Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 05 June 2018 and 19 March 2019 (date of thyroid hormone phase report). The in-life phase of the study was conducted between 14 August 2018 (first day of treatment) and 07 October 2018 (final day of necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
purity >85 % (w/w)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
males: 28 days; females: 54 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
10 males and 10 females per dose and control group
Control animals:
yes, concurrent vehicle
Positive control:
No

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable). Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 7 post partum.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
uring the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14. Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: all surviving adult females on day 14 post partum. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in capitals were preserved from all remaining animals: Adrenals, Pancreas, Bone & bone marrow (sternum), PITUITARY, Brain (including cerebrum, cerebellum and pons), PROSTATE, Cecum, Rectum, Colon, Sciatic nerve, Duodenum, EPIDIDYMIDES (preserved in modified Davidson's fluid), SEMINAL VESICLES (WITH COGATULATING GLAND), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes (fixed in Davidson's fluid), GROSS LESIONS, Spleen, Heart, Stomach, Ileum (including peyer’s patches), TESTES (preserved in modified Davidson's fluid), Jejunum, Thyroid/Parathyroid, Kidneys, Trachea, Liver, Thymus, Lungs (with bronchi; inflated to approximately normal inspriatory volume with buffered 10% formalin before immersion in fixative), Urinary bladder, Lymph nodes (axillary and mesenteric), UTERUS & CERVIX (WITH OVIDUCTS), Muscle (skeletal), VAGINA.
The tissues from five selected control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in CAPITALS from all animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

OTHER:
Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Litter observations:
During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only). On Day 13 of age, where possible, for one male and one female offspring per litter, the thyroid/parathyroids were retained in buffered 10% formalin.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 31 or 32.
- Female animals: Females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

POST-MORTEM EXAMINATIONS
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed
Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in captials were weighed from all remaining animals: Adrenals, Ovaries, Brain, PROSTATE, EPIDIDYMIDIS, Heart, Kidney, Liver, SEMINCAL VESICLE (WITH COAGULATING GLAND), Spleen, TESTES, Liver, Thymus.

Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in capitals were preserved from all remaining animals: Adrenals, Pancreas, Bone & bone marrow (sternum), PITUITARY, Brain (including cerebrum, cerebellum and pons), PROSTATE, Cecum, Rectum, Colon, Sciatic nerve, Duodenum, EPIDIDYMIDES (preserved in modified Davidson's fluid), SEMINAL VESICLES (WITH COGATULATING GLAND), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes (fixed in Davidson's fluid), GROSS LESIONS, Spleen, Heart, Stomach, Ileum (including peyer’s patches), TESTES (preserved in modified Davidson's fluid), Jejunum, Thyroid/Parathyroid, Kidneys, Trachea, Liver, Thymus, Lungs (with bronchi; inflated to approximately normal inspriatory volume with buffered 10% formalin before immersion in fixative), Urinary bladder, Lymph nodes (axillary and mesenteric), UTERUS & CERVIX (WITH OVIDUCTS), Muscle (skeletal), VAGINA.

The tissues from five selected control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in CAPITALS from all animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed at day 13 of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Statistics:
Where appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Data were analyzed using the decision tree from the Provantis. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric)
Reproductive indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval:
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices:
For each group the following were calculated: Mating Index (%) = (Number of animals mated / Number of animals paired) x 100, Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100.
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%) Group mean percentile post-implantation loss was calculated for each female/litter as follows: Post-implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / number of implantation sites) x 100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on day 1 / Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offpring alive on Day 1) x 100
Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offpring alive on Day 4) x 100; Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum
iii. Sex Ratio
Sex ratio was calculated for each litter value on days 1, 4 and 13 post partum, using the following formula: (Number of male offspring / total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
gross pathology
other: sexual maturation

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No Observed Effect Level (NOEL) in an oral screening study for reproductive toxicity was at 1000 mg/kg bw/day in rats.
Executive summary:

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening test in rats via the oral route, conducted according to current OECD guideline 422 and in compliance with GLP, no changes attributable to treatment were noted for the examined reproductive toxicity parameters at 100, 300 or 1000 mg/kg bw/day. There was no effect of treatment on estrous cycle during the pre-pairing phase of the study or at termination. Mating performance as assessed by the number of paired animals that mated was unaffected. There was no effect on fertility, as assessed by the number of females that achieved pregnancy. The intergroup distribution of gestation length observed during the study did not indicate any obvious effect of treatment. The NOEL was 1000 mg/kg bw/day.