3,5-di(undecan-2-yl)heteropolycycle

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-12 to 2019-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 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

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
- 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. 14-15 weeks old
- Body weight at the allocation of the animals to the experimental groups: males: 348 - 400 g (mean: 371.62 g); females: 224 - 250 g (mean: 237.28 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.

HUSBANDRY
- 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 were made. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estrous 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

Remarks:

Sigma-Aldrich Batch No.: MKCD 1021, MKCL 9871, Expiry Date: 18 July 2018 (MKCD 1021), 11 August 2018 (MKCL 9871)

Details on exposure:

PREPARATION OF THE TEST ITEM FORMULATIONS
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline. The vehicle in this study was corn oil.
Based on the results of stability testing (Eurofins Munich Study No. 178290), the test item formulations were prepared at least once every 10 days (within stability time frame as given by Eurofins Munich Study No. 178290). An emulsion with the vehicle was prepared by weighing the test item into a tared plastic vial on a suitable precision balance and adding the vehicle give the appropriate final concentration of the test item. The formulation was kept under magnetic stirring for 10 min. After homogenization the formulation was overlaid with argon to avoid air contact and thereby prevent water uptake. The prepared formulation was stored at room temperature and protected from light. In case of a solidification of the test item formulation it was liquefied by warming the formulation at 37 °C in the incubator before the administration procedure. The vehicle was also used as control item.

EXPERMINETAL GROUPS AND DOSES
According to the results of a previous dose range finding study (BSL Munich Study No. 178287) 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 is completed.

C 0 mg/kg bw/ day (Male No.:1-10/ Female No.:41-50) Conc. 0 mg/mL
LD 100 mg/kg bw/ day (Male No.:11-20/ Female No.:51-60) Conc. 25 mg/mL
MD 300mg/kg bw/ day (Male No.:21-30/ Female No.:61-70) Conc. 75 mg/mL
HD 1000 mg/kg bw/ day (Male No.:31-40/ Female No.:71-80) Conc. 250 mg/mL

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
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

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. 178290). Study pre start stability analysis was 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 at below -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.

The test item was shown to be homogenous according to Eurofins Study No. 178290 (after at least 30 min without stirring). Consequently, 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. 178292) and until then stored under appropriate conditions based on available stability data. The B-samples were retained below -15 °C at BSL Munich (test facility) and discarded after completion of the final study 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

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10 animals per sex per dose
In total, 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:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of premating
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 were completed. Animals of an additional control group were handled identically as 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 animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

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. 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. Female no. 74 which was euthanized for animal welfare reasons was 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. Pups from dam numbers 71, 72, and 73 were weighed on PND 5 instead of PND 4.
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.

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. From dam numbers 71, 72, and 73 blood samples from pups were collected on PND 5. 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). Pup blood was pooled by litter for thyroid hormone analysis.
Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary as no test item related effect observed on T4 hormone levels of males and day 13 pups.
Two pups per litter were sacrificed on day 4 (except from dam numbers 71, 72, and 73 from which pups were sacrificed on day 5) after birth and blood samples are taken for possible serum hormone assessments.
The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated as litter size dropped below 8 pups in litters of dam numbers 42, 46, 49, 51, 54, 58, 61, 69 and 70. As there was only one pup available above a litter size of 8, only one pup was sacrificed in litters of dam numbers 44, 53, 57, 59, and 64.

Postmortem examinations (parental animals):

PATHOLOGY
Males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using an anesthesia (e.g. 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 from the day of mating or from the last day of mating period. 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. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. If appropriate and possible, 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 (all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole, testes, uterus with cervix, vagina, thyroid/parathyroid glands) from all male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol. Inadvertently testes and epididymides of animal nos. 6-10, 16-20, 26-30 and 36-40 were preserved in 4 % neutral-buffered formaldehyde.
All animals found dead and/or 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, vagina).
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved for histopathological examination, but not evaluated.

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), was 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 found dead or 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/parathyroid glands from pups and from the adult animals were not evaluated as it was not considered necessary as there was no test item related effect observed on thyroid weights in parental animals, pups 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 died during the study or which were euthanised due to morbidity. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicles with coagulating glands) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin 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 were 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, Buchsweg 56, 4625 Oberbuchsiten, 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 was performed according to the corresponding SOP’s of the test sites.

Postmortem examinations (offspring):

All surviving pups were killed 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.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data were 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 analysed 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 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

Estrous Cycles, precoital interval, gestation length, copulation index, fertility index, and delivery index

Offspring viability indices:

Number of live births and post-implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, AGD, litter size and litter weights, viability index day 0-4 and day 4-13

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:

no effects observed

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

see Details on results P0 (in: mortality)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

see Details on results P0 (in: histopathology)

Reproductive performance:

no effects observed

Description (incidence and severity):

see Details on results P0: Precoital Interval and Duration of Gestation and Reproductive Indices

Details on results (P0)

Mortality
Mortality and morbidity was observed in 2/10 females of the HD group. Female no. 77 was found dead on premating day 8 and female no. 74 was euthanized in a moribund condition on gestation day 18. Microscopic changes recorded in the heart and thymus of moribund female no. 74 were considered to be the possible cause of its morbidity and were deemed to be most likely due to traumatic injury that may occur during the application procedure. Therefore, it was not considered to be test item related. Histopathologically cause of death of female no. 77 could not be established. All remaining animals survived the scheduled study period.

Clinical Observations
Female no. 74 of the HD group which was euthanized in a moribund condition observed with the clinical findings of piloerection, abnormal breathing, reduced spontaneous activity, increased abdominal respiration, and tachycardia on the day of sacrifice (gestation day 18). Moving the bedding was transiently observed in 10/10 females of the MD group, 10/10 males and 9/10 females of the HD group. Salivation was noted transiently in 1/10 males of the MD group and 7/10 males and 2/10 females of the HD group. The clinical signs of moving the bedding and salivation were seen during the second half of the treatment period in males, during the gestation and lactation period in MD females, and almost throughout the whole treatment period in HD females. Both signs were observed for short duration and were considered to be a sign of a local reaction of the test item rather than adverse systemic effects due to test item administration.
Piloerection of 1/10 males of the HD group was considered incidental. The clinical sign of piloerection was also observed in 5/10 females of the control group, 4/10 females of the LD group, 5/10 females of the MD group, and 9/10 females of the HD group. This finding showed no dose-dependency and occurred mainly at the end of the gestation/at the beginning of the lactation period. It is not considered to be toxicologically relevant. Low incidences of slight clinical signs like alopecia in 4/10 females of the MD and 1/10 females of the HD group, crust in 4/10 females of the MD group, scratch in 1/10 females of the MD group, a swelling on the snout of 1/10 females of the MD group, broken incisor in 1/10 females of the HD group, overgrown teeth in 1/10 males of the LD group were seen without dose-dependency and were not considered to be test item-related. Regurgitation of the vehicle/test item was observed in 1/10 females of the control group, 3/10 females of the LD group, and 2/10 females of the HD group and is not considered related to toxicity but this gavage related reflux/regurgitation observes occasionally in rat with or without slight salivation.

Body Weight Development
There were no statistically significant differences observed for body weight and body weight gain between males of the test item-treated groups and the control group throughout the study period. In females, a slightly but statistically significant higher body weight gain was observed in females of the LD and MD group compared to the controls during the second week of treatment (premating day 7-premating day 14). As this finding was attributed to low group mean body weight gain in control group mainly caused by the body weight loss of two females of the control group (females no. 42 and 43) and there was no significant difference in the HD group compared to the control group, this observation is not considered toxicologically relevant.

Food Consumption
During the entire study period (premating period in males, premating, gestation, and lactation period in females) there was no considerable and statistically significant difference observed in food consumption of male and female animals between the test item-treated groups and the respective control group. Observed marginal differences between the groups were within the normal range of variation and without toxicological relevance.

Pathology- Macroscopic Findings
Males no. 31 and 35 were observed with a pelvis dilatation (right sided) and male no. 5 was noted with abnormal colored (pale, bilateral) and size reduced seminal vesicles (both sides). In absence of histopathological correlation, these findings were considered to be spontaneous in nature and not toxicologically relevant. Prematurely euthanized female no. 77 of the HD group due to animal welfare reasons was found to be autolyzed and was observed with a fluid filled (red) thoracic cavity and an abnormal colored (red) lung. No further gross lesions were observed in any other animal of test item-treated groups or the control group.

Organ Weight
No statistically significant differences were observed in organ weights of male and female test item-treated groups when compared to the corresponding controls. Slight differences followed no dose dependency and were within the normal range of variation of this age and strain of animals.

Histopathology
There were no microscopic findings in the male and female reproductive organs that could be attributed to the treatment with the test item. In female no. 74, which was euthanized in moribund conditions during gestation, a minimal epicardial inflammation was recorded in the heart and a slight chronic-active inflammation in the connective tissue of the thymus. These findings were deemed to be most likely due to traumatic injury that may occur during the application procedure and, therefore, were not considered to be test item related. Secondary stress-related findings consisted of moderate thymus atrophy and slight hypertrophy of the Zona fasciculata of the adrenal cortices. Female no. 77 that died spontaneously during the pre-mating period showed macroscopically abnormal red coloured lungs (correlating microscopically with slight congestion) and a thoracic cavity filled with red fluid. These changes are related to its spontaneous death and autolysis. The assessment of the integrity of the spermatogenetic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides did not provide any evidence of impaired spermatogenesis.All other findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age and in this study type.

Estrous Cycles
The test item had no biologically significant effect on the estrous cycle analysed during the 2 weeks premating period and after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed once during the screening phase in two females of the control group (female no. 43 and female no. 50 - less than 4 day cycle), 1 female of LD group (female no. 60 - less than 4 days) and 4 females of the MD group (female no. 62, female no. 65, female no. 67 and female no. 70 - less than 4 day cycle). Since estrous cyclicity data is based on actual smear stages observed at the time of observation and duration of one cycle is considered as days between 2 estrous stages, it appears as an abnormal cycle but as such it was not. As this effect on estrous cyclicity was observed in just few animals of the control, LD, and MD without dose dependency and it was considered as biological variation and not related to the treatment with the test item.

Precoital Interval and Duration of Gestation
The precoital interval and the duration of gestation were not affected by treatment with Sa 190. There were no statistically significant or biologically relevant differences when comparing the test item-treated groups with the control group.

Reproductive Indices
There were no test item-related effects on the reproductive indices (copulation index, fertility index, and delivery index) in the dose groups when compared to the control group. All indices were at 100% in all groups. Viability index from PND 0 to PND 4 was 88% in the control group, 89% in the LD group, 95% in the MD group and 99% in the HD group. Lower viability index in the control and LD group thereby was mainly caused by one single female in both cases (female no. 43 in the control and female no. 54 in the LD group, in both females the viability index was 0%). Slight differences in the viability index from PND 4 - PND 13 were within the normal range of variation and without toxicological relevance.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: general toxicity and reproductive toxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pup External Findings, see Details on Results F1

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Body weight and weight changes:

no effects observed

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:

no effects observed

Description (incidence and severity):

see Details on results F1

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pup External Findings, see Details on Results F1

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Litter Data, see Deatils on Results F1

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Pre- and Post-Natal Data
The pre-natal and post-natal parameters including the number of corpora lutea, the number of implantations and the number of live pups on PND 0, 4 and 13 showed no statistically significant or relevant differences between test item-treated groups and the controls. Values were comparable between all test item-treated groups and the control group.

Litter Data
The litter parameters including the total number of pups, still births and runts born on PND 0, the number of live pups, number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 were not statistically significantly different in test item groups compared to the control group.

Mean Viability Indices
Viability index from PND 0 to PND 4 was 88% in the control group, 89% in the LD group, 95% in the MD group and 99% in the HD group. Lower viability index in the control and LD group thereby was mainly caused by one single female in both cases (female no. 43 in the control and female no. 54 in the LD group, in both females the viability index was 0%). Slight differences in the viability index from PND 4 - PND 13 were within the normal range of variation and without toxicological relevance.

Litter Weight Data
Sa 190 had no statistically significant or toxicologically relevant effect on pup mean weight on PND 0, 4 and 13. Total litter weight, male litter weight and female litter weight were not affected in test item groups compared to the control group.

Pup Survival Data
The survival of the pups from PND 0 to PND 4 and from PND 4 to PND 13 was not affected in test item groups when compared to the control group. Slightly higher pup mortality from PND 0 to PND 4 in the control and LD group were mainly caused by higher mortality rate of pups from one single dam in each group (dam no. 43 in the control group, dam no 54 in the LD group). However, HD group showed no increased pup mortality and increased mortality in the control and LD group was not considered toxicologically relevant.
 
Anogenital Distance and Nipple Retention
There were no relevant differences in male pup weight or nipple retention of male pups of test item-treated groups compared to the control group. Male pups of the LD group showed slightly but statistically significantly higher anogenital distance and relative anogenital distance compared to male pups of the control group. Males of the MD and HD group showed no difference in anogenital distance compared to the controls. Due to this lack of dose dependency the slight difference between male pups of the LD and the control group is not considered test item-related.
Female pups of the LD group were observed with statistically significantly higher weight, cube root of weight, anogenital distance and relative anogenital distance compared to the control pups. However, as females of HD group showed no relevant differences compared to the control group this finding was not considered toxicologically relevant.

Pup External Findings
No test item related gross external abnormalities of toxicological relevance were observed on PND 0-12 in the pups of any of the groups. One pup in the control group was observed cold to touch and showed reduced spontaneous activity (dam no. 43, pup no. 4) and one pup in the MD group (dam no. 66, pup no. 12) was observed with a tail which was black and partly missing. However, these findings are considered to be incidental and not related to treatment with Sa 190.

Mean thyroid/parathyroid weight
Mean thyroid/parathyroid weight of male and female pups of the test item-treated groups were comparable to mean thyroid/parathyroid weight of corresponding control pups. Differences between the groups were within the normal range of variation and were not caused by treatment with Sa 190. No test item-related effect of toxicological relevance or statistical significance was observed on male and PND 13 pup thyroxine hormone (T4) levels in the test item-treated groups when compared to the controls.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse treatment-related effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

On the basis of this reproduction/ developmental toxicity screening test with Sa 190 in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

Mortality and morbidity was observed in 2/10 females of the HD group. Morbidity of female no. 74 most likely occurred due to traumatic injury that may occur during

application procedure. Histopathologically cause of mortality of female no. 77 could not be established.

No adverse effects of test item were found on male and female clinical observations, body weight development, food consumption, estrous cyclicity, litter data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight, pup external findings, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups.

Applicant's summary and conclusion

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with Sa 190 in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
The NOAEL of Sa 190 in this study for general toxicity and reproductive toxicity screening is considered to be 1000 mg/kg body weight/day.

Executive summary:

Summary

The aim of this study was to assess the possible effects of Sa 190 on male and female fertility and embryofetal development after repeated dose administrationin Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, 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, 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 were completed. Animals of an additional control group were handled identically as 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 animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                 0        mg/kg body weight/day

Low Dose:             100     mg/kg body weight/day

Medium Dose:       300     mg/kg body weight/day

High Dose:            1000   mg/kg body weight/day

The test item formulation was prepared at least once every 10 days. The test item was emulsified in corn oil and administered daily. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg bodyweight.

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, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 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 study day 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 carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues (all gross lesions, testes, epididymides, ovaries, uterus with cervix, vagina, prostate and seminal vesicle with coagulating gland, thyroid/parathyroid glands) was performed on high dose and control animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. 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. All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

Mortality and morbidity was observed in 2/10 females of the HD group. Morbidity of female no. 74 most likely occurred due to traumatic injury that may occur during application procedure. Histopathologically cause of mortality of female no. 77 could not be established.

There were no clinical signs indicating systemic toxicity in this study. The clinical signs of piloerection, abnormal breathing, reduced spontaneous activity, increased abdominal respiration, and tachycardia which were observed in prematurely sacrificed HD female no. 74 were considered to be incidental. Moving the bedding and increased salivation were noted in MD and HD groups and were assumed to be a sign of a local reaction of the test item but not toxicologically relevant. The clinical sign of piloerection was observed in few animals in all test item-treated groups and the control group. As this finding occurred mainly in females and was noted transiently at the end of the gestation/at the beginning of the lactation period it is assumed to be related to the process of giving birth. Piloerection in 1/10 males of the HD group was considered incidental. Low incidences of other clinical signs were observed without dose-dependency and thus were not considered test item-related.

Treatment with Sa 190 had no statistically significant effect on body weight development of males throughout the study period. Mean body weight gain of LD and MD females was slightly but statistically significantly higher compared to the control females. However, this finding was not considered toxicologically relevant as body weight development of HD females was comparable to controls.Treatment with Sa 190 had no statistically significant effect on food consumption in any of the male or female dose groups throughout the study period.

The test item had no biologically significant effect on the estrous cycle analysed during the 2 weeks premating period and after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

The precoital interval and the duration of gestation were not affected by treatment with Sa 190.

Pre-natal and post-natal parameters including the number of corpora lutea, the number of implantations and the number of live pups on PND 0, 4, and 13 showed no relevant differences between the test item-treated groups and the controls.

No statistically significant or toxicologically relevant effects were found in the reproductive indices including copulation index, fertility index, delivery index, and viability index when comparing test item-treated animals and control animals.

The litter parameters including the total number of pups, still births and runts born on PND 0, the number of live pups, number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 showed no significant or relevant differences in test item groups compared to the control group.

Sa 190 had no statistically significant or toxicologically relevant effect on pup mean weight on PND 0, 4 and 13. Total litter weight, male litter weight and female litter weight were not affected in test item groups compared to the control group.

Pup survival showed no statistically significant differences in any of the test item treated groups when compared to the control group. Slight differences between the groups were not considered toxicologically relevant.

Treatment with Sa 190 had no toxicologically relevant on anogenital distance of male and female pups. Slightly but statistically significant higher anogenital distance in male pups of the LD group compared to the control group was not considered relevant due to the lack of dose dependency. Nipple retention was not statistically significantly affected in test item-treated male pup groups compared to the control group.

Sa 190 had no statistically significant or toxicologically relevant effect on serum T4 levels of parental males or the 13 day old pups and on thyroid/parathyroid weight of 13 day old male and female pups.

Isolated external pup findings (cold, reduced spontaneous activity in one pup of the control group and a black and partly missing tail in one pup of the MD group) were observed without dose dependency and were not considered to be toxicologically relevant.

No statistically significant differences were observed in organ weights of male and female test item-treated groups when compared to the corresponding controls. Slight differences followed no dose dependency and thus were not considered to be toxicologically relevant.

There were no gross lesions or histological findings that could be attributed to treatment with the test item

There were no microscopic findings in the male and female reproductive organs that could be attributed to the treatment with the test item. Single findings were considered incidental.

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period during each occasions (study week 1, 3, 5 and in the last week of the study) as measured concentrations were within acceptance criterion of 15 %.

Conclusion

On the basis of this reproduction/ developmental toxicity screening test withSa 190 in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

Mortality and morbidity was observed in 2/10 females of the HD group. Morbidity of female no. 74 most likely occurred due to traumatic injury that may occur during application procedure. Histopathologically cause of mortality of female no. 77 could not be established.

No adverse effects of test item were found on male and female clinical observations, body weight development, food consumption, estrous cyclicity, litter data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight, pup external findings, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups.

The NOAEL of Sa 190 in this study for general toxicity and reproductive toxicity screening is considered to be 1000 mg/kg body weight/day.