Decyldimethylamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 APRIL 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline 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)

Principles of method if other than guideline:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 50 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).

Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

GLP compliance:

yes

Specific details on test material used for the study:

Test item : Genamin 10R 302 D

CAS No. : 1120-24-7

Chemical name (IUPAC) : N,N-dimethyl-decan-1-amine

Physical Appearance : Clear, colourless liquid

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19.2 to 24.3°C and relative humidity between 60 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.6– 12.9 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

Route of administration:

oral: gavage

Details on route of administration:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.

Vehicle:

corn oil

Details on oral exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
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 to each rat was at an equivolume of 5 mL/kg body weight throughout the study. 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 an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

Duration of treatment / exposure:

Daily

Frequency of treatment:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
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 to each rat was at an equivolume of 5 mL/kg body weight throughout the study. 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 an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups: 10/sex/dose
Recovery groups: 5/sex/dose

Control animals:

yes

Details on study design:

Group No. Group Colour of
cage card Dose
(mg/kg/day) Concent-ration
(mg/mL) Dose volume
(mL/kg) No. of
Rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle control White 0 0 5 10
10 M
F Rab4141
Rab4151 Rab4150
Rab4160
G2 Low dose Yellow 25 5 5 10
10 M
F Rab4161
Rab4171 Rab4170
Rab4180
G3 Mid dose Green 50 10 5 10
10 M
F Rab4181
Rab4191 Rab4190
Rab4200
G4 Mid-intermediate Pink 100 20 5 10
10 M
F Rab4201
Rab4211 Rab4210
Rab4220
G5 High Red 150 30 5 10
10 M
F Rab4221
Rab4231 Rab4230
Rab4240
Recovery Groups
G1R Vehicle control recovery White 0 0 5 5
5 M
F Rab4241
Rab4246 Rab4245
Rab4250
G5R High dose recovery Red 150 30 5 5
5 M
F Rab4251
Rab4256 Rab4255
Rab4260
M: Male; F: Female; Rab: Prefix code for rat numbers
Note: Females were selected with typical 4-5 days oestrous cycles.

Observations and examinations performed and frequency:

10.1 Clinical Signs, Morbidity and Mortality
All rats were observed for morbidity and mortality twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays.
10.1 Clinical signs
All rats were observed for clinical signs once daily during the treatment and recovery period. Frequent observations were performed (twice) due to clinical sign of salivation. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
10.2 Detailed Clinical Examination
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period.
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).
10.3 Functional Observation Battery Tests (FOB)
The following neurological examinations were performed on LD 13 for randomly selected 5 main group parental females, on Day 50 for randomly selected 5 main group parental males and at the end of the recovery period (Day 64) for all the recovery group animals.
10.3.1 Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
10.3.2 Observations during Removal of Animal from Home Cage and Handling
The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.
10.3.3 Open Field Observation
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, the rat was evaluated as it moved about freely/unperturbed and the following observations were made and was recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- bizarre behaviour
- urination
- defecation
- rearing
- abnormal vocalizations
10.3.4 Functional Tests
Functional testing included motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
10.3.4.1 Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, distance travelled in inches and resting time in seconds were monitored. The Opto-Varimex 5 motor activity measurement system provided the data analysed at 10 minutes interval and the same was reported.
10.3.4.2 Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
- approach response
- touch response
- click response
- tail-pinch response
- pupil response
- aerial righting reflex

10.3.4.3 Landing Hindlimbs Footsplay
The landing hindlimbs footsplay was assessed by dropping the rat on to a horizontal surface of the table top from a short height and measured the distance between the hind feet upon landing. The heel portion of each hind foot of each rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on a SOP format, which contained the details such as study no., animal no, group, sex and date.
A clean recording SOP format was used for each rat. A total of 3 readings (Trial 1, Trial 2 and Trial 3) were recorded for each rat and average of 3 footsplay values are presented in the report along with the individual footsplay values.
10.3.4.4 Grip Performance
Hindlimbs and forelimbs grip performance was tested using dual grip strength meter (Model: Columbus Instruments). Three trials (Trial 1, Trial 2 and Trial 3) were conducted for each rat i.e., three trials each for forelimbs and hindlimbs. Average of three trials for both forelimbs and hindlimbs were calculated and presented in the report along with the individual grip strength values.
10.3.5 Physiological Observations
Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer.
At the end of the functional test, body weight of each rat was measured.
10.4 Body Weights
i) Individual body weight of males were recorded on Day 1 and at weekly (±1 Day) intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.

10.5 Food Consumption
Food consumption (g) was measured at weekly intervals (±1 Day) during treatment and recovery period.
Food consumption was not measured during the cohabitation period.

Sacrifice and pathology:

10.10.1 Necropsy
All adult animals and pups were subjected to detailed necropsy and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anesthesia. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals which were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death.
For apparently non-pregnant rats, the uteri were stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of embryos (by staining the implantation sites) for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.
10.10.2 Tissue Collection
On completion of the gross pathology examination, the tissues/organs listed in the below table were collected and preserved in 10% neutral buffered formalin from all adults (parents) including recovery groups animals unless otherwise specified. The organ weights as percentage of body weights were determined and presented in the report.
Tissue/organ Organ
Weighing Collection and
Preservation Microscopic Examination
Gross Lesion/ Mass/ Nodule X X
Cervix X X
Epididymis X X X
Ovary X X X
Oviduct X X
Uterus1 X X X
Vagina X X
Gland, Prostate2 X X X
Gland, Seminal vesicle/ Gland, Coagulating2 X X X
Testis3 X X X
Gland, Thyroid/ Gland, Parathroid4 X X X
Muscle, Levator ani/ Bulbospongiosus X X
Gland, Bulbourethral X X
Penis, Glans X X
1: Weighed along with cervix
2: Prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands
3: Collected in modified Davidson’s fluid
4: Weighed after fixation
X: Activity performed.
In addition, the following tissues were collected, weighed and preserved from 5 males and 5 females randomly selected from the main groups and all animals from the recovery groups. Organ weights as percentage of body weights were determined and are presented in the report.
Tissue/organ Organ
Weighing Collection and
Preservation Microscopic Examination
Brain (cerebrum, cerebellum, medulla oblongata and pons) X X X
Bone marrow, Femur5 X X
Bone, Femur/ Joint, Femorotibial9 X X
Bone marrow, Sternum X X
Bone, Sternum 9 X X
Large intestine, Cecum X X
Large intestine, Colon X X
Small intestine, Duodenum X X
Eye6 X X
Muscle, Skeletal X X
Gland, Adrenal X X X
Gland, Mammary X X
Gut-associated lymphoid tissue X X
Heart X X X
Small intestine, Ileum X X
Small intestine, Jejunum X X
Kidney X X X
Liver X X X
Lung7 X X
Lymph node, Mandibular X X
Lymph node, Mesenteric X X
Gland, Pituitary8 X X X
Large intestine, Rectum X X
Nerve, Sciatic X X
Spinal cord X X
Spleen X X X
Stomach X X
Thymus X X X
Trachea X X
Urinary bladder7 X X
X: Activity performed.
5: Prepared from femur marrow and stained using Giemsa stain.
6: Collected in Davidson’s fluid
7: Inflated with 10 % Neutral Buffered Formalin before immersion in fixative
8: Weighed after formalin fixation
9 : Decalcified prior to sectioning
On LD 13, thyroid gland was collected from available one randomly selected male and female pup from each litter and preserved in 10% NBF for the histopathological examination. The thyroid weighing for the pups was performed after fixation.
10.10.3 Histopathology
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 the testes also involved qualitative assessment of the stages of spermatogenesis.
The reproductive organs (including thyroid/parathyroid) were not suspected of showing test item related changes in high dose group and hence the reproductive tissues were not examined in the remaining 5 animals of control and high dose groups and also in all respective lower dose group rats and recovery dose group rats.
Further, liver in males was suspected of showing test item related histopathological changes in the high dose group (G5) and were also examined in the five randomly selected lower dose group rats and all recovery group rats.
The reproductive organs of non-pregnant animals were also examined in all the dose groups.
All gross lesions were examined in all the groups.
The tissues were processed for routine paraffin embedding and 4 – 5 micron thickness sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, oestrous cycle, pre-coital interval , ano-genital distance, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (motor activity, thyroid profile) data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like mating and fertility indices were analysed using Chi-square test. When Chi-square is found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
For two groups, the comparisons of mean between treatment and control group was done using student’s t-test.
Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
There was no mortality observed at any of the doses tested. There were no abnormalities observed in pups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect on the mean fasting body weights and reproductive organ weights of adult rats at all the dose levels compared to their control counter parts in both the sexes. There were no significant changes in thyroid weight of pups on lactation Day 13. Administration of test item resulted in higher liver weight in parental males at all dose levels and was associated with hepatocyte hypertrophy, histologically at dose level of ≥ 100 mg/kg/day. However, the weight increase was not dose related. The liver weight remained unaffected in recovery rats. Females treated with the test item did not show any liver specific effects.

Gross pathological findings:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes.
There were no test item-related histopathological changes noted in reproductive tissues of the parental male/female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.
Reproductive tissues from non-pregnant rats did not show any microscopic changes.
There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect found up to the highest dose level.

Critical effects observed:

no

Conclusions:

The results of the study indicated that the oral administration of test item GENAMIN 10 R 3202 D for 2 weeks prior to mating, during mating, and post mating did not cause any toxicological effect on general health, body weights and food consumption in males at 25, 50, 100 and 150 mg/kg/day and at all the tested doses in females. There were no mortalities observed during the treatment at all the tested doses.
At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.
The test item administration did not reveal any treatment related changes in the hematology, coagulation, clinical chemistry, urinalysis parameters, terminal fasting body weights and gross pathology endpoints in the parental rats of either sex. There were no developmental and non-developmental gross findings noted in male or female pups.
Microscopically, hepatocyte hypertrophy was noted in ≥100 mg/kg/day males and was accompanied by higher liver weight at all the doses (non-dose related). The liver findings showed complete reversal at the end of 14-day recovery phase.
There were no test item-related histopathological changes noted in reproductive tissues in any of the parental male and female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.
Reproductive tissues from non-pregnant rats did not show any microscopic changes.
There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.
No Observed Adverse Effect Level

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, following 14 days post treatment.

The test item was weighed and suspended in vehicle [Corn oil] and administered at the graduated dose levels of 25, 50, 100 and 150 mg/kg bwt/day for low dose (G2), mid dose (G3), Mid-intermediate dose (G4) and High dose(G5)/high dose recovery (G5R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 5 mL/kg bwt/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G22702 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2^nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 51 and towards the end of recovery period (Day 64) for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each main group at the end of two weeks pre-mating period, towards the end of recovery period for all animals after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 51), parental females (LD 14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external genitals for signs of altered development.

Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The reproductive organs were examined from the non-pregnant females.

 

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

Clinical signs and Mortality: There were no clinical signs observed at 25, 50 and 100 mg/kg/day. At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There was no mortality observed at any of the doses tested.

The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.

There were no mortalities observed during the treatment at all the tested doses. There were no abnormalities observed in pups.

Functional Observation Battery: No treatment-related neurological abnormalities were observed at any of the doses tested.

 

Body weights: The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.

Food consumption: Treatment did not affect the food consumption at any of the tested doses in either sex.

Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry and urine parameters at all the doses tested in both sexes.

Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.

Terminal fasting body weights, organ weights and its ratios: There was no effect on the mean fasting body weights and reproductive organ weights of adult rats at all the dose levels compared to their control counter parts in both the sexes. There were no significant changes in thyroid weight of pups on lactation Day 13. Administration of test item resulted in higher liver weight in parental males at all dose levels and was associated with hepatocyte hypertrophy, histologically at dose level of ≥ 100 mg/kg/day. However, the weight increase was not dose related.  The liver weight remained unaffected in recovery rats. Females treated with the test item did not show any liver specific effects.

 

Gross and histopathology:

There were no test item-related gross lesions observed in both the sexes. There were no developmental and non-developmental gross findings noted in male or female pups.

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes. 

There were no test item-related histopathological changes noted in reproductive tissues of the parental male/female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.

There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.

 

In view of the results observed:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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)

Principles of method if other than guideline:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 50 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).

Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

GLP compliance:

yes

Justification for study design:

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 also provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, for 14 days post treatment.

Test material

Test material information

Constituent 1

Reference substance name:

Decyldimethylamine

EC Number:

214-302-7

EC Name:

Decyldimethylamine

Cas Number:

1120-24-7

Molecular formula:

C12H27N

IUPAC Name:

decyldimethylamine

Test material form:

liquid

Details on test material:

Chemical Name: N,N-dimethyl-decan-1-amine
CAS: 1120-24-7

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Hylasco Biotechnology Pvt. Ltd., Plot 4B, MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19.2 to 24.3°C and relative humidity between 60 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.6– 12.9 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
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 to each rat was at an equivolume of 5 mL/kg body weight throughout the study. 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 an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

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 was evidence of sperms in the vaginal smear. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. Not-littered females were sacrificed after 26 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as GD 0. The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
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 to each rat was at an equivolume of 5 mL/kg body weight throughout the study. 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 an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Frequency of treatment:

Daily

Details on study schedule:

Experimental starting date: 30 June 2021
Acclimatization: Start: 30 June 2021
End: 04 July 2021
Pre-treatment period: Start: 05 July 2021
End: 18 July 2021
Treatment: Start: 19 July 2021
End: 15 September 2021
Experiment completion: 30 October 2021
Submission of draft report: 31 October 2021
Study completion: 04 April 2022

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups : 10 males and 10 females
Recovery groups : 5 males and 5 females

Control animals:

yes

Details on study design:

The selected male and female rats were assigned to vehicle control and different treatment groups as shown below:
Group No. Group Colour of
cage card Dose
(mg/kg/day) Concent-ration
(mg/mL) Dose volume
(mL/kg) No. of
Rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle control White 0 0 5 10
10 M
F Rab4141
Rab4151 Rab4150
Rab4160
G2 Low dose Yellow 25 5 5 10
10 M
F Rab4161
Rab4171 Rab4170
Rab4180
G3 Mid dose Green 50 10 5 10
10 M
F Rab4181
Rab4191 Rab4190
Rab4200
G4 Mid-intermediate Pink 100 20 5 10
10 M
F Rab4201
Rab4211 Rab4210
Rab4220
G5 High Red 150 30 5 10
10 M
F Rab4221
Rab4231 Rab4230
Rab4240
Recovery Groups
G1R Vehicle control recovery White 0 0 5 5
5 M
F Rab4241
Rab4246 Rab4245
Rab4250
G5R High dose recovery Red 150 30 5 5
5 M
F Rab4251
Rab4256 Rab4255
Rab4260
M: Male; F: Female; Rab: Prefix code for rat numbers
Note: Females were selected with typical 4-5 days oestrous cycles.
From the respective groups, males and females were mated in a 1: 1 ratio (one male to one female) to get at least 8 pregnant rats at or near term/per group.
However, the recovery groups were kept only for observation of reversibility, persistence or delayed occurrence of systemic toxic effects. These animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study was started from the day of first scheduled kill of dam. Recovery animals were sacrificed after completion of 14 Day recovery period.

Examinations

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 females with regular
4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating 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.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. Each day in the morning, 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 LDs 0 and 4 were recorded.

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

d. 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. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.

e. After standardization, the individual pup body weight was measured on LD13.

f. The number of nipples/areolae in male pups was counted on LD 13.

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

h. The litters were observed daily to note the number of alive, dead and cannibalized pups.

i. In addition to daily clinical observations, all pups were observed for any abnormal behaviour.

j. Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.

Postmortem examinations (parental animals):

All adult animals and pups were subjected to detailed necropsy and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anesthesia. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals which were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death.
For apparently non-pregnant rats, the uteri were stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of embryos (by staining the implantation sites) for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, oestrous cycle, pre-coital interval , ano-genital distance, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (motor activity, thyroid profile) data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like mating and fertility indices were analysed using Chi-square test. When Chi-square is found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
For two groups, the comparisons of mean between treatment and control group was done using student’s t-test.
Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Reproductive indices:

11.1 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/impregnated a female
= ----------------------------------------------------------------- x 100
Number of males with evidence of mating

c. Female mating index (%)

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

d. Female fertility index (%)

Number of pregnant females
= ------------------------------------------- x 100
Number of females with evidence of 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



11.2 Litter Data
a. Mean litter size per group

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

b. Mean viable litter size

No. of viable pups
= -----------------------------------------
Total Number of littered animals

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/ Percentage of male offspring (%)

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
There was no mortality observed at any of the doses tested. There were no abnormalities observed in pups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

gross pathology

histopathology: non-neoplastic

reproductive performance

Results: F1 generation

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect found up to the highest dose.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The results of the study indicated that the oral administration of test item GENAMIN 10 R 3202 D for 2 weeks prior to mating, during mating, and post mating did not cause any toxicological effect on general health, body weights and food consumption in males at 25, 50, 100 and 150 mg/kg/day and at all the tested doses in females. There were no mortalities observed during the treatment at all the tested doses.
At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.
There were no significant changes in neurological parameters, maternal body weights and food consumption during gestation and lactation, pre-coital time, gestation length, mating and fertility parameters. There were no treatment-related effects on the uterine/implantation data and mean litter size. 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. 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, clinical chemistry, urinalysis parameters, terminal fasting body weights and gross pathology endpoints in the parental rats of either sex. There were no developmental and non-developmental gross findings noted in male or female pups.
Microscopically, hepatocyte hypertrophy was noted in ≥100 mg/kg/day males and was accompanied by higher liver weight at all the doses (non-dose related). The liver findings showed complete reversal at the end of 14-day recovery phase.
There were no test item-related histopathological changes noted in reproductive tissues in any of the parental male and female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.
Reproductive tissues from non-pregnant rats did not show any microscopic changes.
There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.
No Observed Adverse Effect Level

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, following 14 days post treatment.

The test item was weighed and suspended in vehicle [Corn oil] and administered at the graduated dose levels of 25, 50, 100 and 150 mg/kg bwt/day for low dose (G2), mid dose (G3), Mid-intermediate dose (G4) and High dose(G5)/high dose recovery (G5R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 5 mL/kg bwt/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G22702 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2^nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.

The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13.

The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 51 and towards the end of recovery period (Day 64) for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each main group at the end of two weeks pre-mating period, towards the end of recovery period for all animals after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 51), parental females (LD 14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external genitals for signs of altered development.

Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The reproductive organs were examined from the non-pregnant females.

 

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

Clinical signs and Mortality: There were no clinical signs observed at 25, 50 and 100 mg/kg/day. At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There was no mortality observed at any of the doses tested.

The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.

There were no mortalities observed during the treatment at all the tested doses. There were no abnormalities observed in pups.

Functional Observation Battery: No treatment-related neurological abnormalities were observed at any of the doses tested.

 

Body weights: The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.

 

Food consumption: Treatment did not affect the food consumption at any of the tested doses in either sex.

 

Maternal body weights and food consumption: The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.

 

Fertility parameters: Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.

 

Litter parameters: 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, ano-genital ratio, pup body weights were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on LD 13 at any of the doses tested.

 

Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry and urine parameters at all the doses tested in both sexes.

 

Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.

 

Terminal fasting body weights, organ weights and its ratios: There was no effect on the mean fasting body weights and reproductive organ weights of adult rats at all the dose levels compared to their control counter parts in both the sexes. There were no significant changes in thyroid weight of pups on lactation Day 13. Administration of test item resulted in higher liver weight in parental males at all dose levels and was associated with hepatocyte hypertrophy, histologically at dose level of ≥ 100 mg/kg/day. However, the weight increase was not dose related.  The liver weight remained unaffected in recovery rats. Females treated with the test item did not show any liver specific effects.

 

Gross and histopathology:

There were no test item-related gross lesions observed in both the sexes. There were no developmental and non-developmental gross findings noted in male or female pups.

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes. 

There were no test item-related histopathological changes noted in reproductive tissues of the parental male/female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.

Reproductive tissues from non-pregnant rats did not show any microscopic changes.

There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.

 

In view of the results observed:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.