Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-18 to 2018-03-16
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-acetylhexanelactam
EC Number:
217-565-6
EC Name:
N-acetylhexanelactam
Cas Number:
1888-91-1
Molecular formula:
C8H13NO2
IUPAC Name:
N-acetylhexanelactam
Test material form:
liquid
Details on test material:
Batch: not yet known
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Clariant Iberica Production, S.A., Tarragona Site, 43110 La Canonja, Spain
- Batch No.of test material: ESD0024613
- Expiration date of the batch: After 06 May 2018
- Manufacture date: 06 May 2016
- Purity as per Certificate of Analysis: 98.10 GC area%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+21 to +29°C)
- Stability under test conditions: The stability and homogeneity of the test item in the vehicle was established at 1 and 200 mg/mL under Eurofins Advinus Study No. G13342. Based on the results, the test item was stable and re-suspendible in the vehicle up to 4 days when stored at room temperature.

Test animals

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:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: (P) x 13-14 weeks
- Weight at study initiation: (P)
Males Females
G1: 325.81 ± 14.75 215.29 ± 8.59
G2: 325.06 ± 15.02 217.13 ± 8.57
G3: 324.74 ± 14.65 216.23 ± 7.60
G4: 325.86 ± 13.53 217.09 ± 9.64

- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes.
Mating and post mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.
Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. This was changed along with cage twice a week. The enrichment was provided both during pre-mating and post mating period for males and pre-mating period for females. During the experimental period, animals were housed in a single experimental room (SC-18).

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet – Pellet (Certified) manufactured by Envigo, P.O.Box 44220, Madison WI 53744-4220, was provided ad libitum to the rats.

- 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 Ltd., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: 19 August 2017 to 23 August 2017

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 58 to 68%
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 07 September 2017 To: 09 November 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item was weighed in to a pre-calibrated beaker and the vehicle (Corn oil) was added up to the pre-mark and mixed using glass rod. The final concentration achieved was 20, 60 and 200 mg/mL for the G2, G3 and G4 groups, respectively. The suspensions were mixed well by stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the solubility test, the test item formed a clear solution in Corn oil, while it could not be dissolved/suspended in Milli-Q water and 0.1% carboxymethyl cellulose sodium salt (medium viscosity). Hence, Corn oil was used as vehicle for dose formulation preparation.
- Concentration in vehicle: G2: 20; G3: 60 and G4: 200 mg/mL
- Amount of vehicle (if gavage): Dose volume administered to each rat was at an equivolume of 5 mL/kg body weight throughout the study. Dose volume was adjusted based on the most recent body weight of individual rat.
Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: 21 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: One per cage
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd month of the treatment period and analysed in-house. For each set, duplicate sample was drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No. G13342. One set of samples were analysed for concentration (a.i) analysis.
Dose formulations were considered acceptable as the overall mean results were within ± 15.0% of the claimed concentration and the overall relative standard deviation (RSD) was less than 10.0%.
The unused back up samples was disposed as analysis results of the first set of samples were within the acceptable limits.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to specific group of rats at approximately the same time each day (varying by ± 3 hours), for 39 days (which included two weeks prior to mating, during the mating period and post mating) after which they were sacrificed.
Females: The dose formulation was administered orally by gavage to the specific group of rats at approximately the same time each day (varying by ± 3 hours), two weeks prior to the mating period and was continued through mating, pregnancy and up to lactation day 13 for G1, G2 and G3 groups. On Lactation Day 14, the females were sacrificed after overnight fasting. Due to observation of clinical signs and mortality in G4 group females, all surviving females were sacrificed on treatment Day 37 during various stages of gestation (gestation Day 17 to Day 21) without allowing for delivery.
The animals in the vehicle control group were handled in an identical manner to the treatment group but were administered vehicle only.

Frequency of treatment:
once Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 15 to 16 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels of 100 (G2), 300 (G3) and 1000 (G4) mg/kg/day were selected for this study based on the results of a 28-Day Repeated Dose Oral Toxicity Study in Wistar rats with 14-Day Recovery Period (Study No. G13343) and in consultation with the Sponsor.
In addition to the test doses, vehicle control group was included. Animals in the vehicle control group were handled in a manner similar to the treatment groups except for test item administration.

- Rationale for animal assignment (if not random):
Grouping was done by the method of body weight stratification and distribution. On the day of randomization (which was performed prior to start of treatment), based on the given temporary animal identification number, each animal with normal oestrous cyclicity (4-5 day cycle) was weighed and the corresponding body weights were recorded. The data was transferred (temporary identification number and body weight) into an excel spread sheet. The body weights recorded were stratified in ascending order.
The statistical analysis was performed to ensure that the weight variation is minimal and inter group variation did not exceed ± 20% of the mean body weight in each group and sex. Rats with extreme body weights were discarded. Grouping was done one day prior to start of treatment.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily once
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating. 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.
Sperm parameters (parental animals):
Parameters examined in [all/P] male parental generations:
Evaluation of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) and AGD ratio (calculated by dividing the ano-genital distance from cube root of body weight), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated blow were prepared for microscopic examination and weighed, respectively.
All gross lesions, Epididymides, Ovaries with oviducts, Thyroid with parathyroids (weighed after fixation), Uterus with cervix, Vagina, Prostrate, Seminalvesicles and coagulation glands, Testes (collected in modified Davidson's fluid), Liver, Kidneys. Levator ani bulbocavernosus muscle complex, Cowper’s glands, Glans penis were weighted preserved.
Further, all gross lesions were examined in the terminally and pre-terminally dead/ sacrificed animals. In addition to tissues/organs indicated in the study plan, stomach (including forestomach and glandular stomach), adrenals, spleen and thymus were collected from all animals sacrificed (terminal and pre-terminally sacrificed) and subjected for histopathological evaluation.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
On Day 13, thyroid gland from available one male and female pup from each litter (randomly selected) was collected and preserved in 10% NBF for the histopathological examination. The thyroid weight was determined after fixation.
Statistics:
Data captured using ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data were entered retrospectively in ProvantisTM) and Clinical Chemistry data were analysed using Provantis built-in statistical tests.
Statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Version 12.0. All data of quantitative variables like body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance and organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-Way Analysis of Variance (ANOVA). In case of non-optimal (non-normal or heteroschedastic) data, ANOVA was done using suitable transformation. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test, if the overall treatment ‘F’ test was found to be significant.
Post implantation loss (%), number of nipples/areolae in male pups, number of implantations, pre-coital interval (days), mean litter size, sex ratio and gestation length (days) were analysed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done if the group differences are found significant.
Z test was performed for testing the differences in proportions for mating, fertility and survival indices.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group
Reproductive indices:
a. Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x 100
b. Male fertility index (%) = (Number of males siring a litter / Number of males cohabited) x 100
c. Female mating index (%) = (Number of females mated / Number of females cohabited) x 100
d. Female fertility index (%) = [Number of pregnant females (confirmed at necropsy) / Number of females used for mating] x 100
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 / Number of implantations) x 100
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. Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born) x 100
d. Sex Ratio (%) = (No. of male pups born / Total No. of pups born) x 100
e. Ano-genital Distance Ratio (mm/g1/3) = (Ano-genital distance / Cube root of body weight)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs viz., hypoactivity, slight piloerection, red discharge from vagina and hindlimbs with exaggerated and splayed movements were observed at 1000 mg/kg Bwt/day females starting from treatment Day 32-37 (GD 14 -16) of treatment.
There were no clinical signs observed at 100 and 300 mg/kg Bwt/day in both sexes.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
- Due to severe clinical signs and mortality at 1000 mg/kg Bwt/day females (5/10 females during treatment days 32-34, i.e. gestation day 14-18), all remaining females were sacrificed on treatment Day 37 (i.e. gestation day 17 to 21).
- One male at the high dose died during blood collection, which could be due to over anaesthesia and not related to the treatment.
- One female at 300 mg/kg Bwt/day, delivered 13 pups after completing 24 days of gestation period. At first observation during morning hours, the dam and all pups were found dead. At necropsy, glandular foci was observed in the stomach.

- No mortalities observed at 100 mg/kg Bwt/day in both sexes.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg Bwt/day, the mean body weights were significantly lower in males on Days 29, 36 and 39 of treatment period with the reduction of 6.2 to 8.2% when compared to vehicle control group. The body weight gain (Days 1-39) was also significantly lower with the reduction of 55.1%, when compared to vehicle control group.
The mean body weights and net body weight gains were unaffected by the treatment at 100 and 300 mg/kg Bwt/day doses in males and at all the doses tested in females during pre-mating period, when compared to vehicle control.
Thus, the treatment resulted in decrease in the body weights in males at 1000 mg/kg Bwt/day when compared to vehicle control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg Bwt/day, the food consumption was significantly lower in males measured during Days 8-15 and 36-39, with the reduction of 19.6-26.5%, when compared to vehicle control group.
The food consumption was unaffected by the treatment at 100 and 300 mg/kg Bwt/day doses in males and at all the doses tested in females during pre-mating period when compared to vehicle control. However, the lower food consumption observed at 1000 mg/kg Bwt/day in females during Days 1-8 was considered toxicologically insignificant as the body weights were unaffected.
The decrease in the food consumption in males at 1000 mg/kg Bwt/day was considered test item-related finding which was associated with decrease in body weights.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Decreased absolute reticulocyte count observed in females (67%) at 1000 mg/kg Bwt/day was considered as test item-related change. Stress resulting from decreased body weights gain during in-life phase could have contributed to reduction in reticulocyte count and was associated microscopically with atrophy/ decreased red pulp cellularity in spleen (grossly small).
There were no test item-related changes in coagulation parameters in males and females at all the doses tested.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related clinical chemistry findings at all the tested dose levels in either sex.
A few variations in clinical chemistry parameters that reached statistical significance were not considered to be toxicologically relevant as they were likely to be due to random biological variation and/or there was no dose correlation.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related histopathological changes were observed in liver, stomach, thyroid, spleen and thymus.
> In liver, test item-related minimal to mild hepatocellular hypertrophy with centrilobular distribution was observed in males at ≥ 100 mg/kg Bwt/day and in female adult rats dosed at 1000 mg/kg Bwt/day and was considered as adaptive response to test item administration.
> In thyroid, follicular epithelial hypertrophy of minimal to mild in severity was noted in males at ≥ 300 mg/kg Bwt/day. This lesion was of diffuse pattern involving many follicles with increase in size/ height of epithelial cells. The change observed in thyroid gland was considered secondary to hepatocellular hypertrophy in liver. Hepatocellular hypertrophy in liver was often seen concomitantly in rats with thyroid gland follicular epithelial cell hypertrophy as indicated in Tanja S. et al., 2011 and Kuei-Meng Wu, 2006.
> In thymus, minimal to severe degree of atrophy was observed in female rats dosed at ≥ 100 mg/kg Bwt/day. Further, in one male at 1000 mg/kg Bwt/day minimal degree of hemorrhage was also observed. These findings were considered as treatment related secondary change and could be related to decreased feed consumption and/or decreased body weight gain (in-life phase) leading to stress in 1000mg/kg Bwt/day dose group.
> In spleen, moderate degree of atrophy was observed in female rats dosed at ≥ 300 mg/kg Bwt/day. Microscopically, decreased red pulp cellularity was observed in few female rats at 1000 mg/kg Bwt/day dose. This change was considered as treatment related secondary change consequence to decreased feed intake and lack of body weight gain at 1000 mg/kg Bwt/day.
> The non-glandular stomach showed epithelial hyperplasia in male rats dosed at 1000 mg/kg Bwt/day and in female rats dosed at ≥100 mg/kg Bwt/day. In addition in some rats mucosal necrosis was observed in glandular/non-glandular mucosa. This change was associated with presence of minimal infiltration of leukocytes involving mainly in non-glandular submucosa.

> There were no test item-related histopathological changes noted in reproductive organs in males. The staging of spermatogenesis did not reveal any stage specific changes. In all treated males, the spermatogenic cycles observed in the different seminiferous tubules of testes were complete.
> There was no test item-related microscopic changes observed in thyroid gland of pups at all the dose levels tested.
In pre-terminally dead and sacrificed females at 1000 mg/kg Bwt/day and in one female at 300 mg/kg Bwt/day, degeneration of implantation sites were observed in the gravid uteri of a few rats. Further, cell debris in lumen was observed in one female rat (Ru2368-all pups found dead) at 300 mg/kg Bwt/day.
All other lesions were considered background changes, and therefore, not considered as treatment related.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
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 4.03, 4.00, 4.00 and 4.11 days in vehicle control, 100, 300 and 1000 mg/kg Bwt/day dose groups, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological changes noted in reproductive organs in males. Evaluation of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The staging of spermatogenesis did not reveal any stage specific changes. In all treated males, the spermatogenic cycles observed in the different seminiferous tubules of testes were complete.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on the mean pre-coital time, mating and fertility indices of sires and dams at all the doses tested. The gestation length at 100 and 300 mg/kg Bwt/day doses was comparable to vehicle control group. However, the male and female fertility index were significantly lower (10%) in the low and high dose groups, when compared to vehicle control group. These significant differences were considered incidental as the difference is within the historical control data (HD range: 80 to 100 %).

Adverse effect on reproductive performance was observed at the 1000 mg/kg Bwt/day dose group, due to reduced mean no. of implantation when compared to vehicle control. There were no effect on implantations at 100 and 300 mg/kg bwt/d compared to control group. An increase in post-implantation loss (%) was observed at 100 and 300 mg/kg Bwt/day groups with 15.1 and 27.2 %, respectively, compared to control group with 9.6 %.

Details on results (P0)

During the treatment period, adverse clinical signs such as hypoactivity, slight piloerection, red discharge from vagina and hindlimbs with exaggerated and splayed movements were observed at 1000 mg/kg Bwt/day dose females starting from treatment Day 32 (GD 14). A total of 5 females were found dead at 1000 mg/kg Bwt/day during GD 14-18 (treatment Days 32- 34). Hence, all the remaining surviving females in the high dose group were sacrificed on treatment Day 37 during various stages of gestation (gestation Day 14 to Day 21) without allowing for delivery.
After 39 days of treatment, all male rats were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis. The animals were subjected to detailed necropsy at sacrifice after overnight fasting and study plan specified tissues were collected. Female rats (dams) were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis. Gross necropsy was performed on all dams on LD 14 for G1, G2 and G3 groups and study plan specified tissues were collected. In G4 females, due to the presence of clinical signs and mortality, clinical pathology investigations were conducted for the surviving females on treatment Day 37 (gestation Day 17 to Day 21). All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pups from each litter was collected for histopathological examination.
Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Evaluation of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. Further, liver and thyroid in both sexes and uterus with cervix, vagina and ovaries in females were examined in the lower dose groups as higher incidence of test item related changes were noted at high dose. In addition, the reproductive organs of non-pregnant rats were examined. Further, all gross lesions were examined in the terminally and pre-terminally dead/ sacrificed animals. Stomach, adrenals, spleen and thymus were subjected for histopathological evaluation from all animals sacrificed terminally and pre-terminally.
Under the experimental conditions employed, the following results were obtained:
• Clinical Signs and Mortalities: Clinical signs such as hypoactivity, slight piloerection, red discharge from vagina and hindlimbs with exaggerated and splayed movements were observed at 1000 mg/kg Bwt/day females starting from treatment Day 32 (GD 14) until sacrifice (treatment Day 37). A total of 5 females were found dead at 1000 mg/kg Bwt/day during GD 14-18 (treatment Days 32- 34) and one female at 300 mg/kg Bwt/day after delivery of dead pups. Due to the observation of clinical signs and mortality at 1000 mg/kg Bwt/day females, all remaining surviving females were sacrificed on treatment Day 37 (GD 14-21).
• Body Weights and Food Consumption: At 1000 mg/kg Bwt/day, the mean body weights and net body weight gains were significantly lower in males which were associated with decreased food consumption. The body weights and food consumption were significantly lower during gestation period up to GD 14. The body weights and food consumption were unaffected during pre-mating, gestation and lactation periods at 100 and 300 mg/kg Bwt/day doses.
• Fertility Parameters: Treatment had no effect on pre-coital time, oestrous cycle length and fertility indices of sires and dams at all the tested doses. The gestation length at 100 and 300 mg/kg Bwt/day doses was comparable to vehicle control group. At 1000 mg/kg Bwt/day, mean no. of implantations was significantly lower when compared to vehicle control. There were no effect on implantations at 100 and 300 mg/kg bwt/d compared to control group. The post-implantation loss (%) observed in control (G1), 100 and 300 mg/kg Bwt/day were 9.6, 15.1 and 27.2%, respectively. The post-implantation loss in the control animals was ranged from 0 to 25%. The loss was contributed by 5/10 dams with an average loss of 9.6%, which is within the historical control data. At 100 mg/kg Bwt/day, the post-implantation loss was ranged from 0 to 46.7%. The loss was attributed to 4/9 dams, of which the loss of 3 dams were more than 35%. At 300 mg kg Bwt/day, the post-implantation loss was ranged from 0 to 100%. The loss was attributed to 7/9 dams, of which the loss of 4 dams were more than 35%. Considering the dose related higher percentage of post-implantation loss and number of animals affected at 100 and 300 mg/kg Bwt/day, a treatment related effect cannot be excluded.
• Litter Data: Day 0 survival index at 300 mg/kg Bwt/day and Day 4 survival index at 100 and 300 mg/kg Bwt/day were significantly lower, due to the total death/cannibalism of pups observed in dams from Day 0 and 1-4. No treatment-related changes in the ano-genital distance and ano-genital distance ratio were observed at any of the doses tested when compared to the control group. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.
• Thyroid Hormone Profile: Increased TSH and decreased T4 activity were observed in adult males at ≥ 300 mg/kg Bwt/day and in females at 1000 mg/kg Bwt/day. Alteration in hormone profile was associated with follicular epithelial hypertrophy in thyroid gland (increased weights) on microscopic examination in males at ≥ 300 mg/kg Bwt/day. Decreased thyroid stimulating hormone (TSH) was observed in pups (LD13) at ≥ 100 mg/kg Bwt/day without any alterations in T4 levels and associated thyroid organ weights/ratios and the gross/microscopic findings.
• Clinical Pathology: The significantly lower reticulocyte count observed in females at 1000 mg/kg Bwt/day was associated with stress resulting from decreased body weights during in-life phase. Microscopically, the reduction in reticulocyte count was associated with atrophy/decreased red pulp cellularity in spleen (grossly small).
• Fasting Body Weights, Organ Weights and its Ratios: Decreased terminal body weights were observed in males at 1000 mg/kg Bwt/day. Increased liver weight in males at ≥ 100 mg/kg Bwt/day and in females at 1000 mg/kg Bwt/day was associated with hepatocellular hypertrophy. Increased thyroid weight (absolute and relative) at 1000 mg/kg Bwt/day in both males and females was microscopically associated with follicular epithelial cell hypertrophy.
• Gross Pathology: Test item-related gross lesions such as, not prominent/small thymus, small spleen, multiple focus and/ or thickening in glandular/non-glandular stomach were observed across the treated males at 1000 mg/kg Bwt/day and females at ≥100 mg/kg Bwt/day dose.
• Histopathology: Test item-related microscopic lesions in atrophy of thymus in one male at 1000 mg/kg Bwt/day and in females at ≥100 mg/kg Bwt/day, atrophy of spleen / decreased red pulp cellularity in females at ≥300 mg/kg Bwt/day and mucosal necrosis/epithelial hyperplasia/ leukocytic infiltration-sub mucosa -glandular and/or non-glandular in stomach of males at 1000 mg/kg Bwt/day and in females at ≥100 mg/kg Bwt/day dose. The hepatocellular hypertrophy with centrilobular distribution in liver observed in males at ≥100 mg/kg Bwt/day and in female adult rats at 1000 mg/kg Bwt/day was considered as adaptive response to test item administration. In thyroid, follicular epithelial hypertrophy noted in males at ≥300 mg/kg Bwt/day was considered secondary to hepatocellular hypertrophy in liver and not an adverse findings. There were no test item-related changes in male and female reproductive organs.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
<= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
<= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
other:
Remarks on result:
other: A reliable no observed adverse effect level (NOAEL) for reproductive toxicity could not be determined, due to reduced mean no. of implantations at 1000 mg/kg Bwt/day and the tendency of higher post-implantation loss at 100 and 300 mg/kg Bwt/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Day 4 survival index was significantly lower at 100 and 300 mg/kg Bwt/day, due to the total death/cannibalism of pups observed in dams from Day 0 and 1-4. Other reproductive parameters were not affected.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean number and body weight of male and female (and total number) pups per litter were not affected by the treatment at 100 and 300 mg/kg/Bwt/day doses
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Decreased thyroid stimulating hormone (TSH) level noted in pups (LD13) at 100 and 300 mg/kg Bwt/day was considered as test item related effect without any alterations in T4 levels and associated microscopic findings in thyroids.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter and viable litter sizes. There were no external abnormalities in live or dead pups in any of the groups.
Day 4 survival index was significantly lower at 100 and 300 mg/kg Bwt/day, due to the total death/cannibalism of pups observed in dams from postnatal Day 0 and 1-4. Other reproductive parameters were not affected.
No treatment-related changes in the ano-genital distance and ano-genital distance ratio were observed at any of the doses tested when compared to the control group. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.
Decreased thyroid stimulating hormone (TSH) level noted in pups (LD13) at 100 and 300 mg/kg Bwt/day was considered as test item related effect without any alterations in T4 levels and associated microscopic findings in thyroids.

Effect levels (F1)

Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
<= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
Remarks on result:
other: No clinical signs, no gross pathological or histopathology changes observed at pups of all dose groups. Male pups did not exhibit areola/nipple retention on PND 13. Decrease of anogenital distance ratio at 100mg/kg bw considered as incidental.

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Test item-related gross changes with corresponding microscopic lesions were observed in thymus, spleen and stomach (animal-wise) have been presented in the table below:

Tissue

Group and Sex

Animal no.

Gross lesions

Microscopic correlates

Thymus

 

G4M

Ru2375

Small/ Discoloration red

Atrophy/ Hemorrhage

G4F

Ru2381; Ru2383; Ru2384; Ru2385; Ru2386; Ru2387; Ru2388 and Ru2389

Small / Not prominent

Atrophy

G3F

Ru2367

G2F

Ru2345

No abnormality detected

Spleen

G4F

Ru2383; Ru2385; Ru2387 and Ru2388

Small

Atrophy

G3F

Ru2367

G2F

Ru2345

G4F

Ru2381; Ru2384; Ru2386;

Small

Decreased red pulp cellularity

Stomach

G4M

Ru2375 and Ru2377

Non-glandular – Thickening

Epithelial hyperplasia-non-glandular ; Leukocytic infiltration-submucosa

G3F

Ru2367

Glandular – Focus(i)

Could not be confirmed due to autolysis

G2, G3, G4 = i.e. Dose groups 100, 300, 1000 mg/kg Bwt/day, respectively.

F = Female; M = Male

Applicant's summary and conclusion

Conclusions:
Based on the findings of an OECD 421 screening study in rats a concern for reproductive toxicity is given. A LOAEL of 100mg/kg Bwt/day is determined.
Executive summary:

The purpose of this study in Wistar rats was to generate limited information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was weighed and suspended in vehiclei.e.,Corn oil and administered to rats at the graduated dose levels of 100, 300, and 1000 mg/kg Bwt/day to low (G2), mid (G3), and high (G4) group rats, respectively. The rats in the vehicle control group (G1) received vehicle alone. The dose volume administered was 5 mL/kg body weight.

Each group in the experiment was comprised of ten male and ten female rats.The prepared 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. During the treatment period,adverse clinical signs such ashypoactivity, slight piloerection, red discharge from vagina and hindlimbs with exaggerated and splayed movementswere observed at 1000 mg/kg Bwt/day dosefemales startingfromtreatmentDay32 (GD 14). A total of 5 females were found dead at 1000 mg/kg Bwt/day during GD 14-18 (treatment Days 32- 34). Hence, all the remaining surviving females in the high dose group were sacrificed on treatment Day 37 during various stages of gestation(gestation Day 14 to Day 21)without allowing for delivery.

 Each rat in the experiment was observed for clinical signs, mortality and morbidity. Body weight was recorded prior to start of treatment and weekly thereafter. Food consumption was recorded at weekly intervals except during the cohabitation period.Female rats were also weighed on Gestation 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 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 surviving male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13.After 39 days of treatment, all male rats were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis. The animals were subjected to detailed necropsy at sacrifice after overnight fasting and study plan specified tissues were collected. Female rats (dams) were fasted overnight (water allowed) and blood was collected prior to necropsy for clinical pathology investigations and thyroid hormone analysis. Gross necropsy was performed on all dams on LD 14 for G1, G2 and G3 groups and study plan specified tissues were collected. In G4 females,due to the presence of clinical signs and mortality, clinical pathology investigations were conducted for the surviving females on treatment Day 37 (gestation Day 17 to Day 21). All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pups from each litter was collected for histopathological examination.

Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Evaluation of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. Further, liver and thyroid in both sexes and uterus with cervix, vagina and ovaries in females were examined in the lower dose groups as higher incidence of test item related changes were noted at high dose. In addition, the reproductive organs of non-pregnant rats were examined. Further, all gross lesions were examined in the terminally and pre-terminally dead/ sacrificed animals.Stomach, adrenals, spleen and thymus were subjected for histopathological evaluation from all animals sacrificed terminally and pre-terminally.

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

·        Clinical Signs and Mortalities:Clinical signs such as hypoactivity, slight piloerection, red discharge from vagina and hindlimbs with exaggerated and splayed movements were observed at1000 mg/kg Bwt/day females starting from treatment Day32 (GD 14) until sacrifice (treatment Day 37). A total of 5 females were found dead at 1000 mg/kg Bwt/day during GD 14-18 (treatment Days 32- 34) and one female at 300 mg/kg Bwt/day after delivery of dead pups. Due to the observation of clinical signs and mortality at 1000 mg/kg Bwt/day females, all remaining surviving females were sacrificed on treatment Day 37(GD 14-21).

·        Body Weights and Food Consumption: At 1000 mg/kg Bwt/day, the mean body weights and net body weight gains were significantly lower in males which were associated with decreased food consumption. The body weights and food consumption were significantly lower during gestation period up to GD 14. The body weights and food consumption were unaffected during pre-mating, gestation and lactation periods at 100 and 300 mg/kg Bwt/day doses.

·        Fertility Parameters: Treatment had no effect on pre-coital time, oestrous cycle length and fertility indices of sires and dams at all the tested doses. The gestation length at 100 and 300 mg/kg Bwt/day doses was comparable to vehicle control group. At 1000 mg/kg Bwt/day, mean no. of implantation was significantly lower when compared to vehicle control. There were no effect on implantations at 100 and 300 mg/kg Bwt/day. The post-implantation loss (%) observed in control, 100 and 300 mg/kg Bwt/day groups were 9.6, 15.1 and 27.2 %, respectively.

·        Litter Data: Day 0 survival index at 300 mg/kg Bwt/day and Day 4 survival index at 100 and 300 mg/kg Bwt/day were significantly lower, due to the total death/cannibalism of pups observed in dams from Day 0 and 1-4. No treatment-related changes in the ano-genital distance and ano-genital distance ratio were observed at any of the doses tested when compared to the control group. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

·        Thyroid Hormone Profile: Increased TSH and decreased T4 activity were observed in adult males at ≥ 300 mg/kg Bwt/day and in females at 1000 mg/kg Bwt/day. Alteration in hormone profile was associated with follicular epithelial hypertrophy in thyroid gland (increased weights) on microscopic examination in males at ≥ 300mg/kg Bwt/day. Decreased thyroid stimulating hormone (TSH) was observed in pups (LD13) at ≥ 100 mg/kg Bwt/day without any alterations in T4 levels and associated thyroid organ weights/ratios and the gross/microscopic findings.

·        Clinical Pathology: The significantly lower reticulocyte count observed in females at 1000mg/kg Bwt/day was associated with stress resulting from decreased body weights during in-life phase. Microscopically, the reduction in reticulocyte count was associated with atrophy/decreased red pulp cellularity in spleen (grossly small). 

·        Fasting Body Weights, Organ Weights and its Ratios: Decreased terminal body weights were observed in males at 1000 mg/kg Bwt/day. Increased liver weight in males at ≥ 100 mg/kg Bwt/day and in females at 1000 mg/kg Bwt/day was associated with hepatocellular hypertrophy. Increased thyroid weight (absolute and relative) at 1000 mg/kg Bwt/day in both males and females was microscopically associated with follicular epithelial cell hypertrophy.

·        Gross Pathology:Test item-related gross lesions such as not prominent/small thymus, small spleen, multiple focus and/ or thickening in glandular/non-glandular stomach were observed across the treated males at 1000 mg/kg Bwt/day and females at ≥100 mg/kg Bwt/day dose.

·        Histopathology: Test item-related microscopic lesions in atrophy of thymus in one male at 1000 mg/kg Bwt/day and in females at ≥100 mg/kg Bwt/day, atrophy of spleen / decreased red pulp cellularity in females at ≥300 mg/kg Bwt/day and mucosal necrosis/epithelial hyperplasia/leucocytic infiltration-sub mucosa-glandular and/or non-glandular in stomach of males at 1000 mg/kg Bwt/day and in females at ≥100 mg/kg Bwt/day dose. The hepatocellular hypertrophy with centrilobular distribution in liver observed in males at ≥100 mg/kg Bwt/day and in female adult rats at 1000 mg/kg Bwt/day was considered as adaptive response to test item administration. In thyroid, follicular epithelial hypertrophy noted in males at ≥300mg/kg Bwt/day was considered secondary to hepatocellular hypertrophy in liver and not an adverse findings. There were no test item-related changes in male and female reproductive organs.

Based on study Directors conclusion:

Daily oral (gavage) administration of N-Acetylhexanelactam 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 (females) resulted in:

•  Changes observed in males were restricted to the highest dose level tested (i.e. 1000 mg/kg Bwt/day) and included: decreased body weight and food consumption, gross pathological changes such as not prominent/small thymus, small spleen, and multiple focus and/ or thickening in glandular/non-glandular stomach. Microscopically, atrophy of thymus and mucosal necrosis/epithelial hyperplasia/leukocytic infiltration-sub mucosa -glandular and/or non-glandular in stomach were observed. Based on these observations, the no observed adverse effect level (NOAEL) for toxicity in males is considered to be 300 mg/kg Bwt/day.

•  Changes observed in females includes, mortalities at ≥ 300 mg/kg Bwt/day, clinical signs, decreased body weight, food consumption and reticulocytes count.at 1000 mg/kg Bwt/day, gross pathological changes such as not prominent/small thymus, small spleen, and multiple focus and/ or thickening in glandular/non-glandular stomach at ≥ 100 mg/kg Bwt/day. Microscopically, atrophy of thymus and mucosal necrosis/epithelial hyperplasia/ leukocytic infiltration-sub mucosa-glandular and/or non-glandular in stomach at ≥ 100 mg/kg Bwt/day and atrophy/ decreased red pulp cellularity in spleen at ≥ 300 mg/kg Bwt/day were observed. Based on these observations, the no observed adverse effect level (NOAEL) for maternal toxicity could not be determined.

•  No adverse effects on fertility was observed up to the highest dose of 1000 mg/kg Bwt/day. Adverse effect on reproductive performance was observed at the 1000 mg/kg Bwt/day dose group, where the mean number of implantation was significantly lower, when compared to vehicle control. There were no effect on implantations at 100 and 300 mg/kg Bwt/day dose groups. The post-implantation loss (%) observed in control, 100 and 300 mg/kg Bwt/day dose groups were 9.6, 15.1 and 27.2 %, respectively. Due to reduced mean no. of implantations at 1000 mg/kg Bwt/day and considering the tendency of dose related higher percentage of post-implantation loss and number of animals affected at 100 and 300 mg/kg Bwt/day, a treatment related effect cannot be excluded and the no observed adverse effect level (NOAEL) for reproductive toxicity could not be determined.

•  Due to significantly reduced postnatal survival rates (Day 0 and 4 survival indices) of pups at 100 and 300 mg/kg Bwt/day dose groups, the no observed adverse effect level (NOAEL) for developmental toxicity could not be determined.

Based on these findings a concern for reproductive toxicity is given and a reproduction toxicity potential of the test substance registered cannot be excluded.