3,5-bis(2,4-dimethylcyclohex-3-en-1-yl)polyheterocycle

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-13 to 2020-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 12-13 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 342 - 388 g (Mean: 364.45 g, ± 20 % = 291.56 – 437.34 g)
females: 210 - 240 g (Mean: 226.78 g, ± 20 % = 181.42 – 272.13 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes. This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage (type IV, polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels were provided for all males and for females until GD 18
- Nesting material were provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
Each animal was marked with its identification number by individual ear tattoo or tail marking.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Preparation of the Test Item Formulations
The vehicle was selected in consultation with the sponsor based on the test item’s characteristics and testing guideline. The test item was emulsified in corn oil. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. In case of a solidification of the test item it was liquefied by warming the test item at 37 °C in the incubator before preparation of the formulation.
The formulation was kept under magnetic stirring for 10 min until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to avoid air contact and thereby prevent water uptake.
Based on the results of stability testing (Eurofins Munich Study No. 178332), the test item formulations were prepared at least once every 10 days. The prepared formulation was stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.
 
Experimental Groups and Doses
According to the results of a previous dose range finding study (BSL Munich Study No. 178329, non GLP) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose). The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

C: 0 mg/kg bw/ day (Male No.: 1-10 / Female No.: 41-50)
LD: 100 mg/kg bw/ day (Male No.: 11-20 / Female No.: 51-60)
MD: 300 mg/kg bw/ day (Male No.: 21-30 / Female No.: 61-70)
HD: 1000 mg/kg bw/ day (Male No.: 31-40 / Female No.: 71-80)
C = control, LD = low dose, MD = medium dose, HD = high dose, bw = body weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

Administration of Doses
The test item and vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight. Animal no. 51 of the LD grpoup was accidentally dosed twice on gestation day 7. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Concentrations:
C: 0 mg/mL
LD: 25 mg/mL
MD: 75 mg/mL
HD: 250 mg/mL
C = control, LD = low dose, MD = medium dose, HD = high dose

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 178332).

Study pre start stability analysis were included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8°C) and 10 day < -15°C.

Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 178332 (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).

Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 178334) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at <-15°C at BSL Munich (test facility) and discarded after completion of the final study report. The results are described in a report which has been attached in the appendix of the final report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Experimental Completion Date: 25 July 2016
Completion Date of Delegated Phase (Histopathology): 31 May 2017
Completion Date of Delegated Phase (Formulation Analysis): 29 March 2017
Arrival of the Test Item: 08 August 2017
Study Initiation Date: 11 December 2017
Amendment to Study Plan: 15 December 2017
2nd Amendment to Study Plan: 27 April 2018
Delivery of Animals: 19 December 2017
Acclimatisation Period: 19 December 2017 to 25 December 2017
Experimental Starting Date: 26 December 2017
Treatment Period: 09 January 2018 to 03 March 2018
Necropsies: 30 January 2018, 02 February 2018, 06 - 07 February 2018, 27 - 28 February 2018, 01 – 04 March 2018
Experimental Completion Date: 05 March 2018
Completion Date of Delegated Phase (Histopathology): 24 July 2018
Completion Date of Delegated Phase (Formulation Analysis): 25 July 2018

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

control group: vehicle

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

LD: low dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

MD: medium dose group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

HD: high dose group

No. of animals per sex per dose:

100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females / group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Selection of Doses
On the basis of a dose range finding study for reproduction/ developmental toxicity screening test after oral administration in Wistar rats with Sa 57 in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day (BSL Munich Study No.: 178329, non-GLP) the following conclusions can be made:
Treatment with Sa 57 was not associated with any systemic, reproductive or developmental toxicity at the tested doses of 100, 300 and 1000 mg/kg bw/day.
Thus, dose levels suggested for the subsequent main study are 100, 300 and 1000 mg/kg body weight/day.

Examinations

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups. All animals were weighed at termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

as part of Histopathology:
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Litter observations:

The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. 
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Clinical Biochemistry
From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes. The two PND 4 pups per litter were female pups. No pups were taken when litter size dropped below 8 pups. When there was only one pup available above a litter size of 8, only one pup was sacrificed. PND 4 pup blood was pooled by litter for thyroid hormone analysis. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

Postmortem examinations (parental animals):

Pathology
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on post-natal day 13 along using an anaesthesia (ketamine/xylazin). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.  
Special attention was paid to the organs of the reproductive system. The following tissues from all male and female animals were preserved in 4 % neutral-buffered formaldehyde except for testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol - and subsequently examined histopathologically: all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole, testes, uterus with cervix, vagina and thyroid/parathyroid glands. The latter were not examined histopathologically.
All animals intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination: adrenal glands, all gross lesions, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), ovaries, oviducts, prostate and seminal vesicles with coagulating glands as a whole, rectum, spleen, stomach, testes, thymus, thyroid/parathyroid glands, trachea, urinary bladder, uterus with cervix and vagina.


Organ Weights
The wet weight of the reproductive organs [epididymides, testes, ovaries, uterus with cervix, prostate, seminal vesicles and coagulating glands, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females), were weighed after fixation] of all sacrificed adult males and females from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of animals euthanised for animal welfare reasons were not recorded.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.



Histopathology
A full histopathology was carried out on the preserved organs and tissues (see above) of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid gland from pups and from the adult animals was not evaluated as it was not considered necessary as there was no test item related effect observed on thyroid weights in parental animals and T4 hormone level in parental males and pups sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon completion of the study.

Postmortem examinations (offspring):

All surviving pups were killed on PND 4 (see Clinical Biochemistry) or by cervical dislocation on PND 13.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved but not examined histopathologically.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

see Results F1 section

Offspring viability indices:

see Results F1 section

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Mortality:

mortality observed, non-treatment-related

Description (incidence):

see Details on results P0

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

see Details on results P0

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:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone (T4) Analysis: see Details on results P0

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

see Details on results P0

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Details on results (P0)

Mortality
During the treatment period, on gestation day 20 (Study Day 40), female animal No. 70 of the MD group was euthanized as it was having problems in littering. Histopathological evaluation did not provide evidence for the cause. In absence of any other indicators it was considered to be most likely incidental, although a relation to the test item cannot be excluded. Besides, no test item related mortality was observed during the study period in any of the groups.

Clinical Observations
Male and female animals of the HD group and females of the MD group were seen moving the bedding immediately after test item administration. In males, this was observed at the end of the pre-mating and during the mating period. In females, it occurred during gestation and post-natal periods.
Slight to moderate salivation was observed in 3/10 males and females of the MD group and 7/10 males and females of the HD group. Additionally, piloerection was observed in 1/10 females of the control group, 2/10 of the MD and 10/10 of the HD group as well as in 1/10 male LD group animals.
All above-mentioned clinical signs were observed immediately after the dose administration and therefore are considered to be signs of a local reaction to the test item rather than a systemic adverse effect and are not toxicologically relevant.
Single or few findings, i.e. abnormal breathing, alopecia, crust (chest, ear), dehydration, half eye closure, hypothermia, long/missing/regrowing incisors and scratch (snout) were observed transiently in single or in few animals and are not assumed to be test item related.
Animal no. 70 of the MD group did not litter and showed dehydration, half eye-lid closure and hypothermia in the late gestation period. This single case is considered incidental and not related to the test item.

Body Weight and Body Weight Gain
Sa 57 had no adverse effect on body weight development in this study.
A tendency towards lower body weights in male animals at the HD level (6 % below controls at terminal sacrifice) due to statistically significant lower weight gain in the first premating and mating weeks, compared to controls, is considered test item related but not adverse. It has to be considered that mean body weight of HD group males was minimally lower than in controls at the start of treatment.
An isolated finding of a slight but statistically significantly lower body weight gain of male animals of the LD group during the first mating week and female animals of the LD group during the first pre-mating week are considered incidental, due to lack of dose-dependency.
 
Food Consumption
Sa 57 had no effect on food consumption in this study.

Estrous Cycles
The test item had no significant effect on the estrous cycle analyzed during the two weeks premating period after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or number of abnormal/normal cycles between the dose groups and the control group.

Precoital Interval and Duration of Gestation
There was no test item related effect on the duration of pre-coital interval and the duration of the gestation in all dose groups compared with the control.

Reproductive Indices
There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. A lower fertility observed in the HD group (3/10 non-pregnant females) is not considered adverse.

Thyroid Weight and Serum T4 Levels
Thyroxine (T4) levels of male adult animals were slightly but statistically significantly lower in the HD group than in the control group. However, values for T4 were within the range of biological variation and historical control data. Thus, this is not considered adverse.

Pathology
There were no macroscopic findings observed in any of the dose groups at necropsy.
One incidental gross pathological change was recorded in HD animal no. 77 as fluid-filled cyst at the corpus luteum which is not considered to be test item-related.

Organ Weight
In males and female, there were no statistically significant differences in the absolute and relative organ weights of all dose groups when compared to the control group. A non-statistically significantly higher ovary weight in the HD group (approx. 25 % above controls) is not considered toxicologically relevant, as it is was within the range of historical control data.

Histopathology
All recorded findings in the decedent animal (Animal no. 70) were deemed to be incidental. In survivors, no test item related changes were noticed.
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina.
The testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.
The treatment with Sa 57 did not induce histomorphological effects in the reproductive organs of the non-pregnant females 60, 74, 77 and 79 and their mating partner male nos. 20, 34, 37 and 39 respectively.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group were between 96.1 % and 106.7 % of the nominal value, between 95.6 % and 98.1 % for the MD dose group and between 98.5 % and 105.5 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 99.7 %, 97.0 %, and 101.0 % of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period as measured concentrations were within acceptance criterion of 20 %.

Sex:

male/female

Remarks on result:

other: No adverse effect was observed at the highest dose of treatment (1000 mg/kg bw/day). A histomorphological NOEL (no observed effect level) could be established at 1000 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

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:

no effects observed

Description (incidence and severity):

Thyroid Hormone (T4) Analysis: see Details on results F1

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

see Details on Results F1

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Litter Observations
There were no test item treatment related effects on litter data including total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13 and all parameters were comparable between dose groups and control group. The finding of a slight but statistically significantly lower number of female pups on PND 0 and 4 in the MD group, when compared to controls, is not assumed to be toxicologically relevant, but more likely incidental. This also relates to a statistically significantly lower total number of live pups on PND 4 and 13 in the MD group that is also considered incidental.

Litter Weight Data
There was no test item related effect on pup mean weight observed in this study. In MD group the total litter weight was slightly but statistically significantly lower on PND 13 and female litter weight statistically significant lower on PND 0 and 4 than in the control group. These effects are related to litter observations described in 12.6 and not considered toxicologically relevant.

Pre- and Post-Natal Data
There was no test item treatment related effect on the number of corpora lutea, number of implantation sites, number of live pups (PND 0, PND 4 and PND 13) and percentage of pre- and post-implantation loss in the dose groups when compared to the control group.

Pup Survival Data
There was no statistically significant effect on the survival of the pups from PND 1 through PND 13 in all dose groups when compared to control group.

Anogenital Distance and Nipple Retention
Male pup weight (and its cube root) was slightly but statistically significantly higher in the HD group, when compared to controls. This coincided with a lower number of male pups in this group.There were no toxicologically relevant changes in mean absolute and relative anogenital distance and pup nipple retention on PND 12. These parameters were within the historical range.

Thyroid Weight and Serum T4 Levels
There were no test item related adverse effects on thyroid gland weight of male and female pups.

Pup External Findings
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period as measured concentrations were within acceptance criterion of 20 %.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

The test item had an effect on group mean pup weight and total mean litter weights in HD group on PND 0, 4 and 13.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of this reproduction/developmental toxicity screening test with Sa 57 in male and female Wistar rats at the dose levels of 100, 300, and 1000 mg/kg/day the following conclusions can be made:
There were no adverse signs of general systemic toxicity and anatomic pathology found up to the dose levels of 1000 mg/kg/day. Therefore, the NOEL of Sa 57 in this reproduction/developmental toxicity screening study is considered to be 1000 mg/kg body weight/day.

Executive summary:

Summary

The objective of this study was to assess the possible effects of Sa 57on reproduction and development, after repeated dose administration in Wistar rats and also intended to get information at an early stage of assessing the toxicological properties of Sa 57.

The test item was administered daily in graduated doses to 3 groups, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females rats, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days. Control group rats were handled similar to that of the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.Before dosing, all females were screened for two weeks for regular estrous cyclicity and rats (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study. Control, low, mid and high dose group rats received the dose at 0, 100, 300 and 1000 mg/kg/day respectively, as repeated dose at the dose volume of 4 mL/kg.

The test item formulation was prepared at least once every 10 days. The test item was emulsified in corn oil and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg body weight. Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in study weeks 1, 3, 5 and in the last week of the study.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, still births, live births, runts and the presence of gross abnormalities.

Live pups were counted, sexed and litters weighed within 24 hours of parturition (PND 0), on PND 4 and PND 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment days 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on post-natalday 4 or 13 and those found dead, were examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues (all gross lesions, epididymides, ovaries, prostate and seminal vesicle with coagulating gland, testes, uterus with cervix, vagina andthyroid/parathyroid glands) was performed on high dose and control parental animals and in non-pregnant female animals of the LD and MD group.

Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in the mating partners of the non-pregnant female LD and MD animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Summary Results

Dose formulation analysis for nominal concentration revealed that the mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 99.7 %, 97.0 %, and 101.0 % of the nominal concentration, respectively.

A single female of the MD group was euthanized as it was having problem in littering. Although most likely incidental, a relation to the test item cannot be excluded. Besides, no test item related mortality was observed in this study.

There were no test item related clinical findings during the study, except moving the bedding, slight to moderate salivation and slight piloerection that were considered to besigns of local reaction to the test item rather than signs of systemic toxicity. Unsuccessful littering and associated clinical signs, i.e.half eye-lid closure and hypothermia in the late gestation period in a single MD group animal are considered incidental and not related to the test item.

The test item had no effect on body weight or body weight gain in both sexes.

The test item had no effect on food consumption of the animals in both sexes.

The test item had no significant effect on estrous cycle analysed during the 2 weeks premating periodwhen compared to the controls. There were no considerable differences in the length or number of normal/abnormal cycles between the dose groups and the control group.

There were no test item related effects on litters which includes total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13 and all parameters were found to be comparable to control. Occasional slight but statistically significantly lower numbers of alive pups in the MD group are not considered toxicologically relevant.

As a result, the mean total litter weight of the MD group was statistically slightly lower than the control on PND 13 and statistically lower mean female litter weight on PND 0 and 4 in MD group. This is not considered toxicologically relevant. Besides, there were no test item related effects in pup mean weight observed in this study.

There was no test item related effector statistically significant effectsobserved in the duration of pre-coital interval and the duration of gestation in all dose groups compared with the control.

There were no test item treatment related effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0, PND 4 and PND 13) and percentage of pre- and post-implantation loss in the dose groups when compared to the control group.

There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. A slightly lower fertility observed in the HD group is not considered adverse.There were no statistical significant effects on the survival of the pups from PND 1 through PND 13 in all dose groups when compared to control group.

There were no toxicologically relevant changes in mean absolute and relative anogenital distance, pup nipple retention on PND12, mean pup weight. These parameters were within the historical range.

There were no test item related effects in the thyroid gland weight of male and female pups. However, mean values of males thyroxine (T4) levels was statistically significantly lower in the HD group than in the control group, however, all mean group values for T4 were within the range of biological variation and historical control data.

No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. There were no macroscopic findings observed in any of the dose groups at necropsy.

In males and female, there were no statistically significant differences in the absolute and relative organ weights of all dose groups when compared to the control group. Mean values of the dose groups were comparable to mean control values.

There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina.

The testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed. The treatment with Sa 57 did not induce histomorphological effects in the reproductive organs of the non-pregnant females 60, 74, 77 and 79 and their mating partners (males no. 20, 34, 37 and 39), respectively.