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REACH

Methyl 2-hexyl-3-oxocyclopentanecarboxylate

EC number: 253-379-1 | CAS number: 37172-53-5

Life Cycle description
Uses advised against

Toxicological information

Toxicity to reproduction

Administrative data

Endpoint:: screening for reproductive / developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 16 Aug 2018 to 07 Nov 2018
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Aug 2018 to 07 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 29 July 2016

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: Males group means: 331 to 334 g. Females group means: 260 to 261 g.
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm). Clean sterilized paddy husk was provided as bedding material. A maximum of 3 animals of the same sex were caged together during acclimatization and 2 animals of the same sex and group during pre-mating. During mating, two animals (one male and one female) of the same group were housed together. Post-mating, females were housed individually and males were housed with their former cage mates. A maximum of three animals of the same sex were housed in the same cage for recovery group animals.
- Diet: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum.
- Water: Water was provided ad libitum.
- Acclimation period: Healthy and young animals were acclimatized initially for five days to experimental room conditions. Further the females were subjected to screening for estrous cycles in a two weeks (14 days) pre-treatment period. The males and females were observed for clinical signs once daily and veterinary examination of all the animals was performed on the day of receipt and on the day of randomization and grouping.

DETAILS OF FOOD AND WATER QUALITY: Food and water was checked for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 to 23.1
- Humidity (%): 47 to 67
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
16 Aug 2018 to 7 Nov 2018

Route of administration:

oral: gavage

Details on route of administration:

The vehicle and test item formulations were administered through oral (gavage) route as it is the probable route of exposure to human.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item formulations were prepared daily before administration. The required quantity of the test item was weighed into a clean beaker. A small volume of corn oil (vehicle) was added into the beaker, mixed well with a glass rod and the mixture was transferred into the measuring cylinder. The beaker was rinsed using a small volume of corn oil and the volume was transferred to the measuring cylinder. The rinsing procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was supplemented with vehicle to the required quantity to get a desired concentration of 10, 30 and 100 mg/mL of test item for the low, mid and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected as a vehicle for the preparation of test item formulations as the test item was not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL (the highest dose concentration) as evidenced by the in-house miscibility test results.
- Concentration in vehicle: 10, 30 and 100 mg/mL corresponding to a dose of 100, 300 and 1000 mg/kg bw, respectively.
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: A1712001

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations was performed during the first week of treatment and during week 5 of the treatment. The samples were collected in duplicate from the top, middle and bottom layer from the low, mid and high dose concentrations, and in duplicate from the middle layer from the vehicle control.

Duration of treatment / exposure:

Males: The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Females: The females were treated for a two week pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13. Females (dams) were sacrificed on lactation day 14 after overnight fasting.

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Low dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4: High dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 recovery (G4R): High dose

No. of animals per sex per dose:

Treatment groups: 12
Recovery groups: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of the treatment and recovery groups were based on the results of the dose range finding study of the test item when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley rats (Bioneeds Study No.: BIO-TX 3392). These doses were selected as the test item when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight (water available)
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Day 1 (before treatment) and weekly thereafter.
- Parameters checked: Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT:
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION:
- Food consumption was measured for main group animals once a week during premating and for main group males during post mating period. Food consumption was not measured during mating period for both males and females. Thereafter, food consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Food consumption was measured for recovery group animals once a week throughout the experimental period.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Once before treatment for all animals, during the end of the dosing period for males (on day 31) and during the lactation period for females (on lactation day 13) of vehicle control and high dose main group animals and during the last week (day 64) for recovery group animals.
- Dose groups that were examined: The examination was not extended to lower dose groups as there were no treatment related changes noted in high dose group animals.

HAEMATOLOGY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked in Table 1 in ‘Any other information on materials and methods incl. tables’ were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked in Table 2 in ‘Any other information on materials and methods incl. tables’ were examined.

URINALYSIS:
- Time schedule for collection of urine: At termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: The animals had no access to food, water was however available.
- How many animals: Urine was collected from five randomly selected males of each main group and for all recovery animals at termination.
- Parameters checked in Table 3 in ‘Any other information on materials and methods incl. results’ were examined. In addition, pH, nitrite and specific gravity were also analysed and urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Males on day 36, females on lactation day 13 and shortly prior to scheduled sacrifice on day 64 for the recovery animals.
- Dose groups that were examined: All groups
- Battery of functions tested: See Table 4 in ‘Any other information on materials and methods incl. tables’.

Sacrifice and pathology:

GROSS PATHOLOGY: Table 5 in ‘Any other information on materials and methods incl. tables’.
The animals were euthanized using carbon dioxide asphyxiation followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination. Relative organ weights were calculated against fasting body weight.

HISTOPATHOLOGY: Table 5 in ‘Any other information on materials and methods incl. tables’.
Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.
All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. The testes were sectioned at 3 to 4 micrometers and stained with hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain was aided in spermatogenesis evaluation.
The investigations were not extended to the lower dose groups and recovery groups as there were no treatment related effects noted at the high dose level.
Bone marrow smear (from one femur) was prepared at the time of necropsy.

Other examinations:

T4 HORMONE ANALYSIS:
Blood samples were collected from all the main group animals for measurement of serum T4 levels according to the following schedule:
a. All dams at termination (lactation day 14).
b. All adult males, at termination (after completion of 36 days of treatment).

The collected blood samples were centrifuged at 5000 rpm for 10 minutes, the serum was separated and stored at -80°C for estimation of serum levels of thyroid hormones (T4) by ELISA method using commercial assay kits. The serum samples from lactation day 13 pups and the adult males were assessed for serum T4 levels. Assessment of T4 in blood samples from the dams and day 4 pups was not performed as there were no treatment related changes noted in males and lactation day 13 pups at any of the tested dose groups.
Blood from lactation day 4 pups was collected by jugular vein incision, whereas blood from lactation day 13 pups and adult animals was collected using the retro-orbital plexus puncture method under Isoflurane anaesthesia with the help of a fine capillary tube.
The assessment of serum T4 levels was not performed for recovery group animals.

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22. All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity in any of the main and recovery group animals of either sex throughout the experimental period. The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity was observed in any of the main and recovery group animals of either sex throughout the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at any of the tested dose main and recovery group animals of either sex.
There were no treatment related changes in body weight and percentage change in body weight during the gestation period in all animals treated with the test substance.
There were no changes in body weight and percentage change in body weight during the lactation period in group G2 (100 mg/kg) and G3 (300 mg/kg) when compared with control group animals. A reduction in mean body weight and percentage change in body weight during lactation in G4 (1000 mg/kg) was noted. This change can be considered as treatment related as the reduction in the percentage body weight change is statistically significant during PND 1 to 4 and PND 4 to 7 when compared with control group animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes noted in mean feed consumption at any of the tested dose levels of both main and recovery group animals and of either sex. However, a statistically significant increase in mean feed consumption for group G2 (100 mg/kg) males during the post mating period was noted, when compared with vehicle control group males. This change is considered as incidental and not treatment related due to lack of dose dependency. In addition, no effects were noted in mean body weight and percent change in body weight during this period at this dose level.
There were no treatment related changes in the feed consumption during the gestation period at all the tested dose levels. However, a statistically significant reduction in feed consumption during gestation day 7 to 14 was noted in group G4 (1000 mg/kg) animals when compared with vehicle control group animals. Feed consumption on GD0-7 and GD7-14 was somewhat lower than the historical group mean control data, but on GD14-21 the feed consumption was higher. This observation is considered as not adverse effect as the reduction is not consistent.
There were no changes in the feed consumption during the lactation period at all the tested dose levels when compared with control group animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in G1 (0 mg/kg) and G4 (1000 mg/kg) group animals of either sex during the ophthalmological examination conducted towards the end of the dosing period and no ocular changes were observed in G1R (0 mg/kg) and G4R (1000 mg/kg) group animals of either sex during the ophthalmological examination conducted at the end of the recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in haematology parameters in any of the animals of either sex in both main or recovery groups treated with the test substance. However, a statistically significant increase in absolute basophils in group G4 (1000 mg/kg) males; a statistically significant decrease in prothrombin time in all the main group males; a statistically significant decrease in activated partial thromboplastin time in group G2 (100 mg/kg) and G3 (300 mg/kg) males; a statistically significant decrease in haematocrit in group G4R (1000 mg/kg) females was noted when compared with their concurrent control group animals. These changes are considered as incidental and not treatment related, as the obtained values are within the in-house historical control data of the species and additionally, the same changes were not observed in the other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in clinical chemistry parameters in any of the animals of either sex in both main or recovery groups treated with the test substance. However, a statistically significant decrease in glucose and chloride levels in group G2 (100 mg/kg) males; a statistically significant decrease in urea and blood urea nitrogen levels in all the main group males; a statistically significant increase in total cholesterol and triglycerides in group G4 (1000 mg/kg) males; a statistically significant increase in chloride levels and decrease in calcium in group G4R (1000 mg/kg) males and a reduction in aspartate aminotransferase in group G4R (1000 mg/kg) females was noted when compared with their concurrent control group animals. These changes are considered as incidental and not treatment related, as the obtained values are within the in-house historical control data of the species and additionally, the same changes were not observed in the other sex.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment related changes in urine analysis parameters in any of the main group males and recovery group animals of either sex.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The results of the neurological/functional examination conducted for five randomly selected main group males and females from each group prior to scheduled sacrifice and for all recovery group animals at the end of recovery period did not indicate any treatment related effect. However, a statistically significant reduction in mean motor activity in group G4 (1000 mg/kg) females; a statistically significant increase in landing foot splay in group G4R (1000 mg/kg) females was noted when compared with concurrent control group females.
These changes are considered as incidental and not treatment related as there were no effects noted in other functional observation battery parameters and no clinical signs of toxicity were noted in all the animals of these dose levels.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in the absolute and relative organ weights for any of the animals of either sex in all main and recovery groups treated with the test substance.
However, the following statistically significant changes were noted:
- decrease in absolute brain weight at group G2 males;
- decrease in relative brain weight at group G4 males;
- increase in absolute testes weight at group G4 males;
- decrease in absolute heart weight at group G2 and G4 females;
- decrease in absolute liver weight at group G2 females;
- decrease in absolute prostate weight and relative testes weight at group G4R males
These changes are considered as incidental and not treatment related, as there were no gross and microscopic findings noted in these organs. In addition, these observations were not noted in other sex.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes during necropsy in any of the main and recovery group animals of either sex.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related histopathological findings were noticed in the study. Lesions considered to be spontaneous and incidental were observed in treated and control rats. These lesions consisted of interstitial mononuclear cell infiltration and concretions in prostate gland, and presence of calculi in kidney.
One female from the high dose group (G4) revealed mononuclear cell infiltration at submucosa of the vagina. This lesion was considered spontaneous because it was not observed in any of the other females. The microscopic findings (Coagulative necrosis of renal tubules, dilated pelvis of kidney, physeal dysplasia in femur bone) observed only in vehicle control animals, hence were considered as spontaneous.
The females from the vehicle control group (GI) and high dose group (G4) revealed the presence of a placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

T4 LEVELS: There were no treatment related changes in serum Thyroxine (T4) hormone levels in all males.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

TABLE 1. SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD
Group, Sex & Dose (mg/kg body weight)			Feed Consumption (g/animal/day)
Group, Sex & Dose (mg/kg body weight)		Week 1 (Day 1 to 7)	Week 2 (Day 7 to 14)	During Post-mating (Day 35 to 37)
	Mean	21.23	22.42	25.73
G1, M & 0	±SD	2.53	2.16	0.55
	n	12	12	12
	Mean	21.15	21.53	27.14*
G2, M & 100	±SD	2.79	2.43	0.83
	n	12	12	12
	Mean	21.51	22.20	26.67
G3, M & 300	±SD	2.52	2.04	1.00
	n	12	12	12
	Mean	21.05	22.65	25.27
G4, M & 1000	±SD	1.95	0.93	0.98
	n	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals
* Statistically significant (P<0.05) change than the vehicle control group.

TABLE 2. SUMMARY OF HAEMATOLOGY RECORD
Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count (Retic) (10⁹cells/L)	Absolute Neutrophils Neut (10³cells/µL)	Absolute Lymphocytes Lymph (10³cells/µL)	Absolute Monocytes Mono (10³cells/µL)	Absolute Eosinophils Eos (10³cells/µL)	Absolute Basophils Baso (10³cells/µL)	Prothrombin Time PT (Seconds)	Activated Prothrombin Time APTT (Seconds)
Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count (Retic) (10⁹cells/L)	Absolute Neutrophils Neut (10³cells/µL)	Absolute Lymphocytes Lymph (10³cells/µL)	Absolute Monocytes Mono (10³cells/µL)	Absolute Eosinophils Eos (10³cells/µL)	Absolute Basophils Baso (10³cells/µL)	Prothrombin Time PT (Seconds)	Activated Prothrombin Time APTT (Seconds)
	Mean	136.42	1.84	5.83	0.23	0.11	0.02	26.78	38.12
G1,M & 0	±SD	16.23	0.73	1.45	0.08	0.05	0.01	5.76	13.71

	n	5	5	5	5	5	5	5	5
	Mean	165.04	1.89	6.26	0.20	0.10	0.02	18.54*	18.72*
G2,M & 100	±SD	23.22	0.38	0.85	0.06	0.03	0.01	1.88	4.54

	n	5	5	5	5	5	5	5	5
	Mean	247.46	1.90	5.89	0.18	0.07	0.01	21.28*	22.30*
G3,M & 300	±SD	166.08	0.43	0.82	0.05	0.03	0.01	2.26	9.16

	n	5	5	5	5	5	5	5
	Mean	185.24	2.87	7.91	0.20	0.09	0.03*	18.04*	31.58
G4, M & 1000	±SD	44.01	1.99	2.73	0.07	0.03	0.01	0.73	6.17

	n	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3. SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Glucose (GLU) (mg/dL)	Urea (mg/dL)	Creatinine (CRE) (mg/dL)	Total Cholesterol (CHO) (mg/dL)	Triglycerides (TRI) (mg/dL)	Total Protein (TPR) (g/dL)	Albumin (ALB) (g/dL)	Alanine aminotransferase (ALT) (U/L)	Aspartate aminotransferase (AST) (U/L)	Alkaline phosphatase (ALP) (U/L)
	Mean	152.00	39.24	0.42	37.40	31.40	6.20	3.64	41.60	84.00	122.60
G1,M&	±SD	61.60	8.08	0.04	8.79	10.64	0.35	1.14	5.18	15.12	20.40
0
	n	5	5	5	5	5	5	5	5	5	5
	Mean	92.60*	26.44*	0.39	37.80	26.80	6.14	3.22	38.20	91.80	124.40
G2,M & 100	±SD	10.69	2.87	0.03	6.53	9.58	0.23	0.07	11.95	23.06	33.89
	n	5	5	5	5	5	5	5	5	5	5
	Mean	99.40	27.64*	0.40	38.40	31.00	6.22	3.25	39.60	77.60	119.60
G3,M &	±SD	6.54	3.04	0.03	8.79	8.60	0.20	0.13	5.77	7.47	30.04
300
	n	5	5	5	5	5	5	5	5	5	5
	Mean	121.20	28.66*	0.40	52.20*	48.80*	6.38	3.24	34.20	73.40	102.80
G4,M &	±SD	14.62	3.05	0.02	7.16	10.06	0.47	0.17	4.97	12.60	21.97
1000
	n	5	5	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3 (Contd...). SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Total Bilirubin (BIT) (mg/dL)	Calcium (CAL) (mg/dL)	Phosphorous (PHO) (mg/dL)	Globulin (GLO) (g/dL)	Blood Urea Nitrogen (BUN) (mg/dL)	Sodium (Na) (mmol/L)	Potassium (K) (mmol/L)	Chloride (CLO) (mmol/L)
	Mean	0.02	9.94	6.30	2.56	18.31	153.00	4.64	117.84
G1, M & 0	±SD	0.01	0.33	0.44	1.03	3.77	2.46	0.55	2.32
	n	5	5	5	5	5	5	5	5
	Mean	0.03	9.94	6.44	2.92	12.34*	148.82	4.14	113.26*
G2, M & 100	±SD	0.01	0.22	0.53	0.24	1.34	2.87	0.14	1.47
	n	5	5	5	5	5	5	5	5
	Mean	0.02	10.16	6.52	2.97	12.90*	154.06	4.31	115.70
G3, M & 300	±SD	0.01	0.11	0.59	0.12	1.42	2.71	0.15	2.13
	n	5	5	5	5	5	5	5	5
	Mean	0.02	10.20	6.42	3.14	13.38'	152.28	4.56	115.62
G4, M & 1000	±SD	0.00	0.32	0.38	0.30	1.42	4.56	0.48	2.28
	n	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3 (Contd...). SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Total Bilirubin (BIT) (mg/dL)	Calcium (CAL) (mg/dL)	Phosphorous (PHO) (mg/dL)	Globulin (GLO) (g/dL)	Blood Urea Nitrogen (BUN) (mg/dL)	Sodium (Na) (mmol/L)	Potassium (K) (mmollL)	Chloride (CLO) (mmol/L)
	Mean	0.06	3.40	6.04	3.09	11.93	145.00	3.35	106.06
G1R, M & 0	±SD	0.02	0.89	0.42	0.29	2.11	1.22	0.17	0.72
	n	5	5	5	5	5	5	5	5
	Mean	0.06	2.04*	6.62	2.90	11.78	145.12	3.56	107.28*
G4R, M & 1000	±SD	0.01	0.78	2.65	0.17	1.87	1.19	0.51	0.55
	n	5	5	5	5	5	5	5	5
	Mean	0.04	3.80	3.78	3.27	12.41	145.22	3.48	108.72
G1R, F & 0	±SD	0.02	135	0.34	0.29	2.10	0.55	0.19	1.48
	n	5	5	5	5	5	5	5	5
	Mean	0.06	3.66	4.28	3.29	12.45	145.68	3.39	109.22
G4R, F & 1000	±SD	0.02	0.93	1.20	0.23	1.92	1.93	0.22	1.22
	n	5	5	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 4. SUMMARY OF FEED CONSUMPTION (g/animal/day) DURING GESTATION RECORD
Group & Dose (mg/kg body weight)		Feed Consumption (g/animal/day) during Gestation Day (GD)
Group & Dose (mg/kg body weight)		0 to 7	7 to 14	14 to 20
	Mean	18.85	21.83	26.63
G1 & 0	±SD	2.17	1.84	2.57
	n	12	12	12
	Mean	19.41	21.71	26.91
G2 & 100	±SD	1.84	1.49	2.01
	n	11	11	11
	Mean	17.71	20.47	26.59
G3 & 300	±SD	1.38	1.16	1.56
	n	10	10	10
	Mean	17.91	19.46*	26.15
G4 & 1000	±SD	1.46	1.51	1.44
	n	11	11	11
SD: Standard Deviation; n: Number of Animals (Females)
Note: The data of non-pregnant animals is excluded for mean calculations
* Statistically significant (P<0.05) change than the vehicle control group

TABLE 5. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) DURING LACTATION
Group & Dose (mg/kg body weight)		Percent Change in body weight (%) during Lactation Day (LD)
Group & Dose (mg/kg body weight)		1 to 4	4 to 7	7 to 13
	Mean	3.63	3.60	4.73
G1&0	±SD	2.10	2.13	2.44
	n	12	12	12
	Mean	2.38	2.83	4.96
G2 & 100	±SD	1.13	1.47	2.75
	n	11	11	11
	Mean	2.00	3.34	6.59
G3 & 300	±SD	1.58	1.84	2.79
	n	10	10	10
	Mean	1.56*	1.70*	2.73
G4 & 1000	±SD	2.36	0.72	1.91
	n	11	11	11

SD: Standard Deviation; n: Number of Animals (Females)
*: Statistically significant (P<0.05) change than the vehicle control group

TABLE 6. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g)
Group, Sex & Dose (mg/kg body weight)		Adrenals	Thymus	Spleen	Epididymis	Testes	Heart	Kidneys	Brain	Liver	Seminal vesicles with coagulating glands	Prostate	Thyroid along with para Thyroid^#
	Mean	0.0713	0.2317	0.7647	1.4503	3A266	1.5315	3.2600	2.2221	12.4578	1.1707	1.5446	0.0300
G1, M &	±SD	0.0072	0.0728	0.0251	0.0834	0.1983	0.0630	0.2829	0.1213	1.2105	0.1223	0.2312	0.0058
0
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0687	0.2370	0.6694	1.4054	3.5192	1.4584	3.2948	1.9582*	12.7096	1.2885	1.6538	0.0307
G2, M &	±SD	0.0072	0.0181	0.0721	0.0299	0.1183	0.1374	0.1119	0.1195	0.8988	0.1325	0.1603	0.0058
100
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0736	0.2497	0.7327	1.3987	3.4107	1.5027	3.2212	2.1377	13.0107	1.2355	1.5937	0.0305
G3,M &	±SD	0.0074	0.0542	0.0858	0.1347	0.1277	0.0545	0.1518	0.0540	0.8457	0.1650	0.2038	0.0047
300
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0738	0.3436	0.8034	1.4557	3.6099*	1.5936	3.5667	2.1674	13.2262	1.1669	1.5059	0.0306
G4, M &	±SD	0.0093	0.1033	0.0965	0.0555	0.2536	0.1800	0.2385	0.0776	1.1703	0.0616	0.1868	0.0036
1000
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0798	0.3982	0.7043	1.4680	3.8296	1.5621	3.4492	2.1940	13.4046	1.3151	1.8776	0.0303
G1R, M	±SD	0.0271	0.0667	0.1032	0.1436	0.2945	0.2125	0.2566	0.2091	0.3531	0.2166	0.1603	0.0027
&0
	n	5	5	5	5	5	5	5	5	5	5	5	5
	Mean	0.0786	0.3841	0.7040	1.4336	3.4943	1.6005	3.5935	2.2144	13.8807	1.2083	1.6840*	0.0287
G4R, M	±SD	0.0157	0.0484	0.0457	0.0762	0.1706	0.1084	0.2094	0.0757	0.8751	0.1330	0.0575	0.0050
& 1000
	n	5	5	5	5	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (Females); #: Weighed post fixation
*: Statistically significant (P<0.05) change than the vehicle control group

TABLE 6 (Contd...). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g)
Group, Sex & Dose (mg/kg body weight)		Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Thyroid along with para Thyroid^#
	Mean	0.1009	0.2834	0.6195	1.3311	2.6962	2.0879	16.3846	0.0310
G1, F &	±SD	0.0198	0.0522	0.1368	0.0744	0.2709	0.0384	1.5298	0.0063
0
		5	5	5	5	5	5	5	12
	Mean	0.0961	0.2881	0.6405	1.1601*	2.6774	2.1819	14.3037*	0.0308
G2, F &	±SD	0.0054	0.0930	0.0969	0.0817	0.2466	0.0266	0.7461	0.0049
100
		5	5	5	5	5	5	5	12
	Mean	0.0937	0.3049	0.7649	1.2065	2.7635	2.1132	16.9891	0.0302
G3, F &	±SD	0.0149	0.0946	0.0725	0.0876	0.1476	0.0502	1.0569	0.0033
300
		5	5	5	5	5	5	5	12
	Mean	0.1032	0.2809	0.6447	1.1626*	2.6732	2.1717	15.8345	0.0317
G4, F &	±SD	0.0128	0.0952	0.2021	0.1021	0.1996	0.1646	1.2621	0.0048
1000
	n	5	5	5	5	5	5	5	12
	Mean	0.0928	0.3907	0.5817	1.1948	2.3005	2.1197	8.9081	0.0289
G1R, F &	±SD	0.0145	0.0527	0.0180	0.1490	0.1729	0.0663	0.7555	0.0035
0
	n	5	5	5	5	5	5	5	5
	Mean	0.1017	0.4139	0.5713	1.1680	2.2110	2.0927	8.9462	0.0279
G4R, F &	±SD	0.0147	0.0418	0.0599	0.0705	0.1374	0.0531	1.0503	0.0033
1000
	n	5	5	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (Females); #: Weighed post fixation
*: Statistically significant (P<0.05) change than the vehicle control group

Conclusions:

In the absence of adverse parental systemic effects, the NOAEL for parental toxicity was placed at 1000 mg/kg (the highest concentration tested).

Executive summary:

The objective of this GLP compliant OECD 422 study was to provide data on the possible effects of the test substance Dihydro Isojamonate on general toxicity, reproductive performance and development of pups. The test substance was suspended in corn oil and administered by oral gavage at 0, 100, 300 and 1000 mg/kg body weight/day. The test substance was administered during a pre-mating period of 2 weeks, during mating, gestation and lactation. Male animals were sacrificed after the mating period after completion of 36 days of treatment. Parental female animals were sacrificed at day 14 of lactation.

In the absence of adverse parental systemic effects, the NOAEL for parental toxicity was placed at 1000 mg/kg (the highest concentration tested).

Cross-reference 2

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Aug 2018 to 07 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, Number 422, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”

Version / remarks:

Adopted 29 July 2016

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occurred or until two weeks had elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
The recovery group animals were not mated and therefore not cohabitated.

Duration of treatment / exposure:

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Low dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4: High dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 recovery (G4R): High dose

No. of animals per sex per dose:

Treatment groups: 12
Recovery groups: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of the treatment and recovery groups were based on the results of the dose range finding study of the test item when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 3392). These doses were selected as the test item when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight (water available)
- Post-exposure recovery period in satellite groups: 14 days

Maternal examinations:

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

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Day 1 (before treatment) and weekly thereafter.
- Parameters checked: Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT:
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION:
- Food consumption was measured for main group animals once a week during premating and for main group males during post mating period. Food consumption was not measured during mating period for both males and females. Thereafter, food consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Food consumption was measured for recovery group animals once a week throughout the experimental period.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Once before treatment for all animals, during the end of the dosing period for males (on day 31) and during the lactation period for females (on lactation day 13) of vehicle control and high dose main group animals and during the last week (day 64) for recovery group animals.
- Dose groups that were examined: The examination was not extended to lower dose groups as there were no treatment related changes noted in high dose group animals.

HAEMATOLOGY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked in Table 1 in ‘Any other information on materials and methods incl. tables’ were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked in Table 2 in ‘Any other information on materials and methods incl. tables’ were examined.

URINALYSIS:
- Time schedule for collection of urine: At termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: The animals had no access to food, water was however available.
- How many animals: Urine was collected from five randomly selected males of each main group and for all recovery animals at termination.
- Parameters checked in Table 3 in ‘Any other information on materials and methods incl. results’ were examined. In addition, pH, nitrite and specific gravity were also analysed and urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Males on day 36, females on lactation day 13 and on shortly prior to scheduled sacrifice on day 64 for the recovery animals.
- Dose groups that were examined: All groups
- Battery of functions tested: See Table 4 in ‘Any other information on materials and methods incl. tables’.

GROSS PATHOLOGY: Table 5 in ‘Any other information on materials and methods incl. tables’.
The animals were euthanized using carbon dioxide asphyxiation followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination. Relative organ weights were calculated against fasting body weight.

HISTOPATHOLOGY: Table 5 in ‘Any other information on materials and methods incl. tables’.
Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.
All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. The testes were sectioned at 3 to 4 micrometers and stained with hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain was aided spermatogenesis evaluation
The investigations were not extended to the lower dose groups and recovery groups as there were no treatment related effects noted at the high dose level.
Bone marrow smear (from one femur) was prepared at the time of necropsy.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05) (see Table 7 in 'Any other information on materials and methods incl. tables' for an overview of the statistical tests used).

Indices:

See Table 6 in 'Any other information on materials and methods incl. tables'.

Clinical signs:

no effects observed

Description (incidence and severity):

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity was observed in any of the main and recovery group animals of either sex throughout the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in haematology parameters in any of the animals of either sex in both main or recovery groups treated with the test substance. However, a statistically significant decrease in haematocrit in group G4R (1000 mg/kg) females was noted when compared with their concurrent control group animals. These changes are considered as incidental and not treatment related, as the obtained values are within the in-house historical control data of the species and additionally, the same changes were not observed in the other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in clinical chemistry parameters in any of the animals of either sex in both main or recovery groups treated with the test substance. However, a reduction in aspartate aminotransferase in group G4R (1000 mg/kg) females was noted when compared with their concurrent control group animals. These changes are considered as incidental and not treatment related, as the obtained values are within the in-house historical control data of the species and additionally, the same changes were not observed in the other sex.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment related changes in urine analysis parameters in any of the main group males and recovery group animals of either sex.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects observed in the neurological/functional examination conducted for five randomly selected main group females from each group prior to scheduled sacrifice and for all recovery group animals at the end of recovery period. However, statistically significant increase in mean defecation at group G3 (300 mg/kg) females; statistically significant reduction in mean motor activity at group G4 (1000 mg/kg) females; statistically significant increase in landing foot splay at group G4R (1000 mg/kg) females was noted when compared with concurrent control group females. These changes are considered as incidental and not treatment related as there were no effects were noted in other functional observation battery parameters and no clinical signs of tocixity was noted at all the animals of this dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during necropsy at any of the tested dose main and recovery group animals of either sex.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in number of corpora lutea, implantations, resorptions and pre-implantation loss at all the tested dose groups. However, a statistically significant increase in the number of corpora lutea and implantations in group G3 (300 mg/kg) cannot to be considered treatment related as the change is due to increase in litter size at this dose level.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in number of corpora lutea, implantations, resorptions and pre-implantation loss at all the tested dose groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in number of corpora lutea, implantations, resorptions and pre-implantation loss at all the tested dose groups.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

There were no changes observed in the gestation length, litter delivered, number of pups delivered, sex ratio, live birth index for group G2 (100 mg/kg) animals when compared with vehicle control group animals.
A slight reduction in live birth index (93.81%) in G3 (300 mg/kg) animals was observed when compared with the vehicle control group animals. The observed live birth index was slightly lower than the historical group mean, but not statistically significantly different. The slight reduction in live birth index is attributable to only 2 animals (Rd0754 and Rd0757). Their individual live birth index is 71.4 and 66.7, respectively, which is well within the historical control range (60-100). Hence, this observation is considered to be not biologically relevant. No statistically significant differences were found for the number of dead and cannibalized pups when compared with the vehicle control group animals.
A statistically significant reduction in litter size and live birth index was noted for G4 (1000 mg/kg) animals. The group mean litter size was also slightly lower than the historical group mean. Litter size of all individual animals (6-13) however, is within the historical control range, which is 5-16. The reduction in live birth index is predominantly attributable to 2 animals (Rd0760 and Rd0761) and to a lesser extent to a third animal (Rd0763). Their individual live birth indices are 10.0, 16.7 and 72.7, respectively. The values of the first two animals is clearly below the historical control range, whereas the index of the third animal is within this range. Hence, the reduction in live birth index should be considered a treatment related adverse effect. An increased number of dead and cannibalized pups reflects the reduced live birth index.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no changes observed in the gestation length.

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

dead fetuses

pre and post implantation loss

other: significant reduction in live birth index

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean of all the male and female pups per dam was calculated initially and subsequently the overall mean pup weight of each sex from all the dams of one group was calculated. The group mean values obtained from each tested dose group was compared with the vehicle control group. There were no changes in mean pup weight of either sex during the lactation period for group G2 (100 mg/kg) when compared with the vehicle control group. Statistically significant reductions in mean male pup weight on PND 1 in group G3 (300 mg/kg) and in mean pup weight (both male and female) on PND 4, 7 and 13 for group G3 (300 mg/kg) and G4 (1000 mg/kg) were noted when compared with the vehicle control. Although the reductions reported for group G3 (300 mg/kg) are statistically significantly different, they are still higher than the historical group mean and thus considered not biologically relevant. Only in male pups on LD 13 the mean pup weight is slightly below the historical mean. However, all mean pup weights/dam were within the historical control range. No individual weights were below the lower limit of the range; in one litter several pups had a weight which was higher than the historical control range. Therefore, this observation is considered to be a chance finding. In addition, although the reductions reported for group G4 (1000 mg/kg) are statistically significantly different, there is no dose response up to LD4 for both males and females and more importantly, the reported values are still higher than the historical group mean and thus considered not biologically relevant. Only in male pups on LD 13 the mean pup weight is slightly below the historical mean. However, all mean pup weights/dam were within the historical control range. Only three individuals of the in total 77 pups, weighed slightly below the lower limit of the range. Therefore, this observation is considered to be a chance finding.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

OUD: As statistical significant reduction in litter size, live birth index and increase in number of dead pups were noted at G4 (1000 mg/kg). Occurrence of cannibalized pups also noted at at this dose level.
These changes are considered as treatment related due to occurrence of dead and cannibalized pups during lactation period and statistical significant reduction in mean pup weight throughout the lactation period at these dose levels.

NIEUW: There were no treatment related changes in litter observation parameters for group G2 (100 mg/kg) and G3 (300 mg/kg) animals when compared with vehicle control group animals during lactation period. The number of dead and cannibalized pups for G3 (300 mg/kg) animals is lower than the historical control value. However, an increased number of dead and cannibalized pups during the lactation period was observed for group G4 (1000 mg/kg) animals, resulting in a clear reduction in pup survival index in this group at PND 1 to 4 and PND 7 to 13 when compared with vehicle control group animals, which is statistically significant at PND 1 to 4. Due to the large standard deviation however, this reduction is not statistically significant. This observed reduction is attributable to three animals in the group, namely Rd0760, Rd0761 and Rd0763. Their individual pup survival index is 0, 0 and 12.5% on LD 1 to 4, respectively. On LD7 to 13, the pup survival index of animal Rd0763 is 0%. All these values are severely below the historical control range (60-100% on LD1 to 4 and 100-100% on LD7 to 13). Hence, this reduction in pup survival index should be considered a treatment related adverse effect.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in litter size and live birth index was noted for G4 (1000 mg/kg) animals. The group mean litter size was also slightly lower than the historical group mean. Litter size of all individual animals (6-13) however, is within the historical control range, which is 5-16.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related changes in litter observation parameters for group G2 (100 mg/kg) and G3 (300 mg/kg) animals when compared with vehicle control group animals during lactation period. The number of dead and cannibalized pups for G3 (300 mg/kg) animals is lower than the historical control value. However, an increased number of dead and cannibalized pups during the lactation period was observed for group G4 (1000 mg/kg) animals, resulting in a clear reduction in pup survival index in this group at PND 1 to 4 and PND 7 to 13 when compared with vehicle control group animals, which is statistically significant at PND 1 to 4. Due to the large standard deviation however, this reduction is not statistically significant. This observed reduction is attributable to three animals in the group, namely Rd0760, Rd0761 and Rd0763. Their individual pup survival index is 0, 0 and 12.5% on LD 1 to 4, respectively. On LD7 to 13, the pup survival index of animal Rd0763 is 0%. All these values are severely below the historical control range (60-100% on LD1 to 4 and 100-100% on LD7 to 13). Hence, this reduction in pup survival index should be considered a treatment related adverse effect.

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in postnatal survival

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

TABLE 1. SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD
Group, Sex & Dose (mg/kg body weight)			Feed Consumption (g/animal/day)
Group, Sex & Dose (mg/kg body weight)		Week 1 (Day 1 to 7)	Week 2 (Day 7 to 14)	During Post-mating (Day 35 to 37)
	Mean	21.23	22.42	25.73
G1, M & 0	±SD	2.53	2.16	0.55
	n	12	12	12
	Mean	21.15	21.53	27.14*
G2, M & 100	±SD	2.79	2.43	0.83
	n	12	12	12
	Mean	21.51	22.20	26.67
G3, M & 300	±SD	2.52	2.04	1.00
	n	12	12	12
	Mean	21.05	22.65	25.27
G4, M & 1000	±SD	1.95	0.93	0.98
	n	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals
* Statistically significant (P<0.05) change than the vehicle control group.

TABLE 2. SUMMARY OF HAEMATOLOGY RECORD
Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count (Retic) (10⁹cells/L)	Absolute Neutrophils Neut (10³cells/µL)	Absolute Lymphocytes Lymph (10³cells/µL)	Absolute Monocytes Mono (10³cells/µL)	Absolute Eosinophils Eos (10³cells/µL)	Absolute Basophils Baso (10³cells/µL)	Prothrombin Time PT (Seconds)	Activated Prothrombin Time APTT (Seconds)
Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count (Retic) (10⁹cells/L)	Absolute Neutrophils Neut (10³cells/µL)	Absolute Lymphocytes Lymph (10³cells/µL)	Absolute Monocytes Mono (10³cells/µL)	Absolute Eosinophils Eos (10³cells/µL)	Absolute Basophils Baso (10³cells/µL)	Prothrombin Time PT (Seconds)	Activated Prothrombin Time APTT (Seconds)
	Mean	136.42	1.84	5.83	0.23	0.11	0.02	26.78	38.12
G1,M & 0	±SD	16.23	0.73	1.45	0.08	0.05	0.01	5.76	13.71

	n	5	5	5	5	5	5	5	5
	Mean	165.04	1.89	6.26	0.20	0.10	0.02	18.54*	18.72*
G2,M & 100	±SD	23.22	0.38	0.85	0.06	0.03	0.01	1.88	4.54

	n	5	5	5	5	5	5	5	5
	Mean	247.46	1.90	5.89	0.18	0.07	0.01	21.28*	22.30*
G3,M & 300	±SD	166.08	0.43	0.82	0.05	0.03	0.01	2.26	9.16

	n	5	5	5	5	5	5	5
	Mean	185.24	2.87	7.91	0.20	0.09	0.03*	18.04*	31.58
G4, M & 1000	±SD	44.01	1.99	2.73	0.07	0.03	0.01	0.73	6.17

	n	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3. SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Glucose (GLU) (mg/dL)	Urea (mg/dL)	Creatinine (CRE) (mg/dL)	Total Cholesterol (CHO) (mg/dL)	Triglycerides (TRI) (mg/dL)	Total Protein (TPR) (g/dL)	Albumin (ALB) (g/dL)	Alanine aminotransferase (ALT) (U/L)	Aspartate aminotransferase (AST) (U/L)	Alkaline phosphatase (ALP) (U/L)
	Mean	152.00	39.24	0.42	37.40	31.40	6.20	3.64	41.60	84.00	122.60
G1,M&	±SD	61.60	8.08	0.04	8.79	10.64	0.35	1.14	5.18	15.12	20.40
0
	n	5	5	5	5	5	5	5	5	5	5
	Mean	92.60*	26.44*	0.39	37.80	26.80	6.14	3.22	38.20	91.80	124.40
G2,M & 100	±SD	10.69	2.87	0.03	6.53	9.58	0.23	0.07	11.95	23.06	33.89
	n	5	5	5	5	5	5	5	5	5	5
	Mean	99.40	27.64*	0.40	38.40	31.00	6.22	3.25	39.60	77.60	119.60
G3,M &	±SD	6.54	3.04	0.03	8.79	8.60	0.20	0.13	5.77	7.47	30.04
300
	n	5	5	5	5	5	5	5	5	5	5
	Mean	121.20	28.66*	0.40	52.20*	48.80*	6.38	3.24	34.20	73.40	102.80
G4,M &	±SD	14.62	3.05	0.02	7.16	10.06	0.47	0.17	4.97	12.60	21.97
1000
	n	5	5	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3 (Contd...). SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Total Bilirubin (BIT) (mg/dL)	Calcium (CAL) (mg/dL)	Phosphorous (PHO) (mg/dL)	Globulin (GLO) (g/dL)	Blood Urea Nitrogen (BUN) (mg/dL)	Sodium (Na) (mmol/L)	Potassium (K) (mmol/L)	Chloride (CLO) (mmol/L)
	Mean	0.02	9.94	6.30	2.56	18.31	153.00	4.64	117.84
G1, M & 0	±SD	0.01	0.33	0.44	1.03	3.77	2.46	0.55	2.32
	n	5	5	5	5	5	5	5	5
	Mean	0.03	9.94	6.44	2.92	12.34*	148.82	4.14	113.26*
G2, M & 100	±SD	0.01	0.22	0.53	0.24	1.34	2.87	0.14	1.47
	n	5	5	5	5	5	5	5	5
	Mean	0.02	10.16	6.52	2.97	12.90*	154.06	4.31	115.70
G3, M & 300	±SD	0.01	0.11	0.59	0.12	1.42	2.71	0.15	2.13
	n	5	5	5	5	5	5	5	5
	Mean	0.02	10.20	6.42	3.14	13.38'	152.28	4.56	115.62
G4, M & 1000	±SD	0.00	0.32	0.38	0.30	1.42	4.56	0.48	2.28
	n	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3 (Contd...). SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Total Bilirubin (BIT) (mg/dL)	Calcium (CAL) (mg/dL)	Phosphorous (PHO) (mg/dL)	Globulin (GLO) (g/dL)	Blood Urea Nitrogen (BUN) (mg/dL)	Sodium (Na) (mmol/L)	Potassium (K) (mmollL)	Chloride (CLO) (mmol/L)
	Mean	0.06	3.40	6.04	3.09	11.93	145.00	3.35	106.06
G1R, M & 0	±SD	0.02	0.89	0.42	0.29	2.11	1.22	0.17	0.72
	n	5	5	5	5	5	5	5	5
	Mean	0.06	2.04*	6.62	2.90	11.78	145.12	3.56	107.28*
G4R, M & 1000	±SD	0.01	0.78	2.65	0.17	1.87	1.19	0.51	0.55
	n	5	5	5	5	5	5	5	5
	Mean	0.04	3.80	3.78	3.27	12.41	145.22	3.48	108.72
G1R, F & 0	±SD	0.02	135	0.34	0.29	2.10	0.55	0.19	1.48
	n	5	5	5	5	5	5	5	5
	Mean	0.06	3.66	4.28	3.29	12.45	145.68	3.39	109.22
G4R, F & 1000	±SD	0.02	0.93	1.20	0.23	1.92	1.93	0.22	1.22
	n	5	5	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 4. SUMMARY OF FEED CONSUMPTION (g/animal/day) DURING GESTATION RECORD
Group & Dose (mg/kg body weight)		Feed Consumption (g/animal/day) during Gestation Day (GD)
Group & Dose (mg/kg body weight)		0 to 7	7 to 14	14 to 20
	Mean	18.85	21.83	26.63
G1 & 0	±SD	2.17	1.84	2.57
	n	12	12	12
	Mean	19.41	21.71	26.91
G2 & 100	±SD	1.84	1.49	2.01
	n	11	11	11
	Mean	17.71	20.47	26.59
G3 & 300	±SD	1.38	1.16	1.56
	n	10	10	10
	Mean	17.91	19.46*	26.15
G4 & 1000	±SD	1.46	1.51	1.44
	n	11	11	11
SD: Standard Deviation; n: Number of Animals (Females)
Note: The data of non-pregnant animals is excluded for mean calculations
* Statistically significant (P<0.05) change than the vehicle control group

TABLE 5. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) DURING LACTATION
Group & Dose (mg/kg body weight)		Percent Change in body weight (%) during Lactation Day (LD)
Group & Dose (mg/kg body weight)		1 to 4	4 to 7	7 to 13
	Mean	3.63	3.60	4.73
G1&0	±SD	2.10	2.13	2.44
	n	12	12	12
	Mean	2.38	2.83	4.96
G2 & 100	±SD	1.13	1.47	2.75
	n	11	11	11
	Mean	2.00	3.34	6.59
G3 & 300	±SD	1.58	1.84	2.79
	n	10	10	10
	Mean	1.56*	1.70*	2.73
G4 & 1000	±SD	2.36	0.72	1.91
	n	11	11	11

SD: Standard Deviation; n: Number of Animals (Females)
*: Statistically significant (P<0.05) change than the vehicle control group

TABLE 6. SUMMARY OF GESTATION LENGTH AND DELIVERY DATA RECORD
Group & Dose (mg/kg body weight)		Gestation Length (Days)	Litter Size (No.)	Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)				Sex Ratio (M/F) at Birth	Live Birth Index (%)
Group & Dose (mg/kg body weight)		Gestation Length (Days)	Litter Size (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Undetermined (No.)	Male (No.)	Female (No.)	Total (No.)	Sex Ratio (M/F) at Birth	Live Birth Index (%)
	Mean	22.17	11.25	6.17	5.08	11.25	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.43	100.00
G1 & 0	±SD	0.58	2.34	1.59	2.07	2.34	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.73	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	22.09	12.09	6.91	5.18	12.09	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.46	100.00
G2 & 100	±SD	0.54	2.02	1.87	1.54	2.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.68	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	21.70	13.40	6.30	6.30	12.60	0.00	0.30	0.30	0.20	0.30	0.00	0.50	1.17	93.81
G3 & 300	±SD	0.48	2.17	3.23	1.95	2.88	0.00	0.67	0.67	0.63	0.95	0.00	1.08	0.85	13.10
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	21.91	10.45*	4.27	4.45	8.73	0.73	0.91	1.64*	0.00	0.00	0.09	0.09	1.02	79.94*
G4 & 1000	±SD	0.30	1.92	2.69	2.34	4.15	1.42	1.58	2.94	0.00	0.00	0.30	0.30	1.01	34.01
	n	11	11	I 1	11	11	11	11	11	11	11	11	11	11	11

M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females)
*: Statistically significant (P<0.05) change than the vehicle control group

TABLE 7. SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group & Dose (mg/kg body weight)		Mean No. of Live Pups At Birth					During LD 1 to 4						Sex Ratio (M/F) at LD 4	No. of Survived Pups during LD 1 to 4	Pup Survival Index (%) LD 1 to 4
			Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)
			Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Un- determi ned (No.)	Male (No.)	Female (No.)	Total (No.)
	Mean	11.25	6.17	5.08	11.25	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.43	11.25	100.00
G1 & 0	±SD	2.34	1.59	2.07	2.34	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.73	2.34	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	12.09	6.82	5.09	11.91	0.09	0.09	0.18	0.00	0.00	0.00	0.00	1.47	11.91	98.26
G2 & 100	±SD	2.02	2.04	1.51	2.12	0.30	0.30	0.40	0.00	0.00	0.00	0.00	0.71	2.12	4.09
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	12.60	6.10	6.20	1230	0.20	0.00	0.20	0.00	0.00	0.10	0.10	1.18	12.30	97.91
G3 & 300	±SD	2.88	3.14	2.04	2.67	0.42	0.00	0.42	0.00	0.00	0.32	0.32	0.87	2.67	3.41
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	8.73	4.00	3.73	7.73	0.09	0.09	0.18	0.00	0.18	0.64	0.82	1.21	7.73	72.41*
G4 & 1000	±SD	4.15	2.83	2.65	4.90	0.30	0.30	0.40	0.00	0.60	1.50	2.09	1.01	4.90	44.06
	n	11	11	11	11	11	11	11	11	11	11	11	911	11	11

M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); LD: Lactation Day
*: Statistically significant (P<0.05) change than the vehicle control group

#: Two females from G4 were found with no live pups on LD4.

Note: The group mean number of live pups at birth: The mean number of live pups per litter at birth (a) was calculated first, then the group mean of mean number of pups per litter (b) was calculated using (a) to present the "Group mean number of live pups at birth".

TABLE 7 (Contd...). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group & Dose (mg/kg body weight)		Mean no. of Live Pups (No.) on LD 4			Pups Sacrificed for Blood Collection on LD 4 (No.)			Live Pups on LD4 after Blood Collection (No.)
Group & Dose (mg/kg body weight)		Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)
		Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)
	Mean	6.17	5.08	11.25	0.00	1.42	1.42	6.17	3.67	9.83
G1 & 0	±SD	1.59	2.07	2.34	0.00	0.90	0.90	1.59	1.72	1.64
	n	12	12	12	12	12	12	12	12	12
	Mean	6.82	5.09	11.91	0.00	1.45	1.45	6.82	3.64	10.45
G2 & 100	±SD	2.04	1.51	2.12	0.00	0.93	0.93	2.04	1.63	1.37
	n	11	11	11	11	11	11	11	11	11
	Mean	6.10	6.20	12.30	0.00	1.20	1.20	6.10	5.00	11.10
G3 & 300	±SD	3.14	2.04	2.67	0.00	0.92	0.92	3.14	2.16	1.85
	n	10	10	10	10	10	10	10	10	10
	Mean	4.00	3.73	7.73	0.00	0.64	0.64	4.00	3.09	7.09
G4 & 1000	±SD	2.83	2.65	4.90	0.00	0.92	0.92	2.83	2.17	4.37
	n	11	11	11	11	11	11	11	11	11
M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); NAD: No abnormality detected; LD: Lactation Day

Note: The group mean number of live pups on LD 4: The mean number of live pups per litter on LD 4 (a) was calculated first, then the group mean of mean number of pups per litter on LD 4 (b) was calculated using (a) to present the "Group mean number of live pups on LD 4".

TABLE 7 (Contd...). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group &						During LD 4 to 7							No. of	Pup Survival
Group &		Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)					No. of	Pup Survival
Dose (mg/kg body	Animal No.	Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)				Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
Dose (mg/kg body	Animal No.	Male	Female	Total	Male	Female	Total	Total	Total	Total	Total	Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
weight)		(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)		LD 4 to 7	LD 4 to 7

	Mean	6.17	3.67	9.83	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.16	9.83	100.00
G1 & 0	±SD	1.59	1.72	1.64	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.31	1.64	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	6.82	3.64	10.45	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.67	10.45	100.00
G2 & 100	±SD	2.04	1.63	1.37	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.40	1.37	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	6.10	5.00	11.10	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.87	11.10	100.00
G3 & 300	±SD	3.14	2.16	1.85	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.83	1.85	0.00
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	4.00	3.09	7.09	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.52	8.67	100.00
G4 & 1000	±SD	2.83	2.17	4.37	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.33	2.92	0.00
	n	11	11	11	11	11	11	11	11	11	11	9	9	9

M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); NAD: No abnormality detected; LD: Lactation Day

Note: The group mean number of live pups on LD 4: The mean number of live pups per liner on LD 4 (a) was calculated first, then the group mean of mean number of pups per litter on LD 4 (b) was calculated using (a) to present the "Group mean number of live pups on LD 4".


Note: The number of pups sacrificed for blood collection on LD 4 were excluded for the mean calculations during LD 4 to 7 and 7 to 13.

TABLE 7 (Contd...). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group &		Live Pups (No.) on			During LD 7 to 13											No. of	Pup Survival
Group &		Live Pups (No.) on			Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)					No. of	Pup Survival
Dose (mg/kg body	Animal No.	LD 7			Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
Dose (mg/kg body	Animal No.	Male	Female	Total	Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total	Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
weight)		(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)		LD 7 to 13	LD 7 to
																	13
	Mean	6.17	3.67	9.83	6.17	3.67	9.83	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.16	9.83	100.00
G1 & 0	±SD	1.59	1.72	1.64	1.59	1.72	1.64	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.31	1.64	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	6.82	3.64	10.45	6.82	3.64	10.45	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.67	10.45	100.00
G2 & 100	±SD	2.04	1.63	1.37	2.04	1.63	1.37	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.40	1.37	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	6.10	5.00	11.10	6.10	5.00	11.10	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.87	11.10	100.00
G3 & 300	±SD	3.14	2.16	1.85	3.14	2.16	1.85	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.83	1.85	0.00
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	4.89	3.78	8.67	4.89	3.67	8.56	0.00	0.00	0.00	0.11	0.00	0.00	0.11	1.71	8.56	88.89
G4 & 1000	±SD	2.26	1.72	2.92	2.26	1.94	3.24	0.00	0.00	0.00	0.33	0.00	0.00	0.33	1.29	3.24	33.33
	n	9	9	9	9	9	9	9	9	9	9	9	9	9	8^#	9	9

M:Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); LD: Lactation Day
N:Two females from G4 were found with no live pups on LD 1 to 4 and one female was found with no live pups during LD7 to 13.

Note: The group mean number of live pups on LD 7: The mean number of live pups per liner on LD 7 (a) was calculated first and then the group mean of mean number of pups per litter on LD 7 (b) was calculated using (a) to present the "Group mean number of live pups on LD 7".

TABLE 8. SUMMARY RECORD OF MEAN PUP WEIGHT (g) DURING LACTATION PERIOD
Group & Dose (mg/kg body weight)		Mean^#Pup Weight (g) on LDI		Mean^#Pup Weight (g) on LD 4		Mean^#Pup Weight (g) on LD 7		Mean^#Pup Weight (g) on LD 13
Group & Dose (mg/kg body weight)		Male	Female	Male	Female	Male	Female	Male	Female
	Mean	6.62	5.93	11.67	10.96	14.99	14.12	25.66	24.21
G1 & 0	±SD	0.24	0.23	0.31	0.28	0.37	0.31	0.92	0.67
	n	12	12	12	12	12	12	12	12
	Mean	6.58	6.19	11.48	10.84	14.43	13.97	24.78	23.78
G2 & 100	±SD	0.25	0.27	0.48	0.53	0.66	0.63	0.81	0.50
	n	11	11	11	11	11	11	11	11
	Mean	6.20*	5.72	9.94*	9.31*	13.60*	12.91*	22.88*	21.87*
G3 & 300	±SD	0.41	0.56	0.80	1.02	0.99	1.05	2.11	1.42
	n	10	10	10	10	10	10	10	10
	Mean	6.82	6.12	10.78*	9.84*	13.51*	12.29*	21.65*	20.61*
G4 & 1000	±SD	0.26	0.79	0.65	0.92	0.82	1.39	1.02	1.01
	n	9	11	8	9	8	9	8	8

SD: Standard Deviation; n: Number of Animals (Females); LD: Lactation Day
*: Statistically significant (P<0.05) change than the vehicle control group.
^#: The mean of all male and female pups per dam was calculated first and then group mean was calculated for all the dams. The presented value is group mean value

TABLE 9. SUMMARY OF THE STUDY
		Group & Dose (mg/kg body weight)
Parameters		G1 & 0	G2 & 100	G3 & 300	G4 & 1000
Pairs started (No.)		12 (1M + 1F)	12 (1M + 1F)	12 (1M + 1F)	12 (1M + 1F)
Females with Evidence of Copulation (No.)		12	12	12	12
Conceiving Days 1 to 5 (No.)		9	9	9	12
Conceiving Days ≥6 (No.)		3	3	3	0
Females achieving Pregnancy (No.)		12	11	10	11
Pregnancy ≤21 days (No.)		0	1	3	I
Pregnancy = 22 days (No.)		7	8	7	10
Pregnancy ≥23 days (No.)		5	2	0	0
Male Mating index (%)		100.00	100.00	100.00	100.00
Male Fertility Index (%)		100.00	91.67	83.33	91.67
Female Mating Index (%)		100.00	100.00	100.00	100.00
Females Fertility Index (%)		100.00	91.67	83.33	91.67
Parturition (%)		100.00	91.67	83.33	91.67
Dams with live young horn on LD 1 (No.)		12	11	10	11
Dams with live young on LD 4 (No.)		12	11	10	11
Dams with live young on LD 7 (No.)		12	11	10	11
Dams with live young on LD 13 (No.)		12	11	10	11
Implants/dam	Mean	11.25	12.09	13.40	10.45
Implants/dam	±SD	2.34	2.02	2.17	1.92
Pre-Implantation Loss (%)	Mean	0.00	0.00	0.00	0.00
Pre-Implantation Loss (%)	±SD	0.00	0.00	0.00	0.00
Post-Implantation Loss (%)	Mean	0.00	0.00	6.19	20.06
Post-Implantation Loss (%)	±SD	0.00	0.00	13.10	34.01
Live pups/dam at birth	Mean	11.25	12.09	12.60	8.73
Live pups/dam at birth	±SD	2.34	2.02	2.88	4.15
Live pups/dam at LD 4	Mean	11.25	11.91	12.30	7.73
Live pups/dam at LD 4	±SD	2.34	2.12	2.67	4.90
Sex ratio (m/f) at birth	Mean	1.43	1.46	1.17	1.02
Sex ratio (m/f) at birth	±SD	0.73	0.68	0.85	1.01
Sex ratio (m/f) at LD 4	Mean	1.43	1.47	1.18	1.21
Sex ratio (m/f) at LD 4	±SD	0.73	0.71	0.87	1.01
Male Pup weight at birth (g)	Mean	6.62	6.58	6.20	6.82
Male Pup weight at birth (g)	±SD	0.24	0.25	0.41	0.26
Female Pup weight at birth (g)	Mean	5.93	6.19	5.72	6.12
Female Pup weight at birth (g)	±SD	0.23	0.27	0.56	0.79
Male Pup weight (g) at the time of AGD measurement (LD 4)	Mean	11.67	11.48	9.94	10.78
Male Pup weight (g) at the time of AGD measurement (LD 4)	±SD	0.31	0.48	0.80	0.65
Female Pup weight (g) at the time of AGD measurement (LD 4)	Mean	10.96	10.84	9.31	9.84
Female Pup weight (g) at the time of AGD measurement (LD 4)	±SD	0.28	0.53	1.02	0.92
Male Pup weight LD 7 (g)	Mean	14.99	14.43	13.60	13.51
Male Pup weight LD 7 (g)	±SD	0.37	0.66	0.99	0.82
Female Pup weight LD 7 (g)	Mean	14.12	13.97	12.91	12.29
Female Pup weight LD 7 (g)	±SD	0.31	0.63	1.05	1.39
Male Pup weight LD 13 (g)	Mean	25.66	24.78	22.88	21.65
Male Pup weight LD 13 (g)	±SD	0.92	0.81	2.11	1.02
Female Pup weight LD 13 (g)	Mean	24.21	23.78	21.87	20.61
Female Pup weight LD 13 (g)	±SD	0.67	0.50	1.42	1.01
Male Pup AGD Ratio on the LD 4	Mean	2.29	2.28	2.25	2.27
Male Pup AGD Ratio on the LD 4	±SD	0.05	0.05	0.09	0.05
Female Pup AGO Ratio on the LD 4	Mean	0.91	0.92	0.95	0.95
Female Pup AGO Ratio on the LD 4	±SD	0.03	0.04	0.03	0.05
Male Pup Nipple Retention on LD 13	Mean	0.00	0.00	0.00	0.00
Male Pup Nipple Retention on LD 13	±SD	0.00	0.00	0.00	0.00
Male Scrum Thyroxine Hormone (T4) Levels (ng/mL)	Mean	80.566	84.355	83.872	82.829
Male Scrum Thyroxine Hormone (T4) Levels (ng/mL)	±SD	4.064	6.958	12.800	6.804
LD 13 Pup Serum Thyroxine Hormone (T4) Levels (ng/mL)	Mean	74.427	74.807	72.458	67.127
LD 13 Pup Serum Thyroxine Hormone (T4) Levels (ng/mL)	±SD	3.017	2.026	4.422	3.537
ABNORMAL PUPS

Dams with 0 (No.)		12	11	10	11
Dams with 1 (No.)		0	0	0	0
Dams with ≥2 (No.)		0	0	0	0
LOSS OF OFFSPRING
Pre-natal (implantations minus live births)
Females with 0 (No.)		12	11	10	11
Females with 1 (No.)		0	0	0	0
Females with 2 (No.)		0	0	0	0
Females with ≥ 3 (No.)		0	0	0	0
Post-natal loss during lactation period
Females with 0 pups (No.)		12	9	5	4
Females with 1 pup (No.)		0	2	3	4
Females with 2 pups (No.)		0	0	0	0
Females with ≥ 3 pups (No.)		0	0	2	3

No.: Number; SD: Standard Deviation; ≥: Greater than equal to;≤: Less than equal to; =: equal to; %: Percentage;
ng/mL: nano gram per milli liter; m/f: male/female; AGD: Ano-genital distance; LD: Lactation Day

TABLE 10. SUMMARY OF UTERI OBSERVATION RECORD
Group&Dose (mg/kg body weight)		No. of Corporalutea	No. of Implantations	Implantation Index (%)	Pre- Implantation Loss (%)	Post- Implantation Loss (%)	Pre-natal Loss (No.)	Post-natal Loss (At birth to LD 13) (%)	Post-natal Loss (At birth to LD 13) (No.)	No. of Early Resorptions	No. of Late Resorptions
	Mean	11.25	11.25	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
G1 & 0	±SD	2.34	2.34	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
		12	12	12	12	12	12	12	12	12	12
	Mean	12.09	12.09	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
G2 & 100	±SD	2.02	2.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
		11	11	11	11	11	11	11	11	11	11
	Mean	13.40*	13.40*	100.00	0.00	6.19	0.80	0.00	0.00	0.00	0.00
G3 & 300	±SD	2.17	2.17	0.00	0.00	13.10	1.69	0.00	0.00	0.00	0.00
		10	10	10	10	10	10	10	10	10	10
	Mean	10.45	10.45	100.00	0.00	20.06*	1.73*	27.27	0.91	0.00	0.00
G4 & 1000	±SD	1.92	1.92	0.00	0.00	34.01	2.90	46.71	2.39	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11

SD: Standard Deviation; n: Number of Animals (Females)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 11. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g)
Group, Sex & Dose (mg/kg body weight)		Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Thyroid along with para Thyroid^#
	Mean	0.1009	0.2834	0.6195	1.3311	2.6962	2.0879	16.3846	0.0310
G1, F &	±SD	0.0198	0.0522	0.1368	0.0744	0.2709	0.0384	1.5298	0.0063
0
		5	5	5	5	5	5	5	12
	Mean	0.0961	0.2881	0.6405	1.1601*	2.6774	2.1819	14.3037*	0.0308
G2, F &	±SD	0.0054	0.0930	0.0969	0.0817	0.2466	0.0266	0.7461	0.0049
100
		5	5	5	5	5	5	5	12
	Mean	0.0937	0.3049	0.7649	1.2065	2.7635	2.1132	16.9891	0.0302
G3, F &	±SD	0.0149	0.0946	0.0725	0.0876	0.1476	0.0502	1.0569	0.0033
300
		5	5	5	5	5	5	5	12
	Mean	0.1032	0.2809	0.6447	1.1626*	2.6732	2.1717	15.8345	0.0317
G4, F &	±SD	0.0128	0.0952	0.2021	0.1021	0.1996	0.1646	1.2621	0.0048
1000
	n	5	5	5	5	5	5	5	12
	Mean	0.0928	0.3907	0.5817	1.1948	2.3005	2.1197	8.9081	0.0289
G1R, F &	±SD	0.0145	0.0527	0.0180	0.1490	0.1729	0.0663	0.7555	0.0035
0
	n	5	5	5	5	5	5	5	5
	Mean	0.1017	0.4139	0.5713	1.1680	2.2110	2.0927	8.9462	0.0279
G4R, F &	±SD	0.0147	0.0418	0.0599	0.0705	0.1374	0.0531	1.0503	0.0033
1000
	n	5	5	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (Females); #: Weighed post fixation
*: Statistically significant (P<0.05) change than the vehicle control group

Conclusions:

In the absence of adverse parental systemic effects, fertility parameters and reproductive performance, the NOAEL for parental and reproduction was placed at 1000 mg/kg (the highest concentration tested).
Based on the effects on post-implantation and post-natal loss, live birth index and pup survival index, the NOAEL for developmental effects was placed at 300 mg/kg (mid concentration).

Executive summary:

The objective of this GLP compliant OECD 422 study was to provide data on the possible effects of the test substance Dihydro Isojamonate on general toxicity, reproductive performance and development of pups. The test substance was suspended in corn oil and administered by oral gavage at 0, 100, 300 and 1000 mg/kg body weight/day. The test substance was administered during a pre-mating period of 2 weeks, during mating, gestation and lactation. Male animals were sacrificed after the mating period after completion of 36 days of treatment. Pups were sacrificed at day 13 of lactation. Parental female animals were sacrificed at day 14 of lactation.

There was no treatment related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of Dihydro lsojamonate. There were no relevant changes in ophthalmology, red blood cell variables and clotting potential, clinical chemistry, urinalysis, organ weight, gross pathology or histopathology. During lactation, a treatment related reduction in the percentage body weight change was observed during PND 1 to 4 and PND 4 to 7. During gestation, a treatment related reduction in feed consumption was observed during GD 7 to 14. In the absence of any other evidence of parental toxicity, these observations were not considered to be adverse.There were no adverse effects of the test substance on fertility and developmental parameters in the G2 (100 mg/kg) and G3 (300 mg/kg) groups. The reductions in litter size and pup weight observed in group G4 (1000 mg/kg) was not considered to be adverse, since values for all individual animals were within the historical control range. Treatment related increases in the percentage post-implantation and post-natal loss and an increased number of dead and cannibalized pups were observed. This is reflected in reductions in live birth index and pup survival index and these findings are considered to be adverse.There were no treatment related effects on Thyroxine (T4) levels between the exposed groups and the control groups for male adults and for 13-day old male and female pups.

In the absence of adverse parental systemic effects, fertility parameters and reproductive performance, the NOAEL for parental and reproduction was placed at 1000 mg/kg (the highest concentration tested).Based on the effects on post-implantation and post-natal loss, live birth index and pup survival index, the NOAEL for developmental effects was placed at 300 mg/kg (mid concentration).

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2019
Report date:: 2019

Materials and methods

Test guideline

Qualifier:: according to guideline
Guideline:: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:: Adopted 29 July 2016
Deviations:: no

GLP compliance:: yes (incl. QA statement)
Limit test:: no

Test material

Test material information

Test material form:: liquid

Specific details on test material used for the study:: Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Test animals

Species:: rat
Strain:: Sprague-Dawley
Details on species / strain selection:: The rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: Males group means: 331 to 334 g. Females group means: 260 to 261 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm). Clean sterilized paddy husk was provided as bedding material. A maximum of 3 animals of the same sex were caged together during acclimatization and 2 animals of the same sex and group during pre-mating. During mating, two animals (one male and one female) of the same group were housed together. Post-mating, females were housed individually and males were housed with their former cage mates. A maximum of three animals of the same sex were housed in the same cage for recovery group animals.
- Diet: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum.
- Water: Water was provided ad libitum.
- Acclimation period: Healthy and young animals were acclimatized initially for five days to experimental room conditions. Further the females were subjected to screening for estrous cycles in a two weeks (14 days) pre-treatment period. The males and females were observed for clinical signs once daily and veterinary examination of all the animals was performed on the day of receipt and on the day of randomization and grouping.

DETAILS OF FOOD AND WATER QUALITY: Food and water was checked for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 to 23.1
- Humidity (%): 47 to 67
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
16 Aug 2018 to 7 Nov 2018

Administration / exposure

Route of administration:: oral: gavage
Vehicle:: corn oil
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
Test item formulations were prepared daily before administration. The required quantity of the test item was weighed into a clean beaker. A small volume of corn oil (vehicle) was added into the beaker, mixed well with a glass rod and the mixture was transferred into the measuring cylinder. The beaker was rinsed using a small volume of corn oil and the volume was transferred to the measuring cylinder. The rinsing procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was supplemented with vehicle to the required quantity to get a desired concentration of 10, 30 and 100 mg/mL of test item for the low, mid and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected as a vehicle for the preparation of test item formulations as the test item was not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL (the highest dose concentration) as evidenced by the in-house miscibility test results.
- Concentration in vehicle: 10, 30 and 100 mg/mL corresponding to a dose of 100, 300 and 1000 mg/kg bw, respectively.
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: A1712001
Details on mating procedure:: - M/F ratio per cage: 1/1
- Length of cohabitation: until mating occurred or until two weeks had elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
The recovery group animals were not mated and therefore not cohabitated.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Sampling and analysis of formulations was performed during the first week of treatment and during week 5 of the treatment. The samples were collected in duplicate from the top, middle and bottom layer from the low, mid and high dose concentrations, and in duplicate from the middle layer from the vehicle control.
Duration of treatment / exposure:: Males: The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Females: The females were treated for a two week pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13. Females (dams) were sacrificed on lactation day 14 after overnight fasting.
Frequency of treatment:: Once daily

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 100 mg/kg bw/day (actual dose received)
Remarks:: Group 2: Low dose

Doses / concentrations 2

Dose / conc.:: 300 mg/kg bw/day (actual dose received)
Remarks:: Group 3: Mid dose

Doses / concentrations 3

Dose / conc.:: 1 000 mg/kg bw/day (actual dose received)
Remarks:: Group 4: High dose

Doses / concentrations 4

Dose / conc.:: 1 000 mg/kg bw/day (actual dose received)
Remarks:: Group 4 recovery (G4R): High dose

No. of animals per sex per dose:: Treatment groups: 12
Recovery groups: 5
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: The dose levels of the treatment and recovery groups were based on the results of the dose range finding study of the test item when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 3392). These doses were selected as the test item when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight (water available)
- Post-exposure recovery period in satellite groups: 14 days

Examinations

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

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Day 1 (before treatment) and weekly thereafter.
- Parameters checked: Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT:
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION:
- Food consumption was measured for main group animals once a week during premating and for main group males during post mating period. Food consumption was not measured during mating period for both males and females. Thereafter, food consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Food consumption was measured for recovery group animals once a week throughout the experimental period.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Once before treatment for all animals, during the end of the dosing period for males (on day 31) and during the lactation period for females (on lactation day 13) of vehicle control and high dose main group animals and during the last week (day 64) for recovery group animals.
- Dose groups that were examined: The examination was not extended to lower dose groups as there were no treatment related changes noted in high dose group animals.

HAEMATOLOGY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked in Table 1 in ‘Any other information on materials and methods incl. tables’ were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked in Table 2 in ‘Any other information on materials and methods incl. tables’ were examined.

URINALYSIS:
- Time schedule for collection of urine: At termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: The animals had no access to food, water was however available.
- How many animals: Urine was collected from five randomly selected males of each main group and for all recovery animals at termination.
- Parameters checked in Table 3 in ‘Any other information on materials and methods incl. results’ were examined. In addition, pH, nitrite and specific gravity were also analysed and urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Males on day 36, females on lactation day 13 and shortly prior to scheduled sacrifice on day 64 for the recovery animals.
- Dose groups that were examined: All groups
- Battery of functions tested: See Table 4 in ‘Any other information on materials and methods incl. tables’.

OTHER: T4 levels
- Time of schedule for examinations: one day before scheduled sacrifice
- Animals: all adult males (after completion of 36 days of treatment)
Oestrous cyclicity (parental animals):: Vaginal smears to evaluate the estrous cycle length and normality were made daily in all females during the pre-treatment period until the start of the treatment period. Only females with normal estrous cyclicity (4 to 5 days) were selected for the treatment. Vaginal smears were also made and evaluated daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. Estrous cyclicity was also monitored on the day of sacrifice.
Recovery group females were not evaluated for estrous cyclicity
Sperm parameters (parental animals):: Parameters examined in P male parental generations:
epididymis weight, stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:: PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams, sires and pup survival index and sex ratio at birth was calculated. T4 levels were evaluated on PND 4 and 13.
Postmortem examinations (parental animals):: GROSS PATHOLOGY: Table 5 in ‘Any other information on materials and methods incl. tables’.
The animals were euthanized using carbon dioxide asphyxiation followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination. Relative organ weights were calculated against fasting body weight.

HISTOPATHOLOGY: Table 5 in ‘Any other information on materials and methods incl. tables’.
Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.
All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. The testes were sectioned at 3 to 4 micrometers and stained with hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain was aided spermatogenesis evaluation.
The investigations were not extended to the lower dose groups and recovery groups as there were no treatment related effects noted at the high dose level.
Bone marrow smear (from one femur) was prepared at the time of necropsy.
Postmortem examinations (offspring):: SACRIFICE
- Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case the litter size was 11 pups/litter.
- The remaining pups were sacrificed at PND 13.
- These animals were examined for gross abnormalities and the findings were recorded.

GROSS EXAMINATION OF PUPS:
The surviving pups were sacrificed on lactation day 13 and the sacrificed pups (on lactation day 4 and 13) and dead/cannabalized pups were examined for gross abnormalities and the findings were recorded.
Statistics:: The raw data was subjected to computer statistical processing. The computer printout of the data was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05) (see Table 7 in 'Any other information on materials and methods incl. tables' for an overview of the statistical tests used).
Reproductive indices:: See Table 6 in 'Any other information on materials and methods incl. tables'.
Offspring viability indices:: See Table 6 in 'Any other information on materials and methods incl. tables'.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:: no effects observed
Description (incidence and severity):: There were no clinical signs of toxicity in any of the main and recovery group animals of either sex throughout the experimental period. The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.
Dermal irritation (if dermal study):: not examined
Mortality:: no mortality observed
Description (incidence):: No mortality/morbidity was observed in any of the main and recovery group animals of either sex throughout the experimental period.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at any of the tested dose main and recovery group animals of either sex.
There were no treatment related changes in body weight and percentage change in body weight during the gestation period in all animals treated with the test substance.
There were no changes in body weight and percentage change in body weight during the lactation period in group G2 (100 mg/kg) and G3 (300 mg/kg) when compared with control group animals. A reduction in mean body weight and percentage change in body weight during lactation in G4 (1000 mg/kg) was noted. This change can be considered as treatment related as the reduction in the percentage body weight change is statistically significant during PND 1 to 4 and PND 4 to 7 when compared with control group animals.
Food consumption and compound intake (if feeding study):: effects observed, non-treatment-related
Description (incidence and severity):: There were no treatment related changes noted in mean feed consumption at any of the tested dose levels of both main and recovery group animals and of either sex. However, a statistically significant increase in mean feed consumption for group G2 (100 mg/kg) males during the post mating period was noted, when compared with vehicle control group males. This change is considered as incidental and not treatment related due to lack of dose dependency. In addition, no effects were noted in mean body weight and percent change in body weight during this period at this dose level.
There were no treatment related changes in the feed consumption during the gestation period at all the tested dose levels. However, a statistically significant reduction in feed consumption during gestation day 7 to 14 was noted in group G4 (1000 mg/kg) animals when compared with vehicle control group animals. Feed consumption on GD0-7 and GD7-14 was somewhat lower than the historical group mean control data, but on GD14-21 the feed consumption was higher. This observation is considered as not adverse effect as the reduction is not consistent.
There were no changes in the feed consumption during the lactation period at all the tested dose levels when compared with control group animals.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: no effects observed
Description (incidence and severity):: There were no ocular changes observed in G1 (0 mg/kg) and G4 (1000 mg/kg) group animals of either sex during the ophthalmological examination conducted towards the end of the dosing period and no ocular changes were observed in G1R (0 mg/kg) and G4R (1000 mg/kg) group animals of either sex during the ophthalmological examination conducted at the end of the recovery period.
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: There were no treatment related changes in haematology parameters in any of the animals of either sex in both main or recovery groups treated with the test substance. However, a statistically significant increase in absolute basophils in group G4 (1000 mg/kg) males; a statistically significant decrease in prothrombin time in all the main group males; a statistically significant decrease in activated partial thromboplastin time in group G2 (100 mg/kg) and G3 (300 mg/kg) males; a statistically significant decrease in haematocrit in group G4R (1000 mg/kg) females was noted when compared with their concurrent control group animals. These changes are considered as incidental and not treatment related, as the obtained values are within the in-house historical control data of the species and additionally, the same changes were not observed in the other sex.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: There were no treatment related changes in clinical chemistry parameters in any of the animals of either sex in both main or recovery groups treated with the test substance. However, a statistically significant decrease in glucose and chloride levels in group G2 (100 mg/kg) males; a statistically significant decrease in urea and blood urea nitrogen levels in all the main group males; a statistically significant increase in total cholesterol and triglycerides in group G4 (1000 mg/kg) males; a statistically significant increase in chloride levels and decrease in calcium in group G4R (1000 mg/kg) males and a reduction in aspartate aminotransferase in group G4R (1000 mg/kg) females was noted when compared with their concurrent control group animals. These changes are considered as incidental and not treatment related, as the obtained values are within the in-house historical control data of the species and additionally, the same changes were not observed in the other sex.
Urinalysis findings:: no effects observed
Description (incidence and severity):: There were no treatment related changes in urine analysis parameters in any of the main group males and recovery group animals of either sex.
Behaviour (functional findings):: effects observed, non-treatment-related
Description (incidence and severity):: The results of the neurological/functional examination conducted for five randomly selected main group males and females from each group prior to scheduled sacrifice and for all recovery group animals at the end of recovery period did not indicate any treatment related effect. However, a statistically significant reduction in mean motor activity in group G4 (1000 mg/kg) females; a statistically significant increase in landing foot splay in group G4R (1000 mg/kg) females was noted when compared with concurrent control group females.
These changes are considered as incidental and not treatment related as there were no effects noted in other functional observation battery parameters and no clinical signs of toxicity were noted in all the animals of these dose levels.
Immunological findings:: not examined
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):: No treatment related histopathological findings were noticed in the study. Lesions considered to be spontaneous and incidental were observed in treated and control rats. These lesions consisted of interstitial mononuclear cell infiltration and concretions in prostate gland, and presence of calculi in kidney.
One female from the high dose group (G4) revealed mononuclear cell infiltration at submucosa of the vagina. This lesion was considered spontaneous because it was not observed in any of the other females. The microscopic findings (Coagulative necrosis of renal tubules, dilated pelvis of kidney, physeal dysplasia in femur bone) observed only in vehicle control animals, hence were considered as spontaneous.
The females from the vehicle control group (GI) and high dose group (G4) revealed the presence of a placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary.
Histopathological findings: neoplastic:: no effects observed
Other effects:: no effects observed
Description (incidence and severity):: T4 LEVELS: There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:: no effects observed
Description (incidence and severity):: There were no treatment related changes in the estrous cyclicity of females at any of the doses tested during pre-mating treatment, mating treatment and on lactation day 14.
Reproductive function: sperm measures:: no effects observed
Reproductive performance:: effects observed, treatment-related
Description (incidence and severity):: There were no changes observed in the gestation length, litter delivered, number of pups delivered, sex ratio, live birth index for group G2 (100 mg/kg) animals when compared with vehicle control group animals.
A slight reduction in live birth index (93.81%) in G3 (300 mg/kg) animals was observed when compared with the vehicle control group animals. The observed live birth index was slightly lower than the historical group mean, but not statistically significantly different. The slight reduction in live birth index is attributable to only 2 animals (Rd0754 and Rd0757). Their individual live birth index is 71.4 and 66.7, respectively, which is well within the historical control range (60-100). Hence, this observation is considered to be not biologically relevant. No statistically significant differences were found for the number of dead and cannibalized pups when compared with the vehicle control group animals.
A statistically significant reduction in litter size and live birth index was noted for G4 (1000 mg/kg) animals. The group mean litter size was also slightly lower than the historical group mean. Litter size of all individual animals (6-13) however, is within the historical control range, which is 5-16. The reduction in live birth index is predominantly attributable to 2 animals (Rd0760 and Rd0761) and to a lesser extent to a third animal (Rd0763). Their individual live birth indices are 10.0, 16.7 and 72.7, respectively. The values of the first two animals is clearly below the historical control range, whereas the index of the third animals is within this range. Hence, the reduction in live birth index should be considered a treatment related adverse effect. An increased number of dead and cannibalized pups reflects the reduced live birth index.

Effect levels (P0)

open all close all

1

: Key result
Dose descriptor:: NOAEL
Remarks:: Systemic toxicity
Effect level:: > 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable due to absence of adverse toxic effects

2

: Key result
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: > 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: female
Remarks on result:: not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

: Key result
Critical effects observed:: no

Results: F1 generation

General toxicity (F1)

Clinical signs:: no effects observed
Description (incidence and severity):: There were no changes noted in clinical and external daily observations of pups at all the tested dose levels during the lactation period.
Dermal irritation (if dermal study):: not examined
Mortality / viability:: mortality observed, treatment-related
Description (incidence and severity):: There were no treatment related changes in litter observation parameters for group G2 (100 mg/kg) and G3 (300 mg/kg) animals when compared with vehicle control group animals during lactation period. The number of dead and cannibalized pups for G3 (300 mg/kg) animals is lower than the historical control value. However, an increased number of dead and cannibalized pups during the lactation period was observed for group G4 (1000 mg/kg) animals, resulting in a clear reduction in pup survival index in this group at PND 1 to 4 and PND 7 to 13 when compared with vehicle control group animals, which is statistically significant at PND 1 to 4. Due to the large standard deviation however, this reduction is not statistically significant. This observed reduction is attributable to three animals in the group, namely Rd0760, Rd0761 and Rd0763. Their individual pup survival index is 0, 0 and 12.5% on LD 1 to 4, respectively. On LD7 to 13, the pup survival index of animal Rd0763 is 0%. All these values are severely below the historical control range (60-100% on LD1 to 4 and 100-100% on LD7 to 13). Hence, this reduction in pup survival index should be considered a treatment related adverse effect.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: The mean of all the male and female pups per dam was calculated initially and subsequently the overall mean pup weight of each sex from all the dams of one group was calculated. The group mean values obtained from each tested dose group was compared with the vehicle control group. There were no changes in mean pup weight of either sex during the lactation period for group G2 (100 mg/kg) when compared with the vehicle control group. Statistically significant reductions in mean male pup weight on PND 1 in group G3 (300 mg/kg) and in mean pup weight (both male and female) on PND 4, 7 and 13 for group G3 (300 mg/kg) and G4 (1000 mg/kg) were noted when compared with the vehicle control. Although the reductions reported for group G3 (300 mg/kg) are statistically significantly different, they are still higher than the historical group mean and thus considered not biologically relevant. Only in male pups on LD 13 the mean pup weight is slightly below the historical mean. However, all mean pup weights/dam were within the historical control range. No individual weights were below the lower limit of the range; in one litter several pups had a weight which was higher than the historical control range. Therefore, this observation is considered to be a chance finding. In addition, although the reductions reported for group G4 (1000 mg/kg) are statistically significantly different, there is no dose response up to LD4 for both males and females and more importantly, the reported values are still higher than the historical group mean and thus considered not biologically relevant. Only in male pups on LD I 3 the mean pup weight is slightly below the historical mean. However, all mean pup weights/dam were within the historical control range. Only three individuals of the in total 77 pups, weighed slightly below the lower limit of the range. Therefore, this observation is considered to be a chance finding.
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Sexual maturation:: not examined
Anogenital distance (AGD):: effects observed, non-treatment-related
Description (incidence and severity):: There were no changes noted in anogenital distance of both male and female pups at any of the tested doses. However, a statistically significant increase in mean female pup ano-genital distance was noted at group G3 (300 mg/kg) and G4 (1000 mg/kg) pups. This occurrence was considered as incidental as the obtained values were within biological range of this species.
Nipple retention in male pups:: no effects observed
Description (incidence and severity):: There were no occurrences of nipples in male pups at any of the tested doses on lactation day 13.
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: no effects observed
Description (incidence and severity):: The pups which were found dead, sacrificed on lactation day 4 for blood collection and pups sacrificed on lactation day 13 were subjected to gross pathological examination. There were no gross pathological changes noted in any of the pups (both sacrificed and found dead). The pups found dead were either cannibalized or found autolysed except for one female pup from G4 group which was found in its amniotic sac.
Histopathological findings:: not examined
Other effects:: effects observed, non-treatment-related
Description (incidence and severity):: A statistically significant decrease in T4 levels of lactation day 13 pups in group G4 was noted when compared with vehicle control group. The variation observed in the T4 levels could not be attributed to treatment as there were no microscopic observations in thyroid gland and no effects were noted in thyroid weight for all dose levels. The T4 levels obtained in animals/pups across the groups are within historical control range. Hence, the variation observed in the T4 levels is considered to be incidental.

Developmental neurotoxicity (F1)

Behaviour (functional findings):: not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:: not examined

Effect levels (F1)

: Key result
Dose descriptor:: NOAEL
Remarks:: Developmental toxicity
Generation:: F1
Effect level:: 300 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: viability; mortality

Target system / organ toxicity (F1)

: Key result
Critical effects observed:: no

Overall reproductive toxicity

: Key result
Reproductive effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day (actual dose received)
Treatment related:: yes
Relation to other toxic effects:: reproductive effects in the absence of other toxic effects
Dose response relationship:: yes
Relevant for humans:: yes

Any other information on results incl. tables

TABLE 1. SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD
Group, Sex & Dose (mg/kg body weight)			Feed Consumption (g/animal/day)
Group, Sex & Dose (mg/kg body weight)		Week 1 (Day 1 to 7)	Week 2 (Day 7 to 14)	During Post-mating (Day 35 to 37)
	Mean	21.23	22.42	25.73
G1, M & 0	±SD	2.53	2.16	0.55
	n	12	12	12
	Mean	21.15	21.53	27.14*
G2, M & 100	±SD	2.79	2.43	0.83
	n	12	12	12
	Mean	21.51	22.20	26.67
G3, M & 300	±SD	2.52	2.04	1.00
	n	12	12	12
	Mean	21.05	22.65	25.27
G4, M & 1000	±SD	1.95	0.93	0.98
	n	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals
* Statistically significant (P<0.05) change than the vehicle control group.

TABLE 2. SUMMARY OF HAEMATOLOGY RECORD
Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count (Retic) (10⁹cells/L)	Absolute Neutrophils Neut (10³cells/µL)	Absolute Lymphocytes Lymph (10³cells/µL)	Absolute Monocytes Mono (10³cells/µL)	Absolute Eosinophils Eos (10³cells/µL)	Absolute Basophils Baso (10³cells/µL)	Prothrombin Time PT (Seconds)	Activated Prothrombin Time APTT (Seconds)
Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count (Retic) (10⁹cells/L)	Absolute Neutrophils Neut (10³cells/µL)	Absolute Lymphocytes Lymph (10³cells/µL)	Absolute Monocytes Mono (10³cells/µL)	Absolute Eosinophils Eos (10³cells/µL)	Absolute Basophils Baso (10³cells/µL)	Prothrombin Time PT (Seconds)	Activated Prothrombin Time APTT (Seconds)
	Mean	136.42	1.84	5.83	0.23	0.11	0.02	26.78	38.12
G1,M & 0	±SD	16.23	0.73	1.45	0.08	0.05	0.01	5.76	13.71

	n	5	5	5	5	5	5	5	5
	Mean	165.04	1.89	6.26	0.20	0.10	0.02	18.54*	18.72*
G2,M & 100	±SD	23.22	0.38	0.85	0.06	0.03	0.01	1.88	4.54

	n	5	5	5	5	5	5	5	5
	Mean	247.46	1.90	5.89	0.18	0.07	0.01	21.28*	22.30*
G3,M & 300	±SD	166.08	0.43	0.82	0.05	0.03	0.01	2.26	9.16

	n	5	5	5	5	5	5	5
	Mean	185.24	2.87	7.91	0.20	0.09	0.03*	18.04*	31.58
G4, M & 1000	±SD	44.01	1.99	2.73	0.07	0.03	0.01	0.73	6.17

	n	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3. SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Glucose (GLU) (mg/dL)	Urea (mg/dL)	Creatinine (CRE) (mg/dL)	Total Cholesterol (CHO) (mg/dL)	Triglycerides (TRI) (mg/dL)	Total Protein (TPR) (g/dL)	Albumin (ALB) (g/dL)	Alanine aminotransferase (ALT) (U/L)	Aspartate aminotransferase (AST) (U/L)	Alkaline phosphatase (ALP) (U/L)
	Mean	152.00	39.24	0.42	37.40	31.40	6.20	3.64	41.60	84.00	122.60
G1,M&	±SD	61.60	8.08	0.04	8.79	10.64	0.35	1.14	5.18	15.12	20.40
0
	n	5	5	5	5	5	5	5	5	5	5
	Mean	92.60*	26.44*	0.39	37.80	26.80	6.14	3.22	38.20	91.80	124.40
G2,M & 100	±SD	10.69	2.87	0.03	6.53	9.58	0.23	0.07	11.95	23.06	33.89
	n	5	5	5	5	5	5	5	5	5	5
	Mean	99.40	27.64*	0.40	38.40	31.00	6.22	3.25	39.60	77.60	119.60
G3,M &	±SD	6.54	3.04	0.03	8.79	8.60	0.20	0.13	5.77	7.47	30.04
300
	n	5	5	5	5	5	5	5	5	5	5
	Mean	121.20	28.66*	0.40	52.20*	48.80*	6.38	3.24	34.20	73.40	102.80
G4,M &	±SD	14.62	3.05	0.02	7.16	10.06	0.47	0.17	4.97	12.60	21.97
1000
	n	5	5	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3 (Contd...). SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Total Bilirubin (BIT) (mg/dL)	Calcium (CAL) (mg/dL)	Phosphorous (PHO) (mg/dL)	Globulin (GLO) (g/dL)	Blood Urea Nitrogen (BUN) (mg/dL)	Sodium (Na) (mmol/L)	Potassium (K) (mmol/L)	Chloride (CLO) (mmol/L)
	Mean	0.02	9.94	6.30	2.56	18.31	153.00	4.64	117.84
G1, M & 0	±SD	0.01	0.33	0.44	1.03	3.77	2.46	0.55	2.32
	n	5	5	5	5	5	5	5	5
	Mean	0.03	9.94	6.44	2.92	12.34*	148.82	4.14	113.26*
G2, M & 100	±SD	0.01	0.22	0.53	0.24	1.34	2.87	0.14	1.47
	n	5	5	5	5	5	5	5	5
	Mean	0.02	10.16	6.52	2.97	12.90*	154.06	4.31	115.70
G3, M & 300	±SD	0.01	0.11	0.59	0.12	1.42	2.71	0.15	2.13
	n	5	5	5	5	5	5	5	5
	Mean	0.02	10.20	6.42	3.14	13.38'	152.28	4.56	115.62
G4, M & 1000	±SD	0.00	0.32	0.38	0.30	1.42	4.56	0.48	2.28
	n	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 3 (Contd...). SUMMARY OF CLINICAL CHEMISTRY RECORD
Group, Sex & Dose (mg/kg body weight)		Total Bilirubin (BIT) (mg/dL)	Calcium (CAL) (mg/dL)	Phosphorous (PHO) (mg/dL)	Globulin (GLO) (g/dL)	Blood Urea Nitrogen (BUN) (mg/dL)	Sodium (Na) (mmol/L)	Potassium (K) (mmollL)	Chloride (CLO) (mmol/L)
	Mean	0.06	3.40	6.04	3.09	11.93	145.00	3.35	106.06
G1R, M & 0	±SD	0.02	0.89	0.42	0.29	2.11	1.22	0.17	0.72
	n	5	5	5	5	5	5	5	5
	Mean	0.06	2.04*	6.62	2.90	11.78	145.12	3.56	107.28*
G4R, M & 1000	±SD	0.01	0.78	2.65	0.17	1.87	1.19	0.51	0.55
	n	5	5	5	5	5	5	5	5
	Mean	0.04	3.80	3.78	3.27	12.41	145.22	3.48	108.72
G1R, F & 0	±SD	0.02	135	0.34	0.29	2.10	0.55	0.19	1.48
	n	5	5	5	5	5	5	5	5
	Mean	0.06	3.66	4.28	3.29	12.45	145.68	3.39	109.22
G4R, F & 1000	±SD	0.02	0.93	1.20	0.23	1.92	1.93	0.22	1.22
	n	5	5	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 4. SUMMARY OF FEED CONSUMPTION (g/animal/day) DURING GESTATION RECORD
Group & Dose (mg/kg body weight)		Feed Consumption (g/animal/day) during Gestation Day (GD)
Group & Dose (mg/kg body weight)		0 to 7	7 to 14	14 to 20
	Mean	18.85	21.83	26.63
G1 & 0	±SD	2.17	1.84	2.57
	n	12	12	12
	Mean	19.41	21.71	26.91
G2 & 100	±SD	1.84	1.49	2.01
	n	11	11	11
	Mean	17.71	20.47	26.59
G3 & 300	±SD	1.38	1.16	1.56
	n	10	10	10
	Mean	17.91	19.46*	26.15
G4 & 1000	±SD	1.46	1.51	1.44
	n	11	11	11
SD: Standard Deviation; n: Number of Animals (Females)
Note: The data of non-pregnant animals is excluded for mean calculations
* Statistically significant (P<0.05) change than the vehicle control group

TABLE 5. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) DURING LACTATION
Group & Dose (mg/kg body weight)		Percent Change in body weight (%) during Lactation Day (LD)
Group & Dose (mg/kg body weight)		1 to 4	4 to 7	7 to 13
	Mean	3.63	3.60	4.73
G1&0	±SD	2.10	2.13	2.44
	n	12	12	12
	Mean	2.38	2.83	4.96
G2 & 100	±SD	1.13	1.47	2.75
	n	11	11	11
	Mean	2.00	3.34	6.59
G3 & 300	±SD	1.58	1.84	2.79
	n	10	10	10
	Mean	1.56*	1.70*	2.73
G4 & 1000	±SD	2.36	0.72	1.91
	n	11	11	11

SD: Standard Deviation; n: Number of Animals (Females)
*: Statistically significant (P<0.05) change than the vehicle control group

TABLE 6. SUMMARY OF GESTATION LENGTH AND DELIVERY DATA RECORD
Group & Dose (mg/kg body weight)		Gestation Length (Days)	Litter Size (No.)	Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)				Sex Ratio (M/F) at Birth	Live Birth Index (%)
Group & Dose (mg/kg body weight)		Gestation Length (Days)	Litter Size (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Undetermined (No.)	Male (No.)	Female (No.)	Total (No.)	Sex Ratio (M/F) at Birth	Live Birth Index (%)
	Mean	22.17	11.25	6.17	5.08	11.25	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.43	100.00
G1 & 0	±SD	0.58	2.34	1.59	2.07	2.34	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.73	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	22.09	12.09	6.91	5.18	12.09	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.46	100.00
G2 & 100	±SD	0.54	2.02	1.87	1.54	2.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.68	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	21.70	13.40	6.30	6.30	12.60	0.00	0.30	0.30	0.20	0.30	0.00	0.50	1.17	93.81
G3 & 300	±SD	0.48	2.17	3.23	1.95	2.88	0.00	0.67	0.67	0.63	0.95	0.00	1.08	0.85	13.10
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	21.91	10.45*	4.27	4.45	8.73	0.73	0.91	1.64*	0.00	0.00	0.09	0.09	1.02	79.94*
G4 & 1000	±SD	0.30	1.92	2.69	2.34	4.15	1.42	1.58	2.94	0.00	0.00	0.30	0.30	1.01	34.01
	n	11	11	I 1	11	11	11	11	11	11	11	11	11	11	11

M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females)
*: Statistically significant (P<0.05) change than the vehicle control group

TABLE 7. SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group & Dose (mg/kg body weight)		Mean No. of Live Pups At Birth					During LD 1 to 4						Sex Ratio (M/F) at LD 4	No. of Survived Pups during LD 1 to 4	Pup Survival Index (%) LD 1 to 4
			Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)
			Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Un- determi ned (No.)	Male (No.)	Female (No.)	Total (No.)
	Mean	11.25	6.17	5.08	11.25	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.43	11.25	100.00
G1 & 0	±SD	2.34	1.59	2.07	2.34	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.73	2.34	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	12.09	6.82	5.09	11.91	0.09	0.09	0.18	0.00	0.00	0.00	0.00	1.47	11.91	98.26
G2 & 100	±SD	2.02	2.04	1.51	2.12	0.30	0.30	0.40	0.00	0.00	0.00	0.00	0.71	2.12	4.09
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	12.60	6.10	6.20	1230	0.20	0.00	0.20	0.00	0.00	0.10	0.10	1.18	12.30	97.91
G3 & 300	±SD	2.88	3.14	2.04	2.67	0.42	0.00	0.42	0.00	0.00	0.32	0.32	0.87	2.67	3.41
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	8.73	4.00	3.73	7.73	0.09	0.09	0.18	0.00	0.18	0.64	0.82	1.21	7.73	72.41*
G4 & 1000	±SD	4.15	2.83	2.65	4.90	0.30	0.30	0.40	0.00	0.60	1.50	2.09	1.01	4.90	44.06
	n	11	11	11	11	11	11	11	11	11	11	11	911	11	11

M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); LD: Lactation Day
*: Statistically significant (P<0.05) change than the vehicle control group

#: Two females from G4 were found with no live pups on LD4.

Note: The group mean number of live pups at birth: The mean number of live pups per litter at birth (a) was calculated first, then the group mean of mean number of pups per litter (b) was calculated using (a) to present the "Group mean number of live pups at birth".

TABLE 7 (Contd...). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group & Dose (mg/kg body weight)		Mean no. of Live Pups (No.) on LD 4			Pups Sacrificed for Blood Collection on LD 4 (No.)			Live Pups on LD4 after Blood Collection (No.)
Group & Dose (mg/kg body weight)		Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)
		Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)
	Mean	6.17	5.08	11.25	0.00	1.42	1.42	6.17	3.67	9.83
G1 & 0	±SD	1.59	2.07	2.34	0.00	0.90	0.90	1.59	1.72	1.64
	n	12	12	12	12	12	12	12	12	12
	Mean	6.82	5.09	11.91	0.00	1.45	1.45	6.82	3.64	10.45
G2 & 100	±SD	2.04	1.51	2.12	0.00	0.93	0.93	2.04	1.63	1.37
	n	11	11	11	11	11	11	11	11	11
	Mean	6.10	6.20	12.30	0.00	1.20	1.20	6.10	5.00	11.10
G3 & 300	±SD	3.14	2.04	2.67	0.00	0.92	0.92	3.14	2.16	1.85
	n	10	10	10	10	10	10	10	10	10
	Mean	4.00	3.73	7.73	0.00	0.64	0.64	4.00	3.09	7.09
G4 & 1000	±SD	2.83	2.65	4.90	0.00	0.92	0.92	2.83	2.17	4.37
	n	11	11	11	11	11	11	11	11	11
M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); NAD: No abnormality detected; LD: Lactation Day

Note: The group mean number of live pups on LD 4: The mean number of live pups per litter on LD 4 (a) was calculated first, then the group mean of mean number of pups per litter on LD 4 (b) was calculated using (a) to present the "Group mean number of live pups on LD 4".

TABLE 7 (Contd...). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group &						During LD 4 to 7							No. of	Pup Survival
Group &		Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)					No. of	Pup Survival
Dose (mg/kg body	Animal No.	Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibalized Pups per litter (No.)				Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
Dose (mg/kg body	Animal No.	Male	Female	Total	Male	Female	Total	Total	Total	Total	Total	Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
weight)		(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)		LD 4 to 7	LD 4 to 7

	Mean	6.17	3.67	9.83	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.16	9.83	100.00
G1 & 0	±SD	1.59	1.72	1.64	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.31	1.64	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	6.82	3.64	10.45	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.67	10.45	100.00
G2 & 100	±SD	2.04	1.63	1.37	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.40	1.37	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	6.10	5.00	11.10	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.87	11.10	100.00
G3 & 300	±SD	3.14	2.16	1.85	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.83	1.85	0.00
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	4.00	3.09	7.09	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.52	8.67	100.00
G4 & 1000	±SD	2.83	2.17	4.37	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.33	2.92	0.00
	n	11	11	11	11	11	11	11	11	11	11	9	9	9

M: Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); NAD: No abnormality detected; LD: Lactation Day

Note: The group mean number of live pups on LD 4: The mean number of live pups per liner on LD 4 (a) was calculated first, then the group mean of mean number of pups per litter on LD 4 (b) was calculated using (a) to present the "Group mean number of live pups on LD 4".


Note: The number of pups sacrificed for blood collection on LD 4 were excluded for the mean calculations during LD 4 to 7 and 7 to 13.

TABLE 7 (Contd...). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD
Group &		Live Pups (No.) on			During LD 7 to 13											No. of	Pup Survival
Group &		Live Pups (No.) on			Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)					No. of	Pup Survival
Dose (mg/kg body	Animal No.	LD 7			Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
Dose (mg/kg body	Animal No.	Male	Female	Total	Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total	Sex Ratio (M/F) at LD 7	Survived Pups during	Index (%) during
weight)		(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)	(No.)		LD 7 to 13	LD 7 to
																	13
	Mean	6.17	3.67	9.83	6.17	3.67	9.83	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.16	9.83	100.00
G1 & 0	±SD	1.59	1.72	1.64	1.59	1.72	1.64	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.31	1.64	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Mean	6.82	3.64	10.45	6.82	3.64	10.45	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.67	10.45	100.00
G2 & 100	±SD	2.04	1.63	1.37	2.04	1.63	1.37	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2.40	1.37	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
	Mean	6.10	5.00	11.10	6.10	5.00	11.10	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.87	11.10	100.00
G3 & 300	±SD	3.14	2.16	1.85	3.14	2.16	1.85	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.83	1.85	0.00
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Mean	4.89	3.78	8.67	4.89	3.67	8.56	0.00	0.00	0.00	0.11	0.00	0.00	0.11	1.71	8.56	88.89
G4 & 1000	±SD	2.26	1.72	2.92	2.26	1.94	3.24	0.00	0.00	0.00	0.33	0.00	0.00	0.33	1.29	3.24	33.33
	n	9	9	9	9	9	9	9	9	9	9	9	9	9	8^#	9	9

M:Male: F: Female; SD: Standard Deviation; n: Number of Animals (Females); LD: Lactation Day
N:Two females from G4 were found with no live pups on LD 1 to 4 and one female was found with no live pups during LD7 to 13.

Note: The group mean number of live pups on LD 7: The mean number of live pups per liner on LD 7 (a) was calculated first and then the group mean of mean number of pups per litter on LD 7 (b) was calculated using (a) to present the "Group mean number of live pups on LD 7".

TABLE 8. SUMMARY RECORD OF MEAN PUP WEIGHT (g) DURING LACTATION PERIOD
Group & Dose (mg/kg body weight)		Mean^#Pup Weight (g) on LDI		Mean^#Pup Weight (g) on LD 4		Mean^#Pup Weight (g) on LD 7		Mean^#Pup Weight (g) on LD 13
Group & Dose (mg/kg body weight)		Male	Female	Male	Female	Male	Female	Male	Female
	Mean	6.62	5.93	11.67	10.96	14.99	14.12	25.66	24.21
G1 & 0	±SD	0.24	0.23	0.31	0.28	0.37	0.31	0.92	0.67
	n	12	12	12	12	12	12	12	12
	Mean	6.58	6.19	11.48	10.84	14.43	13.97	24.78	23.78
G2 & 100	±SD	0.25	0.27	0.48	0.53	0.66	0.63	0.81	0.50
	n	11	11	11	11	11	11	11	11
	Mean	6.20*	5.72	9.94*	9.31*	13.60*	12.91*	22.88*	21.87*
G3 & 300	±SD	0.41	0.56	0.80	1.02	0.99	1.05	2.11	1.42
	n	10	10	10	10	10	10	10	10
	Mean	6.82	6.12	10.78*	9.84*	13.51*	12.29*	21.65*	20.61*
G4 & 1000	±SD	0.26	0.79	0.65	0.92	0.82	1.39	1.02	1.01
	n	9	11	8	9	8	9	8	8

SD: Standard Deviation; n: Number of Animals (Females); LD: Lactation Day
*: Statistically significant (P<0.05) change than the vehicle control group.
^#: The mean of all male and female pups per dam was calculated first and then group mean was calculated for all the dams. The presented value is group mean value

TABLE 9. SUMMARY OF THE STUDY
		Group & Dose (mg/kg body weight)
Parameters		G1 & 0	G2 & 100	G3 & 300	G4 & 1000
Pairs started (No.)		12 (1M + 1F)	12 (1M + 1F)	12 (1M + 1F)	12 (1M + 1F)
Females with Evidence of Copulation (No.)		12	12	12	12
Conceiving Days 1 to 5 (No.)		9	9	9	12
Conceiving Days ≥6 (No.)		3	3	3	0
Females achieving Pregnancy (No.)		12	11	10	11
Pregnancy ≤21 days (No.)		0	1	3	I
Pregnancy = 22 days (No.)		7	8	7	10
Pregnancy ≥23 days (No.)		5	2	0	0
Male Mating index (%)		100.00	100.00	100.00	100.00
Male Fertility Index (%)		100.00	91.67	83.33	91.67
Female Mating Index (%)		100.00	100.00	100.00	100.00
Females Fertility Index (%)		100.00	91.67	83.33	91.67
Parturition (%)		100.00	91.67	83.33	91.67
Dams with live young horn on LD 1 (No.)		12	11	10	11
Dams with live young on LD 4 (No.)		12	11	10	11
Dams with live young on LD 7 (No.)		12	11	10	11
Dams with live young on LD 13 (No.)		12	11	10	11
Implants/dam	Mean	11.25	12.09	13.40	10.45
Implants/dam	±SD	2.34	2.02	2.17	1.92
Pre-Implantation Loss (%)	Mean	0.00	0.00	0.00	0.00
Pre-Implantation Loss (%)	±SD	0.00	0.00	0.00	0.00
Post-Implantation Loss (%)	Mean	0.00	0.00	6.19	20.06
Post-Implantation Loss (%)	±SD	0.00	0.00	13.10	34.01
Live pups/dam at birth	Mean	11.25	12.09	12.60	8.73
Live pups/dam at birth	±SD	2.34	2.02	2.88	4.15
Live pups/dam at LD 4	Mean	11.25	11.91	12.30	7.73
Live pups/dam at LD 4	±SD	2.34	2.12	2.67	4.90
Sex ratio (m/f) at birth	Mean	1.43	1.46	1.17	1.02
Sex ratio (m/f) at birth	±SD	0.73	0.68	0.85	1.01
Sex ratio (m/f) at LD 4	Mean	1.43	1.47	1.18	1.21
Sex ratio (m/f) at LD 4	±SD	0.73	0.71	0.87	1.01
Male Pup weight at birth (g)	Mean	6.62	6.58	6.20	6.82
Male Pup weight at birth (g)	±SD	0.24	0.25	0.41	0.26
Female Pup weight at birth (g)	Mean	5.93	6.19	5.72	6.12
Female Pup weight at birth (g)	±SD	0.23	0.27	0.56	0.79
Male Pup weight (g) at the time of AGD measurement (LD 4)	Mean	11.67	11.48	9.94	10.78
Male Pup weight (g) at the time of AGD measurement (LD 4)	±SD	0.31	0.48	0.80	0.65
Female Pup weight (g) at the time of AGD measurement (LD 4)	Mean	10.96	10.84	9.31	9.84
Female Pup weight (g) at the time of AGD measurement (LD 4)	±SD	0.28	0.53	1.02	0.92
Male Pup weight LD 7 (g)	Mean	14.99	14.43	13.60	13.51
Male Pup weight LD 7 (g)	±SD	0.37	0.66	0.99	0.82
Female Pup weight LD 7 (g)	Mean	14.12	13.97	12.91	12.29
Female Pup weight LD 7 (g)	±SD	0.31	0.63	1.05	1.39
Male Pup weight LD 13 (g)	Mean	25.66	24.78	22.88	21.65
Male Pup weight LD 13 (g)	±SD	0.92	0.81	2.11	1.02
Female Pup weight LD 13 (g)	Mean	24.21	23.78	21.87	20.61
Female Pup weight LD 13 (g)	±SD	0.67	0.50	1.42	1.01
Male Pup AGD Ratio on the LD 4	Mean	2.29	2.28	2.25	2.27
Male Pup AGD Ratio on the LD 4	±SD	0.05	0.05	0.09	0.05
Female Pup AGO Ratio on the LD 4	Mean	0.91	0.92	0.95	0.95
Female Pup AGO Ratio on the LD 4	±SD	0.03	0.04	0.03	0.05
Male Pup Nipple Retention on LD 13	Mean	0.00	0.00	0.00	0.00
Male Pup Nipple Retention on LD 13	±SD	0.00	0.00	0.00	0.00
Male Scrum Thyroxine Hormone (T4) Levels (ng/mL)	Mean	80.566	84.355	83.872	82.829
Male Scrum Thyroxine Hormone (T4) Levels (ng/mL)	±SD	4.064	6.958	12.800	6.804
LD 13 Pup Serum Thyroxine Hormone (T4) Levels (ng/mL)	Mean	74.427	74.807	72.458	67.127
LD 13 Pup Serum Thyroxine Hormone (T4) Levels (ng/mL)	±SD	3.017	2.026	4.422	3.537
ABNORMAL PUPS

Dams with 0 (No.)		12	11	10	11
Dams with 1 (No.)		0	0	0	0
Dams with ≥2 (No.)		0	0	0	0
LOSS OF OFFSPRING
Pre-natal (implantations minus live births)
Females with 0 (No.)		12	11	10	11
Females with 1 (No.)		0	0	0	0
Females with 2 (No.)		0	0	0	0
Females with ≥ 3 (No.)		0	0	0	0
Post-natal loss during lactation period
Females with 0 pups (No.)		12	9	5	4
Females with 1 pup (No.)		0	2	3	4
Females with 2 pups (No.)		0	0	0	0
Females with ≥ 3 pups (No.)		0	0	2	3

No.: Number; SD: Standard Deviation; ≥: Greater than equal to;≤: Less than equal to; =: equal to; %: Percentage;
ng/mL: nano gram per milli liter; m/f: male/female; AGD: Ano-genital distance; LD: Lactation Day

TABLE 10. SUMMARY OF UTERI OBSERVATION RECORD
Group&Dose (mg/kg body weight)		No. of Corporalutea	No. of Implantations	Implantation Index (%)	Pre- Implantation Loss (%)	Post- Implantation Loss (%)	Pre-natal Loss (No.)	Post-natal Loss (At birth to LD 13) (%)	Post-natal Loss (At birth to LD 13) (No.)	No. of Early Resorptions	No. of Late Resorptions
	Mean	11.25	11.25	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
G1 & 0	±SD	2.34	2.34	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
		12	12	12	12	12	12	12	12	12	12
	Mean	12.09	12.09	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
G2 & 100	±SD	2.02	2.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
		11	11	11	11	11	11	11	11	11	11
	Mean	13.40*	13.40*	100.00	0.00	6.19	0.80	0.00	0.00	0.00	0.00
G3 & 300	±SD	2.17	2.17	0.00	0.00	13.10	1.69	0.00	0.00	0.00	0.00
		10	10	10	10	10	10	10	10	10	10
	Mean	10.45	10.45	100.00	0.00	20.06*	1.73*	27.27	0.91	0.00	0.00
G4 & 1000	±SD	1.92	1.92	0.00	0.00	34.01	2.90	46.71	2.39	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11

SD: Standard Deviation; n: Number of Animals (Females)
*: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 11. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g)
Group, Sex & Dose (mg/kg body weight)		Adrenals	Thymus	Spleen	Epididymis	Testes	Heart	Kidneys	Brain	Liver	Seminal vesicles with coagulating glands	Prostate	Thyroid along with para Thyroid^#
	Mean	0.0713	0.2317	0.7647	1.4503	3A266	1.5315	3.2600	2.2221	12.4578	1.1707	1.5446	0.0300
G1, M &	±SD	0.0072	0.0728	0.0251	0.0834	0.1983	0.0630	0.2829	0.1213	1.2105	0.1223	0.2312	0.0058
0
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0687	0.2370	0.6694	1.4054	3.5192	1.4584	3.2948	1.9582*	12.7096	1.2885	1.6538	0.0307
G2, M &	±SD	0.0072	0.0181	0.0721	0.0299	0.1183	0.1374	0.1119	0.1195	0.8988	0.1325	0.1603	0.0058
100
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0736	0.2497	0.7327	1.3987	3.4107	1.5027	3.2212	2.1377	13.0107	1.2355	1.5937	0.0305
G3,M &	±SD	0.0074	0.0542	0.0858	0.1347	0.1277	0.0545	0.1518	0.0540	0.8457	0.1650	0.2038	0.0047
300
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0738	0.3436	0.8034	1.4557	3.6099*	1.5936	3.5667	2.1674	13.2262	1.1669	1.5059	0.0306
G4, M &	±SD	0.0093	0.1033	0.0965	0.0555	0.2536	0.1800	0.2385	0.0776	1.1703	0.0616	0.1868	0.0036
1000
	n	5	5	5	12	12	5	5	5	5	12	12	12
	Mean	0.0798	0.3982	0.7043	1.4680	3.8296	1.5621	3.4492	2.1940	13.4046	1.3151	1.8776	0.0303
G1R, M	±SD	0.0271	0.0667	0.1032	0.1436	0.2945	0.2125	0.2566	0.2091	0.3531	0.2166	0.1603	0.0027
&0
	n	5	5	5	5	5	5	5	5	5	5	5	5
	Mean	0.0786	0.3841	0.7040	1.4336	3.4943	1.6005	3.5935	2.2144	13.8807	1.2083	1.6840*	0.0287
G4R, M	±SD	0.0157	0.0484	0.0457	0.0762	0.1706	0.1084	0.2094	0.0757	0.8751	0.1330	0.0575	0.0050
& 1000
	n	5	5	5	5	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (Females); #: Weighed post fixation
*: Statistically significant (P<0.05) change than the vehicle control group

TABLE 11 (Contd...). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g)
Group, Sex & Dose (mg/kg body weight)		Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Thyroid along with para Thyroid^#
	Mean	0.1009	0.2834	0.6195	1.3311	2.6962	2.0879	16.3846	0.0310
G1, F &	±SD	0.0198	0.0522	0.1368	0.0744	0.2709	0.0384	1.5298	0.0063
0
		5	5	5	5	5	5	5	12
	Mean	0.0961	0.2881	0.6405	1.1601*	2.6774	2.1819	14.3037*	0.0308
G2, F &	±SD	0.0054	0.0930	0.0969	0.0817	0.2466	0.0266	0.7461	0.0049
100
		5	5	5	5	5	5	5	12
	Mean	0.0937	0.3049	0.7649	1.2065	2.7635	2.1132	16.9891	0.0302
G3, F &	±SD	0.0149	0.0946	0.0725	0.0876	0.1476	0.0502	1.0569	0.0033
300
		5	5	5	5	5	5	5	12
	Mean	0.1032	0.2809	0.6447	1.1626*	2.6732	2.1717	15.8345	0.0317
G4, F &	±SD	0.0128	0.0952	0.2021	0.1021	0.1996	0.1646	1.2621	0.0048
1000
	n	5	5	5	5	5	5	5	12
	Mean	0.0928	0.3907	0.5817	1.1948	2.3005	2.1197	8.9081	0.0289
G1R, F &	±SD	0.0145	0.0527	0.0180	0.1490	0.1729	0.0663	0.7555	0.0035
0
	n	5	5	5	5	5	5	5	5
	Mean	0.1017	0.4139	0.5713	1.1680	2.2110	2.0927	8.9462	0.0279
G4R, F &	±SD	0.0147	0.0418	0.0599	0.0705	0.1374	0.0531	1.0503	0.0033
1000
	n	5	5	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (Females); #: Weighed post fixation
*: Statistically significant (P<0.05) change than the vehicle control group

Applicant's summary and conclusion

Conclusions:

Executive summary:

The objective of this GLP compliant OECD 422 study was to provide data on the possible effects of the test substance Dihydro Isojamonate on general toxicity, reproductive performance and development of pups. The test substance was suspended in corn oil and administered by oral gavage at 0, 100, 300 and 1000 mg/kg body weight/day. The test substance was administered during a pre-mating period of 2 weeks, during mating, gestation and lactation. Male animals were sacrificed after the mating period after completion of 36 days of treatment. Pups were sacrificed at day 13 of lactation. Parental female animals were sacrificed at day 14 of lactation.

In the absence of adverse parental systemic effects, fertility parameters and reproductive performance, the NOAEL for parental and reproduction was placed at 1000 mg/kg (the highest concentration tested).Based on the effects on post-implantation and post-natal loss, live birth index and pup survival index, the NOAEL for developmental effects was placed at 300 mg/kg (mid concentration).

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Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Toxicity to reproduction

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Results: F1 generation

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