 12 (during embryogenesis) in dose concentrati as a single dose of 508 mg/kg.GD 1 was defined as the day sperm were observed in a vaginal smear. On GD 20, the female rats were euthanized, and the fetuses were removed for further analysis (Wilson’s sectioning or alizarin red S-staining). Fetal viability and body weight were unaffected. No adverse effect on fetal ossification occurred when test material was administered preimplantation. Small delays in some bone ossification sites occurred in test material treated fetuses, as compared with controls, when the test material was administered during the period of organogenesis, observations compatible with the small but biologically unimportant reduction in fetal body that also occurred in these fetuses. No other effects were observed .Hence NOAEL for maternal and developmental toxicity was considered to be 508mg/kg bw . When female rats were treated with test material orally.

Based on the data available from study report test chemical howed reproductive toxicityat dose concentration 4.3 mg/kg bw/day.When rats were treated with test material orally.Hence the test chemical is likely to classify as a reproductive toxicant as per the criteria mentioned in CLP regulation.

Endpoint:
toxicity to reproduction
Remarks:
other: one-generation study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from NTP report
Qualifier:
according to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
The study was performed to evaluate carcinogenic, reproductive and developmental effect of the test chemical in rats.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
Not Specified
Species:
rat
Strain:
other: CrL:COBS CD (SD) BR strain
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 170 - 242 g (group average range)
- Housing: Animals were housed individually in a controlled environment in suspended galvanised metal cages equipped with solid sides and back, wire mesh front, floor and top.
- Diet (e.g. ad libitum): S.F. Laboratory Diet No 1, ad libitum
- Water (e.g. ad libitum):Ad libitum
- Other details: On the day prior to initiation of treatment, electric clippers were used to remove the fur from an area approximately 7 x 5 cm of the intrascapular region of each animal.
Daily dermal application commenced on Day 6 of pregnancy and continued daily up to and including Day 15 of pregnancy of rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 2°C
- Humidity (%): 53% ± 11%
- Air changes (per hr): 13 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle (light phase 8.00 - 20.00 hours)
Route of administration:
dermal
Type of inhalation exposure (if applicable):
not specified
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 7 x 5 cm
- % coverage: covering at least the shaved dorsal area of dose application)
- Type of wrap if used: aluminium foil patch (covering at least the shaved dorsal area of dose application) which was held in place by a medical-type
adhesive bandage
- Time intervals for shavings or clipplings: Chemical depilation was not employed nor was the skin scarified but shaving was repeated as necessary throughout the treatment period .

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The skin was not washed between applications .
- Time after start of exposure: NA

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 0.14, 0.43 or 1.40 ml/kg per day
- Concentration (if solution): 100%
- Constant volume or concentration used: no. Dosage volumes were calculated for individual animals on Day 6 of pregnancy and adjusted according to bodyweight on Days 8, 10, 12 and 14 .
- For solids, paste formed: NA

VEHICLE - not applicable
- Justification for use and choice of vehicle (if other than water):
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
-According to A.M. Api et.al. (RIFM fragrance ingredient safety assessment, α-methylbenzyl alcohol, CAS registry number 98-85-1) systemic absorption was assumed to be 100% by oral, dermal and inhalation route.
Details on mating procedure:
- M/F ratio per cage: Time-mated rats were delivered from the vendor.
- Length of cohabitation: Not Available
- Proof of pregnancy: The day of mating, as judged by the appearance of sperm in the vaginal smear or by the presence of a vaginal plug was considered as Day 0 of pregnancy.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not Available
- Further matings after two unsuccessful attempts: [no / yes (explain)] Not Available
- After successful mating each pregnant female was caged (how): Not Available
- Any other deviations from standard protocol: Not Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purity of samples was checked by direct injection onto a flame ionization gas chromatograph.
Duration of treatment / exposure:
10 days
Frequency of treatment:
Daily, on Day 6 and up to and including Day 15 of pregnancy.
Details on study schedule:
Test commenced on Day 6 of pregnancy of rats and continue till day 20 of pregnancy i.e. day of sacrifice.
Remarks:
Doses / Concentrations:
0, 0.14, 0.43 or 1.40 ml/kg/day = 140 mg/Kg bw, 430 mg/kg bw or 1400 mg/kg bw per day
Basis:
nominal conc.
No. of animals per sex per dose:
120 females
0 mg/kg: 25 females
140 mg/kg: 35 females
430 mg/kg: 25 females
1400 mg/kg: 35 females
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Clinical signs
All animals were regularly handled and observed daily for obvious changes or signs of reaction to treatment. Any local skin irritation resulting from treatment was scored on each day of the dosing period on a numerical basis.

Food consumption:
Food consumption was measured from weighday to weighday.

Body weights:
All rats were weighed initially (=Day 1 of gestation) and on Days 3 and 6, thereafter, on alternate days through to Day 20 of pregnancy.

Test substance intake:
The test chemical was applied dermally.

Reproductive function: estrous cycle
No data available

Reproductive function: sperm measures
No data available

Reproductive performance:
The ovaries and uteri were examined immediately to determine the number of corpora lutea, the number and distribution of live young, the number and distribution of embryonic/fetal deaths and individual fetal weight from which the litter weight was calculated.

Organ weights:
The liver and kidneys of 15 control and 13 high dose rats were weighed.

Gross pathology:
On Day 20 of pregnancy the animals were killed, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs.

Histopathology:
The liver and kidneys of 15 control and 13 high dose rats were investigated by histological examination.

Other findings:
Hematology:
On the morning of sacrifice, samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of 15 control and 13 high dose rats (as two high dose rats in this batch had already died).
Parameters examined:
Packed cell volume (PCV), Haemoglobin (Hb), Red cells count (RDC), Mean corpuacular haemogiobin concentration (MCHC), Mean corpuscular volume, Total white cell count (WBC Total), Platelet count (PLTS), Differential WBC counts and Cell morphology.

Clinical chemistry:
On the morning of sacrifice, samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of 15 control and 13 high dose rats (as two high dose rats in this batch had already died).
Parameters examined:
Total Protein, Albumin (Alb), Globulin (Glob), Urea nitrogen (Urea N),Creatinine (Creat), sodium (Na), Potassium (K), Calcium (Ca), Inorganic phosphorus (P), Chloride (Cl), Cholesterol (Chol), Glucose, Alkaline phosphatese (AP), Glutamic-pyruvic transaminase (GPT) and Glutamic-oxaloacatic transaminase (GOT).
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
The ovaries and uteri were examined immediately to determine:
(a) number of corpora lutea
(b) number and distribution of live young
(c) number and distribution of embryonic/fetal deaths
(d) individual foetal weight from which the litter weight was calculated
(e) fetal abnormalities
Postmortem examinations (parental animals):
- Sacrifice on gestation day 20 (Any animals that died or were killed for humane reasons were weighed and subjected to post mortem examination.)
- Organs examined: The liver and kidneys of 15 control and 13 high dose rats were weighed and preserved using the same rats as used for the laboratory investigations . The organs were preserved in 10% buffered formalin against the contingency of subsequent histological examination
Postmortem examinations (offspring):
Half the pups in each litter were preserved in Bouin's solution for subsequent free-hand sectioning to discover visceral abnormalities (Wilson technique), the remainder were fixed in 74 OP industrial methylated spirit for subsequent macroscopic examination, evisceration and determination of sex prior to clearing and alizarin staining (modified Dawson technique) for skeletal examination .
Statistics:
Intergroup differences regarding embryonic deaths, mean foetal weight etc were compared with controls using Kruskal-Wallis test and Fisher’s exact test. Differences between groups were analyzed by ANOVA followed by t-test.
Reproductive indices:
Pup Survival Index
Offspring viability indices:
Resorption Index, Litter survival
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). Signs started to develop on about Day 12 of pregnancy, six days after start of treatment and escalated during the remainder of the dosing period. Other less frequently observed signs included suspected blood on the undercage tray paper, slight edema in the dosing area and perineal staining.
Towards the end of the dosing period a brownish colored deposit became apparent in the dosing area of all the highest dosage group animals.
With the exception of a single non-pregnant animal treated with 430 mg/kg/day which had a hunched posture and was walking on its toss on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage.
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Subsequent mean weight gain of rats treated with 430 or 1400 mg/kg/day did not show any substantial difference from control throughout the remainder of the study. In contrast, mean weight gain of animals treated at 1400 mg/kg/day was markedly retarded. Although substantial recovery of body weight occurred after dosing ceased, parity with the control group was not regained and mean bodyweight at termination was noticeably depressed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mean ovulation rate (as assessed by corpora lutea count) and mean pre-implantation loss of the treatment groups compared favorably with control, resulting in higher mean implantation rates. The overall pregnancy rate was similar for all groups.
Clinical signs:
Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). Signs started to develop on about Day 12 of pregnancy, six days after start of treatment and escalated during the remainder of the dosing period. Other less frequently observed signs included suspected blood on the undercage tray paper, slight edema in the dosing area and perineal staining.
Towards the end of the dosing period a brownish colored deposit became apparent in the dosing area of all the highest dosage group animals.
With the exception of a single non-pregnant animal treated with 430 mg/kg/day which had a hunched posture and was walking on its toss on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage.

Body weights:
Subsequent mean weight gain of rats treated with 430 or 1400 mg/kg/day did not show any substantial difference from control throughout the remainder of the study. In contrast, mean weight gain of animals treated at 1400 mg/kg/day was markedly retarded. Although substantial recovery of body weight occurred after dosing ceased, parity with the control group was not regained and mean bodyweight at termination was noticeably depressed.
Food consumption:
Food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment.
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: F0: Female F1. Male/Female No effects observed.
Remarks on result:
other: Generation: F0 & F1 (migrated information)
Dose descriptor:
LOAEL
Effect level:
1 400 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Body weight, hematology and level of embryo-fetal deaths.
Critical effects observed:
yes
Lowest effective dose / conc.:
1 400 mg/kg bw/day (nominal)
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1400 mg/kg/day, the incidence of embryo-fetal deaths was significantly increased and extended to total litter loss in 5/23 litters. Death predominantly occurred early in pregnancy.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight: At 1400 mg/kg/day, there was a consequent significant reduction in litter size and in litter weight. Reduction in the litter weight was further enhanced by significantly lower mean weight of surviving fetuses.
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, treatment-related
Description (incidence and severity):
At 1400 mg/kg/day, morphological change was observed in 160 of the 161 fetuses. A variety of skeletal and soft tissue changes were observed and the degree of change varied between individuals, including anoph-thalmia/micro-phthalmia, ventricular septal defects, and defects of the thoracic ribs and occurrence of cervical rib(s).
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
The NOAEL for the test chemical was observed to be 140 mg/kg bw/day on the rats.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
140 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
Remarks on result:
other: No toxic effects observed
Critical effects observed:
not specified
Lowest effective dose / conc.:
1 400 mg/kg bw/day (nominal)
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 400 mg/kg bw/day (nominal)
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Dose Effect on Parents (mg/kg/day):

 

Category

Number of animals in groups

 

1

(Control)

2

(Dose 140)

3

(Dose 430)

4

(Dose 1400)

Mated

25

35

25

35

Non-Pregnant

4

1

3

5

Total resorption

0

0

0

5

Killed

 

 

 

1

Died

 

 

 

2

With Live young day 20

21

30

22

18

With live young day 15

0

4

0

4

Conclusions:
LOAEL was considered to be 1400 mg/kg/day for the maternal generation, while NOAEL was considered to be 140 mg/kg/day for both the maternal generation and the F1 generation after exposure to phenylethyl alcohol dermally.
Executive summary:

In a developmental and reprotoxicity study, the toxic effect of phenylethyl alcohol were evaluated in time-mated female CrL:COBS CD (SD) BR rats. The test chemical was administered dermally at a dosage of 0, 140, 430 or 1400 mg/kg per day during days 6-15 of pregnancy. In emergence of other data, according to Api et.al. (RIFM fragrance ingredient safety assessment, alpha-methylbenzyl alcohol, CAS Registry number 98 -85 -1) systemic absorption of the test chemical was assumed to be 100% through oral, dermal and inhalation route.

The study was terminated and the animals killed at day 20 of gestation. Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). With the exception of a single non-pregnant animal treated with 430 mg/kg/day which had a hunched posture and was walking on its toes on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage. Body weight and food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment. Subsequent mean weight gain of rats treated with 430 or 1400 ml/kg/day did not show any substantial difference from control. The results from the hematology showed a higher value for white cell count in animals treated with 1400 mg/kg/day. Despite the marked clinical response observed at 1400 mg/kg/day, there were no findings in gross pathology amongst surviving animals in any group at termination which were considered to be related to treatment. When investigating the litters from females treated with 1400 mg/kg/day, the incidence of embryo-fetal deaths was significantly increased and extended to total litter loss in 5/23 litters. Death predominantly occurred early in pregnancy. There was also a consequent significant reduction in litter size and in litter weight. Reduction in the litter weight was further enhanced by significantly lower mean weight of surviving fetuses. In addition, morphological change was observed in 160 of the 161 fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals. No such changes of results were observed in litters from females treated with 140 or 430 mg/kg/day. Therefore, LOAEL was considered to be 1400 mg/kg/day for the maternal generation, while NOAEL was considered to be 140 mg/kg/day for the maternal generation and the F1 generation after exposure to phenylethyl alcohol.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
4.3 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is Klimicsh 2 and from authoritative database
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is Klimicsh 2 and from authoritative database
Additional information

Reproductive toxicity study

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical.The studies are as mentioned below:

Via oral route :

Study1.

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 material 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 item 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 methyl phenylacetate (CAS 101-41-7).

Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material in Wistar Rats with 14 days recovery, where in 0, 308, 556 and 1000 mg/kg body weight, doses were tested; No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with methyl phenylacetate (CAS 101-41-7)orally.

Study2.

Reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed on Crl:COBSCD(SD)BR female rats. The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet in dose concentration 0, 1000, 3000 or 10,000 ppm (calculated doses were 83, 266 and 799 mg/kg for the respective exposure groups). The concentrations tested were based on a pilot study in which dietary inclusion of up to 9000 ppm test material was well tolerated in pregnant rats and did not result in unacceptable loss of diet palatability. High performance liquid chromatographic (HPLC) analyses were used to validate concentration, homogeneity and stability of test material throughout the study. 28 female rats per dose group were exposed to test material on GDs 6 through 15, In all the animals Feed consumption was measured daily, and body weights were taken on GDs 1, 3, 6 and then on alternate days until necropsy on GD 20, when in utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for external, soft tissue and skeletal alterations (malformations and variations).

No deaths occurred during the study.No obvious clinical signs of toxicity was noted. No effects were observed at 83mg/kg and 266mg/kg dose group . At 799mg/kg dose group , reduced maternal feed consumption and slight weight loss occurred on treatment days 1 and 2 (GDs 6–7, 7–8). Calculated mean group intake of test material was 0, 83, 266 and 799 mg/kg/day for the pregnant rats in the 0, 1000, 3000 and 10,000 ppm groups, respectively. mean values for embryo-fetal loss, litter sizes, sex ratios were equivalent in control and test group also the mean fetal body weights, was equivalent in control and test groups.The number, type and distribution of fetal malformations, anomalies and skeletal variations also were unaffected by maternal exposure to test material at concentrationsas high as 10,000 ppm (799 mg/kg/day), although at799mg/kg dose group there was a possible marginal delay in fetal ossification(a slightly higher incidence of fetuses with incomplete ossification of some sites, such as the sacro-caudal vertebral arches).Hence No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 799mg/kg. When female rats were treated with  2-phenylethan-1-ol CAS 60-12 -8 by orally for GDs 6 through 15

Study 3.

The reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed on pregnant Long- Evans rats. The aqueous suspension of test material was administered by gavage at dose concentration 4.3, 43 and 432 mg/kg to rats on GDs 6 through 15. All doses were administered at a volume of 20 mL/kg,Group sizeswere 19, 7, 7 and 5 pregnant rats in the control (distilled water),4.3, 43 and 432 mg/kg dose groups, respectively. Doses were selected to be 0.24, 2.4 and 24% of the LD50 value published by Owston et al. (1981).The dams were observed daily for signs of toxicity and lethality. Body weights were recorded on GDs 0 through 15 and 20. All dams were overdosed with ether on GD 20, and their fetuses were delivered by Cesarean-section. Observations were made for uterine weight, litter weight, individual pup weight, and numbers of live and dead pups, resorptions, implantations, sex distribution, crown-rump length and corpora lutea. All live pups were examined for gross external malformations. As report that 224, 51, 65 and 60 fetuses in the four respective dose groups were evaluated for both soft tissue and skeletal examinations. It is unclear how this number was attained, because soft tissue evaluation was by sectioning (Wilson’s method), precluding skeletal evaluation of the same specimen (alcian blue-alizarin red S-staining).

Severe maternal intoxication was observed at the 432 mg/kg dose group immediately after administration and persisted overnight on each day of treatment. No clinical effects were observed at the 4.3 and 43 mg/kg doses but reduced weight gain was evident at the 43 mg/kg dose. Mean litter size was reduced at the 43 and 430 mg/kg dose levels .Fetal growth retardation (reduced body weight and crown-rump length) was observed at the 4.3 and 432 mg/kg doses, with runting (body weight <2.7 g) occurring in 5 of 30, 0 of 61 and 32 of 51 fetuses at the 0.43, 43 and 432 mg/ kg dose levels, respectively. This absence of dose-dependency also was probably related to the relatively few litters evaluated and thepattern of postimplantation loss (resorption)

The percentage of dead or malformed fetuses per total implants was 55%, 97% and 100% at 4.3, 43 and 432 mg/kg, respectively. A dose–related increase in malformations was reported, although how the number of fetuses affected was determined is unclear, because the number of fetuses evaluated by each methodology and the total number of fetuses available for evaluation were not provided. Reduced cranial- bone ossification, a variant, was observed at a low incidence (0 of 224, 7 of 60, 2 of 65 and 6 of 51 fetuses in the 0, 0.43, 43 and 432 mg/kg dose groups, respectively). Additional retardations of ossification of the limbs, ribs and tail were evident at the 43 and 432 mg/kg dose levels. Microphthalmia (small eyes) and hydronephrosis (enlarged renal pelvis) occurred at 0.43, 4.3 and 432 mg/kg doses. Additional malformations included open eyes, micromelia (small paw) and a low incidence of heart defects at the 43 and 432 mg/kg doses. The 432 mg/kg dose was also associated with spina bifida (open spinal column), phocomelia (short limb), sirenomelia (joined limbs), micrognathia (short jaw) and webfoot/club foot. As there were effects at all three dose levels, a NOAEL could not be determined from this study and is presumed to be < 4.3 mg/kg

Study4.

The reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed onpregnant rats.Test material in sunflower oil was administered to pregnant rats (10 per group) by gavage on GD 4 (preimplantation) or GDs 10 through 12 (during embryogenesis) in dose concentrati as a single dose of 508 mg/kg.GD 1 was defined as the day sperm were observed in a vaginal smear. On GD 20, the female rats were euthanized, and the fetuses were removed for further analysis (Wilson’s sectioning or alizarin red S-staining). Fetal viability and body weight were unaffected. No adverse effect on fetal ossification occurred when test material was administered preimplantation. Small delays in some bone ossification sites occurred in test material treated fetuses, as compared with controls, when the test material was administered during the period of organogenesis, observations compatible with the small but biologically unimportant reduction in fetal body that also occurred in these fetuses. No other effects were observed .Hence LOAEL for maternal and developmental toxicity was considered to be 508mg/kg bw . When female rats were treated with 2-phenylethan-1-ol CAS 60-12-8 orally.

Via dermal route :

Study 5

In a developmental and reprotoxicity study, the toxic effect of phenylethyl alcohol were evaluated in time-mated female CrL:COBS CD (SD) BR rats. The test chemical was administered dermally at a dosage of 0, 140, 430 or 1400 mg/kg per day during days 6-15 of pregnancy. In emergence of other data, according to Api et.al. (RIFM fragrance ingredient safety assessment, alpha-methylbenzyl alcohol, CAS Registry number 98 -85 -1) systemic absorption of the test chemical was assumed to be 100% through oral, dermal and inhalation route.

The study was terminated and the animals killed at day 20 of gestation. Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). With the exception of a single non-pregnant animal treated with 430 mg/kg/day which had a hunched posture and was walking on its toes on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage. Body weight and food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment. Subsequent mean weight gain of rats treated with 430 or 1400 ml/kg/day did not show any substantial difference from control. The results from the hematology showed a higher value for white cell count in animals treated with 1400 mg/kg/day. Despite the marked clinical response observed at 1400 mg/kg/day, there were no findings in gross pathology amongst surviving animals in any group at termination which were considered to be related to treatment. When investigating the litters from females treated with 1400 mg/kg/day, the incidence of embryo-fetal deaths was significantly increased and extended to total litter loss in 5/23 litters. Death predominantly occurred early in pregnancy. There was also a consequent significant reduction in litter size and in litter weight. Reduction in the litter weight was further enhanced by significantly lower mean weight of surviving fetuses. In addition, morphological change was observed in 160 of the 161 fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals. No such changes of results were observed in litters from females treated with 140 or 430 mg/kg/day. Therefore, LOAEL was considered to be 1400 mg/kg/day for the maternal generation, while NOAEL was considered to be 140 mg/kg/day for the maternal generation and the F1 generation after exposure to phenylethyl alcohol.

Study 6

Reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed onpresumed pregnant Crl:COBS CD(SD) BR female rats.10 females were used in each dose group.The test material in unchanged from in dose concentration0.07, 0.14, 0.28, 0.43, or 0.70 mL/kg (70, 140, 280, 430 and 700 mg/kg, respectively) was applied topically on GDs 6 through 15. The application sites were covered with aluminum foil, held in place by an adhesive bandage.Feed and body weights were recorded daily, as well as daily clinical observations. The animals were euthanized on GD 20 and in utero parameters were assessed on the basis of litter values. All live fetuses were examined for gross external alterations; approximately one-third of these live fetuses were also examined for visceral alterations, using a dissection technique. All live fetuses, rather than only ½ of the fetuses, as in the previous evaluation, were examined for skeletal alterations and number of ossification sites after staining with alizarin red S.

No deaths occurred during the study. All doses from 70 to 700 mg/kg/day (0.07 to 0.70 mL/kg) resulted in low levels of erythema and/or desquamation at the application site. No other signs of maternal toxicity were observed at doses of 70 to 430 mg/kg (0.07 to 0.43 mL/kg). At 700 mg/kg (0.7 mL/kg), ptosis and/or urine stained abdominal fur were also observed, as well as reduced feed consumption and body weight gain. No adverse effects on mean live litter sizes or resorptions were seen at doses up to 700 mg/kg (0.7 mL/kg). Dose-dependent significant reductions in fetal body weight were observed at 140 mg/kg (0.14 mL/kg) and higher doses, accompanied by statistically significant increases in reversible delays in ossification. HenceNo Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 70 mg/kg.When femalerats were treated with 2-phenylethan-1-ol CAS 60-12-8by dermal application for GDs 6 through 15.

 Study 7

The reproductive and developmental toxicty study of 2-phenylethan-1-ol CAS 60-12-8 was performed in time-mated Crl:COBSCD(SD) BR female rats. The test material in dose concentration 0.14, 0.43 and 1.40 mL/kg (140, 430 and 1400 mg/kg, respectively) were applied daily to the shaved backs of the rats on GDs 6 through 15. To prevent possible oral ingestion of the control (water) or phenylethyl alcohol, the dermal application sites were occluded by covering with aluminum foil that was held in place by a porous adhesive bandage. Observations were made for signs of toxicity, mortality, body weight and feed consumption (GDs 1, 3, 6 and alternate days until necropsy on GD 20). Liver and kidney weights and hematology and serum chemistry parameters were also determined in 15 control and 13 high dose rats on GD 20. In utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for gross external (all live fetuses), soft tissue (1/2 of the fetuses per litter, using Wilson’s free-hand dissection method) and skeletal changes (1/2 of the fetuses per litter, following alizarin red S-staining).

Mortality (1 moribund euthanasia on GD 11 and 2 deaths on GD 13) were observed almost entirely at the 1400 mg/kg dose. Maternal toxicity was observed as local irritation at the application site, especially in the 1400mg/kg dose group. Irritability,hunched posture, walking on toes, piloerection, periorbital staining. were observed almost entirely at the 1400 mg/kg dose.These signs started around GD 12, became progressively worse during the remainder of the treatment period (until GD15), and essentially resolved during the 5 days of no treatment (GD15 to GD 20). Mean feed consumption and body weight gains weremarkedly reduced at the 1400 mg/kg dose. Liver and kidney weights, and necropsy observations were unaffected. pregnancy incidences were unaffected. Developmental toxicity included statistically significant increases in early resorptions, at the1400 mg/kg dose. At the 140 mg/kg dose (0.14 mL/kg), no maternal toxicity was observed.

A related reduction in mean litter sizes and a marked reduction in mean fetal weight at the 1400 mg/kg dose. At the 140 mg/kg dose (0.14 mL/kg), litter parameters were comparable to control, with the exception of a marginally higher than control incidence of skeletal changes (rudimentary cervical ribs and thoracic vertebral irregularities). These observations showed dose dependent increases at 430 mg/kg (0.43 mL/kg) and 1400 mg/kg ,(1.40 mL/kg) doses. Additional findings at 430 mg/kg consisted of occasional fetuses with soft tissue changes similar to those observed at 1400 mg/kg, as well as moderate degrees of incomplete ossification. At 1400 mg/kg, maternal toxicity, significantly increased embryo-fetal death (resorption), with associated reduced litter size, and significantly reduced fetal body weight were noted. Many morphological changes occurred in 160/161 fetuses at this dose. More than 40% of the fetuses and 70% of the litters had anophthalmia or microphthalmia, ventricular septal defects, defects or irregularities affecting the thoracic, lumbar and sacrocaudal vertebrae, which were associated with short or kinky tail and defects of the thoracic ribs.Hence the maternal NOAEL was considered to be 43 mg/kg (0.43 mL/kg) and the developmental NOAEL was considered to be approximately 140 mg/kg (0.14 mL/kg).When rats were treated with 2-phenylethan-1-ol CAS 60-12-8 via dermal application.

Study 8

The reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed on presumed pregnant female rats.Undiluted test material in dose concentration 140, 430, or 1400 mg/ kg/day (0.14, 0.43, or 1.4 ml/kg) or distilled water (1.4 ml/kg, vehicle control). were applied percutaneously to clipped skin on the back (5 X7 cm), occluded with foil, and secured with micropore tape. After approximately 24 h, the test sites were rinsed and dried with gauze. The rats were also fitted with Elizabethan collars to prevent oral ingestion. One hundred female rats were dosed once daily on gestational days 7 through 20 (GD 7–20). Twenty rats from each dosage group were selected for Cesarean-sectioning on GD 21 and the remaining rats were selected for natural delivery.

 At 1400 mg/kg/day, mortality was significantly increased in comparison to the vehicle control group the deaths were attributed to treatment with test material in rats assigned to Cesarean-sectioning group while rats assigned to natural delivery group At 1400 mg/kg/day, one rat was found dead on DG 13. This death was presumed related to treatment with test material because: (1) similar events occurred in the rats assigned to Cesarean- sectioned; and (2) the observation occurred in the high dosage group.

primary irritancy including flaking and/or erythema, occurred in female rats percutaneously administered 430 and 1400 mg/kg/day during the gestation period. The onset and severity of the skin reactions was, in general, dependent on the dosage of test material . The number of rats with adverse clinical signs was significantly increased at 1400 mg/kg/day in comparison to the vehicle control group in rats assigned to Cesarean-sectioning group while rats assigned to natural delivery group similar clinical signs like primary irritancy (erythema, edema, and/or flaking) occurred during the gestation period in all dosage groups. The onset and severity of the skin reactions was, in general, dependent on the dosage of test material .The number of rats with adverse clinical signs was significantly increased at 1400 mg/kg/day in comparison to the vehicle control group.

 A statistically significant loss in body weight, coupled with reduced or significantly reduced feed consumption values, occurred at 1400 mg/kg/day for the entire dosage period, as compared to the vehicle control group values in rats assigned to Cesarean-sectioning group while rats assigned to natural delivery group body weight gains were significantly reduced for the entire gestation dosage period and the entire gestation period, as compared to the vehicle control group values. In addition, body weight gains were significantly reduced in the 430 and 1400 mg/kg/day dosage groups on Days 1–4 of lactation. Thereafter, body weight gains were comparable among the dosage groups.

Absolute and relative feed consumption values were reduced or significantly reduced at 1400 mg/kg/day on DGs 7–21, in comparison to the vehicle control group values. Corresponding to reductions in body weight gain, absolute and relative feed consumption values were also reduced at 1400 mg/kg/day dosage group on DLs 1–4 and overall for DLs 1–14, in comparison to the vehicle control group values.

Rats assigned to Cesarean-sectioning Pregnancy occurred in 16 to 20 rats in each dosage group. As a result of the increased mortality that occurred in the 1400 mg/kg/ day dosage group, Cesarean-sectioning observations on DG 21 were based on 20, 18, 20 and 9 pregnant rats in Groups I through IV, respectively. At 1400 mg/kg/day, postimplantation loss was increased or significantly increased, in comparison to the vehicle control group values. Seven of the nine rats in this dosage group had total litter loss (i.e., 100% resorbed conceptuses). These increases in postimplantation loss resulted in an overall significant reduction in the averages for litter size and live fetuses at 1400 mg/kg/day, in comparison to the vehicle control group values (1.3 per liter vs. 14.6 per litter in vehicle controls).While Rats assigned to natural delivery Pregnancy occurred in 16–20 female rats in the four dosage groups. All pregnant dams at 140 and 430 mg/kg/day delivered litters, 8 of the 16 pregnant rats delivered a litter at 1400 mg/kg/day. Of these 8 dams, one had a litter with no liveborn pups and four had all pups die before day 4 postpartum. At 1400 mg/kg/day, the duration of gestation was significantly increased and the gestation index was significantly reduced. The averages for the total number of pups delivered and the number of liveborn pups were reduced or significantly reduced in this same dosage group, in comparison to the vehicle control group values. At 1400 mg/kg/day, a significant number of litters had pups with mild dehydration (based on skin turgor), a thread-like tail and that were not nursing. The percentage of pups that died or were presumed cannibalized on day 1 and days 2–4 postpartum was significantly increased at 1400 mg/kg/day, as compared to the vehicle control group values. Reflecting this increase in pup mortality, the viability index at 1400 mg/kg/day was significantly reduced relative to the vehicle control group value (63.2% vs. 99.0% in vehicle controls). The average number of surviving pups per litter was significantly reduced at 1400 mg/kg/day on days 4, 7, 14 and 21 postpartum, in comparison to the vehicle control group values.

 Rats assigned to Cesarean-sectioning At 430 mg/kg/day, the average fetal body weight was significantly lower than the concurrent vehicle control group values. These values were also below the range observed historically at the Testing Facility Rats assigned to natural delivery the average pup weight per litter was significantly lower at 1400 mg/kg/day on day 1 postpartum relative to the vehicle control group value. On postpartum days 4, 7 and 21, pup body weights at 1400 mg/kg/day were 9%, 10% and 7% lower than the corresponding vehicle control group values.

Rats assigned to Cesarean-sectioning all 12 fetuses in the two litters that had live fetuses at 1400 mg/kg/day had one or more gross, soft tissue and/or skeletal alterations. The limited number of fetuses and litters with live fetuses compared to the other dosage groups resulted in significantly increased fetal and litter incidences of each of these alterations. At 1400 mg/kg/day, the average number of ossification sites per fetus per litter was significantly reduced for caudal vertebrae, sternal centra, xiphoid, metacarpals, phalanges, metatarsals and phalanges. Corresponding to the reduction in fetal weight in the 430 mg/kg/day dosage group, ossification site averages for caudal vertebrae, fore- and hindlimb phalanges and metatarsals were also significantly reduced and below the historical control range for this Testing Facility. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased at 430 mg/kg/day, as compared to the vehicle control group values.However, all apparent delays in ossification and increased numbers of cervical ribs that were observed in the Cesarean-delivered fetuses in the 430 mg/kg/day were resolved by DL 21. Hence The maternal no-observable-adverse-effect-level (NOAEL) was considered to be 430 mg/kg/day due to mortality and reductions in body weight and feed consumption at 1400 mg/kg/day. The developmental NOAEL was considered to be 140 mg/kg/day. When female rats were treated with 2-phenylethan-1-ol CAS 60-12-8via dermal application.

Thus, Based on the data available from different studies for target chemical 1-phenylethanol (98-85-1)and its structuraly similar read across substance 2-phenylethan-1-ol CAS 60-12-8 / EC 200-456-2)andMethyl phenyl acetate; methyl phenylacetate (CAS 101-41-7) via oral and dermal route,NOAEL for 1-phenylethanol (98-85-1)was considered to be in range of 4.3 -556 mg/kg bw/day for F0, F1 generation but as adverse effects like markedly decreased pregnancy index / fertility index, In addition, morphological change was observed in fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals at different dose group. Thus, comparing this value with the criteria of CLP regulation 1-phenylethanol (98-85-1)is likely to classify as reproductive and developmental toxicant.

 

Effects on developmental toxicity

Description of key information

Developmental toxicity study

Based on the data available from different studies for target chemical 1-phenylethanol (98-85-1)and its structuraly similar read across substance 2-phenylethan-1-ol CAS 60-12-8 / EC 200-456-2)andMethyl phenyl acetate; methyl phenylacetate (CAS 101-41-7) via oral and dermal route,LOAEL for 1-phenylethanol (98-85-1)was considered to be in range of 4.3 -556 mg/kg bw/day for F0, F1 generation but as adverse effects like markedly decreased pregnancy index / fertility index, In addition, morphological change was observed in fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals at different dose group. Thus, comparing this value with the criteria of CLP regulation 1-phenylethanol (98-85-1)is likely to classify as reproductive and developmental toxicant.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from NTP report.
Qualifier:
according to guideline
Guideline:
other: refer below principle
Principles of method if other than guideline:
The study was performed to evaluate carcinogenic, reproductive and developmental effect of phenylethyl alcohol in rats.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
No data
Species:
rat
Strain:
other: CrL.3 COBS CD (SD) BR strain
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited.
- Age at study initiation: No data available
- Weight at study initiation: 170 - 242 g (group average range)
- Fasting period before study: No data available
- Housing: Animals were housed individually in a controlled environment in suspended galvanised metal cages equipped with solid sides and back, wire mesh front, floor and top.
- Diet (e.g. ad libitum): S.F. Laboratory Diet No 1, ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimatization period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2°C
- Humidity (%):53 ± 11%
- Air changes (per hr): 13 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: To: No data available
Route of administration:
dermal
Type of inhalation exposure (if applicable):
not specified
Vehicle:
unchanged (no vehicle)
Remarks on MMAD:
No data
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): Water
- Concentration in vehicle: 0, 140, 430 or 1400 mg/kg
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
-According to A.M. Api et.al. (RIFM fragrance ingredient safety assessment, α-methylbenzyl alcohol, CAS registry number 98-85-1) systemic absorption was assumed to be 100% by oral, dermal and inhalation route.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purity of samples from each of the four different sources was checked by direct injection onto a flame ionization gas chromatography. A Fluka standard of 99.5% was used as a reference.
.
Details on mating procedure:
- M/F ratio per cage: Time-mated rats were delivered from the vendor.
- Length of cohabitation: Not Available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: The day of mating, as judged by the appearance of sperm in the vaginal smear or by the presence of a vaginal plug was considered as Day 0 of pregnancy.
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility.:No data
- Further matings after two unsuccessful attempts: [no / yes (explain)]:No data
- After successful mating each pregnant female was caged (how):No data
- Any other deviations from standard protocol:No data
Duration of treatment / exposure:
15 days, i.e. from Day 6 of gestation until sacrifice on Day 20 or gestation.
Frequency of treatment:
Daily, on Day 6 and up to and including Day 15 of pregnancy.
Duration of test:
15 days
Remarks:
Doses / Concentrations:
0, 0.14, 0.43 or 1.40 ml/kg/day = 0, 140, 430 or 1400 mg/kg bw
Basis:nominal conc.
No. of animals per sex per dose:
120 females
0 mg/kg: 25 females
140 mg/kg: 35 females
430 mg/kg: 25 females
1400 mg/kg: 35 females
Control animals:
yes, concurrent vehicle
Details on study design:
Further details on study design
- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): The 120 animals were assigned to four groups by computerised stratified randomisation to give approximately equal initial mean bodyweights
- Other: No data available
Maternal examinations:
Clinical signs
All animals were regularly handled and observed daily for obvious changes or signs of reaction to treatment. Any local skin irritation resulting from treatment was scored on each day of the dosing period on a numerical basis.

Mortality:
Any animals that died or were killed for humane reasons were weighed and subjected to post mortem examination.

Food consumption:
Food consumption was measured from weighday to weighday.

Body weights:
All rats were weighed initially (=Day 1 of gestation) and on Days 3 and 6, thereafter, on alternate days through to Day 20 of pregnancy.

Hematology:
On the morning of sacrifice, samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of 15 control and 13 high dose rats (as two high dose rats in this batch had already died).
Parameters examined:
Packed cell volume (PCV), Haemoglobin (Hb), Red call count (RDC), Mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume, Total white cell count (WBC Total), Platelet count (PLTS), Differential WBC counts and Cell morphology.

Clinical chemistry:
On the morning of sacrifice, samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of 15 control and 13 high dose rats (as two high dose rats in this batch had already died).
Parameters examined:
Total Protein, Albumin (Alb), Globulin (Glob), Urea nitrogen (Urea N),Creatinine (Creat), sodium (Na), Potassium (K), Calcium (Ca), Inorganic phosphorus (P), Chloride (Cl), Cholesterol (Chol), Glucose, Alkaline phosphatese (AP), Glutamic-pyruvic transaminase (GPT) and Glutamic-oxaloacatic transaminase (GOT).

Gross pathology:
On Day 20 of pregnancy the animals were killed, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs. The liver and kidneys of 15 control and 13 high dose rats were weighed.

Histopathology:
The liver and kidneys of 15 control and 13 high dose rats were investigated by histological examination.
Ovaries and uterine content:
The ovaries and uteri were examined immediately to determine the number of corpora lutea, the number and distribution of live young, the number and distribution of embryonic/fetal deaths and individual fetal weight from which the litter weight was calculated.
Fetal examinations:
The fetuses were weighed and was examined externally, and was examined for fetal abnormalities and malfunctions as well as for skeletal examinations.
Statistics:
Statistical analysis of incidences of abnormalities was considered unnecessary given the results obtained.

Statistical analyses were, however, performed routinely on litter data, using the litter as the basic sample unit and non-parametric tests (Jonckheere and Kruskal-Wallis) as these values rarely follow a 'normal' distritution. Analysis of covariance followed by William’s test was used for assessing intergroup differencas in mean organ weights. The Kruskal-Wallis test was also used to analyse intergroup differences in the results of the Biochemical and Haematological examinations.
Indices:
Not Available
Historical control data:
Not Available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). Signs started to develop on about Day 12 of pregnancy, six days after start of treatment and escalated during the remainder of the dosing period. Other less frequently observed signs included suspected blood on the undercage tray paper, slight edema in the dosing area and perineal staining.

Towards the end of the dosing period a brownish colored deposit became apparent in the dosing area of all the highest dosage group animals.

With the exception of a single non-pregnant animal treated with 430mg/kg/day which had a hunched posture and was walking on its toss on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
At 1400 mg/kq/day, two animals were found dead on Day 13 of pregnancy (seven days after start of treatment) and one had already been killed on Day 12 due to moribund condition. All three showed signs of reaction typical for this group. In addition, the latter animal had shown rapid respiration, blood in the urine and was apparently unable to walk. At autopsy, however, the only macroscopic observation appeared to be slight congestion of the dorsal subcutis of one of the animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Subsequent mean weight gain of rats treated with 430 or 1400 mg/kg/day did not show any substantial difference from control throughout the remainder of the study. In contrast, mean weight gain of animals treated at 1400 mg/kg/day was markedly retarded. Although substantial recovery of body weight occurred after dosing ceased, parity with the control group was not regained and mean bodyweight at termination was noticeably depressed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A higher value for white cell count were observed in animals treated with 1400 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
As compared to control, lower levels in urea nitrogen, creatinine, GPT, GOT and alkaline phosphatase were observed when animals were treated with 1400 mg/kg/day.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between control and test groups in respect of liver and kidney weights adjusted for differences in body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Despite the marked clinical response observed at 1400 mg/kg/day, there were no findings amongst surviving animals in any group at termination which were considered to be related to treatment.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:Yes
Details on maternal toxic effects:

Clinical signs: Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). Signs started to develop on about Day 12 of pregnancy, six days after start of treatment and escalated during the remainder of the dosing period. Other less frequently observed signs included suspected blood on the undercage tray paper, slight edema in the dosing area and perineal staining.Towards the end of the dosing period a brownish colored deposit became apparent in the dosing area of all the highest dosage group animals. With the exception of a single non-pregnant animal treated with 430mg/kg/day which had a hunched posture and was walking on its toss on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage.

Mortality: At 1400 mg/kq/day, two animals were found dead on Day 13 of pregnancy (seven days after start of treatment) and one had already been killed on Day 12 due to poor condition. All three showed signs of reaction typical for this group. In addition, the latter animal had shown rapid respiration, blood in the urine and was apparently unable to walk. At autopsy, however, the only macroscopic observation appeared to be slight congestion of the dorsal subcutis of one of the animals.

Food consumption: Food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment.

Body weights: Subsequent mean weight gain of rats treated with 430 or 1400 mg/kg/day did not show any substantial difference from control throughout the remainder of the study. In contrast, mean weight gain of animals treated at 1400 mg/kg/day was markedly retarded. Although substantial recovery of body weight occurred after dosing ceased, parity with the control group was not regained and mean bodyweight at termination was noticeably depressed.

Hematology: A higher value for white cell count were observed in animals treated with 1400 mg/kg/day.

Clinical chemistry: As compared to control, lower levels in urea nitrogen, creatinine, GPT, GOT and alkaline phosphatase were observed when animals were treated with 1400 mg/kg/day.

Organ weights: There were no statistically significant differences between control and test groups in respect of liver and kidney weights adjusted for differences in body weight.

Gross pathology: Despite the marked clinical response observed at 1400 mg/kg/day, there were no findings amongst surviving animals in any group at termination which were considered to be related to treatment.

Histopathology: No data available
Dose descriptor:
LOAEL
Effect level:
1 400 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: toxic effects observed
Dose descriptor:
NOAEL
Effect level:
430 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
other: Maternal toxicity
Remarks on result:
other: No developmental toxic effects observed
Abnormalities:
not specified
Localisation:
not specified
Fetal body weight changes:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean values for litter weight and mean fetal weight were essentially comparable with those of controls in dose group 140 or 430 mg/kg/day but There was a consequent significant reduction in litter weight. Reduction in the litter weight was further enhanced by significantly lower mean weight of surviving fetuses in dose group 1400mg/kg /day
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
After treatment with 140 or 430 mg/kg/day:There were no instances of total litter loss at either dosage and the overall incidence of embryo-fetal deaths was similar to, or below, that of the control group.
There was a consequent significant reduction in litter size at dose 1400mg/kg/day.
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Morphological change was observed in 160 of the 161 fetuses. A variety of skeletal and soft tissue changes were observed and the degree of change varied between individuals, including anophthalmia/microphthalmia, ventricular septal defects, and defects of the thoracic ribs and occurrence of cervical rib(s).
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: No effects

Details on embryotoxic / teratogenic effects:

Examination of maternal uterus:

Mean ovulation rate (as assessed by corpora lutea count) and mean pre-implantation loss of the treatment groups compared favorably with control, resulting in higher mean implantation rates. The overall pregnancy rate was similar for all groups.

After treatment with 1400 mg/kg/day: The incidence of embryo-fetal deaths was significantly increased and extended to total litter loss in 5/23 litters. Death predominantly occurred early in pregnancy.

There was a consequent significant reduction in litter size and in litter weight. Reduction in the litter weight was further enhanced by significantly lower mean weight of surviving fetuses.

Morphological change was observed in 160 of the 161 fetuses. A variety of skeletal and soft tissue changes were observed and the degree of change varied between individuals, including anophthalmia/microphthalmia, ventricular septal defects, and defects of the thoracic ribs and occurrence of cervical rib(s).

After treatment with 140 or 430 mg/kg/day: There were no instances of total litter loss at either dosage and the overall incidence of embryo-fetal deaths was similar to, or below, that of the control group.

Mean values for litter size, litter weight and mean fetal weight were essentially comparable with those of controls.
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
external malformations
other: Developmental toxicity : Morphological changes in fetus
Remarks on result:
other: No developmental toxic effects observed
Dose descriptor:
LOAEL
Effect level:
430 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
external malformations
other: Developmental toxicity :morphological changes in fetuses
Remarks on result:
other: morphological chenges observed
Abnormalities:
not specified
Localisation:
other: not specified
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 400 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
NOEAL and LOEAL for maternal toxicity in female rats were considered to be 430 and 1400 mg/kg/day, respectively. NOAEL and LOEL for developmental toicity was considered to be 140 and 430 mg/kg/day after exposure to phenylethyl alcohol.
Executive summary:

In a developmental and reprotoxicity study, the toxic effect of phenylethyl alcohol were evaluated in time-mated female CrL:COBS CD (SD) BR rats. The test chemical was administered dermally at a dosage of 0, 140, 430 or 1400 mg/kg per day during days 6-15 of pregnancy. In an emergence of an evidence,according to Api et.al. (RIFM fragrance ingredient safety assessment, alpha-methylbenzyl alcohol, CAS registry number 98 -85 -1), systemic absorption of the test chemical was assumed to be 100% by oral, dermal and inhalation route.

The study was terminated and the animals killed at day 20 of gestation.Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). With the exception of a single non-pregnant animal treated with 430 mg/kg/day which had a hunched posture and was walking on its toss on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage. Body weight and food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment. Subsequent mean weight gain of rats treated with 430 or 140 mg/kg/day did not show any substantial difference from control. The results from the hematology showed a higher value for white cell count in animals treated with 1400 mg/kg/day. Despite the marked clinical response observed at 1400/kg/day, there were no findings in gross pathology amongst surviving animals in any group at termination which were considered to be related to treatment. When investigating the litters from females treated with 1400mg/kg/day, the incidence of embryo-fetal deaths was significantly increased and extended to total litter loss in 5/23 litters. Death predominantly occurred early in pregnancy. There was also a consequent significant reduction in litter size and in litter weight. Reduction in the litter weight was further enhanced by significantly lower mean weight of surviving fetuses. In addition, morphological change was observed in 160 of the 161 fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals. No such changes of results were observed in litters from females treated with 140 or 430 mg/kg/day. Therefore, NOEAL and LOEAL for maternal toxicity in female rats were considered to be 430 and 1400 mg/kg/day, respectively. NOAEL and LOEL for developmental toicity was considered to be 140 and 430 mg/kg/day after exposure to phenylethyl alcohol.

Endpoint:
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 from various test chemicals
Justification for type of information:
Weight of evidence approach based on the available information from various test 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
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 reproductive toxicity studies on rats
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: 1.Wistar 2.Crl:COBSCD(SD)BR 3.Long- Evans rats
Details on test animals or test system and environmental conditions:
Study 1.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. Bedding material of batch No. SPAR-30/2015 (Sparconn Life Sciences Bangalore) was used in this study and a copy of report of microbial and chemical contaminants analysed periodically by manufacturer of bedding material are incorporated in the raw data.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet of batch no. 004915, 041215 and 041015 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: A conventional laboratory pelleted diet of batch no. 004915, 041215 and 041015 from approved supplier (Nutrivet Life Sciences, Pune) was offered. Aqua guard filtered drinking water in bottles was offered.

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: November 16, 2015 to March 26, 2016
Route of administration:
other: 1,3& 4.oral: gavage 2.oral: feed
Vehicle:
other: 1.corn oil 4.sunflower oil
Details on exposure:
Study1.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
Study 2.Details on exposure
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet
- Storage temperature of food:
Study 3.Details on exposure
PREPARATION OF DOSING SOLUTIONS: test material was suspended in distilled water

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:4.3, 43 and 432 mg/kg
- Amount of vehicle (if gavage):20 mL/kg
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study1.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.
Study2.High performance liquid chromatographic (HPLC) analyses were used to validate concentration, homogeneity and stability of test material throughout the study
Details on mating procedure:
Study1.- M/F ratio per cage: One male and one female
- 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.
- 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
Study 3.Pregnant female rats were used
Duration of treatment / exposure:
Study1.
64 days
Study2.& 3.
10 days ( from day 6 through day 15)
Study4.
4 days ( GD 4 (preimplantation) or GDs 10 through 12 (during embryogenesis)
Frequency of treatment:
Daily
Duration of test:
Study 1:.64 days
Study 2,3&4:20 days
Remarks:
Study1.Test animals:
0 (G1-control), 308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4)
Recovery animals: 0 (G1-R) or 1000 (G4-R) mg/Kg bw
Study2.
0, 1000, 3000 or 10,000 ppm (calculated doses were 83, 266 and 799 mg/kg for the respective exposure groups).
Study3
.0,4.3, 43 and 432 mg/kg
Study4.
0,508 mg/kg
No. of animals per sex per dose:
Study1.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
Study2.Total:112
0mg/kg bw/day:28 female
83mg/kg bw/day:28 female
266mg/kg bw/day:28 female
799mg/kg bw/day:28 female
Study3.Total:38
0mg/kg bw/day:19female
4.3mg/kg bw/day:7female
43mg/kg bw/day:7female
432mg/kg bw/day:5 female
Study4.Total:20
0mg/kg bw/day:10female
508mg/kg bw/day:10female

Control animals:
yes, concurrent vehicle
Details on study design:
- 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
Maternal examinations:
Study1.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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other:
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: Yes:
- Head examinations: No data
Statistics:
study 1.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
Indices:
Pregnancy index/fertility index was determined
Historical control data:
No data
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Study1.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.
Study2.No obvious clinical signs of toxicity was noted.
Study3.Severe maternal intoxication was observed at the 432 mg/kg dose group immediately after administration and persisted overnight on each day of treatment. No clinical effects were observed at the 4.3 and 43 mg/kg doses.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
Study1.No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period.
Study2.No deaths occurred during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Study1.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.

These changes observed were inconsistent, hence not considered as effect of the test item administration.
Study2.No effects were observed at 83mg/kg and 266mg/kg dose group . At 799mg/kg dose group , reduced maternal feed consumption and slight weight loss occurred on treatment days 1 and 2 (GDs 6–7, 7–8).
Study3.reduced weight gain was evident at the 43 mg/kg dose
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.

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):
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
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
Description (incidence and severity):
At the end of treatment and recovery period, absolute and relative weight of organs of treated rats of either sex did not differ significantly except a significant increase in relative wieght of Adrenal of G4-R male group when compared to the respective control group rats
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
External Findings: External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance.

Internal Findings: Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.
Neuropathological findings:
not specified
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
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
1.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.
4.No adverse effect on fetal ossification occurred when test material was administered preimplantation
Dose descriptor:
LOAEL
Effect level:
> 4.3 - <= 799 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Remarks on result:
other: decrease in pregancy index was observed in 1000mg/kg bw dose group
Abnormalities:
not specified
Localisation:
not specified
Fetal body weight changes:
no effects observed
Description (incidence and severity):
1.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
2.The mean fetal body weights, was equivalent in control and test groups.
3.Fetal growth retardation (reduced body weight and crown-rump length) was observed at the 4.3 and 432 mg/kg doses, with runting (body weight <2.7 g) occurring in 5 of 30, 0 of 61 and 32 of 51 fetuses at the 0.43, 43 and 432 mg/ kg dose levels, respectively. This absence of dose-dependency also was probably related to the relatively few litters evaluated and the pattern of postimplantation loss (resorption
4.Fetal body weight were unaffected.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
4.Fetal viability were unaffected.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
1.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.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
3.Mean litter size was reduced at the 43 and 430 mg/kg dose levels
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
1.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)
3.The percentage of dead or malformed fetuses per total implants was 55%, 97% and 100% at 4.3, 43 and 432 mg/kg, respectively. A dose–related increase in malformations was reported, although how the number of fetuses affected was determined is unclear, because the number
of fetuses evaluated by each methodology and the total number of fetuses available for evaluation were not provided. Reduced cranial-
bone ossification, a variant, was observed at a low incidence (0 of 224, 7 of 60, 2 of 65 and 6 of 51 fetuses in the 0, 0.43, 43 and 432 mg/kg dose groups, respectively). Additional retardations of ossification of the limbs, ribs and tail were evident at the 43 and 432 mg/kg dose levels. Microphthalmia (small eyes) and hydronephrosis (enlarged renal pelvis) occurred at 0.43, 4.3 and 432 mg/kg doses. Additional malformations included open eyes, micromelia (small paw) and a low incidence of heart defects at the 43 and 432 mg/kg doses. The 432 mg/kg dose was also associated with spina bifida (open spinal column), phocomelia (short limb), sirenomelia (joined limbs), micrognathia (short jaw) and webfoot/club foot.
4.Small delays in some bone ossification sites occurred in treated fetuses, as compared with controls, when the test material
was administered during the period of organogenesis, observations compatible with the small but biologically unimportant reduction in fetal body that also occurred in these fetuses. No other effects were observed

Skeletal malformations:
no effects observed
Description (incidence and severity):
The number, type and distribution of fetal malformations, anomalies and skeletal variations also were unaffected by maternal exposure to test material at concentrations as high as 10,000 ppm (799 mg/kg/day), although at 799mg/kg dose group there was a possible marginal delay in fetal ossification (a slightly higher incidence of fetuses with incomplete ossification of some sites, such as the sacro-caudal vertebral arches)
Visceral malformations:
not specified
Other effects:
not specified
Dose descriptor:
LOAEL
Effect level:
> 4.3 - <= 799 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
external malformations
Remarks on result:
other: No developmental toxic effects was observed
Abnormalities:
not specified
Localisation:
other: not specified
Developmental effects observed:
yes
Lowest effective dose / conc.:
4.3 mg/kg bw/day (nominal)
Treatment related:
not specified
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
Low Observed Adverse Effect Level (LOAEL) is considered to be 4.3-799 mg/kg bw. When male and female wistar rats were treated with1-phenylethanol (98-85-1) orally.
Executive summary:

Data available from different studies were reviewed to determine the developmental toxicity of  1-phenylethanol (98-85-1).The studies are as mentioned below:

Study 1.

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 material 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 item 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 Item.

Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material in Wistar Rats with 14 days recovery, where in 0, 308, 556 and 1000 mg/kg body weight, doses were tested;No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with test material orally.

Study 2

Reproductive and developmental toxicity study of test materialwas performed onCrl:COBSCD(SD)BR female rats. The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet in dose concentration 0, 1000, 3000 or 10,000 ppm (calculated doses were 83, 266 and 799 mg/kg for the respective exposure groups). The concentrations tested were based on a pilot study in which dietary inclusion of up to 9000 ppm test material was well tolerated in pregnant rats and did not result in unacceptable loss of diet palatability. High performance liquid chromatographic (HPLC) analyses were used to validate concentration, homogeneity and stability of test material throughout the study. 28 female rats per dose group were exposed to test material on GDs 6 through 15, In all the animals Feed consumption was measured daily, and body weights were taken on GDs 1, 3, 6 and then on alternate days until necropsy on GD 20, when in utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for external, soft tissue and skeletal alterations (malformations and variations).

No deaths occurred during the study.No obvious clinical signs of toxicity was noted. No effects were observed at 83mg/kg and 266mg/kg dose group . At 799mg/kg dose group , reduced maternal feed consumption and slight weight loss occurred on treatment days 1 and 2 (GDs 6–7, 7–8). Calculated mean group intake of test material was 0, 83, 266 and 799 mg/kg/day for the pregnant rats in the 0, 1000, 3000 and 10,000 ppm groups, respectively. mean values for embryo-fetal loss, litter sizes, sex ratios were equivalent in control and test group also the mean fetal body weights, was equivalent in control and test groups.The number, type and distribution of fetal malformations, anomalies and skeletal variations also were unaffected by maternal exposure to test material at concentrations

as high as 10,000 ppm (799 mg/kg/day), although at799mg/kg dose group there was a possible marginal delay in fetal ossification(a slightly higher incidence of fetuses with incomplete ossification of some sites, such as the sacro-caudal vertebral arches).HenceNo Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 799mg/kg. Whenfemalerats were treated withtest material by orally for GDs 6 through 15.

Study 3

The reproductive and developmental toxicity study of test material was performed onpregnant Long- Evans rats. The aqueous suspension of test material was administered by gavage at dose concentration 4.3, 43 and 432 mg/kg to rats on GDs 6 through 15. All doses were administered at a volume of 20 mL/kg,Group sizeswere 19, 7, 7 and 5 pregnant rats in the control (distilled water),4.3, 43 and 432 mg/kg dose groups, respectively. Doses wereselected to be 0.24, 2.4 and 24% of the LD50 value published byOwston et al. (1981).The dams were observed daily for signs of toxicity and lethality. Body weights were recorded on GDs 0 through 15 and 20. All dams were overdosed with ether on GD 20, and their fetuses were delivered by Cesarean-section. Observations were made for uterine weight, litter weight, individual pup weight, and numbers of live and dead pups, resorptions, implantations, sex distribution, crown-rump length and corpora lutea. All live pups were examined for gross external malformations. As report that 224, 51, 65 and 60 fetuses in the four respective dose groups were evaluated for both soft tissue and skeletal examinations. It is unclear how this number was attained, because soft tissue evaluation was by sectioning (Wilson’s method), precluding skeletal evaluation of the same specimen (alcian blue-alizarin red S-staining).

 Severe maternal intoxication was observed at the 432 mg/kg dose group immediately after administration and persisted overnight on each day of treatment. No clinical effects were observed at the 4.3 and 43 mg/kg doses but reduced weight gain was evident at the 43 mg/kg dose. Mean litter size was reduced at the 43 and 430 mg/kg dose levels .Fetal growth retardation (reduced body weight and crown-rump length) was observed at the 4.3 and 432 mg/kg doses, with runting (body weight <2.7 g) occurring in 5 of 30, 0 of 61 and 32 of 51 fetuses at the 0.43, 43 and 432 mg/ kg dose levels, respectively. This absence of dose-dependency also was probably related to the relatively few litters evaluated and thepattern of postimplantation loss (resorption)

The percentage of dead or malformed fetuses per total implants was 55%, 97% and 100% at 4.3, 43 and 432 mg/kg, respectively. A dose–related increase in malformations was reported, although how the number of fetuses affected was determined is unclear, because the number of fetuses evaluated by each methodology and the total number of fetuses available for evaluation were not provided. Reduced cranial- bone ossification, a variant, was observed at a low incidence (0 of 224, 7 of 60, 2 of 65 and 6 of 51 fetuses in the 0, 0.43, 43 and 432 mg/kg dose groups, respectively). Additional retardations of ossification of the limbs, ribs and tail were evident at the 43 and 432 mg/kg dose levels. Microphthalmia (small eyes) and hydronephrosis (enlarged renal pelvis) occurred at 0.43, 4.3 and 432 mg/kg doses. Additional malformations included open eyes, micromelia (small paw) and a low incidence of heart defects at the 43 and 432 mg/kg doses. The 432 mg/kg dose was also associated with spina bifida (open spinal column), phocomelia (short limb), sirenomelia (joined limbs), micrognathia (short jaw) and webfoot/club foot. As there were effects at all three dose levels, a NOAEL could not be determined from this study and is presumed to be < 4.3 mg/kg

Study 4

The reproductive and developmental toxicity study of test material was performed onpregnant rats.Test material in sunflower oil was administered to pregnant rats (10 per group) by gavage on GD 4 (preimplantation) or GDs 10 through 12 (during embryogenesis) in dose concentrati as a single dose of 508 mg/kg.GD 1 was defined as the day sperm were observed in a vaginal smear. On GD 20, the female rats were euthanized, and the fetuses were removed for further analysis (Wilson’s sectioning or alizarin red S-staining). Fetal viability and body weight were unaffected.

No adverse effect on fetal ossification occurred when test material was administered preimplantation. Small delays in some bone ossification sites occurred in test material treated

fetuses, as compared with controls, when the test material was administered during the period of organogenesis, observations compatible with the small but biologically unimportant reduction in fetal body that also occurred in these fetuses. No other effects were observed .Hence NOAEL for maternal and developmental toxicity was considered to be 508mg/kg bw .When female rats were treated with test material orally.

 

Based on the data available from different studies,test material showed developmental toxicity at dose concentration 4.3 mg/kg bw/day by oral route .Hence the test chemical is likely to classify as a reproductive and developmental toxicant as per the criteria mentioned in CLP regulation.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
4.3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is Klimicsh 2 and from authoritative database
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is Klimicsh 2 and from authoritative database
Additional information

Developmental toxicity study

Data available from different studies were reviewed to determine the developmental toxicity of test chemical.The studies are as mentioned below:

Via oral route :

Study1.

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 material 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 item 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 Item.

Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material in Wistar Rats with 14 days recovery, where in 0, 308, 556 and 1000 mg/kg body weight, doses were tested; No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with test material orally.

Study2.

Reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed onCrl:COBSCD(SD)BR female rats. The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet in dose concentration 0, 1000, 3000 or 10,000 ppm (calculated doses were 83, 266 and 799 mg/kg for the respective exposure groups). The concentrations tested were based on a pilot study in which dietary inclusion of up to 9000 ppm test material was well tolerated in pregnant rats and did not result in unacceptable loss of diet palatability. High performance liquid chromatographic (HPLC) analyses were used to validate concentration, homogeneity and stability of test material throughout the study. 28 female rats per dose group were exposed to test material on GDs 6 through 15, In all the animals Feed consumption was measured daily, and body weights were taken on GDs 1, 3, 6 and then on alternate days until necropsy on GD 20, when in utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for external, soft tissue and skeletal alterations (malformations and variations).

No deaths occurred during the study.No obvious clinical signs of toxicity was noted. No effects were observed at 83mg/kg and 266mg/kg dose group . At 799mg/kg dose group , reduced maternal feed consumption and slight weight loss occurred on treatment days 1 and 2 (GDs 6–7, 7–8). Calculated mean group intake of test material was 0, 83, 266 and 799 mg/kg/day for the pregnant rats in the 0, 1000, 3000 and 10,000 ppm groups, respectively. mean values for embryo-fetal loss, litter sizes, sex ratios were equivalent in control and test group also the mean fetal body weights, was equivalent in control and test groups.The number, type and distribution of fetal malformations, anomalies and skeletal variations also were unaffected by maternal exposure to test material at concentrationsas high as 10,000 ppm (799 mg/kg/day), although at799mg/kg dose group there was a possible marginal delay in fetal ossification(a slightly higher incidence of fetuses with incomplete ossification of some sites, such as the sacro-caudal vertebral arches).HenceNo Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 799mg/kg. When female rats were treated with 2-phenylethan-1-ol CAS 60-12-8 by orally for GDs 6 through 15.

Study 3.

The reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed onpregnant Long- Evans rats. The aqueous suspension of test material was administered by gavage at dose concentration 4.3, 43 and 432 mg/kg to rats on GDs 6 through 15. All doses were administered at a volume of 20 mL/kg,Group sizeswere 19, 7, 7 and 5 pregnant rats in the control (distilled water),4.3, 43 and 432 mg/kg dose groups, respectively. Doses wereselected to be 0.24, 2.4 and 24% of the LD50 value published byOwston et al. (1981).The dams were observed daily for signs of toxicity and lethality. Body weights were recorded on GDs 0 through 15 and 20. All dams were overdosed with ether on GD 20, and their fetuses were delivered by Cesarean-section. Observations were made for uterine weight, litter weight, individual pup weight, and numbers of live and dead pups, resorptions, implantations, sex distribution, crown-rump length and corpora lutea. All live pups were examined for gross external malformations. As report that 224, 51, 65 and 60 fetuses in the four respective dose groups were evaluated for both soft tissue and skeletal examinations. It is unclear how this number was attained, because soft tissue evaluation was by sectioning (Wilson’s method), precluding skeletal evaluation of the same specimen (alcian blue-alizarin red S-staining).

 Severe maternal intoxication was observed at the 432 mg/kg dose group immediately after administration and persisted overnight on each day of treatment. No clinical effects were observed at the 4.3 and 43 mg/kg doses but reduced weight gain was evident at the 43 mg/kg dose. Mean litter size was reduced at the 43 and 430 mg/kg dose levels .Fetal growth retardation (reduced body weight and crown-rump length) was observed at the 4.3 and 432 mg/kg doses, with runting (body weight <2.7 g) occurring in 5 of 30, 0 of 61 and 32 of 51 fetuses at the 0.43, 43 and 432 mg/ kg dose levels, respectively. This absence of dose-dependency also was probably related to the relatively few litters evaluated and thepattern of postimplantation loss (resorption)

The percentage of dead or malformed fetuses per total implants was 55%, 97% and 100% at 4.3, 43 and 432 mg/kg, respectively. A dose–related increase in malformations was reported, although how the number of fetuses affected was determined is unclear, because the number of fetuses evaluated by each methodology and the total number of fetuses available for evaluation were not provided. Reduced cranial- bone ossification, a variant, was observed at a low incidence (0 of 224, 7 of 60, 2 of 65 and 6 of 51 fetuses in the 0, 0.43, 43 and 432 mg/kg dose groups, respectively). Additional retardations of ossification of the limbs, ribs and tail were evident at the 43 and 432 mg/kg dose levels. Microphthalmia (small eyes) and hydronephrosis (enlarged renal pelvis) occurred at 0.43, 4.3 and 432 mg/kg doses. Additional malformations included open eyes, micromelia (small paw) and a low incidence of heart defects at the 43 and 432 mg/kg doses. The 432 mg/kg dose was also associated with spina bifida (open spinal column), phocomelia (short limb), sirenomelia (joined limbs), micrognathia (short jaw) and webfoot/club foot. As there were effects at all three dose levels, a NOAEL could not be determined from this study and is presumed to be < 4.3 mg/kg

Study4.

The reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed onpregnant rats.Test material in sunflower oil was administered to pregnant rats (10 per group) by gavage on GD 4 (preimplantation) or GDs 10 through 12 (during embryogenesis) in dose concentrati as a single dose of 508 mg/kg.GD 1 was defined as the day sperm were observed in a vaginal smear. On GD 20, the female rats were euthanized, and the fetuses were removed for further analysis (Wilson’s sectioning or alizarin red S-staining). Fetal viability and body weight were unaffected. No adverse effect on fetal ossification occurred when test material was administered preimplantation. Small delays in some bone ossification sites occurred in test material treated fetuses, as compared with controls, when the test material was administered during the period of organogenesis, observations compatible with the small but biologically unimportant reduction in fetal body that also occurred in these fetuses. No other effects were observed .Hence LOAEL for maternal and developmental toxicity was considered to be 508mg/kg bw . When female rats were treated with 2-phenylethan-1-ol CAS 60-12-8orally.

Via dermal route :

Study 5

In a developmental and reprotoxicity study, the toxic effect of phenylethyl alcohol were evaluated in time-mated female CrL:COBS CD (SD) BR rats. The test chemical was administered dermally at a dosage of 0, 140, 430 or 1400 mg/kg per day during days 6-15 of pregnancy. In emergence of other data, according to Api et.al. (RIFM fragrance ingredient safety assessment, alpha-methylbenzyl alcohol, CAS Registry number 98 -85 -1) systemic absorption of the test chemical was assumed to be 100% through oral, dermal and inhalation route.

The study was terminated and the animals killed at day 20 of gestation. Signs of reaction to treatment were seen for the highest dosage group (1400 mg/kg/day). With the exception of a single non-pregnant animal treated with 430 mg/kg/day which had a hunched posture and was walking on its toes on Day 16 of the study, there were no overt signs of reaction to treatment at the intermediate or low dosage. Body weight and food intake of rats treated with 1400 mg/kg/day became noticeably suppressed towards the end of the treatment period, but approached control levels after cessation of treatment. Subsequent mean weight gain of rats treated with 430 or 1400 ml/kg/day did not show any substantial difference from control. The results from the hematology showed a higher value for white cell count in animals treated with 1400 mg/kg/day. Despite the marked clinical response observed at 1400 mg/kg/day, there were no findings in gross pathology amongst surviving animals in any group at termination which were considered to be related to treatment. When investigating the litters from females treated with 1400 mg/kg/day, the incidence of embryo-fetal deaths was significantly increased and extended to total litter loss in 5/23 litters. Death predominantly occurred early in pregnancy. There was also a consequent significant reduction in litter size and in litter weight. Reduction in the litter weight was further enhanced by significantly lower mean weight of surviving fetuses. In addition, morphological change was observed in 160 of the 161 fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals. No such changes of results were observed in litters from females treated with 140 or 430 mg/kg/day. Therefore, LOAEL was considered to be 1400 mg/kg/day for the maternal generation, while NOAEL was considered to be 140 mg/kg/day for the maternal generation and the F1 generation after exposure to phenylethyl alcohol.

Study 6

Reproductive and developmental toxicity study of 2-phenylethan-1-ol CAS 60-12-8 was performed onpresumed pregnant Crl:COBS CD(SD) BR female rats.10 females were used in each dose group.The test material in unchanged from in dose concentration0.07, 0.14, 0.28, 0.43, or 0.70 mL/kg (70, 140, 280, 430 and 700 mg/kg, respectively) was applied topically on GDs 6 through 15. The application sites were covered with aluminum foil, held in place by an adhesive bandage.Feed and body weights were recorded daily, as well as daily clinical observations. The animals were euthanized on GD 20 and in utero parameters were assessed on the basis of litter values. All live fetuses were examined for gross external alterations; approximately one-third of these live fetuses were also examined for visceral alterations, using a dissection technique. All live fetuses, rather than only ½ of the fetuses, as in the previous evaluation, were examined for skeletal alterations and number of ossification sites after staining with alizarin red S.

No deaths occurred during the study. All doses from 70 to 700 mg/kg/day (0.07 to 0.70 mL/kg) resulted in low levels of erythema and/or desquamation at the application site. No other signs of maternal toxicity were observed at doses of 70 to 430 mg/kg (0.07 to 0.43 mL/kg). At 700 mg/kg (0.7 mL/kg), ptosis and/or urine stained abdominal fur were also observed, as well as reduced feed consumption and body weight gain. No adverse effects on mean live litter sizes or resorptions were seen at doses up to 700 mg/kg (0.7 mL/kg). Dose-dependent significant reductions in fetal body weight were observed at 140 mg/kg (0.14 mL/kg) and higher doses, accompanied by statistically significant increases in reversible delays in ossification. HenceNo Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 70 mg/kg.When femalerats were treated with 2-phenylethan-1-ol CAS 60-12-8 by dermal application for GDs 6 through 15.

 Study 7

The reproductive and developmental toxicty study of 2-phenylethan-1-ol CAS 60-12-8 performed in time-mated Crl:COBSCD(SD) BR female rats. The test material in dose concentration 0.14, 0.43 and 1.40 mL/kg (140, 430 and 1400 mg/kg, respectively) were applied daily to the shaved backs of the rats on GDs 6 through 15. To prevent possible oral ingestion of the control (water) or phenylethyl alcohol, the dermal application sites were occluded by covering with aluminum foil that was held in place by a porous adhesive bandage. Observations were made for signs of toxicity, mortality, body weight and feed consumption (GDs 1, 3, 6 and alternate days until necropsy on GD 20). Liver and kidney weights and hematology and serum chemistry parameters were also determined in 15 control and 13 high dose rats on GD 20. In utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for gross external (all live fetuses), soft tissue (1/2 of the fetuses per litter, using Wilson’s free-hand dissection method) and skeletal changes (1/2 of the fetuses per litter, following alizarin red S-staining).

Mortality (1 moribund euthanasia on GD 11 and 2 deaths on GD 13) were observed almost entirely at the 1400 mg/kg dose. Maternal toxicity was observed as local irritation at the application site, especially in the 1400mg/kg dose group. Irritability,hunched posture, walking on toes, piloerection, periorbital staining. were observed almost entirely at the 1400 mg/kg dose.These signs started around GD 12, became progressively worse during the remainder of the treatment period (until GD15), and essentially resolved during the 5 days of no treatment (GD15 to GD 20). Mean feed consumption and body weight gains weremarkedly reduced at the 1400 mg/kg dose. Liver and kidney weights, and necropsy observations were unaffected. pregnancy incidences were unaffected. Developmental toxicity included statistically significant increases in early resorptions, at the1400 mg/kg dose. At the 140 mg/kg dose (0.14 mL/kg), no maternal toxicity was observed.

A related reduction in mean litter sizes and a marked reduction in mean fetal weight at the 1400 mg/kg dose. At the 140 mg/kg dose (0.14 mL/kg), litter parameters were comparable to control, with the exception of a marginally higher than control incidence of skeletal changes (rudimentary cervical ribs and thoracic vertebral irregularities). These observations showed dose dependent increases at 430 mg/kg (0.43 mL/kg) and 1400 mg/kg ,(1.40 mL/kg) doses. Additional findings at 430 mg/kg consisted of occasional fetuses with soft tissue changes similar to those observed at 1400 mg/kg, as well as moderate degrees of incomplete ossification. At 1400 mg/kg, maternal toxicity, significantly increased embryo-fetal death (resorption), with associated reduced litter size, and significantly reduced fetal body weight were noted. Many morphological changes occurred in 160/161 fetuses at this dose. More than 40% of the fetuses and 70% of the litters had anophthalmia or microphthalmia, ventricular septal defects, defects or irregularities affecting the thoracic, lumbar and sacrocaudal vertebrae, which were associated with short or kinky tail and defects of the thoracic ribs.Hence the maternal NOAEL was considered to be 43 mg/kg (0.43 mL/kg) and the developmental NOAEL was considered to be approximately 140 mg/kg (0.14 mL/kg).When rats were treated with 2-phenylethan-1-ol CAS 60-12-8via dermal application.

Study 8

The reproductive and developmental toxicity study of test material was performed on presumed pregnant female rats.Undiluted test material in dose concentration 140, 430, or 1400 mg/ kg/day (0.14, 0.43, or 1.4 ml/kg) or distilled water (1.4 ml/kg, vehicle control). were applied percutaneously to clipped skin on the back (5 X7 cm), occluded with foil, and secured with micropore tape. After approximately 24 h, the test sites were rinsed and dried with gauze. The rats were also fitted with Elizabethan collars to prevent oral ingestion. One hundred female rats were dosed once daily on gestational days 7 through 20 (GD 7–20). Twenty rats from each dosage group were selected for Cesarean-sectioning on GD 21 and the remaining rats were selected for natural delivery.

 At 1400 mg/kg/day, mortality was significantly increased in comparison to the vehicle control group the deaths were attributed to treatment with test material in rats assigned to Cesarean-sectioning group while rats assigned to natural delivery group At 1400 mg/kg/day, one rat was found dead on DG 13. This death was presumed related to treatment with test material because: (1) similar events occurred in the rats assigned to Cesarean- sectioned; and (2) the observation occurred in the high dosage group.

primary irritancy including flaking and/or erythema, occurred in female rats percutaneously administered 430 and 1400 mg/kg/day during the gestation period. The onset and severity of the skin reactions was, in general, dependent on the dosage of test material . The number of rats with adverse clinical signs was significantly increased at 1400 mg/kg/day in comparison to the vehicle control group in rats assigned to Cesarean-sectioning group while rats assigned to natural delivery group similar clinical signs like primary irritancy (erythema, edema, and/or flaking) occurred during the gestation period in all dosage groups. The onset and severity of the skin reactions was, in general, dependent on the dosage of test material .The number of rats with adverse clinical signs was significantly increased at 1400 mg/kg/day in comparison to the vehicle control group.

 A statistically significant loss in body weight, coupled with reduced or significantly reduced feed consumption values, occurred at 1400 mg/kg/day for the entire dosage period, as compared to the vehicle control group values in rats assigned to Cesarean-sectioning group while rats assigned to natural delivery group body weight gains were significantly reduced for the entire gestation dosage period and the entire gestation period, as compared to the vehicle control group values. In addition, body weight gains were significantly reduced in the 430 and 1400 mg/kg/day dosage groups on Days 1–4 of lactation. Thereafter, body weight gains were comparable among the dosage groups.

Absolute and relative feed consumption values were reduced or significantly reduced at 1400 mg/kg/day on DGs 7–21, in comparison to the vehicle control group values. Corresponding to reductions in body weight gain, absolute and relative feed consumption values were also reduced at 1400 mg/kg/day dosage group on DLs 1–4 and overall for DLs 1–14, in comparison to the vehicle control group values.

Rats assigned to Cesarean-sectioning Pregnancy occurred in 16 to 20 rats in each dosage group. As a result of the increased mortality that occurred in the 1400 mg/kg/ day dosage group, Cesarean-sectioning observations on DG 21 were based on 20, 18, 20 and 9 pregnant rats in Groups I through IV, respectively. At 1400 mg/kg/day, postimplantation loss was increased or significantly increased, in comparison to the vehicle control group values. Seven of the nine rats in this dosage group had total litter loss (i.e., 100% resorbed conceptuses). These increases in postimplantation loss resulted in an overall significant reduction in the averages for litter size and live fetuses at 1400 mg/kg/day, in comparison to the vehicle control group values (1.3 per liter vs. 14.6 per litter in vehicle controls).While Rats assigned to natural delivery Pregnancy occurred in 16–20 female rats in the four dosage groups. All pregnant dams at 140 and 430 mg/kg/day delivered litters, 8 of the 16 pregnant rats delivered a litter at 1400 mg/kg/day. Of these 8 dams, one had a litter with no liveborn pups and four had all pups die before day 4 postpartum. At 1400 mg/kg/day, the duration of gestation was significantly increased and the gestation index was significantly reduced. The averages for the total number of pups delivered and the number of liveborn pups were reduced or significantly reduced in this same dosage group, in comparison to the vehicle control group values. At 1400 mg/kg/day, a significant number of litters had pups with mild dehydration (based on skin turgor), a thread-like tail and that were not nursing. The percentage of pups that died or were presumed cannibalized on day 1 and days 2–4 postpartum was significantly increased at 1400 mg/kg/day, as compared to the vehicle control group values. Reflecting this increase in pup mortality, the viability index at 1400 mg/kg/day was significantly reduced relative to the vehicle control group value (63.2% vs. 99.0% in vehicle controls). The average number of surviving pups per litter was significantly reduced at 1400 mg/kg/day on days 4, 7, 14 and 21 postpartum, in comparison to the vehicle control group values.

 Rats assigned to Cesarean-sectioning At 430 mg/kg/day, the average fetal body weight was significantly lower than the concurrent vehicle control group values. These values were also below the range observed historically at the Testing Facility Rats assigned to natural delivery the average pup weight per litter was significantly lower at 1400 mg/kg/day on day 1 postpartum relative to the vehicle control group value. On postpartum days 4, 7 and 21, pup body weights at 1400 mg/kg/day were 9%, 10% and 7% lower than the corresponding vehicle control group values.

Rats assigned to Cesarean-sectioning all 12 fetuses in the two litters that had live fetuses at 1400 mg/kg/day had one or more gross, soft tissue and/or skeletal alterations. The limited number of fetuses and litters with live fetuses compared to the other dosage groups resulted in significantly increased fetal and litter incidences of each of these alterations. At 1400 mg/kg/day, the average number of ossification sites per fetus per litter was significantly reduced for caudal vertebrae, sternal centra, xiphoid, metacarpals, phalanges, metatarsals and phalanges. Corresponding to the reduction in fetal weight in the 430 mg/kg/day dosage group, ossification site averages for caudal vertebrae, fore- and hindlimb phalanges and metatarsals were also significantly reduced and below the historical control range for this Testing Facility. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased at 430 mg/kg/day, as compared to the vehicle control group values.However, all apparent delays in ossification and increased numbers of cervical ribs that were observed in the Cesarean-delivered fetuses in the 430 mg/kg/day were resolved by DL 21. Hence The maternal no-observable-adverse-effect-level (NOAEL) was considered to be 430 mg/kg/day due to mortality and reductions in body weight and feed consumption at 1400 mg/kg/day. The developmental NOAEL was considered to be 140 mg/kg/day. When female rats were treated with 2-phenylethan-1-ol CAS 60-12-8 via dermal application.

Based on the data available from different studies for target chemical 1-phenylethanol (98-85-1)and its structuraly similar read across substance 2-phenylethan-1-ol CAS 60-12-8 / EC 200-456-2)andMethyl phenyl acetate; methyl phenylacetate (CAS 101-41-7) via oral and dermal route,LOAEL for 1-phenylethanol (98-85-1)was considered to be in range of 4.3 -556 mg/kg bw/day for F0, F1 generation but as adverse effects like markedly decreased pregnancy index / fertility index, In addition, morphological change was observed in fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals at different dose group. Thus, comparing this value with the criteria of CLP regulation 1-phenylethanol (98-85-1)is likely to classify as reproductive and developmental toxicant.

 

Justification for classification or non-classification

Thus, Based on the data available from different studies for target chemical 1-phenylethanol (98-85-1)and its structuraly similar read across substance 2-phenylethan-1-ol CAS 60-12-8 / EC 200-456-2)and Methyl phenyl acetate; methyl phenylacetate (CAS 101-41-7) via oral and dermal route,LOAEL for 1-phenylethanol (98-85-1)was considered to be in range of 4.3 -556 mg/kg bw/day for F0, F1 generation but as adverse effects like markedly decreased pregnancy index / fertility index, In addition, morphological change was observed in fetuses, and a variety of skeletal and soft tissue changes were seen, where the degree of change varied between individuals at different dose group. Thus, comparing this value with the criteria of CLP regulation 1-phenylethanol (98-85-1)is likely to classify as reproductive and developmental toxicant.

 

Additional information