Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL for reproductive/developmental toxicity was 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-20 to 2014-09-11
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 section under results
Qualifier:
according to
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
Version / remarks:
July 2000
Deviations:
yes
Remarks:
See overall remarks section under results
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Q30420AAYB
- Expiration date of the lot/batch: 08/08/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: 1.29 mg/L in water
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy Wistar rats (male and female), Crl: WI(Han) (Full Barrier) from Charles River, 97633 Sulzfeld, Germany
Sex:
male/female
Details on test animals and environmental conditions:
Test System
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: males: 10-11 weeks old, females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 268 - 325 g (mean: 297.00 g, ± 20 % = 237.60 – 356.40 g)
females: 186 - 220 g (mean: 200.14 g, ± 20 % = 160.11 – 240.17 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
The animal study was authorized by local government under file no. 55.2-1-54-2532.2-11-11.

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 (lot no. 0801)
- 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 kept pairwise in IVC cages (except during gestation/lactation period when females were housed individually), type III H,
polysulphone cages on Altromin saw fibre bedding (lot no. 131113). During pre-mating period animals were kept in groups of 2 animals of the same gender.
For mating one female was paired with one male. During post-mating period males were housed with their respective pre-mating partners.
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. No animal arriving at BSL showed pathological signs.
Before the first administration all animals used for the study were weighed. Mean body weight of the pairwise kept animals was used to assign all animals to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partner.
Each animal was marked with its identification number by individual ear tattoo.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Experimental Groups and Doses
According to the results of a previous dose range finding study (BSL study no. 140286) 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) and 1 control group (C).
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 54 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 3 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

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

The highest dose level was chosen with the aim of inducing toxic effects, but not 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 corn oil using the same volume as used for the dose groups.

Administration of Doses
The test item formulation and vehicle were administered at a single dose to the animals by oral gavage with disposable polypropylene feeding tubes for rats (78 mm long; 3.0 mm diameter; Instech Laboratories Inc). The application volume for all groups was 5 mL/kgbody 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). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. 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:
Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of 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). Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation, 6 hours after the preparation (at room temperature) and another sample 10 days after the preparation (stored at 2-8 °C), from high and low dose formulations (6 samples). Each sample was retained twice (sample A, sample B). The dose formulation analysis was performed by BSL BIOSERVICE Scientific Laboratories GmbH, in accordance with GLP. All formulation samples were stored at -15 °C to -35 °C. The A samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140289. The exact procedure was described in a phase plan which was amended to the study plan. The B samples will be retained at BSL until the analysis has been performed, 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 54 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 3 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:
Arrival of the Test Item: 07 January 2014
Study Initiation Date: 20 March 2014
Delivery of Animals: 20 March 2014
Acclimatisation Period: 20 March 2014 till 26 March 2014
Experimental Starting Date: 27 March 2014
Experimental Completion Date: 21 May 2014
Completion Date of Delegated Phase (Histopathology): 08 September 2014
Completion Date of Delegated Phase (Formulation Analysis): 01 September 2014
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low Dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose
No. of animals per sex per dose:
10 per sex per dose (Total 80)
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 54 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 3 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.
The test item formulation was prepared freshly at least once every ten days. The test item was suspended in corn oil and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.
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 and on day 4 post-partum.
The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.
Parental animals: Observations and examinations:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day after dosing. 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 behaviourwere recorded.

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period 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) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice. Food consumption was measured weekly 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.
Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. 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 and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Livepups were identified by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
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 4 using anaesthesia (ketamine/xylazin, 2:1, Pharmanovo GmbH, lot no: 24664, expiry date: 06/2015; Serumwerk, lot no: 00513, expiry date: 05/2015 and Serumwerk, lot no: 01213, expiry date: 10/2015) and exsanguinating them. Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating. All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions (including discoloration) of all adult animals were preserved in 4% neutral-buffered formaldehyde (10% neutral buffered formalin), except for testes and epididymides which were preserved in modified Davidson’s Solution for approximately 24 hours and then transferred in 70% ethanol. The macroscopic finding of discoloured yellow adipose tissue of male animals no. 11 – 22 from LD and MD groups was recorded, but no samples of these macroscopic lesions were preserved. The macroscopic finding in the liver (hernia diaphragmatica) in female no. 48 from the control group was not preserved. Organs of one animal (no. 76, HD group) which was euthanized prematurely for animal welfare reasons were preserved and examined histopathologically in order to determine the cause of death (all gross lesions, brain, stomach, small and large intestines (including Peyer’s patches), liver, thymus, spleen, ovaries, uterus with cervix, lung, urinary bladder, lymph nodes (mesenteric and axillary), trachea, kidneys, adrenal glands, heart, vagina). The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland as a whole) of all male adult animals as well as the ovaries and uterus with cervix of all female adult animals were weighed. Paired organs were weighed together. Organ weights of one animal which was euthanized prematurely were not taken.

Histopathology
A full histopathology was carried out on the preserved organs and tissues of one animal which was euthanized due to morbidity. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in control and HD animals and in non-pregnant female animals of the LD (animal no. 51) and MD group (animal no. 67). Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners (no. 11 and no. 27) of the non pregnant female LD and MD animals. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. 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). 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 was 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 sacrificed by decapitation on post-natal day 4. Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Offspring viability indices:
There were no statistically or biologically significant effects on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group. A slightly higher mean mortality of pups between PND 1 and PND 4 was observed in the LD group (9.3%) and the HD group (4.1%) compared to 0.0% in the control group. As this outcome did not achieve statistical significance and did not show a dose response pattern, it is considered incidental and not related to the test item. Regarding female no. 57 of the LD group, 7 pups were missing on PND 1 or 2, two pups were found dead on PND 1 and one pup was euthanized on PND 3 due to animal welfare reasons. Two pups from female no. 72 of the HD group were missing on PND 2. The loss of pups was attributed to cannibalism by the dam.
Clinical signs:
no effects observed
Description (incidence and severity):
However, the skin of all male and female animals of the LD, the MD and the HD group was observed to be slightly discoloured yellowish from approximately the third week of treatment until the end of the study due to the properties of the coloured test item. Occasional incidences of salivation were noted in one male animal of the MD group, most males of the HD group and all females of the HD group. Moving the bedding was noted transitorily in some males and females of the MD group and in all males and females of the HD group, respectively. As the symptoms of salivation and moving the bedding were noted mainly immediately after administration, these signs are considered to be a local effect of the test item applied via gavage.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Deposition of eosinophilic substance in the proximal tubular epithelium and proximal tubular degeneration/regeneration, which were recorded in all or most animals examined histopathologically, and granular casts and/or tubular dilatation recorded in some males. There were not clear dose-relationships in group mean severity of these findings. In addition, since neither tubular degeneration/regeneration nor single tubular cell death was observed in 2 high-dose females despite the presence of eosinophilic substance deposits, it is unlikely that the tubular degenerative changes were caused by direct injurious effect of the test item. From these findings, it was considered that the deposition of eosinophilic substance was the histomorphologic representation indicating physiologic excretion processes that occurred by re-absorption of the test item or its metabolites, and the tubular degeneration/regeneration, as well as granular casts and tubular dilatation, were considered to be the secondary events following the excess re-absorption, and therefore, these renal findings were considered not to be adverse. In female no. 76 of the HD group which was euthanized for animal welfare reasons on gestation day 23, black content was recorded in the stomach. Some other histologic alterations were recorded at a minimum severity or focally in a few animals from both of the control and high-dose groups, but all of these were within the range of normal background lesions which are observed in males of this strain and age. Not considerd as treatment related.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. The fertility index (number of pregnant females/ number of copulated females x 100) (80 % in control group, 90 % in LD, 100 % in MD and 80 % in HD group) was within the normal range of variation for this strain and within historical control data. The cause of the unsuccessful pregnancy and/or infertility of females no. 45 and 50 of control group, no. 51 (LD group), no. 67 (MD group) and no. 71 and 74 of HD group as well as their male pairing partners (no. 5, 10, 11, 27, 31 and 34), respectively, could not be established from the reproductive organs examined histopathologically from these animals. The viability index (no. of live offspring at day 4 / no. of live offspring at birth) X 100) was slightly lower in the LD group (90.7 %) and the HD group (95.9 %) when compared to 100% in the control group. As this was attributed to the death of pups in one single dam per group, respectively, and without dose-dependency, this finding is considered incidental. A slightly reduced delivery index in the HD group (87.5 %) compared to 100 % in the control group was based on pregnant female no. 76 of the HD group which was euthanized for animal welfare reasons before littering. At necropsy 8 dead foetuses were found in its uterus. After histopathological examination of the organs, this animal’s morbidity is not considered to be related to the treatment with the test item. The remaining pregnant females of HD group littered normally.
Mortality
Female no. 76 of the HD group was euthanized on gestation day 23 for animal welfare reasons. At necropsy 8 dead foetuses were found in the uterus. Histomorphologic findings leading directly to this animal’s morbidity (anaemia, apathy, vocalization, moderately reduced spontaneous activity, red nasal discharge and slight piloerection) were not identified. As a result of the histopathological examination, the animal’s morbidity is considered not to be directly related to the treatment with the test item. It was likely caused by dystocia which was also probably the cause of the death of the foetuses. With this exception, all animals survived the treatment period.

Clinical Observations
There were no clinical signs of toxicological relevance in the dose groups, when compared to the control group. However, the skin of all male and female animals of the LD, the MD and the HD group was observed to be slightly discoloured yellowish from approximately the third week of treatment until the end of the study due to the properties of the coloured test item. Slight to severe salivation before and after dose application was noted transitorily in one single male animal of the MD group. There were occasional incidences of slight to moderate salivation in most males of the HD group and severe salivation in one male of the HD group. Slight to moderate salivation was also observed in all females of the HD group and severe salivation in two females of the HD group. Moving the bedding was noted transitorily in 8 males of the MD group and all males of the HD group as well as in 4 females of the MD group and all females of the HD group. As the symptoms of salivation and moving the bedding were noted mainly immediately after administration, these signs are considered to be a local reaction to the test item rather than a systemic effect. Slight piloerection was observed in the control group (one female), the LD group (two females), the MD group (one female) and the HD group (two females). As the clinical sign of piloerection was transitory in nature and did not follow a dose-related pattern, it is not considered to be test item-related. Low incidences of slight clinical signs like alopecia, a crust at the cheek, aggressiveness, slight diarrhea and nasal discharge were noted in isolated males and/or females of the dose groups and/or the control group. As these findings were mostly transient and seen irrespective of the groups in isolated animals, they are considered to be incidental. Prior to euthanasia for animal welfare reasons on gestation day 23, female no. 76 of the HD group showed anaemia, apathy, vocalization, moderately reduced spontaneous activity, red nasal discharge and slight piloerection (see 12.1 and 12.14). The animal’s deteriorated health condition was not noted until gestation day 23. At necropsy 8 dead foetuses were found in the uterus. Therefore, the animal’s morbidity and death of the foetuses was likely caused by parturition difficulties (dystocia).

Body Weight Development
In both males and females, the mean body weight increased with the progress of the study during premating, mating/postmating and gestation period in the control, the LD, the MD and the HD group. There were no test item-related effects of toxicological relevance noted on body weight and body weight gain in both males and females. A slight tendency towards a lower body weight was observed in the female LD and MD group during gestation compared to the control group, but lacked statistical significance and values were within the normal range of variation of historical control data. In the second week of treatment the mean body weight gain of the female HD group was statistically significantly higher compared to the control group (13.20 g compared to 6.40 g). In the first week of gestation body weight gain of the female HD group was also slightly higher compared to controls, but without achieving statistical significance. As during the remaining treatment period no such effect was seen and values were within the normal range of variation for animals of this strain, this is not assumed to be toxicologically relevant.

Food Consumption
There were no biologically significant effects on food consumption during the treatment period in both males and females of the dose groups, when compared to the respective controls. In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in the control, the LD, the MD and the HD group. However, during both weeks of premating, the food consumption in the male LD and the male HD group was slightly (between 6 % and 9 %) lower as compared to the respective control group. Though reaching statistical significance, without following a dose dependent pattern these findings are not considered to be toxicologically relevant and are within a normal biological variation. Females of the MD and the HD group were noted to have a slightly (each 9%) lower food consumption in the first week of premating when compared to the control group. This effect was not seen in the second week of premating and is therefore considered as biological variation. In accordance to an attenuated slightly higher body weight gain of the female HD group in the second week of premating, the food consumption was slightly higher compared to the other dose groups and control group, but did not reach statistical significance. In accordance to the tendency of a marginal lower body weight during the gestation period, a slight, statistically significant, tendency of decreased food consumption during gestation was observed in the females of the LD and MD group compared to the control group. This effect was not observed in the HD group and is therefore not considered to be test item-related.

Pathology- Macroscopic Findings
There were no macroscopic findings of toxicological relevance in any of the groups. However, adipose tissue of each male and female animal of the LD, MD and HD group was noted to be discoloured yellow which was attributable to the treatment with the coloured test item. In addition, yellow discoloration was recorded in pancreas and mammary glands of the euthanized female no. 76 of the HD group. Macroscopic yellow discoloration in these organs and tissues was considered to be due to distribution of the test item (or its metabolites) into the adipose tissues which are present in and around each organ, or to be due to the presence of it in the lympho-vascular system. However, in the adipose tissue, pancreas and mammary glands, there was no histologic alteration that correlated with the macroscopic findings. Discoloured yellow kidneys were observed in 8 males and one female of the LD group, in all males and no females of the MD group and in 7 males and 3 females of the HD group. It was assumed that the test item produces eosinophilic substance deposition in the kidney, which was considered to be related to the excretion process of the test item and/or its metabolites, being followed by the secondary changes including tubular degeneration/regeneration, granular casts and/or tubular dilatation due to excess deposition (re-absorption) without clear dose relationship in severity. These renal changes were not considered to be adverse. In female no. 76 of the HD group which was euthanized for animal welfare reasons during the course of the study, black content was recorded in the stomach. This was considered to be associated with glandular stomach erosion and probably duodenal erosion (inflammatory cell infiltration with hemorrhagic spots in the lamina propria of the pyloric-duodenal junction) that were recorded microscopically. Slight single or occasional findings like pelvic kidney dilatation, hernia diaphragmatica, hemorrhagic testes, fluid filled uterus or discoloured red ovaries were observed in control animals and/or are assumed to be common background findings in this strain and age.

Organ Weight
In females, there were no statistically or biologically significant effects on the absolute and relative organ weights in the dose groups, when compared to the control group. In males, there was a marginal tendency towards a lower mean weight of epididymides in the LD and MD group compared to the control group. Values were within the range of historical control data but achieved statistical significance in the LD and the MD group when related to body weight (10 % below the control group, respectively). Without following a dose response pattern, this is considered to be incidental. There were no effects of toxicological relevance on the mean weight of testes and prostates.

Histopathology
Under the conditions of this study, the test item FAT 93450/Z TE produced no histological evidence of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix, and vagina. In addition, as a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. However, treatment-related histomorphologic changes were observed in the kidneys that were examined as gross lesion. Those consisted of deposition of eosinophilic substance in the proximal tubular epithelium and proximal tubular degeneration/regeneration, which were recorded in all or most animals examined histopathologically, and granular casts and/or tubular dilatation recorded in some males. There were not clear dose-relationships in group mean severity of these findings. In addition, since neither tubular degeneration/regeneration nor single tubular cell death was observed in 2 high-dose females despite the presence of eosinophilic substance deposits, it is unlikely that the tubular degenerative changes were caused by direct injurious effect of the test item. From these findings, it was considered that the deposition of eosinophilic substance was the histomorphologic representation indicating physiologic excretion processes that occurred by re-absorption of the test item or its metabolites, and the tubular degeneration/regeneration, as well as granular casts and tubular dilatation, were considered to be the secondary events following the excess re-absorption, and therefore, these renal findings were considered not to be adverse. Although substance deposition in the tubular epithelium of the kidney might have been involved partially in the macroscopic alteration in the strength or tone of color, as with the other organs and tissues it was mostly considered to be due to distribution of the test item (or its metabolites) into the adipose tissues which are present in and around each organ, or due to the presence of it in the lympho-vasuclar system, and therefore, it was unlikely that macroscopic discoloration in kidneys correlated wholly with histologic eosinophilic substance deposit. In female no. 76 of the HD group which was euthanized for animal welfare reasons on gestation day 23, black content was recorded in the stomach. This was considered to be associated with glandular stomach erosion and probably duodenal erosion (inflammatory cell infiltration with hemorrhagic spots in the lamina propria of the pyloric-duodenal junction) that were recorded microscopically. All lesions, except for eosinophilic substance deposition in the kidney, were non-specific alteration that generally occurs under the stressful condition in the debilitated animals or physiologic responses that occurs during pregnancy, or were within the range of normal background findings which is frequently recorded in animals of this strain and age. Since eosinophilic substance deposition in the kidney was minimal in degree and the tubular degeneration/regeneration was not observed in this animal, such minimal renal change is unlikely to be a cause of the animal’s morbidity. As a result, although histomorphologic findings leading directly to the animal’s morbidity were not identified under the condition of this study, any lesions that could be attributable to treatment with the test item were not observed, and therefore, it was considered that at least the animal’s morbidity was not directly related to treatment with the test item. It was probably caused by parturition difficulties. Some histologic alterations were recorded at a minimum severity or focally in a few animals from both of the control and high-dose groups, but all of these were within the range of normal background lesions which are observed in males of this strain and age. In this context, the cause of the unsuccessful pregnancy and/or infertility of female nos. 45 and 50 (control group), no. 51 (LD group), no. 67 (MD group) and nos. 71 and 74 (HD group) and their male pairing partners nos. 5 and 10 (control group), no. 11 (LD group), no. 27 (MD group) and nos. 31 and 34 (HD group), respectively, could not be established from the reproductive organs examined from these animals.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined in study weeks 1, 3, 5 and in the last week for all dose groups. The mean recoveries observed in the LD, the MD and the HD group were 81.9 %, 85.3 % and 88.9 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as mean of measured concentration did not differ from nominal concentration by more than 20 %. Single values (one sample of the HD group (study week 3) with 77.2 %; and two samples of the LD group (study week 1 and 3) with 78.7 % and 68.0 %, respectively) were slightly beyond 20% difference from the nominal concentration. This is considered to have no influence on the validity of the present study, as no adverse findings were seen in the LD, the MD and the HD group. Regarding the HD group, the difference from the nominal concentration was in one single sample marginally beyond 20 % and mean nominal concentration was confirmed from the first to the last week of the study. Stability of formulation samples was investigated in study week 1 for the LD and the HD group. After 6 hours of storage at room temperature, recovery compared to starting value was 96.6% and 100.2 % for LD and HD group, respectively. After 10 days of storage at 2-8 °C, recovery compared to starting value was 100.3 % and 112.0 % for LD and HD group, respectively. All samples were stable, as concentration after storage did not differ from start value by more than 20 %.
Homogeneity of formulation samples was determined in study week 1 and 5 for the LD and the HD group. The mean recovery observed for the LD group was 82.9% and 102.5 % of the nominal value and 85.3 % and 110.2 % of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 4.4 % and 9.3 % in LD dose group and 10.4 % and 11.2 % in HD dose group. All samples were homogenous, as COV was below 20 %.

Litter Data
There were no effects of toxicological relevance for litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes noted for these litter data. However, there was a very slightly – but not statistically significantly – higher number of still births in the HD group (0.57) when compared to the control group (0.00). As this was attributed to one single dam, this small difference was considered to have no toxicological relevance.

Litter Weight Data
There were no statistically or biologically significant effects on the litter weight data including pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4. Values were within the normal range of variation for animals of this strain.

Precoital Interval and Duration of Gestation
There were no statistically or biologically significant effects on the duration of precoital interval and the duration of gestation in the dose groups, when compared to the control group. Values were within the normal range of variation of historical control data.

Pre- and Post-Natal Data
There were no statistically or biologically significant effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of pre- and post-implantation loss in the dose groups, when compared to the control group. A slight tendency towards a lower post-implantation loss in the dose groups (10 % in the LD, 9 % in the MD, 8 % in the HD group) compared to 14% in the control group is considered incidental.

Reproductive Indices
There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. The fertility index (number of pregnant females/ number of copulated females x 100) (80% in control group, 90 % in LD, 100 % in MD and 80 % in HD group) was within the normal range of variation for this strain and within historical control data. The cause of the unsuccessful pregnancy and/or infertility of females no. 45 and 50 of control group, no. 51 (LD group), no. 67 (MD group) and no. 71 and 74 of HD group as well as their male pairing partners (no. 5, 10, 11, 27, 31 and 34), respectively, could not be established from the reproductive organs examined histopathologically from these animals. The viability index (no. of live offspring at day 4 / no. of live offspring at birth) X 100) was slightly lower in the LD group (90.7 %) and the HD group (95.9 %) when compared to 100 % in the control group. As this was attributed to the death of pups in one single dam per group, respectively, and without dose-dependency, this finding is considered incidental. A slightly reduced delivery index in the HD group (87.5 %) compared to 100 % in the control group was based on pregnant female no. 76 of the HD group which was euthanized for animal welfare reasons before littering. At necropsy 8 dead foetuses were found in its uterus. After histopathological examination of the organs, this animal’s morbidity is not considered to be related to the treatment with the test item. The remaining pregnant females of HD group littered normally.

Pup Survival Data
There were no statistically or biologically significant effects on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group. A slightly higher mean mortality of pups between PND 1 and PND 4 was observed in the LD group (9.3 %) and the HD group (4.1 %) compared to 0.0 % in the control group. As this outcome did not achieve statistical significance and did not show a dose response pattern, it is considered incidental and not related to the test item. Regarding female no. 57 of the LD group, 7 pups were missing on PND 1 or 2, two pups were found dead on PND 1 and one pup was euthanized on PND 3 due to animal welfare reasons. Two pups from female no. 72 of the HD group were missing on PND 2. The loss of pups was attributed to cannibalism by the dam.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Pup external findings:
No gross external abnormalities of toxicological relevance were observed in any of the groups. However, the skin of all pups of the LD, MD and HD group was observed to be discoloured orange since PND 1 or 2 due to the properties of the coloured test item. A dark snout observed in one single pup of the control group is considered incidental. Few additional incidental findings were noted in pups from one isolated female no. 57 of the LD group such as cold pups with milk barely visible and one dehydrated pup. There were few incidental external findings in the MD group (dark bluish nose in pup no. 1 of dam no. 63) and the HD group (bite injury at hindpaw in pup no. 7 of dam no. 72, scaly skin in pup nos. 2-6 of dam no. 72). These findings are considered to be spontaneous and not related to the test item.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect seen
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
On the basis of this reproduction/ developmental toxicity screening test the NOAEL for FAT 93450/Z TE in male and female Wistar rats is considered to be 1000 mg/kg bw/day.
Executive summary:

The objective of this study was to assess the possible effects of FAT 93450/Z TE 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 54 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 3 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.

The following doses were evaluated:

Control:                         0        mg/kg bw/day

Low Dose:                    100     mg/kg bw/day

Medium Dose:              300     mg/kg bw/day

High Dose:                   1000   mg/kg bw/day

The test item formulation was prepared freshly at least once every ten days. The test item was suspended in corn oil and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosedfor 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight . During the period of administration, the animals were observed each day for signs of toxicity. One animal that was euthanized for animal welfare reasons was 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 and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post natal day 4. 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 postnatalday 4 and those found dead or euthanized for animal welfare reasons, were carefully examined for gross external abnormalities. The testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland as a whole) of all male adult animals as well as the ovaries and uterus with cervix of all female adult animals were weighed. A histopathological evaluation of testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs (prostate, seminal vesicle with coagulating gland) was performed on high dose and control animals, as well as in non-pregnant female animals of the LD and MD females and their male mating partners.Any gross lesion macroscopically identified was examined microscopically in all animals. A set of organs of one animal which was euthanized during the course of the study was preserved and examined histopathologically in order to determine the cause of death. Female no. 76 of the HD group was euthanized on gestation day 23 for animal welfare reasons. At necropsy 8 dead foetuses were found in the uterus. Histomorphologic findings leading directly to this animal’s morbidity (anaemia, apathy, vocalization, moderately reduced spontaneous activity, red nasal discharge and slight piloerection) were not identified. As a result of the histopathological examination, the animal’s morbidity is considered not to be directly related to the treatment with the test item. It was likely caused by dystocia which was also probably the cause of the death of the foetuses. With this exception, all animals survived the treatment period. There were no clinical signs of toxicological relevance in the dose groups, when compared to the control group. However, the skin of all male and female animals of the LD, the MD and the HD group was observed to be slightly discoloured yellowish from approximately the third week of treatment until the end of the study due to the properties of the coloured test item. Occasional incidences of salivation were noted in one male animal of the MD group, most males of the HD group and all females of the HD group. Moving the bedding was noted transitorily in some males and females of the MD group and in all males and females of the HD group, respectively. As the symptoms of salivation and moving the bedding were noted mainly immediately after administration, these signs are considered to be a local effect of the test item applied via gavage. No test item-related effects of toxicological relevance were noted for body weight and body weight gain in both males and females of any of the dose groups. There were no test item-related effects of toxicological relevance on food consumption during the treatment period in both males and females.

There were no test item-related effects of toxicological relevance for litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4.

There were no test item-related effects of toxicological relevance for litter weight data including pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4. There were no test item-related effects of toxicological relevance on the duration of precoital interval and the duration of gestation. There were no test item-related effects of toxicological relevance on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of pre and post-implantation loss. There were no test item-related effects of toxicological relevance on the reproductive indices (copulation, fertility, delivery and viability indices). There were no test item-related effects of toxicological relevance on the survival of the pups from PND 0 through PND 4. No gross external abnormalities of toxicological relevance were observed in pups of the dose groups, when compared to the control group. However, the skin of all pups of the LD, MD and HD group was observed to be discoloured orange since PND 1 or 2 due to the properties of the coloured test item. In males and females, there were no differences of toxicological relevance in absolute and relative organ weights between the dose groups and the control group. There were no macroscopic findings of toxicological relevance noted in any of the groups. However, adipose tissue of each male and female animal of the LD, MD and HD group was noted to be discoloured yellow which was attributable to the treatment with the coloured test item. In addition, yellow discoloration was recorded in pancreas and mammary glands of the euthanized female no. 76 of the HD group. Macroscopic yellow discoloration in these organs and tissues was considered to be due to distribution of the test item (or its metabolites) into the adipose tissues which are present in and around each organ, or to be due to the presence of it in the lympho-vascular system. However, in the adipose tissue, pancreas and mammary glands, there was no histologic alteration that correlated with the macroscopic findings. Discoloured yellow kidneys were observed in 8 males and one female of the LD group, in all males and no females of the MD group and in 7 males and 3 females of the HD group. It was assumed that the test item produces eosinophilic substance deposition in the kidney, which was considered to be related to the excretion process of the test item and/or its metabolites, being followed by the secondary changes including tubular degeneration/regeneration, granular casts and/or tubular dilatation due to excess deposition (re-absorption) without clear dose relationship in severity. These renal changes were not considered to be adverse. The test item produced no histological evidence of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix, and vagina. As a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. The cause of the unsuccessful pregnancy and/or infertility of female nos. 45 and 50 (control group), no. 51 (LD group), no. 67 (MD group) and nos. 71 and 74 (HD group) and their male pairing partners nos. 5 and 10 (control group), no. 11 (LD group), no. 27 (MD group) and nos. 31 and 34 (HD group), respectively, could not be established from the reproductive organs examined from these animals. Concentration of FAT 93450/Z TE in formulation samples was determined in study weeks 1, 3, 5 and in the last week for all dose groups. The mean recoveries observed in the LD, the MD and the HD group were 81.9 %, 85.3 % and 88.9 % of the nominal concentration, respectively. Stability of formulation samples was investigated in study week 1 for the LD and the HD group. After 6 hours of storage at room temperature, recovery compared to starting value was 96.6 % and 100.2 % for LD and HD group, respectively. After 10 days of storage at 2-8 °C, recovery compared to starting value was 100.3 % and 112.0 % for LD and HD group, respectively. Homogeneity of formulation samples was determined in study week 1 and 5 for the LD and the HD group. The mean recovery observed for the LD group was 82.9 % and 102.5 % of the nominal value and 85.3 % and 110.2 % of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 4.4 % and 9.3 % in LD dose group and 10.4 % and 11.2 % in HD dose group. Conclusion On the basis of this reproduction/ developmental toxicity screening test with FAT 93450/Z TE 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: Histomorphologically observed renal changes (secondary changes including tubular degeneration/regeneration, granular casts and/or tubular dilatation) with eosinophilic substance deposition without clear dose relationship in severity are not considered to be adverse. Thus, the NOAEL in this study is considered to be 1000 mg/kg bw/day. On the basis of this reproduction/ developmental toxicity screening test the NOAEL for FAT 93450/Z in male and female Wistar rats is considered to be 1000 mg/kg body weight.

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:
GLP study and conducted according to OECD Guideline .
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In order to assess the possible effects of FAT 93450/Z on male and female fertility and embryofetal development, reproduction / developmental toxicity screening test was performed according to OECD guideline 421. There were no clinical signs of toxicological relevance in the dose groups, when compared to the control group. However, the skin of all male and female animals of the LD, the MD and the HD group was observed to be slightly discoloured yellowish from approximately the third week of treatment until the end of the study due to the properties of the coloured test item. Histomorphologically observed renal changes (secondary changes including tubular degeneration/regeneration, granular casts and/or tubular dilatation) with eosinophilic substance deposition without clear dose relationship in severity are not considered to be adverse. No adverse effects on reproduction and developmental parameters was seen. Thus, the NOAEL in this study is considered to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

The NOAEL for reproductive/developmental toxicity was 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
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

No classification for reproductive toxicity is needed according to CLP regulation (EC) No 1272/2008.