N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

To summarize, daily oral (gavage) administration of test item “Hostavin 3206 LIQ (Impoverished Xylene)” to Wistar rats at the dose levels 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, body weights, food intake, pre-coital time, gestation length, mating and fertility parameters. Functional observations did not reveal any test item related changes at all the tested doses. The survival indices were not altered by the treatment. The test item administration did not reveal any changes in the hematology, coagulation and clinical chemistry parameters. There were no test- item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. Gross examination of pups on LD 13 did not reveal any gross changes. There were no microscopic changes observed in both males and females. Further, the male and female reproductive organs did not reveal any changes. The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration. No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of parental rats and pups. The No Observed Adverse Effect Level (NOAEL) of is considered to be 1000 mg/kg Bwt/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Department of Safety Assessment Advinus Therapeutics Limited Bengaluru 560 058, India

- Age at study initiation: 14-16 weeks
- Weight at study initiation: Males: 394 to 526 g Females: 231 to 282 g

- Fasting period before study:
- Housing:
- Use of restrainers for preventing ingestion (if dermal): yes

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, Wi 53744-4420 was provided ad libitum to the animals.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limted., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment. During the acclimatization period, animals were observed once daily for any abnormalities. Only the animals that are determined by the veterinarian to suitable for use were assigned to this study. Female rats used in this study were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 and 24°C and relative humidity between 59 and 68 %. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12 - 15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

- Temperature (°C): 21 and 24°C.
- Humidity (%): 59 and 68 %.
- Air changes (per hr):12 - 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 29 March 2016 To: 04 July 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily prior to start of the treatment.
Required quantities of the test item were weighed in to a pre-calibrated beaker* and small volume of corn oil was added. The resulting pre-mix was warmed. This pre-mix was allowed to thaw to room temperature and the volume was made up with the vehicle to attain desired concentrations of 20, 60 and 200 mg/mL for the G2, G3 and G4/G4R groups, respectively.

Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated cylinder to the final volume of the batch size (70 mL). The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water was marked on the beaker using a marker. Once these lines were marked, the water was discarded and the beaker was dried. The volume was made up to the upper meniscus when preparing the dose formulations.

Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring using a magnetic stirrer.

The volume of dose formulation prepared was varied depending on the requirement and/or body weights of the rats recorded during experimental period.


VEHICLE
Corn oil was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study (Study No. N2825) with the same test item.

Details of components used for vehicle preparation are as follows:

- Name of vehicle: Corn oil
- Manu-factured by: Sigma
- Lot no.: MKBV2080V
- Date of receipt: 19.10.2015
- Date of expiry: 18.10.2020



Name of vehicle Manu-factured by Lot no. Date of receipt Date of expiry
Corn oil Sigma MKBV2080V 19.10.2015 18.10.2020

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there is evidence of sperms in the vaginal smear and /or vaginal plug or for a maximum period of 2 weeks. Subsequently, pregnant females were housed individually until LD 14. The females not mated within 14 days of pairing with the first male was placed with a second proven male of the same group and the presence of sperm in the vaginal smear and/or vaginal plug is not confirmed within 7 days, that female was sacrificed 25 days after last day of mating period. All the mated females were maintained till they litter or for a maximum period of at least 25 days from the last day of cohabitation. Not littered females were sacrificed after 25 days from the day they are found of sperm positive (by vaginal smear examination).

The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulation Analysis
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and once during Week 4 of the treatment and were analysed in-house. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control, duplicate samples from only middle layer was drawn.

The analysis was done as per the method validated under Advinus Study No. G11306. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient conditions for reanalysis purpose as a backup and the second set of samples were discarded, as the analysis results of first set of samples were within the limits.
Formulations were considered acceptable as mean results are within ± 15 % of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.

Duration of treatment / exposure:

Treatment
Males: The dose formulation was administered to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating period and during the post mating period until sacrifice.
Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered for each rat was 5 mL/kg Bwt throughout the study except for rat number Rt3330 (G2F) wherein 5.1 mL was administered from experimental Day 22 to 28. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 5 mL/kg Bwt.
The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.

Frequency of treatment:

Males: The dose formulation was administered to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating period and during the post mating period until sacrifice.

Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered for each rat was 5 mL/kg Bwt throughout the study except for rat number Rt3330 (G2F) wherein 5.1 mL was administered from experimental Day 22 to 28. The dose volume was adjusted based on the most recent body weight of individual rat.

Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 5 mL/kg Bwt.

The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.

Details on study schedule:

Study initiation date: 08 March 2016

Experimental starting date: 10 March 2016

Acclimatization: Start: 10 March 2016
End: 14 March 2016

Pre-treatment period: Start: 15 March 2016
End: 28 March 2016

Treatment: Start: 29 March 2016
End: 11 June 2016

Recovery period: 20 May 2016 to 02 June 2016

Experiment completion: 7 July 2016

Submission of Draft report: 29 August 2016

Study completion: 21 November 2016

Remarks:

Doses / Concentrations:
G1 & G1R '0' mg/kg Bwt/day
Basis:
nominal conc.
0

Remarks:

Doses / Concentrations:
G2 '100' mg/kg Bwt/day
Basis:
nominal conc.
20

Remarks:

Doses / Concentrations:
G3 '300' mg/kg Bwt/day
Basis:
nominal conc.
60

Remarks:

Doses / Concentrations:
G4 & G4R '1000' mg/kg Bwt/day
Basis:
nominal conc.
200

No. of animals per sex per dose:

10 Males and 10 Females per each main groups. 5 Males and 5 Females for recovery group.

Control animals:

yes

Details on study design:

- Dose selection rationale: G1 '0' mg/kg Bwt/day; G2 '100' mg/kg Bwt/day; G3 '300' mg/kg Bwt/day; G4 '1000' mg/kg Bwt/day; G1R '0' mg/kg Bwt/day; G4R '1000' mg/kg Bwt/day

- Rationale for animal assignment : Grouping was done by the method of body weight stratification and distribution. On the day of randomization, based on the given temporary animal identification number, each animal with normal oestrous cyclicity (4-5 day cycle) was weighed and the corresponding body weight was recorded. The data was transferred to EDP (Electronic Data Processing) for data input (temporary identification number and body weight) into an excel spread sheet. The body weight recorded was stratified in ascending order.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs, the observation for morbidity and mortality was carried out once in the morning during holidays. All rats were observed for clinical signs once daily during the treatment and recovery periods.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment and recovery periods.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards.

On the days of detailed clinical examination, general clinical signs were not performed except on treatment Day 1.


BODY WEIGHT:
i) Individual body weights of males were recorded initially and at weekly intervals thereafter. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on GD 0, 7, 14 and 20 and on LD 0, 4 and 13.


FOOD CONSUMPTION AND COMPOUND INTAKE : Cagewise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of lactation period.

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating period to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. The overall pattern of each female was characterized as regular cycling (having recurring 4–5 day cycles) and irregularly cycling (having cycles with a period of diestrus longer than 3 days or a period of estrus more or less than 2 days). Incomplete cycles (having prolonged periods of either estrus or diestrus) were not included in calculating the mean cycle length.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.

b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0, 4 and 13 were recorded.

c. On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Blood samples were collected from two of the surplus pups, pooled, and used for determination of serum T4 levels. In case of insufficient pups in a litter to have two surplus pups, two of the pups that were typically retained was used for blood collection for serum T4 assessments. When litter size dropped below the culling target (8 pups/litter), preference was given to remove two female pups for PND 4 blood samples in order to retain more male pups for observation of nipple retention on PND 13.

d. After standardization, the individual pup body weight was recorded on Day 13 of lactation.

e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from cube root of body weight.

f. The number of nipples/areolae in male pups were counted on PND 13.

g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.

h. Fertility indices for dams, sires as well as the pup survival index till LD 4 were calculated.

Postmortem examinations (parental animals):

Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland collected from LD 13 pups from all the groups were also evaluated. All gross lesions were examined from all the groups.

Postmortem examinations (offspring):

All pups LD 13; including dead pups were examined macroscopically for any structural abnormalities/pathological changes and findings were recorded.

Statistics:

ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data was entered retrospectively in ProvantisTM) and Clinical Chemistry data was analysed using Provantis built-in statistical tests.

The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights), body weight, net weight gain, food consumption, oestrous cycle length, hormone levels, ano-genital distance, body weights, ano-genital index, mean number and weight of pups, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant.

In case of recovery groups, data was analysed using Two sample t-test. Comparison of means between treatment recovery group(s) and vehicle control recovery group was performed.

Post-implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (Days) was analysed after suitable transformation (√ x + ½) of the data. One-way ANOVA was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.

Z test was performed for testing the differences in proportions for Day 4 survival index, mating and fertility indices.
All analyses and comparisons were evaluated at the 5% (P<0.05) level.

Reproductive indices:

9.1 Reproductive Performance Data of Parents

a. Male mating index (%)

Number of males with evidence of mating
----------------------------------------- x 100
Number of males cohabited

b. Male fertility index (%)

Number of males siring a litter
=----------------------------------------- x 100
Number of males cohabited

c. Female mating index (%)

Number of females mated
=-------------------------------- x 100
Number of females cohabited

d. Female fertility index (%)

Number of pregnant females (confirmed at necropsy)
= ----------------------------------------------- x 100
Number of females used for mating

e. Mean number of implantations/group

Total number of implantations
= ----------------------------
Total number of pregnant animals

f. Post implantation loss (%)

Number of implantations - Number of live pups
= ------------------------------------------------ x 100
Number of implantations

Offspring viability indices:

a. Mean litter size per group

Total Number of pups
= -------------------------------------------------
Total Number of littered animals

b. Mean viable litter size

No. of viable pups on Day 1
= -----------------------------------------
No. of females littered

c. Live birth index (%)

No. of viable pups born (at first observation)
= ----------------------------------------------------------x 100
Total no. of pups born (at first observation)


d. Day 4 survival index (%)

Number of viable pups on lactation Day 4
= ----------------------------------------------------- x 100
Number of viable pups born

e. Sex Ratio (%)

No. of male pups born
= -------------------------------- x 100
Total No. of pups born

f. Ano-genital Distance Ratio (mm/g1/3 )

Ano-genital distance
= --------------------------------
Cube root of body weight

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one female rat (Rt3272) in the vehicle control group was died after blood
sample collection during pre-mating period, could be due to over anaesthesia

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

11. RESULTS AND DISCUSSION
Details of experimental design, treatment, clinical pathology investigations and sacrifice schedule are presented in Table 1.

11.1 In-Life Data

11.1.1 Detailed Clinical Examination, Clinical Signs, Morbidity and Mortality

Refer Table 2, Appendices 1, 2 and 15

No treatment-related clinical signs were observed at any of the doses tested. However, the clinical sign of sparse hair loss was observed in one male rat in the control recovery and three female rats in the high dose recovery groups. The observation was considered as spontaneous finding and not related to treatment.

No mortality was observed at any of the doses tested. However, an incidence of one female rat (Rt3272) in the vehicle control group was died after blood sample collection during pre-mating period, could be due to over anaesthesia.

There were no abnormalities observed in pups.

11.1.2 Functional Observation Battery

Refer Tables 3 and 4, Appendices 3, 4 and 5

Home cage and Handling observations: No treatment-related abnormalities were observed in all the tested dose groups in both sexes
Open field observations: No treatment-related abnormalities were observed in any of the doses tested in both sexes.

Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.

Motor Activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:

Males:
Lower: Stereotypic time at interval 1 at the low, mid and high doses, interval 2 and 3 at the mid and high doses and total stereotypic time at the mid and high doses.

Higher: Ambulatory time at interval 1 at the mid, high and high dose recovery, interval 2 at the high dose and total ambulatory time at the mid, high and high dose recovery, horizontal counts at interval 1 and total horizontal counts at the high dose recovery, ambulatory counts at interval 1 and total ambulatory counts at the high dose recovery.

Females:
Higher: Ambulatory time at interval 1 and total ambulatory time at the high dose recovery.

The above observed statistical variations in the motor activity measurement were considered to be incidental as the observed changes did not follow the dose proportion and there were no changes observed in the home cage or open field observations.
Neuromuscular observation:

Landing hind limb footsplay: No significant changes were observed at all the tested doses in both sexes. However, an incidence of significantly lower hind limb footsplay was observed in high dose recovery females. This change was considered toxicologically significant as the similar change was not observed in high dose main toxicity group.

Grip strength: No significant changes were observed at all the tested doses in both sexes.

Physiological observation:
Body temperature: The physiological observation of body temperature was unaffected in both sexes. . However, an incidence of significantly lower body temperature was observed in high dose recovery females. This change was considered toxicologically significant as were no changes observed in the home cage or open field observations.

The body weights recorded at the end of functional tests were comparable with the control group.

11.1.3 Body Weights and Net Weight Gains

Refer Tables 5 and 6, Appendices 6 and 7

The mean body weights and net weight gains unaffected by the treatment at all the doses tested when compared to vehicle control in both sexes. In the high dose recovery group, the body weights were unaffected both during the treatment and recovery period.

Thus, the treatment did not affect the mean body weights at all the tested doses in either sex when compared to vehicle control.

11.1.4 Food Consumption

Refer Tables 7 and 8, Appendix 8 and 9
The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control. However, an incidence of significantly lower food intake on weeks 1, 2 and 6 at 100 mg/kg Bwt/day and on week 1 at 1000 mg/kg Bwt/day. This reduction in food intake was toxicologically insignificant as the mean body weights were not affected. In the high dose recovery group, the food consumption was not affected both during the treatment and recovery period.

11.1.5 Oestrous Cycle Evaluation

Refer to Table 9 and Appendix 10

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation).

The calculated mean oestrous cycle length was 4.04, 4.00, 4.00 and 4.01 days in vehicle control, 100, 300 and 1000 mg/kg Bwt/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

11.1.6 Maternal Body Weights, Weight Change and Food Intake during the Gestation Period

Refer to Tables 10 and 11 and Appendices 11 and 12

Treatment had no effects on the body weights and food intake during different intervals of the gestation period at all the doses tested.

11.1.7 Maternal Body Weights, Weight Change and Food Intake during the Lactation Period

Refer to Tables 12 and 13 and Appendices 13 and 14

Treatment had no effects on the body weights and food intake during different intervals of the lactation period at all the doses tested when compared to vehicle control.


11.1.12 Fertility Index

Refer to Table 18 and Appendices 18 to 21

There were no treatment-related effects on the mean pre-coital time and gestation length at all the tested doses.

No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.

Incidences of higher male mating and fertility index at the high dose, female mating index at all the treated groups and female fertility index at the low and high dose groups were observed. This significance differences could be due to one male rat (Rt3268) which did not impregnate female and one female rat (Rt3272) died in the vehicle control and hence these findings were considered incidental.

11.1.13 Uterine/Implantation Data

Refer to Table 18, Appendices 18 to 21

No treatment-related effects were observed with respect to implantations, and percentage of post implantation loss when compared to control means.

11.1.14 Oestrous Cycle at prior to sacrifice

Refer to Table 19, Appendix 22

Oestrous cycle measured for all female rats prior to sacrifice and the evaluated oestrous cycle were comparable to control group.

11.2 Pathology

For detailed pathology report refer to Appendix 23

Oral administration of Hostavin 3206 LIQ (Impoverished with Xylene) in Wistar rats did not affect the hematology, coagulation, clinical chemistry, terminal fasting body weights/organ weights, gross and histopathology endpoints in adult rats of both the sexes. Further, the male and female reproductive organs did not reveal any changes.

The pups sacrificed on PND 13 did not reveal any gross abnormality.

The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration. No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of parental rats and pups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Generation not specified (migrated information)

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

11.1.8 Mean Number and Mean Body Weight of Pups during the Lactation Period

Refer to Tables 16 and 17 and Appendices 18 to 20

The mean number and body weight of male and female (and total number) pups per litter were not affected by the treatment at all the doses tested.

11.1.9 Ano-genital Distance (AGD)

Refer to Table 14 and Appendix 16

No changes attributable to test item were detected in the AGD, body weight and ratio of AGD to the cube root of body weight of either sex. However, an incidence of significantly higher mean weight of male pups on the day of ano-genital distance measured was observed in all the treated groups. The ratio of AGD to the cube root of body weight was significantly lower at 100 mg/kg Bwt/day. These significant differences were considered to be toxicologically insignificant as the mean ano-genital distance was not affected and the decreased ratio of AGD was not in dose dependent.

11.1.10 Areolae/Nipple Retention in Pups

Refer to Table 15 and Appendix 17

The male pups did not exhibit areola/nipple retention on PND 13.

11.1.11 Survival Data of Pups

Refer to Table 18 and Appendices 18 to 21

Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter size, and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.

No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

no

Reproductive effects observed:

not specified

Key result

Reproductive effects observed:

no

1.1.1             Clinical Chemistry

Plasma was separated following centrifugation of whole blood at 5000 rpm at 4° C for 5 minutes and analysed using Dimension RxL MaX Clinical Chemistry System (Dade Behring Inc. Newark, DE 19714, USA) Automatic Analyser for the following parameters:

Serial No.	Parameters	Abbreviations	Units
1	Alanine Aminotransferase	ALT	U/L
2	Albumin	Alb	g/L
3	Alkaline Phosphatase	ALP	U/L
4	Aspartate Aminotransferase	AST	U/L
5	Albumin/Globulin ratio [calculated values]	A/G Ratio	-
6	Blood urea nitrogen	BUN	mmol/L
7	Total Bile acids	TBA	µmol/L
8	Calcium	Ca	mmol/L
9	Chloride	Cl	mEq/L
10	Creatinine	Creat	µmol/L
11	Gamma Glutamyl Transferase	GGT	U/L
12	Glucose	Glu	mmol/L
13	Globulin [calculated values]	Glob	g/L
14	Inorganic phosphorous	Pi	mmol/L
15	Potassium	K	mEq/L
16	Sodium	Na	mEq/L
17	Total bilirubin	T.Bil	µmol/L
18	Total cholesterol	T.Chol	mmol/L
19	Total plasma protein	T.Pro	g/L
20	Triglycerides	Trig	mmol/L
Note:  Direct and indirect bilirubin were not determined as the total bilirubin levels           were less than 10 µmol/L

1.1.2             Urinalysis

Urine was collected at the end of the pre-mating period, from randomly selected 5 males and 5 females of main groups and at the end of recovery period from all rats, in urine collection tubes. For urine collection, each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis.

Urine samples were analysed for the following parameters:

Serial No.	Parameter
1	Specific gravity^1
2	Nitrite^2
3	pH^2
4	Proteins^2
5	Glucose^2
6	Ketone bodies^2
7	Urobilinogen^2
8	Bilirubin^2
9	Leukocytes^2
10	Erythrocytes^2
11	Appearance (colour and clarity)^3
12	Volume^3
^1: Measured by refractometry ^2: Analyzed with Clinitek status analyzer using Multistix 10 SG strips (Bayer Healthcare LLC,) ^3: Recorded manually

 

1.1.3             Hormone Analysis

Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:

·        Two pups per litter on LD 4 after birth

·        All dams and at least two pups per litter on LD 13

·        All adult males, prior to sacrifice.

 

The blood from pups was pooled by litter for thyroid hormone analyses.

Pups were lightly anaesthetized with isoflurane and incised at jugular vein in the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labeled tubes and kept on bench top for 15-20 min before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4° C. The serum samples were placed in labeled plastic tubes and stored at ~ -70 °C until they were analyzed

1.1.4             Thyroid Profile Hormones

The following thyroid hormones were estimated by Enzyme linked immuno sorbent assay (ELISA) method for the samples as stated using BIO-RAD microplate washer and BIO-RAD model 680 reader.

Serial No.	Parameters	Abbreviations	Units^c
1	Rodent Thyroid Stimulating Hormone	TSH	ng/mL
2	Rodent Thyroxin	T4	ng/mL
^c: expansion of unit: ng/mL: nano grams/ per milli litre

 

The kits manufactured by Endocrine Technologies Inc.,were used for the assay.

Conclusions:

To summarize, daily oral (gavage) administration of test item “Hostavin 3206 LIQ (Impoverished Xylene)” to Wistar rats at the dose levels 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, body weights, food intake, pre-coital time, gestation length, mating and fertility parameters. Functional observations did not reveal any test item related changes at all the tested doses. The survival indices were not altered by the treatment. The test item administration did not reveal any changes in the hematology, coagulation and clinical chemistry parameters. There were no test- item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. Gross examination of pups on LD 13 did not reveal any gross changes. There were no microscopic changes observed in both males and females. Further, the male and female reproductive organs did not reveal any changes. The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration. No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of parental rats and pups. The No Observed Adverse Effect Level (NOAEL) of is considered to be 1000 mg/kg Bwt/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item Hostavin 3206 LIQ (Impoverished of Xylene) was suspended in vehicle (corn oil) and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg Bwt/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. A concurrent control (G1) and a control recovery group (G1R) of rats received vehicle alone. The dose volume administered was 5 mL/kg Bwt/day. The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 (for females). In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams.

The identity of Hostavin 3206 LIQ (Impoverished of Xylene) was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No.G11306. Based on the results, the test item was found to be stable for up to 24 hours at 2 and 200 mg/mL concentrations, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during Week 4 of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the nominal concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period.Female rats were also weighed onGestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were examined for the appearance nipples/areolae on post-natal day (PND) 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group. For recovery groups, functional observation battery was done prior to sacrifice. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. Throxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams at termination on LD 14 and two pups per litter on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland collected from LD 13 pups from all the groups was also evaluated. All gross lesions were examined from all the groups.

Under the experimental conditions employed, the following results were obtained:

• There were no treatment-related clinical signs or mortality observed at any of the doses tested.
• No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.
• The body weights and food consumption were unaffected by the treatment at all the doses tested.
• The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses.
• Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses.
• No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.
• The survival indices were not altered by the treatment at all the doses tested.
• There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size.
• There were no external abnormalities in live or dead pups in any of the groups.
• No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group.
• The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.
• The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females
• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. Gross examination of pups on LD 13 did not reveal any gross changes.
• There were no test item- related gross and microscopic changes in both males and females.
• The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration.
• No test item-related changes were observed in organ weights, gross and histopathology of thyroid gland of parental rats and pups.

No Observed Adverse Effect Level

Daily oral (gavage) administration of Hostavin 3206 LIQ (Impoverished with Xylene) to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects on fertility and reproductive performance. The No Observed Adverse Effect Level (NOAEL) of Hostavin 3206 LIQ (Impoverished with Xylene) for systemic and reproductive toxicity in parental rats is considered to be 1000 mg/kg Bwt/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

not mandatory

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive toxicity is appropriate.

With reference to the OECD 422 studies performed with the registration substance, it is concluded that the test item is not subject to classification and labelling according to Regulation 1272/2008/EC regarding reproductive toxicity.