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REACH

3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

EC number: 222-294-1 | CAS number: 3407-42-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity

The test chemical was given to 7 rats per sex per dose level at 0 (vehicle; corn oil), 125, 375 and 1125 mg/kg/day. The study was performed according to GLP and according to OECD 407 with some additional reproductive-related endpoints included.Results:All animals survived to planned death and there were no clinical signs attributed to the test chemical. No significant changes in body weight were observed, except for a slight increase in male body weight at 1125 mg/kg on day 8 of treatment (mean, 248 g) compared to the control group (mean 237 g). This effect was considered incidental and not toxicologically significant. No significant changes in food intake or water intake were observed. No significant changes in locomotor activity were observed. No abnormalities were found during the ophthalmic examinations. Significant changes in haematology were as follows: significant increase in % monocytes in males treated at 125 mg/kg; significant increase in % basophils in males treated at 125 mg/kg; significant increases in white blood cell counts in females treated at 375 and 1125 mg/kg; significant decreases in red blood cell counts in females treated at 375 and 1125 mg/kg; significant decrease in haematocrit counts in females treated at 1125 mg/kg; significant increase in MCV in females treated at 1125 mg/kg; and significant increases in MCH in females treated at 375 and 1125 mg/kg. The observed changes in haematology were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings. Significant changes in clinical chemistry were as follows: significant increase in total cholesterol in male rats treated at 1125 mg/kg; significant increase in creatinine in male rats treated at 125 mg/kg; significant increase in glucose in male rats treated at 375 mg/kg; significant increases in total proteins in male rats treated at 375 and 1125 mg/kg; significant increase in potassium in female rats treated at 1125 mg/kg; significant increase in sodium in female rats treated at 1125 mg/kg; significant increase in SGPT in female rats treated at 375 and 1125 mg/kg; significant decrease in glucose in female rats treated at 1125 mg/kg; significant increase in total protein in female rats treated at 1125 mg/kg; significant increases in urea nitrogen in female rats treated at 375 and 1125 mg/kg; and a significant increase in bile acid in female rats treated at 375 mg/kg. No significant changes in serum testosterone or serum oestrogen were observed. The observed changes in clinical chemistry were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings. No significant changes in absolute organ weight were observed, with the exception of significant increases in liver weight in female rats treated at 375 and 1125 mg/kg. Significant changes in relative organ weight included a significant decrease in relative heart weight in male rats treated at 125 mg/kg and two significant increases in relative liver weight in female rats treated at 375 and 1125 mg/kg. In male rats, no gross pathological findings were observed. In female rats, gross pathological findings were limited to a slightly increased stomach at 1125 mg/kg and an enlarged uterus with uterine fluid at 1125 mg/kg. Histopathology performed at 0 and 1125 mg/kg showed excess of lymphocytes in the lungs of 2/7 control males, 1/7 high-dosed males, 1/7 control females, and 1/7 high-dosed females and reactive spleens in 3/7 control males, 1/7 high-dosed males, 2/7 control females, and 1/7 high-dosed females. These histopathological findings were consistent with the historical control data of the test facility and were therefore not considered to be toxicologically relevant. The histopathologic examinations did not reveal any significant effects on spermatogenesis.Conclusion:NOAEL was considered at 1125 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:: reproductive toxicity, other
Remarks:: 28-days repeated dose toxicity study with extended reproductive parameters
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Justification for type of information:: Data is from study report
Qualifier:: according to guideline
Guideline:: other: According to OECD Guideline 407 with additional examinations for reproductive performance.
Version / remarks:: Adopted 3 October 2008
Principles of method if other than guideline:: Additional reproductive-related endpoints included serum testosterone, serum estrogen, and spermatogenesis stages during microscopic examinations.
GLP compliance:: yes
Limit test:: no
Justification for study design:: No Data Available
Species:: rat
Strain:: Sprague-Dawley
Sex:: male/female
Details on test animals or test system and environmental conditions:: - Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, 160 062, Punjab, India.
- Age at study initiation: (P) x wks; (F1) x wks: (P) 7 to 8 weeks old
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available
- Fasting period before study: Yes, 2 hrs fasted before dose admonition.
- Housing: Animals were housed in sterilized solid bottom polypropylene cages with stainless steel grill tops with facilities for food, water bottles and bedding of clean paddy husk. The cages were suspended on stainless steel racks. Animal was identified by a unique identification (ID) number. The animals were identified by individual numerical number written on the tail, also specified on individual cage tag.
- Diet (e.g. ad libitum): Standard laboratory sterile extruded pelleted rodent feed, ad libitum.
- Water (e.g. ad libitum): Potable tap water filtered through Reviva Reverse Osmosis System (water filter cum purifier) of Eureka Forbes was provided in polypropylene bottles with stainless steel sipper tubes, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3º C
- Humidity (%): 30-70 %
- Air changes (per hr): 25±5 air changes per hour
- Photoperiod (hrs dark / hrs light): 12hrs dark/ light cycle
Route of administration:: oral: gavage
Type of inhalation exposure (if applicable):: not specified
Vehicle:: corn oil
Details on exposure:: PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in corn oil prior to treatment.

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): Corn oil
- Concentration in vehicle: 0, 125, 375 or 1125 mg/kg/day
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on mating procedure:: No mating was performed.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The test item (125, 375 and 1125 mg) was weighed on balance and dissolved in 5 ml of corn oil. An aliquot was taken from three different layers of the dose mixtures and was extracted in methanol for GCMS analysis. The concentration of the test item in each layer was calculated using the standard curve (5 mg IBC / ml methanol).
Duration of treatment / exposure:: 28 days
Frequency of treatment:: Once per day.
Details on study schedule:: No data available
Dose / conc.:: 0 mg/kg bw/day (actual dose received)
Dose / conc.:: 125 mg/kg bw/day (actual dose received)
Dose / conc.:: 375 mg/kg bw/day (actual dose received)
Dose / conc.:: 1 125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:: 7 rats per sex per dose.
Control animals:: yes, concurrent vehicle
Details on study design:: No data available
Positive control:: No positive control was included.
Parental animals: Observations and examinations:: Mortality, clinical signs (once per day), detailed clinical signs (once per week), body weight (treatment days 1, 8, 15, 22, 28, 29) food intake (once per week), water intake (once per week), ophthalmology (4th week of treatment), locomotor activity (4th week of treatment), hematology (end of treatment), clinical chemistry (end of treatment)

Hematologic parameters: Haemoglobin, RBC, Total and differential leucocyte count, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), and Platelet count

Clinical chemistry parameters: Sodium, Potassium, Glucose, Total cholesterol, Blood urea, Creatinine, Total protein, Albumin, SGPT (Serum glutamic pyruvic transaminase), ALT, SGOT (Serum glutamic oxaloacetic transaminase), AST, Hormones analysis (testosterone and estrogen) and Total bile acids.
Postmortem examinations (parental animals):: The following organs were weighed: liver, adrenals, spleen, heart, kidney, brain, testes, epididymides, ovaries, thymus. The following organs/tissues were examined microscopically: brain, stomach, large intestine, small intestine, liver, kidney, adrenal gland, spleen, heart, thymus, lungs, testis, ovaries, uterus, lymph nodes, perihperal nerve, bone marrow, and gross lesions (if any).
Statistics:: Statistical analysis was carried out by using Microsoft Excel and IBM SPSS statistics version-20.0. All analyses and comparisons were evaluated at the 5 % level, statistically significant differences (p≤0.05) indicated by appropriate notation. The focus was to examine the mean differences and their significance between control and low dose group, control and mid dose group and control and high dose group. The statistical decision was taken by preparing the univariant GLM MODEL procedure was used to check the significance between above mentioned groups. For multiple comparisons Turkey’s HSD test was applied.
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: There were no clinical signs attributed to the test item. Some abnormalities like nasal discharge, slight dullness, hunched posture, fore paw stained red, sneezing, red crust around the nostrils, perineum wet were common to all the groups.
Dermal irritation (if dermal study):: not examined
Mortality:: no mortality observed
Description (incidence):: All animals survived to planned death.
Body weight and weight changes:: effects observed, non-treatment-related
Description (incidence and severity):: No significant changes in body weight were observed, except for a slight increase in male body weight at 1125 mg/kg on day 8 of treatment (mean, 248 g) compared to the control group (mean 237 g). This effect was considered incidental and not toxicologically significant.
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: No significant effects were observed in the study.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: no effects observed
Description (incidence and severity):: No significant effects were observed.
Ophthalmological findings:: no effects observed
Description (incidence and severity):: No significant effects were observed.
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: Significant changes in haematology were as follows: significant increase in % monocytes in males treated at 125 mg/kg; significant increase in % basophils in males treated at 125 mg/kg; significant increases in white blood cell counts in females treated at 375 and 1125 mg/kg; significant decreases in red blood cell counts in females treated at 375 and 1125 mg/kg; significant decrease in haematocrit counts in females treated at 1125 mg/kg; significant increase in MCV in females treated at 1125 mg/kg; and significant increases in MCH in females treated at 375 and 1125 mg/kg. The observed changes in haematology were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: Significant changes in clinical chemistry were as follows: significant increase in total cholesterol in male rats treated at 1125 mg/kg; significant increase in creatinine in male rats treated at 125 mg/kg; significant increase in glucose in male rats treated at 375 mg/kg; significant increases in total proteins in male rats treated at 375 and 1125 mg/kg; significant increase in potassium in female rats treated at 1125 mg/kg; significant increase in sodium in female rats treated at 1125 mg/kg; significant increase in SGPT in female rats treated at 375 and 1125 mg/kg; significant decrease in glucose in female rats treated at 1125 mg/kg; significant increase in total protein in female rats treated at 1125 mg/kg; significant increases in urea nitrogen in female rats treated at 375 and 1125 mg/kg; and a significant increase in bile acid in female rats treated at 375 mg/kg. No significant changes in serum testosterone or serum oestrogen were observed.
Urinalysis findings:: not examined
Behaviour (functional findings):: no effects observed
Description (incidence and severity):: No significant changes in locomotor activity scores were observed.
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: Histopathology performed at 0 and 1125 mg/kg showed excess of lymphocytes in the lungs of 2/7 control males, 1/7 high-dosed males, 1/7 control females, and 1/7 high-dosed females and reactive spleens in 3/7 control males, 1/7 high-dosed males, 2/7 control females, and 1/7 high-dosed females. These histopathological findings were consistent with the historical control data of the test facility and were therefore not considered to be toxicologically relevant. The histopathologic examinations did not reveal any significant effects on spermatogenesis.
Histopathological findings: neoplastic:: no effects observed
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: not examined
Reproductive performance:: not examined
: Key result
Dose descriptor:: NOAEL
Effect level:: 1 125 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical signs; mortality; body weight and weight gain; food consumption and compound intake; ophthalmological examination; haematology; clinical biochemistry; organ weights and organ / body weight ratios; gross pathology; histopathology: non-neoplastic; histopathology: neoplastic
: Key result
Critical effects observed:: no
Remarks on result:: not measured/tested
Critical effects observed:: not specified
: Key result
Reproductive effects observed:: no
Conclusions:: NOAEL was considered to be 1125 mg/kg bw/day in both male and female SD rats.
Executive summary:: The test chemical was given to 7 rats per sex per dose level at 0 (vehicle; corn oil), 125, 375 and 1125 mg/kg/day. The study was performed according to GLP and according to OECD 407 with some additional reproductive-related endpoints included. Results: All animals survived to planned death and there were no clinical signs attributed to the test chemical. No significant changes in body weight were observed, except for a slight increase in male body weight at 1125 mg/kg on day 8 of treatment (mean, 248 g) compared to the control group (mean 237 g). This effect was considered incidental and not toxicologically significant. No significant changes in food intake or water intake were observed. No significant changes in locomotor activity were observed. No abnormalities were found during the ophthalmic examinations. Significant changes in haematology were as follows: significant increase in % monocytes in males treated at 125 mg/kg; significant increase in % basophils in males treated at 125 mg/kg; significant increases in white blood cell counts in females treated at 375 and 1125 mg/kg; significant decreases in red blood cell counts in females treated at 375 and 1125 mg/kg; significant decrease in haematocrit counts in females treated at 1125 mg/kg; significant increase in MCV in females treated at 1125 mg/kg; and significant increases in MCH in females treated at 375 and 1125 mg/kg. The observed changes in haematology were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings. Significant changes in clinical chemistry were as follows: significant increase in total cholesterol in male rats treated at 1125 mg/kg; significant increase in creatinine in male rats treated at 125 mg/kg; significant increase in glucose in male rats treated at 375 mg/kg; significant increases in total proteins in male rats treated at 375 and 1125 mg/kg; significant increase in potassium in female rats treated at 1125 mg/kg; significant increase in sodium in female rats treated at 1125 mg/kg; significant increase in SGPT in female rats treated at 375 and 1125 mg/kg; significant decrease in glucose in female rats treated at 1125 mg/kg; significant increase in total protein in female rats treated at 1125 mg/kg; significant increases in urea nitrogen in female rats treated at 375 and 1125 mg/kg; and a significant increase in bile acid in female rats treated at 375 mg/kg. No significant changes in serum testosterone or serum oestrogen were observed. The observed changes in clinical chemistry were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings. No significant changes in absolute organ weight were observed, with the exception of significant increases in liver weight in female rats treated at 375 and 1125 mg/kg. Significant changes in relative organ weight included a significant decrease in relative heart weight in male rats treated at 125 mg/kg and two significant increases in relative liver weight in female rats treated at 375 and 1125 mg/kg. In male rats, no gross pathological findings were observed. In female rats, gross pathological findings were limited to a slightly increased stomach at 1125 mg/kg and an enlarged uterus with uterine fluid at 1125 mg/kg. Histopathology performed at 0 and 1125 mg/kg showed excess of lymphocytes in the lungs of 2/7 control males, 1/7 high-dosed males, 1/7 control females, and 1/7 high-dosed females and reactive spleens in 3/7 control males, 1/7 high-dosed males, 2/7 control females, and 1/7 high-dosed females. These histopathological findings were consistent with the historical control data of the test facility and were therefore not considered to be toxicologically relevant. The histopathologic examinations did not reveal any significant effects on spermatogenesis. Conclusion: NOAEL was considered at 1125 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 125 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Klimisch 1.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Effects on developmental toxicity

Description of key information

The substance was given by oral gavage to 24 pregnant wistar rats at 0, 250, 500 and 1000 mg/kg bw/day from GD5 to GD19. The study was performed according to OECD 414 (adopted in 2018) and GLP.Results (adults): There were no unscheduled deaths, clinical signs, or gross pathological findings at 250 and 500 mg/kg/day. At 1000 mg/kg/day, one dam (Rz1075) had reddish discharge from the vagina on GD18 which was attributed to test item. No other clinical signs were observed at 1000 mg/kg/day, and there were no unschedulded deaths nor any gross pathological findings at 1000 mg/kg/day. As compared to the vehicle control group, statistically and dose-dependent decreases in maternal body weight (from -5 to -32%), in maternal body weight gain (from -17 to -199%), in adjusted maternal body weight (from -8 to -14%) and in adjusted maternal body weight gain (from -61 to -133%) were observed in all dosed groups. These effects at 250, 500 and 1000 mg/kg/day were attributed to the test item.As compared to vehicle control group, significant and dose-dependent decreases in mean food consumption (from -15 to -53%) were observed in all dosed groups during different periods of gestation.These effects at 250, 500 and 1000 mg/kg/day were attributed to the test item.No significant changes in T3 or T4 levels were observed.Significant increases in TSH levels were observed at 500 and 1000 mg/kg, however these effects were not considered to be of toxicological significance since the TSH levels at 500 and 1000 mg/kg were well within the historical control range of the test facility and since no significant changes in thyroid weight or histology of the thyroid were observed.No significant effects were observed at 250 mg/kg/day.Ten dams of 24 pregnant rats treated at 500 mg/kg/day and 22 dams of 23 pregnant rats treated at 1000 mg/kg/day had complete resorptions (i.e. only implantation sites with no fetuses). Hence, therewere only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation. Significant changes in uterine weight, number of implantations, implantation index, early resorptions, and post-implantation losses were observed at 500 and 1000 mg/kg/day. The effects observed at 500 and 1000 mg/kg/day were attributed to the test item. Gross evaluations of the placenta revealed no remarkable findings.Results (foetus):The total number of fetuses were 293, 302, 150, and 3 at 0, 250, 500 and 1000 mg/kg/day, respectively. No dead fetuses were observed at any dose level. No significant changes in sex ratio or AGD were observed. Significant decreases in male fetal weight and in sex-combined fetal weight were observed at 250 and 500 mg/kg/day and these effects were attributed to the test item. External, visceral and skeletal examinations revealed no teratogenic effects attributed to the test item at 250 and 500 mg/kg/day. There were 14 litters at 500 mg/kg/day and only 1 litter at 1000 mg/kg/day available for evaluation. Due to low sample size at 1000 mg/kg/day, the data from fetal external, visceral and skeletal evaluations were considered insufficient for evaluation of teratological effects.Conclusions:NOAEL for maternal toxicity could not be established due to significant decreases in body weight, body weight gain, and food intake at≥250mg/kg/day.NOAEL for maternal developmental toxicity was considered at250 mg/kg/day.Significant changes in uterine weight, early resorptions, postimplantation loss, and implantation index were observed at≥500mg/kg/dayand this dose level was therefore considered to be LOAEL for maternal developmental toxicity.NOAEL for fetal developmental toxicity could not be established due to significant decreases in fetal weights at≥250mg/kg/day. NOAEL for teratogenicity was considered at500 mg/kg/daysince fetal external, visceral and skeletal examinations did not reveal any signs of teratogenicity at 250 and 500 mg/kg/day.

The observed effects on fetal developmental toxicity (i.e. the small changes in fetal body weight) were considered to be secondary to maternal systemic toxicity whereas the observed effects on maternal developmental toxicity were not considered to be entirely due to maternal systemic toxicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The data is from study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted 25 June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd. 4B, MN Park, Shameerpet Mandal, Turkapally Village, Medchal District, Telangana -500078
- Age at study initiation: 12 weeks
- Weight at study initiation: G1: 259.946 ± 17.79; G2: 256.054 ± 19.41; G3: 258.465 ± 18.33; G4: 259.443 ± 15.27
- Fasting period before study: No data available
- Housing: standard polysulfone rat cages (size: Length 425 mm × Width 266 mm × Height 185 mm) with stainless steel top grill.
- Bedding: Steam sterilized corn cob was used as bedding material and was changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets, ad libitum
- Water (e.g. ad libitum): Deep bore-well water, ad libitum in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: five days before initiation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 58 - 66 %
- Air changes (per hr): 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light and 12-hours dark cycle

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test checmical was dissolved in Corn oil prior to dosing.

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is insoluble in water, but soluble in various oils and organic solvents. The test item was found to be miscible in corn oil and corn oil was therefore selected as vehicle.
- Concentration in vehicle: G1: 0 mg/ml; G2: 62.5 mg/ml; G3: 125 mg/ml; G4: 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
- Lot/batch no. (if required): NA
- Purity: NA

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: yes
- M/F ratio per cage: 1:1
- Length of cohabitation: Two weeks
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No data available

Duration of treatment / exposure:

Two Weeks

Frequency of treatment:

Daily

Duration of test:

GD 5 to GD 19

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24 females per group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20.
- Organs examined: placenta, thoracic and abdominal viscera, uterine content, thyroid gland

OTHER:

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: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Gross evaluation of placenta

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Statistics:

The data on maternal body weight, body weight change in interval, maternal food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, hormone analyses (T4, T3, TSH), and the weight of thyroid gland were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant. Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using ANCOVA taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal-Wallis test for group comparison. Wilcoxon pairwise comparisons of the treated groups with the control group were performed, when the group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran-Armitage trend test and pair-wise comparison will be tested by Fisher’s exact test for group association.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs at at 250 and 500 mg/kg/day. At 1000 mg/kg/day, one dam had reddish discharge from vagina on GD18 which was considered to be treatment-related. No other clinical signs were observed at 1000 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

As compared to the vehicle control group, statistically and dose-dependent decreases in maternal body weight (from -5 to -32%), in maternal body weight gain (from -17 to -199%), in adjusted maternal body weight (from -8 to -14%) and in adjusted maternal body weight gain (from -61 to -133%) were observed during most recorded time points and intervals. These observed effects on body weight, body weight gain, adjusted body weight, and adjusted body weight gain at ≥250 mg/kg/day were considered treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

As compared to the vehicle control group, significant reductions in mean food consumption were noticed following treatment at 250, 500 and
1000 mg/kg/day, during GD 5 - 8, 8 - 11, 11 - 14, 14 – 17 and 17 - 20. The decreases ranged from -15 to -27% at 250 mg/kg/day, from -25 to -30% at 500 mg/kg/day, and from -41 to -53% at 1000 mg/kg/day. Net food intakes during GD 5-20 were -22%, -27%, -46% and during gestation (GD 0-20) they were -17%, -21%, -35% lower at 250, 500 and 1000 mg/kg/day, respectively, when compared to the vehicle control group. The observed effects on food intake at ≥250 mg/kg/day were considered treatment-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in T3 or T4 levels were observed. Significant increases in TSH levels were observed at 500 and 1000 mg/kg, however these effects were not considered to be of toxicological significance since the TSH levels at 500 and 1000 mg/kg were well within the historical control range of the test facility and since no significant changes in thyroid weight or histology of the thyroid were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant changes in thryoid weight were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological effects were observed in the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No significant changes in the histology of the thyroid were observed in the study. The incidences of ectopic thymus were randomly distributed across the groups and were considered incidental background findings and not related to the test item administration.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

No significant effects were observed at 250 mg/kg/day. Ten dams of 24 pregnant rats treated at 500 mg/kg/day and 22 dams of 23 pregnant rats treated at 1000 mg/kg/day had complete resorptions (i.e. only implantation sites with no fetuses). Hence, there were only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

No significant changes in pre- or post-implantation loss were observed at 250 mg/kg/day. At 500 and 1000 mg/kg/day there were significant decreases in the number of implantations by -11% and -19%, respectively; significant decreases in implantation indexes by -14 and -20%, respectively; and significant increases in % post-implantation losses by 55 and 99%, respectively.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 500 and 1000 mg/kg/day there were significant increases in % early resorptions by 50 and 98%, respectively, when compared to the control group. No other significant changes were observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were observed at any dose level.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant rats were 21, 24, 24, 23 at 0, 250, 500 and 1000 mg/kg /day, respectively.

Dose descriptor:

LOAEL

Remarks:

maternal systemic toxicity

Effect level:

<= 250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

dead fetuses

early or late resorptions

maternal abnormalities

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

Dose descriptor:

LOAEL

Remarks:

maternal developmental toxicity

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

early or late resorptions

number of abortions

pre and post implantation loss

total litter losses by resorption

Abnormalities:

no effects observed

Localisation:

placenta

Description (incidence and severity):

Gross evaluation of placenta did not reveal any remarkable findings in any dams at any dose level.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Sex-combined fetal weights were significantly decreased at 250 (by -5%) and 500 mg/kg/day (by -6%) compared to the control data. Significant decreases in male fetal weight were observed at 250 (by -5%) and 500 mg/kg/day (by -8%) compared to the control data, whereas only a significant decrease in female fetal weight was observed at 250 mg/kg/day (by -4%) compared to the control data.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No dead fetuses were observed in the study.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant changes in sex ratio were observed.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

There were only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was one incidence of a fetus (1/302 fetuses) with a thread like tail at 250 mg/kg/day [malformation], one small fetus (1/150 fetuses) at 500 mg/kg/day [anomaly] and two fetuses with hindlimbs turned inwards at 1000 mg/kg/day (2/3 fetuses) [malformation]. These observations at 250 and 500 mg/kg were considered incidental and not treatment-related.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg/day, there was one fetus from one dam (Rz1025) which had absent caudal vertebrae (centrum and arch absent) [malformation]. The same fetus had thread like tail during external examination [malformation]. These malformations at 250 mg/kg/day were considered to be spontaneous in nature since no such malformations were observed at 500 mg/kg/day. At 1000 mg/kg/day, one fetus from one dam (Rz1075) had tarsal bones bent inwards [malformation]. The same fetus had hind limbs turned inwards during external examination [malformation]. There were significant increases in variations of incomplete ossification of skull bones namely parietal, interparital and supraoccipital bones at 500 mg/kg/day. Delayed/incomplete ossifications are generally considered transient changes and these finding denote generalized growth delays which normally ossify late in gestation. They also do not have general predictive value for teratogenicity [Carney and Kimmel (2007)]. A significant increase in the incidene of bilateral wavy ribs (#4 to #11) [anomaly] was observed at 500 mg/kg/day as compared to vehicle control group. Wavy ribs are manifestations of delayed ossification, are transient and reversible and are not of developmental importance since they are expected to resolve during maturation (Hayasaka et al., 1985; Kast, 1994; Mitchard & Stewart, 2014). The observed variations and anomalies at 500 mg/kg/day were considered non-adverse based on the discussions above, and they were likely secondary to maternal systemic toxicity at 500 mg/kg/day.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed during visceral examination at 250 and 500 mg/kg. There was one incidence of moderate bilateral renal pelvis dilation at 250 mg/kg/day (1/145 fetuses) [anomaly]. This finding was considered spontaneous in nature and therefore not treatment-related. All fetal heads from the Wilsons-Razor blade sectioning were found to be normal (i.e. no malformations, anomalies, or variations were observed).

Details on embryotoxic / teratogenic effects:

A total of 14 litters at 500 mg/kg and only 1 litter at 1000 mg/kg were available for evaluation due to the number of complete resorptions observed at 500 and 1000 mg/kg. The results from the external, visceral, and seketal examinations performed at 1000 mg/kg were not considered to be conclusive due to low sample size.

Dose descriptor:

LOAEL

Effect level:

<= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Dose descriptor:

NOAEL

Remarks:

Teratogenicity

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Table for Maternal Survival, Pregnancy Status and Fetus Disposition

Parameters	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	250	500	1000
Total No. of rats found sperm positive / group		24	24	24	24
Duration of treatment		GD 5 to 19 (total 15 days)
Caesarean section (day of presumed gestation)		GD 20
Number of rats sacrificed at caesarean section		24	24	24	24
Number. of rats non-pregnant at caesarean section		3	0	0	1
Number of rats pregnant at caesarean section		21	24	24	23
Number of dams with complete resorptions Number of litters examined		0 21	0 24	10 14	22 1
Total number of fetuses		293	302	150	3
Total number of dead fetuses		0	0	0	0

Number of fetuses evaluated
a. External examination		293	302	150	3
b. Visceral examination		140	145	72	1
c. Skeletal examination		153	157	78	2

Table for Clinical Signs and Mortality

Observation Type: All Types

From Day 0 (Mating (A)) to 20 (Mating)

Female

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

Normal

Killed ‑ terminal kill

Discharge

Values = Number of Animals Affected

Table for Maternal Body Weight of Pregnant Rats

Sex: Female

Day(s) Relative to Mating

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

Body

Weight

(g)

0[a]

Mean

%Diff

258.46

15.81

‑

256.05

19.41

‑0.93

258.47

18.33

0.00

259.76

15.53

0.51

3[a]

Mean

%Diff

274.90

16.40

‑

270.79

20.34

‑1.49

273.00

19.47

‑0.69

275.44

15.62

0.20

5[a]

Mean

%Diff

282.41

16.75

‑

278.79

21.26

‑1.28

281.42

21.02

‑0.35

283.22

16.10

0.29

8[a]

Mean

%Diff

291.59

17.75

‑

279.67

22.14

‑4.09

282.03

20.26

‑3.28

274.09*

19.69

‑6.00

11[a]

Mean

%Diff

306.63

20.97

‑

290.32*

22.28

‑5.32

291.51*

19.37

‑4.93

278.92*

20.20

‑9.04

14[a]

Mean

%Diff

325.57

20.75

‑

302.34*

22.59

‑7.14

299.74*

19.23

‑7.94

279.46*

19.22

‑14.17

17[a]

Mean

%Diff

356.30

27.33

‑

329.30*

26.60

‑7.58

310.98*

27.02

‑12.72

273.96*

22.27

‑23.11

20[a1]

Mean

%Diff

400.72

31.91

‑

366.14*

31.33

‑8.63

329.87*

40.22

‑17.68

274.36*

21.62

‑31.53

[a] ‑ Anova & Dunnett: * = p < 0.05 ; [a1] ‑ Anova & Dunnett(Rank): * = p < 0.05

Table for Maternal Body Weight Gain of Pregnant Rats

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 250 mg/kg/day	G3 500 mg/kg/day	G4 1000 mg/kg/day
Absolute Weight Gain (g)	0 → 3 [a]	Mean SD N %Diff	16.44 4.88 21 -	14.74 5.73 24 ‑10.33	14.53 3.97 24 ‑11.61	15.68 4.57 23 ‑4.61
	3 → 5 [a1]	Mean SD N %Diff	7.52 3.56 21 -	8.00 3.32 24 6.38	8.43 3.84 24 12.12	7.78 3.15 23 3.47
	5 → 8 [a2]	Mean SD N %Diff	9.18 5.21 21 -	0.88* 4.37 24 ‑90.46	0.61* 5.17 24 ‑93.37	‑9.13* 9.25 23 ‑199.44
	8 → 11 [a2]	Mean SD N %Diff	15.04 7.19 21 -	10.66 4.78 24 ‑29.13	9.48* 7.51 24 ‑36.97	4.83* 10.45 23 ‑67.91
	11 → 14 [a]	Mean SD N %Diff	18.94 5.07 21 -	12.02* 5.53 24 ‑36.57	8.23* 5.85 24 ‑56.58	0.54* 7.8 23 ‑97.15
	14 → 17 [a2]	Mean SD N %Diff	30.73 9.08 21 -	26.96 8.46 24 ‑12.25	11.24* 15.43 24 ‑63.41	‑5.50* 8.91 23 ‑117.89
	17 → 20 [a2]	Mean SD N %Diff	44.42 9.78 21 -	36.83* 9.64 24 -17.09	18.90* 18.50 24 -57.46	0.41* 5.87 23 -99.09
	0 → 5 [a]	Mean SD N %Diff	23.95 5.78 21 -	22.74 5.68 24 -5.09	22.96 4.68 24 -4.16	23.46 4.63 23 -2.08
	5 → 20 [a1]	Mean SD N %Diff	118.31 21.77 21 -	87.35* 17.03 24 -26.17	48.45* 40.72 24 -59.05	8.86* 15.43 23 -107.48
	0 → 20 [a1]	Mean SD N %Diff	142.27 25.20 21 -	110.08* 18.56 24 -22.62	71.41* 40.35 24 -49.81	14.60* 17.18 23 -89.74
Adjusted Bodyweight (g)	GD20 GU WT [a]	Mean SD N %Diff	316.02 19.35 21 -	291.78* 23.20 24 -7.67	291.86* 19.67 24 -7.65	272.24* 17.79 23 -13.85
Adjusted BW Gain (g)	ADJ BWT GD5B [a]	Mean SD N %Diff	33.61 8.82 21 -	12.99* 10.08 24 -61.35	10.44* 14.59 24 -68.95	10.98* 11.40 23 -132.65

[a] ‑ Anova & Dunnett: * = p < 0.05; [a1] ‑ Anova & Dunnett(Log); [a1] ‑ Anova & Dunnett(Rank): * = p < 0.05; [a2] ‑ Anova & Dunnett(Rank): * = p < 0.05

Table for Food Consumption of Pregnant Rats

Sex: Female

Day(s) Relative to Mating

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

Food Mean

Consumption

(g/day)

0 → 3 [a]

Mean

%Diff

22.59

2.66

21.59

2.46

‑4.42

22.66

1.93

0.35

22.61

2.71

0.09

3 → 5 [a]

Mean

%Diff

24.06

2.3

23.42

2.42

‑2.66

24.34

1.98

1.14

23.37

2.23

‑2.90

5 → 8 [a]

Mean

%Diff

21.77

3.75

15.87*

2.71

‑27.09

15.18*

3.43

‑30.30

10.21*

3.59

‑53.11

8 → 11 [a1]

Mean

%Diff

21.83

4.26

16.35*

2.26

‑25.11

16.41*

2.6

‑24.83

12.8*

2.62

‑41.39

11 → 14 [a]

Mean

%Diff

25.96

2.87

19.35*

2.92

‑25.48

18.73*

2.69

‑27.85

14.93*

2.90

‑42.52

14 → 17 [a1]

Mean

%Diff

26.83

2.57

22.00*

2.76

‑18.00

19.77*

4.98

‑26.31

13.72*

3.77

‑48.85

17 → 20 [a1]

Mean

%Diff

27.73

2.42

23.67*

3.36

‑14.65

20.28*

4.78

‑26.89

15.32*

2.94

‑44.77

[a] ‑ Anova & Dunnett: * = p < 0.05; [a1] ‑ Anova & Dunnett(Rank): * = p < 0.05

Contd. Table for Food Consumption of Pregnant Rats

Sex: Female

Day(s) Relative to Mating (Litter: A)

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

Food Mean

Consumption

(g/day)

0 → 5 [a]

Mean

%Diff

23.18

2.37

22.32

2.23

‑3.69

23.33

1.67

0.68

22.91

2.37

‑1.15

5 →20 [a1]

Mean

%Diff

24.83

2.72

19.45*

2.29

‑21.66

18.07*

3.00

‑27.20

13.39*

2.24

‑46.05

0 → 20 [a2]

Mean

%Diff

24.41

2.52

20.17*

2.15

‑17.40

19.39*

2.37

‑20.58

15.77*

1.89

‑35.39

[a] ‑ Anova & Dunnett: * = p < 0.05

Table for Maternal Data

Sex: Female

G10

mg/kg/day

G2 250

mg/kg/day

G3 500

mg/kg/day

G4 1000

mg/kg/day

Group size

Pregnant at C/S

Gravid Uterus Weight

[a]

Mean SD

84.702

20.604

74.357

18.785

38.015 *

33.505

2.121 *

5.989

Number of

Corpora Lutea

[k]

Mean

Sum

17.0

2.6

356.0

16.5

1.9

396.0

17.7

2.5

425.0

17.1

2.5

394.0

Number of

Implantation

[k]

Mean SD

Sum

15.3

2.7

321.0

15.3

2.0

368.0

13.6 *

2.5

326.0 *

12.4 *

3.2

285.0 *

Number of dams with Early Resorption

Number of Early Resorptions

[k]

Mean SD

Sum

1.2

1.3

26.0

2.3

2.5

56.0

6.8 *

5.9

162.0 *

12.1 *

3.5

278.0 *

% Early Resorption

/Animal

[k]

Mean SD

9.23

11.06

15.59

17.01

50.46 *

44.36

97.66 *

11.23

Number of dams with Late Resorption

Number of Late Resorptions

[k]

Mean SD

Sum

0.1

0.3

2.0

0.4

0.9

10.0

0.6

1.6

14.0

0.2

0.8

4.0

% Late Resorption

/Animal

[k]

Mean SD

0.68

2.17

2.72

5.59

4.22

12.56

1.34

6.42

Number of dams with

Resorptions

[f]

Total Number of Resorptions (Early + Late)

[f]

Mean SD

Sum

1.3

1.2

28.0

2.8

66.0

7.3

5.6

176.0

12.3

3.3

282.0

Pre-implantation Loss/Animal

[k]

Mean SD

1.67

1.77

35.00

1.17

1.31

28.00

4.13 *

2.64

99.00 *

4.74 *

1.94

109.00 *

% Pre-implantation

Loss

[k]

Mean

9.8

10.2

6.9

7.7

22.8 *

13.7

28.4 *

12.5

Post-implantation Loss/Animal

[k]

Mean SD

1.33

1.20

28.00

2.75

2.77

66.00

7.33 *

5.60

176.00 *

12.26 *

3.25

282.00 *

% Post-implantation

Loss (%)

[k]

Mean

9.9

10.7

18.3

18.7

54.7 *

41.9

99.0 *

4.8

Implantation

Index

[a1]

Mean SD

90.24

10.20

93.05

7.68

77.17 *

13.67

71.57 *

12.52

Note: Gross evaluation of placenta revealed no findings;

[a] - Anova & Dunnett(Rank): * = p < 0.05; [a1] - Anova & Dunnett(Log): * = p < 0.05; [k] - Kruskal-Wallis & Wilcoxon: * = p < 0.05; [f] - Cochran Armitage, Chi-Squared & Fisher's Exact

Table for Litter Data

Sex: Female			G10 mg/kg/day	G2 250 mg/kg/day	G3 500 mg/kg/day	G4 1000 mg/kg/day
Total Number of fetuses		Sum	293	302	150	3
Total Number of Dead Fetuses		Sum	0	0	0	0
Total Number of Live fetuses		Sum	293	302	150	3
Live Male fetus		Sum	150	156	75	3
% Male Fetus			51.2	51.7	50.0	100.0
Mean Fetal Weight- Male (g)	[c]	Mean SD	4.153 0.328	3.933 * 0.415	3.836 * 0.306	4.697 .
Mean AGD- Male (mm)	[c1]	Mean SD	2.67 0.16	2.62 0.13	2.64 0.15	3.00
Live Female fetus		Sum	143	146	75	0
% Female Fetus			48.8	48.3	50.0	0.0
Mean Fetal Weight- Female (g)	[c2]	Mean SD	3.898 0.335	3.728 * 0.428	3.725 0.359	. .
Mean AGD- Female (mm)	[c3]	Mean SD	0.96 0.12	1.00 0.13	0.96 0.10	. .
Mean Fetal Weight -Male+Female (g)	[c4]	Mean SD	4.027 0.318	3.827 * 0.405	3.776 * 0.302	4.697 .
Mean AGD Male + Female (mm)	[c5]	Mean SD	1.83 0.19	1.82 0.31	1.84 0.32	3.00

[c] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Male Fetuses}

[c1] - Ancova/Anova & Dunnett;Covariate(s): Number of LiveMaleFetuses}

[c2] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Female Fetuses}

[c3] - Ancova/Anova & Dunnett;{Covariate(s): Number of Live Female Fetuses}

[c4] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Fetuses}

[c5] - Ancova/Anova & Dunnett;Covariate(s): Number of Live Fetuses}

Table for Fetal External Observations

Exam type: External

Number of Live Fetuses Examined

Number of Litters Evaluated

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

293

302

150

Limbs

Tail, Thread‑like tail – Malformation

Limb, both, Bent Inward – Malformation

General

Fetus, Small Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.3)

1(4.2)

0(0.0)

1(0.7)

1(7.1)

0(0.0)

2(66.7)

1(100.0)

0(0.0)

Fetuses N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact; [Litters N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact

Table for Fetal Visceral Observations

Exam type: Fresh Visceral Body Only

Number of Live Fetuses Examined

Number of Litters Evaluated

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

140

145

Abdomen

Kidney, both, Renal pelvis dilation, Moderate – Anomaly

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.7)

1(4.2)

0(0.0)

Fetuses N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact; [Litters N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact

Table for Fetal Skeletal Observations

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

153

157

Hindlimbs

Tarsal bone, both, Bent – Malformation

Caudal vertebrate

14th caudal centrum, Absent – Anomaly

13th caudal centrum, Absent – Anomaly

12th caudal centrum, Absent – Anomaly

11th caudal centrum, Absent – Anomaly

10th caudal centrum, Absent – Anomaly

9th caudal centrum, Absent – Anomaly

8th caudal centrum, Absent – Malformations

7th caudal centrum, Absent – Malformations

6th caudal centrum, Absent – Malformations

5th caudal centrum, Absent – Malformations

4th caudal centrum, Absent – Malformations

3rd caudal centrum, Absent – Malformations

2nd caudal centrum, Absent – Malformations

1st caudal centrum, Absent – Malformations

6th caudal arch, Absent – Malformation

5th caudal arch, Absent – Malformation

4th caudal arch, Absent – Malformation

3rd caudal arch, Absent – Malformation

2nd caudal arch, Absent – Malformation

1st caudal arch, Absent – Malformation

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

[Fetuses N] Cochran Armitage, Chi Squared & Fisher's Exact; [Litters N] Cochran Armitage, Chi Squared & Fisher's Exact

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

153

157

sacral vertebrae

4th sacral centrum, Absent – Malformation

3rd sacral centrum, Absent – Malformation

2nd sacral centrum, Absent – Malformation

1st sacral centrum, Absent – Malformation

4th sacral arch, Absent – Malformation

4rd sacral arch, Absent – Malformation

4nd sacral arch, Absent – Malformation

1st sacral arch, Absent – Malformation

Lumbar vertebrae

6th lumbar centrum, Absent – Malformation

5th lumbar centrum, Hypoplastic – Anomaly

2nd lumbar centrum, Dumbbell‑shaped – Anomaly

6th lumbar arch, Displaced – Anomaly

6th lumbar arch, Incomplete ossification Anomaly

7th lumbar centrum and arch, Extra – Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

[Fetuses N] Cochran Armitage, Chi Squared & Fisher's Exact; [Litters N] Cochran Armitage, Chi Squared & Fisher's Exact

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

153

157

thoracic vertebrae

13th thoracic centrum, Dumbbell shaped – Anomaly

13th thoracic centrum, Asymmetrical split – Anomaly

12th thoracic centrum, Unilateral Ossification –Anomaly

12th thoracic centrum, Split – Anomaly

12th thoracic centrum, Dumbbell‑shaped – Anomaly

12th thoracic centrum, Asymmetrical dumbbell shaped – Anomaly

12th thoracic centrum, Asymmetrical split – Anomaly

11th thoracic centrum, Unilateral Ossification – Anomaly

11th thoracic centrum, Dumbbell‑shaped – Anomaly

11th thoracic centrum, Asymetrical dumbbell shaped – Anomaly

10th thoracic centrum, Split – Anomaly

10th thoracic centrum, Dumbbell‑shaped – Anomaly

10th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

9th thoracic centrum, Split – Anomaly

9th thoracic centrum, Dumbbell‑shaped – Anomaly

9th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

8th thoracic centrum, Split – Anomaly

8th thoracic centrum, Dumbbell‑shaped – Anomaly

7th thoracic centrum, Split – Anomaly

7th thoracic centrum, Dumbbell‑shaped – Anomaly

6th thoracic centrum, Unilateral Ossification – Anomaly

6th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

5th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

4th thoracic centrum, Dumbbell‑shaped – Anomaly

4th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

3rd thoracic centrum, Dumbbell‑shaped – Anomaly

2nd thoracic centrum, Dumbbell‑shaped – Anomaly

2nd thoracic centrum, Delayed skeletal ossification – Variation

2nd thoracic centrum, Incomplete ossification – Variation

1st thoracic centrum, Delayed skeletal ossification – Variation

1st thoracic centrum, Incomplete ossification – Variation

Cervical vertebrae

Cervical rib, Bilateral, Rudimentary – Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

2(1.3)

2(9.5)

0(0.0)

2(1.3)

2(9.5)

10(6.5)

8(38.1)

2(1.3)

2(9.5)

0(0.0)

11(7.2)

9(42.9)

1(0.7)

1(4.8)

2(1.3)

2(9.5)

23(15.0)

11(52.4)

2(1.3)

2(9.5)

0(0.0)

23(15.0)

11(52.4)

0(0.0)

5(3.3)

5(23.8)

1(0.7)

1(4.8)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

2(1.3)

2(9.5)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

3(1.9)

3(12.5)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

9(5.7)

9(37.5)

0(0.0)

13(8.3)

11(45.8)

2(1.3)

2(8.3)

1(0.6)

1(4.2)

13(8.3)

9(37.5)

2(1.3)

2(8.3)

1(0.6)

1(4.2)

12(7.6)*

9(37.5)*

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)*

0(0.0)

1(0.6)

1(4.2)

0(0.0)

1(1.3)

1(7.1)

4(5.1)

3(21.4)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

4(5.1)

4(28.6)

1(1.3)

1(7.1)

0(0.0)

6(7.7)

3(21.4)

1(1.3)

1(7.1)

0(0.0)

2(2.6)*

2(14.3)*

0(0.0)

3(3.8)

3(21.4)

0(0.0)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

153

157

ribs

14th rib, Right, Rudimentary - Anomaly

14th rib, Right, Accessory – Anomaly

14th rib, Left, Rudimentary – Anomaly

14th rib, Left, Accessory – Anomaly

14th rib, Bilateral, Rudimentary – Anomaly

14th rib, Bilateral, Accessory – Anomaly

14th rib, Bilateral, Extra – Anomaly

13th rib, Right, Wavy Rib, Moderate – Anomaly

13th rib, Left, Wavy Rib, Moderate – Anomaly

13th rib, Bilateral, Wavy Rib, Moderate – Anomaly

12th rib, Left, Wavy Rib, Moderate – Anomaly

12th rib, Right, Wavy Rib, Moderate – Anomaly

12th rib, Bilateral, Wavy Rib, Moderate – Anomaly

11th rib, Left, Wavy Rib, Slight – Anomaly

11th rib, Right, Wavy Rib, Slight – Anomaly

11th rib, Right, Wavy Rib, Moderate – Anomaly

11th rib, Bilateral, Wavy Rib, Slight – Anomaly

11th rib, Bilateral, Wavy Rib, Moderate – Anomaly

10th rib, Right, Wavy Rib, Slight – Anomaly

10th rib, Right, Wavy Rib, Moderate – Anomaly

10th rib, Left, Wavy Rib, Slight – Anomaly

10th rib, Bilateral, Wavy Rib, Slight – Anomaly

10th rib, Bilateral, Wavy Rib, Moderate – Anomaly

9th rib, Left, Wavy Rib, Slight – Anomaly

9th rib, Right, Wavy Rib, Slight – Anomaly

9th rib, Right, Wavy Rib, Moderate – Anomaly

9th rib, Bilateral, Wavy Rib, Slight – Anomaly

9th rib, Bilateral, Wavy Rib, Moderate – Anomaly

8th rib, Left, Wavy Rib, Slight – Anomaly

8th rib, Right, Wavy Rib, Slight – Anomaly

8th rib, Right, Wavy Rib, Moderate – Anomaly

8th rib, Bilateral, Wavy Rib, Slight – Anomaly

8th rib, Bilateral, Wavy Rib, Moderate – Anomaly

7th rib, Right, Wavy Rib, Slight – Anomaly

7th rib, Right, Wavy Rib, Moderate – Anomaly

7th rib, Left, Wavy Rib, Slight – Anomaly

7th rib, Bilateral, Wavy Rib, Slight – Anomaly

7th rib, Bilateral, Wavy Rib, Moderate – Anomaly

6th rib, Left, Wavy Rib, Slight – Anomaly

6th rib, Right, Wavy Rib, Slight – Anomaly

6th rib, Right, Wavy Rib, Moderate – Anomaly

6th rib, Bilateral, Wavy Rib, Slight – Anomaly

6th rib, Bilateral, Wavy Rib, Moderate – Anomaly

5th rib, Right, Wavy Rib, Slight – Anomaly

5th rib, Right, Wavy Rib, Moderate – Anomaly

5th rib, Left, Wavy Rib, Slight – Anomaly

5th rib, Bilateral, Wavy Rib, Slight – Anomaly

5th rib, Bilateral, Wavy Rib, Moderate – Anomaly

4th rib, Right, Wavy Rib, Slight – Anomaly

4th rib, Right, Wavy Rib, Moderate – Anomaly

4th rib, Left, Wavy Rib, Slight – Anomaly

4th rib, Bilateral, Wavy Rib, Slight – Anomaly

4th rib, Bilateral, Wavy Rib, Moderate – Anomaly

3rd rib, Bilateral, Wavy Rib, Moderate – Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

12(7.8)

8(38.1)

2(1.3)

2(9.5)

14(9.2)

11(52.4)

0(0.0)

12(7.8)

9(42.9)

2(1.3)

2(9.5)

0(0.0)

7(4.5)

6(25.0)

1(0.6)

1(4.2)

12(7.6)

9(37.5)

4(2.5)

3(12.5)

17(10.8)

12(50.0)

0(0.0)

1(0.6)

1(4.2)

2(1.3)

2(8.3)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

3(1.9)

3(12.5)

4(2.5)

3(12.5)

0(0.0)

1(0.6)

1(4.2)

7(4.5)*

7(29.2)*

0(0.0)

3(1.9)

2(8.3)

2(1.3)

2(8.3)

9(5.7)*

6(25.0)*

0(0.0)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

6(3.8)*

4(16.7)

0(0.0)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

8(5.1)*

5(20.8)

0(0.0)

1(0.6)

1(4.2)

0(0.0)

7(4.5)*

5(20.8)

0(0.0)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

6(3.8)*

4(16.7)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

2(1.3)

2(8.3)

4(2.5)

4(16.7)

0(0.0)

1(0.6)

1(4.2)

0(0.0)

3(3.8)

3(21.4)

0(0.0)

5(6.4)

4(28.6)

0(0.0)

12(15.4)

7(50.0)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

3(3.8)

3(21.4)

2(2.6)

2(14.3)

3(3.8)*

3(21.4)

4(5.1)*

3(21.4)

10(12.8)*

8(57.1)*

1(1.3)

1(7.1)

6(7.7)*

6(42.9)*

5(6.4)*

4(28.6)*

10(12.8)*

8(57.1)*

3(3.8)*

3(21.4)

2(2.6)

2(14.3)

4(5.1)*

4(28.6)*

3(3.8)*

3(21.4)

1(1.3)

1(7.1)

11(14.1)*

9(64.3)*

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

4(5.1)*

4(28.6)*

3(3.8)*

3(21.4)*

11(14.1)*

9(64.3)

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

2(2.6)

2(14.3)

10(12.8)*

8(57.1)*

5(6.4)*

5(35.7)*

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

4(5.1)*

4(28.6)*

1(1.3)

1(7.1)

8(10.3)*

7(50.0)*

1(1.3)

1(7.1)

4(5.1)

4(28.6)

1(1.3)

1(7.1)

6(7.7)*

5(35.7)*

2(2.6)

2(14.3)

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

3(3.8)*

3(21.4)*

1(1.3)

1(7.1)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

mg/kg/day

250

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

153

157

sternebrae

6th sternebra, Hypoplastic – Anomaly

6th sternebra, Delayed skeletal ossification – Variation

6th sternebra, Incomplete ossification – Variation

5th sternebra, Hypoplastic – Anomaly

5th sternebra, Asymmetrical ossification – Anomaly

5th sternebra, Split – Anomaly

4th sternebra, Asymmetrical ossification – Anomaly

4th sternebra, Incomplete ossification – Variation

4th sternebra, Split – Anomaly

3rd sternebra, Asymmetrical ossification – Anomaly

3rd sternebra, Incomplete ossification – Variation

2nd sternebra, Hypoplastic – Anomaly

2nd sternebra, Split – Anomaly

1st sternebra, Incomplete ossification – Variation

Skull

Supraoccipital, Incomplete ossification – Variation

Parietal, Incomplete ossification – Variation

Interparietal, Incomplete ossification – Variation

Hyoid, Incomplete ossification – Variation

Frontal, Incomplete ossification – Variation

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

7(4.6)

6(28.6)

43(28.1)

18(85.7)

19(12.4)

11(52.4)

1(0.7)

1(4.8)

2(1.3)

2(9.5)

1(0.7)

1(4.8)

13(8.5)

9(42.9)

1(0.7)

1(4.8)

1(0.7)

1(4.8)

7(4.6)

7(33.3)

2(1.3)

2(9.5)

1(0.7)

1(4.8)

1(0.7)

1(4.8)

0(0.0)

2(1.3)

2(9.5)

2(1.3)

2(9.5)

0(0.0)

1(0.6)

1(4.2)

15(9.6)

6(25.0)

50(31.8)

19(79.2)

21(13.4)

13(54.2)

0(0.0)

2(1.3)

2(8.3)

0(0.0)

6(3.8)

4(16.7)

0(0.0)

1(0.6)

1(4.2)

6(3.8)

5(20.8)

1(0.6)

1(4.2)

0(0.0)

20(12.7)*

13(54.2)*

35(22.3)*

15(62.5)*

41(26.1)*

16(66.7)*

0(0.0)

2(2.6)

2(14.3)

34(43.6)

14(100.0)

10(12.8)

8(57.1)

0(0.0)

2(2.6)

2(14.3)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

0(0.0)

5(6.4)

3(21.4)

0(0.0)

1(1.3)

1(7.1)

25(32.1)*

10(71.4)*

31(39.7)*

12(85.7)*

34(43.6)*

12(85.7)*

7(9.0)*

3(21.4)

2(2.6)

1(7.1)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

[Fetuses N] Cochran Armitage, Chi Squared & Fisher's Exact; [Litters N] Cochran Armitage, Chi Squared & Fisher's Exact

Conclusions:

NOAEL for maternal toxicity could not be established due to significant decreases in body weight, body weight gain, and food intake at ≥250mg/kg/day. NOAEL for maternal developmental toxicity was considered at 250 mg/kg/day. Significant changes in uterine weight, early resorptions, postimplantation loss, and implantation index were observed at ≥500 mg/kg/day and this dose level was therefore considered to be LOAEL for maternal developmental toxicity. NOAEL for fetal developmental toxicity could not be established due to significant decreases in fetal weights at ≥250mg/kg/day. NOAEL for teratogenicity was considered at 500 mg/kg/day since fetal external, visceral and skeletal examinations did not reveal any signs of teratogenicity at 250 and 500 mg/kg/day. The observed effects on fetal developmental toxicity (i.e. the small changes in fetal body weight) were considered to be secondary to maternal systemic toxicity whereas the observed effects on maternal developmental toxicity were not considered to be entirely due to maternal systemic toxicity.

Executive summary:

The substance was given by oral gavage to 24 pregnant wistar rats at 0, 250, 500 and 1000 mg/kg bw/day from GD5 to GD19. The study was performed according to OECD 414 (adopted in 2018) and GLP. Results (adults): There were no unscheduled deaths, clinical signs, or gross pathological findings at 250 and 500 mg/kg/day. At 1000 mg/kg/day, one dam (Rz1075) had reddish discharge from the vagina on GD18 which was attributed to test item. No other clinical signs were observed at 1000 mg/kg/day, and there were no unschedulded deaths nor any gross pathological findings at 1000 mg/kg/day. As compared to the vehicle control group, statistically and dose-dependent decreases in maternal body weight (from -5 to -32%), in maternal body weight gain (from -17 to -199%), in adjusted maternal body weight (from -8 to -14%) and in adjusted maternal body weight gain (from -61 to -133%) were observed in all dosed groups. These effects at 250, 500 and 1000 mg/kg/day were attributed to the test item.As compared to vehicle control group, significant and dose-dependent decreases in mean food consumption (from -15 to -53%) were observed in all dosed groups during different periods of gestation.These effects at 250, 500 and 1000 mg/kg/day were attributed to the test item.No significant changes in T3 or T4 levels were observed.Significant increases in TSH levels were observed at 500 and 1000 mg/kg, however these effects were not considered to be of toxicological significance since the TSH levels at 500 and 1000 mg/kg were well within the historical control range of the test facility and since no significant changes in thyroid weight or histology of the thyroid were observed.No significant effects were observed at 250 mg/kg/day. Ten dams of 24 pregnant rats treated at 500 mg/kg/day and 22 dams of 23 pregnant rats treated at 1000 mg/kg/day had complete resorptions (i.e. only implantation sites with no fetuses). Hence, therewere only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation. Significant changes in uterine weight, number of implantations, implantation index, early resorptions, and post-implantation losses were observed at 500 and 1000 mg/kg/day. The effects observed at 500 and 1000 mg/kg/day were attributed to the test item. Gross evaluations of the placenta revealed no remarkable findings.Results (foetus): The total number of fetuses were 293, 302, 150, and 3 at 0, 250, 500 and 1000 mg/kg/day, respectively. No dead fetuses were observed at any dose level. No significant changes in sex ratio or AGD were observed. Significant decreases in male fetal weight and in sex-combined fetal weight were observed at 250 and 500 mg/kg/day and these effects were attributed to the test item. External, visceral and skeletal examinations revealed no teratogenic effects attributed to the test item at 250 and 500 mg/kg/day. There were 14 litters at 500 mg/kg/day and only 1 litter at 1000 mg/kg/day available for evaluation. Due to low sample size at 1000 mg/kg/day, the data from fetal external, visceral and skeletal evaluations were considered insufficient for evaluation of teratological effects. Conclusions: NOAEL for maternal toxicity could not be established due to significant decreases in body weight, body weight gain, and food intake at ≥250mg/kg/day. NOAEL for maternal developmental toxicity was considered at 250 mg/kg/day. Significant changes in uterine weight, early resorptions, postimplantation loss, and implantation index were observed at ≥500mg/kg/dayand this dose level was therefore considered to be LOAEL for maternal developmental toxicity. NOAEL for fetal developmental toxicity could not be established due to significant decreases in fetal weights at ≥250mg/kg/day. NOAEL for teratogenicity was considered at 500 mg/kg/day since fetal external, visceral and skeletal examinations did not reveal any signs of teratogenicity at 250 and 500 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: LOAEL
: 500 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Klimisch 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Justification for classification or non-classification

As per CLP guidance, a comparison between the severity of the effects on fertility and (or) development and the severity of other toxicological findings must made during classification. In the presented OECD 414 study, the observed effects on development were observed in the presence of maternal systemic toxicity. The observed effects on fetal developmental toxicity (i.e. the small changes in fetal body weight) were considered to be secondary to maternal systemic toxicity whereas the observed effects on maternal developmental toxicity were not considered to be entirely due to maternal systemic toxicity.

By taking into account CLP guidance, the substance is regarded to be classified as “H361d: suspected of damaging fertility or the unborn child” (Category 2).

Additional information

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Dose		Testes	Epididymides	Ovaries	Uterus
0 (mg/kg)	Mean	2.6556	1.0201	0.0944	0.5236
	SD	0.6146	0.1210	0.0322	0.1078
	SEM	0.2323	0.0457	0.0122	0.0407
125 (mg/kg)	Mean	2.7550	0.9950	0.0953	0.5293
	SD	0.2371	0.1474	0.0382	0.1607
	SEM	0.0896	0.0557	0.0144	0.0607
375 (mg/kg)	Mean	2.8762	0.9389	0.0725	0.4847
	SD	0.1901	0.1180	0.0209	0.0706
	SEM	0.0718	0.0446	0.0079	0.0267
1125 (mg/kg)	Mean	2.6205	0.9031	0.0693	0.5155
	SD	0.4577	0.2122	0.0290	0.1438
	SEM	0.1730	0.0802	0.0110	0.0543

Dose		Testes	Epididymides	Ovaries	Uterus
0 (mg/kg)	Mean	0.9291	0.3560	0.0383	0.2180
	SD	0.2331	0.0436	0.0136	0.0385
	SEM	0.0881	0.0165	0.0051	0.0146
375 (mg/kg)	Mean	0.9504	0.3462	0.0384	0.2135
	SD	0.1070	0.0756	0.0158	0.0687
	SEM	0.0405	0.0286	0.00601	0.0260
1125 (mg/kg)	Mean	0.9617	0.3139	0.0297	0.1994
	SD	0.0908	0.0451	0.0083	0.0375
	SEM	0.0343	0.0171	0.0031	0.0135
1000 (mg/kg)	Mean	0.9235	0.3176	0.0295	0.2213
	SD	0.1438	0.0678	0.0116	0.0614
	SEM	0.0544	0.0256	0.0044	0.0232

Dose		Testesterone nmol/L	Estrogen ng/L
0 (mg/kg)	Mean	14.11	9.01
	SD	2.94	0.87
	SEM	1.11	0.33
125 (mg/kg)	Mean	15.19	9.14
	SD	1.07	1.14
	SEM	0.40	0.43
375 (mg/kg)	Mean	12.30	10.00
	SD	1.80	2.03
	SEM	0.68	0.77
1125 (mg/kg)	Mean	13.64	10.39
	SD	1.94	1.35
	SEM	0.73	0.51