Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 421: NOAEL (parental/developmental/reproductive) = 1000 mg/kg bw/d

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 421. A detailed read across justification is provided in chapter 13 of this dossier.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-13 to 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
see Overall remarks, attachments
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

Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: 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: 291 - 347 g (mean: 321.38 g, ± 20 % = 257.10 - 385.65 g)
females: 202 - 252 g (mean: 225.50 g, ± 20 % = 180.40 - 270.60 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

Number and Sex of the Animals
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.

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
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

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Specifications provided by the supplier Manufacturer: Name: corn oil Manufacturer: Sigma Aldrich Batch No.: MKCD9871 Physical State: liquid Storage Conditions: at room temperature Expiry Date: 06 August 2018
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. 177727), the test item formulations were prepared at least once
every 10 days (within stability time frame as given by Eurofins Munich Study No. 177727).
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.
The formulation was placed into an ultrasonic bath for approximately 30 min at room temperature and finally vortexed and/or stirred until visual homogeneity was achieved.
The prepared formulation was stored at room temperature and protected from light. 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. 178028 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)
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)

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
TThe 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.
Concentration: C: 0 mg/mL; LD:25 mg/mL; MD: 75 mg/mL; HD: 250 mg/mL


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. 177727).
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 -15 to -35 °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. 177727 (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 5 mL).
The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 177728) and until then stored under appropriate
conditions based on available stability data. The B-samples will be retained at -15 to -35 °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 is completed.
Frequency of treatment:
7 days/week
Details on study schedule:
Arrival of the Test Item: 23 October 2017 / 09 November 2017 / 11 June 2018
Study Initiation Date: 13 April 2018
Amendment to Study Plan: 24 September 2018
Delivery of Animals: 19 April 2018
Acclimatisation Period: 19 April until 23 April 2018
Experimental Starting Date: 24 April 2018
Treatment Period:
males: 08/09 May 2018 to 05/06 June 2018
females: 08/09 May 2018 up to 09 July 2018
Necropsies:
males: 05 June 2018, 06 June 2018
females: 17 June 2018, 20 June 2018, 26 June 2018, 27 June 2018, 28 June 2018, 29 June 2018, 30 June 2018, 01 July 2018, 10 July 2018
Experimental Completion Date: 11 July 2018
Completion Date of Delegated Phase (Histopathology): to follow
Completion Date of Delegated Phase (Formulation Analysis): to follow
Study Completion Date: to follow


Remarks:
0, 100, 300, 1000 mg/kg bw /day
Basis: nominal concentration
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 Dose Selection
According to the results of a previous dose range finding study (BSL Munich Study No. 178028) and in consultation with the sponsor, dose levels had been selected for this study.
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.
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. Prematurely sacrificed female no. 78 of the HD group 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.

Clinical Biochemistry
From all adult males at termination blood samples were collected wherever possible from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).












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 a part of histopathology the following sperm parameters were examined: a detailed qualitative examination of the testes 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.

From 2 female pups/litter on day 4 after birth (if possible), from all dams (except of preliminary euthanized dam no. 78) and 2 pups/litter at termination on day 13
(except of dam numbers 47 and 54 which had no pups and number 78) and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes.
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).
Due to significantly and dose-dependently lower T4 levels in test item-treated parental males and PND 13 pups compared to the corresponding controls, assessment of T4 in blood samples
from the parental females (dams) was done. Pup blood was pooled by litter for thyroid hormone analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments.
The two pups per litter were female pups if possible to reserve male pups for nipple retention evaluations. No pups were eliminated as litter size dropped below 8 pups in litters of
dam numbers 51, 53, and 59 of the LD group, 61, 63, and 70 in the MD group, and 71, 72, 75, and 80 in the HD group. As there was only one pup available above a litter size of 8,
only one pup was sacrificed in the litter of dam number 52 of the LD group.
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 from all females except female numbers 47, 48, 54, and 67 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 female no. 54 of the LD group was sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
Female no. 47 of the control group was not recorded sperm positive and was sacrificed on study day 26.
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 (female no. 47 of the control group) and for any females sacrificed on day 26 post-coitum due to
non-delivery (female no. 54 of the LD group).
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 testes and epididymides
which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.

Due to significantly and dose-dependently lower T4 levels in parental males and PND 13 old pups observed compared to the corresponding controls, the histopathological examination
of thyroid/parathyroid glands of adult males and females and PND 13 pups of the control and HD groups was performed.
Female no. 78 of the HD group which was intercurrently euthanised for animal welfare reasons was 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)


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 female no. 78 of the HD group which was euthanised for animal welfare reasons were not recorded.
Thyroid/parathyroid glands from 1 pup/sex/litter (except from litters no. 47, 54, 76, and 78)/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 (all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole,
testes, uterus with cervix, vagina, thyroid/parathyroid glands) 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 sacrificed on PND 13 and from all adult animals of the control and HD groups were examined as there was test item related effect observed
on T4 hormone level in parental males and pups sacrificed on PND 13. In order to guarantee a fast histopathological examination of LD and MD thyroid/parathyroid glands (if necessary),
the histological processing to microscope slides were performed for LD and MD groups together with the C and HD groups.
A full histopathology was carried out on the preserved organs and tissues of all animals which were euthanised due to morbidity (female no. 78 of the HD group).
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.
These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group.
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, 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 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 the completion of the study.


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:
The reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups were compared to control group indices.
Offspring viability indices:
see Reproductive Indices
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:
not examined
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
Description (incidence and severity):
see Details on results P0
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see Details on results P0
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
see Details on results P0
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone (T4) analysis and Thyroid/parathyroid gland weight: see Details on results 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 under Histopathology, in Details on results P0
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Precoital interval and duration of gestation: see Details on results P0;
Pre-and postnatal data: see Details on results F1;
Reproductive indices: see Details on results F1
Mortality
One female from HD group (Female no. 78) was euthanized in a moribund condition on PND 4.
However, mortality was considered as incidental. All remaining animals survived the scheduled study period.

Clinical Observations
Female no. 78 of the HD group which was euthanized in a moribund condition was observed with clinical signs of piloerection,
sunken flanks, hypothermia and dehydration on the day of sacrifice.
In terminally sacrificed animals, moving the bedding was transiently observed in 1/10 females of the control group, 1/10 males and 2/10 females
of the LD group, 10/10 males and 10/10 females of the MD group, 10/10 males and 10/10 females of the HD group. Salivation was noted transiently
in 2/10 females of the LD group, 5/10 males and 1/10 females of the MD group, 10/10 males and 3/10 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 and during the gestation
and lactation period in 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. Low incidences of slight clinical signs like alopecia in 3/10 males of the control group and 2/10 females
of the HD group, a crust in 2/10 males of the control group and 1/10 males of the LD group, a wound in 1/10 females of the LD group,
abnormal breathing in 1 each female of control and LD group and aggressive behaviour in 1/10 males of the HD group were seen
without dose-dependency and are not considered to be test item-related. The clinical sign of piloerection was observed in 7/10 females
of the control group, 5/10 females of the LD group, 3/10 females of the MD group, and 7/10 females of the HD group.
This finding showed no dose-dependency, occurred only in females of all groups including control and was noted transiently at the
end of the gestation/at the beginning of the lactation period. It is not considered to be toxicologically relevant.
Regurgitation of the test item was observed in 3/10 females of the LD group and 1/10 females of the MD group which could
be attributed to oral gavaging procedure and as such not related to treatment with the test item.

Body Weight Development
The test material had no statistically significant effect on body weight development of male and female animals throughout the study
period except marginal but statistically significantly lower body weight gain during premating period of day 7-14 in HD males when
compared to the controls. During the first week of treatment (premating day 1-7) females of the HD group showed a slight body weight loss
without achieving statistical significance when compared to the control group (mean body weight change controls: 4.90 g, mean body weight
change HD: -0.90). During the second week of treatment (premating day 7-14) the body weight change normalized and females of the HD group
gained weight for the rest of the study period (gestation and lactation period). Females of the other dose groups gained weight comparable to
females of the control group throughout the study period (premating, gestation, lactation). As mean body weight and body weight gain of
male and female treatment groups was in the normal range of variation throughout the treatment period of this study when compared to
the control group, this marginal but statistically significant effect in HD males during premating day 7-14 and in HD female during premating
day 1-7 without achieving statistical significance was not considered to be adverse.

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.

Estrous Cyclicity
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 days cycle in the rat were
observed in one female of control (female no. 42: less than 4 day cycle) and 1 female of the LD group (female no. 58: more than 5 days).
The female of the LD group with abnormal more than 5 day cycle had followed normal proestrus i.e. stage before estrous. The following stage
could not be determined as an estrous due to the absence of cells in the vaginal smear. Since estrous cyclicity data is based on actual smear
stages observed at the time of observation and one cycle duration is considered as days between 2 estrous stages, it appears as an abnormal
cycle but as such it was not and therefore this is considered to be toxicologically irrelevant. Furthermore, as this effect on estrus cyclicity was
observed in just one each animal from control and LD without dose dependency and therefore 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 the test item.
There were no statistically significant differences when comparing the test item-treated groups with the control group.

Pre- and Post-Natal Data
No considerable test item related effects were noted for mean values of number of corpora lutea, implantations sites,
live pups on PND 0, 4 and 13 as well as pre implantation loss and post implantation loss in all dose groups.
However, slightly but statistically significantly lower (p<0.05) mean number of live pups on PND 0, PND 4, and PND 13 was noted in the
LD group when comparing to the controls). This difference showed no dose dependency and was therefore not considered as test item-related.
Furthermore, mean number of alive pups on PND 4 (before interim sacrifice of pups intended for blood sampling) was slightly but statistically
significantly lower (p<0.05) in the HD group compared to the control group). As the mean number of alive pups was not affected on PND 0
and PND 13 this difference was considered as incidental.

Reproductive Indices
There were not test item-related effects on the reproductive indices (copulation index, fertility index, delivery index, and viability index)
in the dose groups when compared to the control group.
Viability index from PND 0 to PND 4 was 100% in the control and MD group, 99.15% in the LD group, and 89.74% in the HD group.
The lower viability index in the HD group was mainly caused by one female (no. 78) and was considered as incidental. From PND 4 to PND 13,
the viability index was 100% in all treatment groups and in the control group.

Thyroid Hormone (T4) Analysis
Mean thyroxine (T4) levels of parental males (42.61 nmol/l in the LD, 40.01 nmol/l in the MD and 35.71 nmol/l in the HD group compared
to 80.52 nmol/l in control group) and 13 day old pups (57.23 nmol/l in the LD, 45.91 nmol/l in the MD and 39.18 nmol/l in the HD group
compared to 91.80 nmol/l in control group) were statistically significantly (p < 0.001)) and dose-dependently lower in test item-treated
groups compared to the corresponding controls. Thus, this effect is considered as test item-related.  
However, toxicological relevance of this finding cannot be decided based on this screening study as no additional finding in the study supporting a mode of action of the test item have been identified. Thus, the findings on T4 cannot be considered as adverse. Furthermore, there was no
test item related effect observed on pup thyroid weight. (Based on T4 results in dams and histopathology of thyroid in males, females and PND 13
pups, this section needs to be adopted) Due to significantly and dose-dependently lower T4 levels in test item-treated males and PND 13 old
pups compared to the corresponding controls assessment of T4 in blood samples from the dams was done.
…results….to be included…
No test item related effect of toxicological relevance or statistical significance was observed and mean weight of thyroid/parathyroid
glands of male and female pups was comparable between test item-treated groups and the corresponding control group.

Pathology
Female no. 54 of the LD group was observed with a fluid filled (dark) uterus at the time of necropsy.
This finding was considered as incidental and not test item-related.
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. Mean prostate weight tended to be slightly higher in the male LD group compared to
control males (deviation from control: 12%). As prostate weight of males in higher dose groups was not affected this difference
was not considered to be toxicologically relevant. Mean thyroid/parathyroid weight of males showed a slight tendency towards lower
weight in test item-treated groups compared to the control group (deviation from control group: 11% in LD, deviation of: 17% in MD and 10 % in HD).
Mean thyroid/parathyroid weight of test item-treated females was observed to be slightly higher compared to control females
(deviation from control group: 9% in LD, 23% in MD and 20% in HD). As differences in mean male and female thyroid/parathyroid weight
showed no dose-dependency and consistency when compared with respective controls, this was not considered to be toxicologically relevant.
Slight differences in the mean ovary (deviation from the control group: 12% in LD and 10% in HD) and mean uterus weight
(deviation of uterus weight of the LD group from the control group: 11%, deviation of uterus weight of the MD group from the control group:
-7%, deviation of uterus weight of the HD group from the control group: 6%) of test item-treated females compared to the control group followed
no dose dependency and were not considered toxicologically relevant as female reproductive organs undergo variable changes depending on
the stage of the estrous cycle. Based on histo evaluation, thyroid interpretation needs to be modified.

Histopathology
There were no microscopic findings in the male and female reproductive organs that could be attributed to the treatment with the test item.
All 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.
The treatment with the test item did not reveal effects on the completeness of sperm stages or cell populations in the HD group
when compared to the control group. There was no indication for maturation arrest, reabsorption of sperm or of any other type of degenerative effects.
All findings recorded (minimal tubular degeneration/atrophy, unilateral in HD male no. 32 and no. 37) are within the range of normal background alterations.
Thyroid histo results of male females and PND 13 pups to be included.

Dose Formulation Analysis
In this study phase 16 samples in total were measured for determination of test item in formulation samples received from
Eurofins Munich / BSL Munich Study No. 177724 (main study).
Concentration 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 was between 90.8% and 101.1 % of the nominal value,
between 93.3 % and 96.9 % for the MD dose group and between 97.3 % and 101.7 % 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 94.6 %, 94.9 %, and 99.6 %
of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations
were within acceptance criterion of 10 %
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pup external findings: see Details on results F1
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
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Pup external findings: see Details on results F1
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone (T4) analysis and thyroid gland weight: see Details on results F1;
Pre- and Post-Natal Data: see Details on results F1;
Reproductive Indices: see Details on results F1
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Litter Data
The litter parameters including runts, and number of males, females and sex ratio on PND 0, 4, and 13 were not affected by treatment
with the test item in any of the dose groups when compared to the controls.
The mean value of still births was slightly but statistically significantly higher in the HD group when compared to the controls
(0.30 in HD compared to 0.00 in control group).This marginal increase in the HD group mean still birth value was attributed to 1 each still birth
from just 3/10 HD females that was considered to be incidental. The mean value of still births was not affected in the LD and MD group
compared to the controls. The mean total number of pups, number of alive pups, number of males (alive & dead) was slightly but statistically
significantly lower in the LD group compared to the control group on PND 0, 4 and PND 13. This difference followed no dose dependency
and is thereby considered incidental. There was also slightly but statistically significantly lower mean number of alive pups on PND 4
(before interim sacrifice) in the HD group observed when compared to the control group which was not considered as test item related but incidental.

Litter Weight Data
Treatment with the test item had no test item related effect on litter weight data on PND 0, 4 and 13 when comparing test item-treated groups and the controls.However, mean pup weight on PND 0 was statistically significantly higher in the LD (6.66 g) and MD (6.59 g) groups compared to the control pups (5.89 g).
This effect could be attributed to slightly lower number of pups in these groups. As this difference followed no dose dependency and is within the
normal range of variation it is not considered to be toxicologically relevant.

Pup Survival Data
Treatment with the test item had no statistically significant effect on pup survival data when comparing test item-treated groups to
the control group. Mean pup mortality from PND 0 to PND 4 was 0.00% in the control group, 0.85% in the LD group, 0.00% in the MD group and 10.26%
in the HD group. Slightly increased pup mortality in the HD group compared to the control group was mainly caused by high pup mortality rate in one
HD female (female no. 78 was observed with 80% pup mortality from PND 0 to PND 4) and was not considered to be toxicologically relevant.
No pup mortality was observed in any of the test item-treated groups or the control group from PND 4 to PND 13.

Anogenital Distance and Nipple Retention
No statistically significant differences were observed in the absolute and relative anogenital distance on PND 0 when comparing male and
female test item-treated groups to the corresponding controls. There was a statistically significantly higher pup weight, cube root of pup weight
observed in all treatment groups in males and LD and MD groups in females when compared with the respective controls.
As no effect on anogenital distance was observed in both males and females, this effect has no toxicological significance in this context.
Mean nipple retention on PND 12 was statistically significantly higher in LD males when compared to the male control group
(control males: 0.00 and LD males: 0.14). Mean nipple retention of the MD and HD groups was comparable to the control group.
Due to the lack of dose dependency the slight difference in the LD group was not considered to be test item-related.

Thyroid Hormone (T4) Analysis
Mean thyroxine (T4) levels of parental males (42.61 nmol/l in the LD, 40.01 nmol/l in the MD and 35.71 nmol/l in the HD group compared
to 80.52 nmol/l in control group) and 13 day old pups (57.23 nmol/l in the LD, 45.91 nmol/l in the MD and 39.18 nmol/l in the HD group
compared to 91.80 nmol/l in control group) were statistically significantly (p < 0.001)) and dose-dependently lower in test item-treated
groups compared to the corresponding controls. Thus, this effect is considered as test item-related. However, toxicological relevance of this finding cannot be decided based on this screening study as no additional finding in the study supporting a mode of action of the test item have been identified. 
Thus, the findings on T4 cannot be considered as adverse. Furthermore, there was no test item related effect observed on pup thyroid weight. (Based on T4 results in dams and histopathology of thyroid in males, females and PND 13 pups, this section needs to be adopted). Due to significantly and dose-dependently lower T4 levels in test item-treated males and PND 13 old pups compared to the corresponding controls assessment of T4 in blood samples from the dams was done.
No test item related effect of toxicological relevance or statistical significance was observed and mean weight of thyroid/parathyroid
glands of male and female pups was comparable between test item-treated groups and the corresponding control group.

Pup External Findings
Treatment with the test item caused no gross external pup findings in any of the test item-treated groups or the control group.
Single pup external findings (one pup with a dark spot on the head in the MD group, one pup of the HD group with a dark skin area on the left hind paw,
one pale pup in the HD group) observed in this study were not considered to be test item-related.
HD dam no. 78 was observed with 10/11 pups showing no indication of suckling. This observation was not assumed to be toxicologically relevant.





Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant effect
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

The aim of this study was to assess the possible effects of the test material on male and female fertility and embryofetal development after repeated dose administration in 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 maximum 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 was 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 formulations were prepared freshly at least once every 10 days. The testitem was dissolved in corn oil and administered daily. Dose volumes were adjustedindividually based on weekly body weight measurements. The administrationvolume was 4 mL/kg body weight.

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 period 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).Due to significantly and dose-dependently lower T4 levels in test item-treated males and PND 13 old pups compared to the corresponding controls assessment of T4 in blood samples from the dams was done.

The males were sacrificed after completion of the mating period on 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 (adrenal glands, testes, epididymides, ovaries, oviducts, uterus with cervix, vagina, prostate and seminal vesicle with coagulating gland) 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. In addition,Due to significantly and dose-dependently lower T4 levels in parental males and PND 13 old pups compared to the corresponding controls, the histopathological examination of thyroid/parathyroid glands of adult males and females and PND 13 pups of the control and HD groups was performed. 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

One female from HD group (Female no. 78) was euthanized in a moribund condition on PND 4.

There were no clinical signs which could be attributed to treatment with the test item in this study. The clinical signs of piloerection, sunken flanks, hypothermia, and dehydration which were observed in prematurely sacrificed HD female no. 78 were considered to be incidental. Moving thebedding and increased salivation were noted in few animals of all test item-treated groups and the control group 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 was noted transiently and was not considered toxicologically relevant. Low incidences of few other clinical signs were observed without dose-dependency and thus were not considered to be test item-related.

Treatment with the test material had no toxicologically relevant effect on body weight development or food consumption in this study. Slight differences between test item-treated groups and the control group were within the normal range of variation and without toxicological relevance.


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 the test material.

There were no statistically significant or toxicologically relevant effects on the reproductive indices including copulation index, fertility index, delivery index, and viability index.

Slight but statistically significantly higher mean value of still births in the HD group was considered to be incidental as it was mainly attributed to single still births in few dams. The mean value of still births was not affected in the LD and MD group compared to the controls.

Statistically significant differences in mean total number of pups, number of alive pups, number of males (alive & dead) andslightly but statistically significantly lowermean number of alive pups followed no dose dependency and were thereby considered incidental.

Treatment with the test item had no toxicologically relevant effect on litter weight data in any of the test item-treated groups or the control group. Statistically significant but slightly higher mean pup weight in the LD and MD group compared to the control group observed on PND 0 was not considered toxicologically relevant.

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

Anogenital distance showed no statistically significant differences between test item-treated male and female pups and the respective controls on PND 0. Slight statistically significant higher mean nipple retention of male pups in the LD group was not considered toxicologically relevant as mean nipple retention of male MD and HD pups was comparable to the value of control males.

Mean T4 levels were shown to be dose-dependently and statistically significant lower in test item-treated adult males and 13-day old pups.Thus, this effect is considered as test item-related.However, toxicological relevance of this finding cannot be decided based on this screening study asT4 is not the only parameter to confirm a possible mode of action and T4 itself needs to be correlated with lot of other parameters in the study like histopathology of parental reproductive organs and their weights, litter size, sex ratio and estrous cyclicity. No additional findings supporting a possible mode of action of the test item have been identified. Thus, there is no conclusive evidence of the mode of action of the test item and as a result of that, the findings on T4 cannot be considered as adverse.

Isolated slight external pup findings followed no dose-dependency and were not considered to be test item-related.

No test item related gross lesions were observed during the macroscopic observation at necropsy.

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 in the mean organ weight of prostate, ovaries and uterus and thyroid/parathyroid glands of test item-treated animals compared to their controls followed no dose dependency and thus were not considered toxicologically relevant.

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

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 10 %.



1.1.       Conclusion

On the basis of this reproduction/ developmental toxicity screening test with the test material 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:

There was one HD female sacrificedin a moribund condition on PND 4due to animal welfare reasons.


There was a test item related effect observed on mean thyroxine (T4) levels in parental males and pups sacrificed on PND 13.However, toxicological relevance of this finding cannot be decided based on this screening study as no additional finding in the study supporting a mode of action of the test item have been identified.


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 group.


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

Conclusions:
On the basis of this reproduction/ developmental toxicity screening test with the test material 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:
There was one HD female sacrificed in a moribund condition on PND 4 due to animal welfare reasons.
There was a test item related effect observed on mean thyroxine (T4) levels in parental males and pups sacrificed on PND 13. However, toxicological relevance of this finding cannot be decided based on this screening study as no additional finding in the study supporting a possible mode of action of the test item.
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 the test material in this study for general toxicity and reproductive toxicity screening is considered to be 1000 mg/kg body weight/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD Guideline study under GLP conditions
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the data provided which are considered to be reliable and suitable, the test item is not classified for reproductive toxicity according to Regulation (EC) No 1272/2008.