Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-09-17 to 2013-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

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
Test material information:
Composition 1
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. 10-11 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 264 - 314 g (mean: 295.53 g, ± 20% = 236.42 – 354.63 g)
females: 183 - 217 g (mean: 199.89 g, ± 20% = 159.91 – 239.86 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.

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. 0702)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 190612)
- 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.
Before the first administration all animals 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.
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
The test item was weighed into a tarred plastic vial on a suitable precision balance and the vehicle (sterile water) was added to give the appropriate final concentration of the test item.
The vehicle was selected as suggested by the sponsor and on the basis of the test item’s characteristics. The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation was recorded for all dosing formulations.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The vehicle was also used as control item.

The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

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

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.
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 copulation. 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. In case of unsuccessful mating,
re-mating of females with proven males of the same group was considered.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration were 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 7 (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 and another sample 6 hours after
the preparation (at room temperature), from high and low dose formulations (4 samples).
All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH on particular day of sampling.
Formulation analysis was performed in accordance with GLP and the procedures followed for the determination of test item concentration
in the dosing formulations and control formulation were described in a separate study phase plan (BSL phase study No. 122442) issued
by the Principal Investigator which was amended to study plan.
Duration of treatment / exposure:
The female animals were treated with the test item formulation or vehicle on 7 days per week basis for approximately 54 days, i.e. during 14 days of pre-mating and 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:

Study Initiation Date: 17 September 2012
1st Amendment to Study Plan: 14 December 2012
Experimental Starting Date: 26 September 2012
Experimental Completion Date: 18 November 2012
Completion Date of
Delegated Phase (Histopathology): 13 February 2013

Completion Date of
Delegated Phase
(Formulation Analysis): 18 February 2013

Date of Draft Report (BSL): 20 March 2013
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg Body weight/day
Basis:
nominal conc.
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
This included the control group animals shared with BSL study No. 122429.
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Time schedule: daily
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of
anticipated effects 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.

BODY WEIGHT:
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:
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.

FOOD EFFICIENCY:
Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and
body weight gain data: Yes

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. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
SACRIFICE
The males were sacrificed any time after the completion of the mating period (minimum total dosing of 28 days). Females were sacrificed on
respective post-partum day 4 along with the pups by using an anaesthesia (ketamine/xylazin, 2:1, medistar Arzneimittel,
lot no: 00312, expiry date: 06/2014 and Serumwerk, lot no: 0312, expiry date: 05/2014) was used.
Females not delivered were sacrificed on day 26 after the sperm-positive vaginal smear as an evidence of mating.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body,
all orifices and the cranial, thoracic and abdominal cavities and their contents.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.
The number of corpora lutea and implantation sites was recorded for any females sacrificed 24 to 26 days after the end of the
pairing period with no evidence of mating and for any females sacrificed on day 25 post-coitum due to non-delivery.
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
of all adult animals were preserved in 10 % neutral buffered formalin, except for eyes, testes and epididymides which were preserved in
modified Davidson’s Solution and then transferred in 10 % neutral buffered formalin.


HISTOPATHOLOGY / ORGAN WEIGHTS
The testes and epididymides of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately. Organ weights of animals found dead were not taken.
Additional organs (all gross lesions, lung, brain, urinary bladder, stomach, lymph nodes (mesenteric and axillary), small and large intestines
(including Peyer´s patches), trachea, liver, kidneys, thymus, adrenal glands, spleen, heart)of animals which died during the course of the study
were preserved and examined histopathologically in order to determine the cause of death (to be agreed with the sponsor and with additional cost).

A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) of testes, epididymides,
ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) from C and HD group
animals and all organs showing gross lesions except discolorations due to the color of the test item were examined.
Female reproductive organs were also evaluated in non-pregnant females.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed
in the high dose group.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the
evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.

Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd. (test site for
tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was performed at the GLP-certified
contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking,
embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
Postmortem examinations (offspring):
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 5.01 software (p<0.05 was considered as statistically significant).
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Clinical Observations
No mortalities were observed at any dose levels of male and female animals.
No test item related clinical signs were observed in male and female animals during the entire study period.
However, few specific findings observed in few male and female animals of treatment groups were slight to severe piloerection,
bradykinesia, abnormal breathing, moving the bedding, alopecia on various body parts and salivation. As these clinical signs were
observed on few days of the treatment period, in very few animals and in absence of any effect on parameters of general health like
body weight and food consumption in treatment groups, these findings were considered to be non adverse.

Body Weight Development
In male and females, no statistically significant effect on body weight and body weight gain was observed throughout the study period in
treatment groups except significant increase during premating day 7-14 in HD males and significant decrease during gestation day 0-7 in
LD and MD group females was observed when compared with controls. Since it was increase in body weight gain in males and due to lack of
dose dependency in decrease in body weight gain during gestation day 0-7 in females, this significant effect was considered to be
toxicologically irrelevant.

Food Consumption
In males, statistical analysis of food consumption data revealed no statistically significant effect in treatment groups when compared with controls.
In females, statistical analysis of food consumption data revealed significant decrease during gestation day 0-7 and 7-14 in LD and MD
group females when compared with controls. As no test item related effect on group mean body weight was observed during that period
and due to lack of dose dependency in decrease in food consumption, this significant effect on food consumption was considered to be
non adverse.

Pathology
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose
of Ketamine/Xylazine (3:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males and females.
Few spontaneous gross pathological findings observed in MD and HD male animals were yellow spots on epididymides, dark discolouration
of organs like kidneys and mesenteric lymph nodes. These findings related to discolouration in males were attributed to the colour of the
test item and as such not a systemic effect due to the test item administration.

Organ Weight
In males, statistical analysis of organ weight data revealed, statistically significant decrease in absolute prostate (with seminal vesicles and
coagulating gland) weight in MD group and relative prostate weight in HD group when compared with the control. Since the difference in group
mean absolute and relative prostate weight was marginal and in the light of absence of histopathological findings, this significant effect on
prostate weight was considered to be non adverse. In females, no statistically significant effect on absolute and relative (to body weight)
organ weights was observed in any treatment group when compared with the controls.


Histopathology
Dark discoloration of kidney and/or mesenteric lymph node was observed in all males treated at 1000 mg/kg/day and a proportion of males
treated at 300 mg/kg/day. At 100 mg/kg/day, mesenteric lymph node of one single male was discolored dark. These color changes were
considered to be caused by the color of the test item itself, but were not evaluated histologically according to the study plan.
Histologically, no test item-related findings were noted in the male and female reproductive organs. Reproductive organs of most females showed
typical post-partum histomorphology. One control female, one female treated at 100 mg/kg/day and three females treated at 1000 mg/kg/day
did not show any indication of recent pregnancy at terminal sacrifice. This was considered to be unrelated to treatment, as histomorphology of
their reproductive organs indicated physiological sexual cycling.
As a conclusion, no test item-related pathological lesions were noted in male and female reproductive organs in this study.
The NOAEL (No Observed Adverse Effect Level) for pathology of reproductive organs is therefore considered to be 1000 mg/kg/day under
the conditions of this study. Macroscopic colour changes of kidney and mesenteric lymph node were considered to be directly caused by the test item, but were not evaluated histologically.


Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No findings of toxicological relevance were observed up to the highest dose tested.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Litter Data
No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth
and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4.
All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Litter Weight Data
Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight,
male litter weight and female litter weight on PND 0 and PND 4 when compared with corresponding controls.

Precoital Interval and Duration of Gestation
No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups.
All pregnancies resulted in normal births. All females in control and treatment groups showed evidence of copulation during 14 days mating
period except one female from MD group (No. 127) did not show evidence of mating through vaginal smear. However, animal was pregnant
and littered normally. Successful mating resulted in 9, 9, 10 and 7 pregnancies in the control, low, mid and high dose respectively.


Pre- and Post-Natal Data
Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0,
percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Reproductive Indices
No treatment related effect on copulation index, delivery index and viability index was observed when compared with controls.
Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in HD (70 %) dose group as compared to C (90%),
LD (90%) and MD groups (100 %).


Pup Survival Data
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.
Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed
in this study except one pup from HD group female 133 (pup No. 4) was found dead on PND 2. Since this pup mortalitiy was observed in one
female and just one pup was dead, this incidence was not considered to be test item related.


Pup External Findings
No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4.
However, following few external findings were observed:
In control females, one pup from female 45 (pup No. 3) was observed for hematoma at head on PND 0.
In LD females, one pup from female 116 (pup No. 2) was observed for redness on the nose on PND 0.
In HD females, one pup from female 140 (pup No. 2) was observed for dark spot at the neck on PND 0.
All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

Dose descriptor:
NOAEL
Remarks:
developmental
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 findings of toxicological relevance were observed up to the highest dose tested.
Reproductive effects observed:
not specified
Conclusions:
In conclusion, the repeated dose administration of FAT 40821/A in sterile water to the male (28 days) and female (maximum 54 days) Wistar rats
at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40821/A, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.
Executive summary:

The aim of this study was to assess the possible effects ofFAT 40821/Aon 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 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 were completed. Animals of an additional control group were handled identically as the dose groups but received

aqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on treatment day 30 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 postnatal day 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) from C and HD Group animals and all organs showing gross lesions except discolorations due to the color of the test item were examined. 

Female reproductive organs were also evaluated in non-pregnant females.

The following doses were evaluated:

Control:  0  mg/kg body weight; Low Dose: 100 mg/kg body weight; Medium Dose: 300 mg/kg body weight, High Dose: 1000 mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved inaqua ad injectionem(sterile water) 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. Theadministration volume was 5 mL/kg body weight.

Summary Results

Mortality

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Signs

No test item related clinical signs were observed in male and female animals during the entire study period. However, few specific findings observed in few male and female animals of treatment groups were slight to severe piloerection, bradykinesia, abnormal breathing, moving the bedding, alopecia on various body parts and salivation.

Body Weight Development

In male and females, no statistically significant effect on body weight and body weight gain was observed throughout the study period in treatment groups except significant increase during premating day 7-14 in HD males and significant decrease during gestation day 0-7 in LD and MD group females was observed when compared with controls.

Food Consumption

In males, statistical analysis of food consumption data revealed no statistically significant effect in treatment groups when compared with controls.

In females, statistical analysis of food consumption data revealed significant decrease during gestation day 0-7 and 7-14 in LD and MD group females when

compared with controls.

Litter Data

No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0

and number of males, number of females, total number of pups and sex ratio on PND 4.

Litter Weight Data

Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with corresponding controls.

Precoital Interval and duration of Gestation

No treatment related effect was observed on precoital interval and duration of gestation and valueswere comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data

Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Reproductive Indices

No treatment related effect on copulation index, delivery index and viability index was observed when compared with controls.

Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in HD (70 %) dose group as compared to C (90%), LD (90%)

and MD groups (100 %).

Pup Survival Data

No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study

except one pup from HD group female 133 (pup No. 4) was found dead on PND 2.

Pup external Findings

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings like

hematoma at head in one pup from control group female, redness on the nose in one pup from LD group female and dark spot at the neck in one pup from HD

group was observed on PND 0.

All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

Pathology

At necropsy of male and females, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females.

Few spontaneous gross pathological findings observed in MD and HD male animals wereyellow spotson epididymides, dark discolouration of organs like kidneys

and mesenteric lymph nodes.

Organ Weight

In males, statistical analysis of organ weight data revealed, statistically significant decrease in absolute prostate (with seminal vesicles and coagulating gland)

weight in MD group and relative prostate weight in HD group when compared with the control.

In females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology

Histologically, no test item-related findings were noted in the male and female reproductive organs. Reproductive organs of most females showed typical post-partum histomorphology. One control female, one female treated at 100 mg/kg/day and three females treated at 1000 mg/kg/day did not show any indication of recent pregnancy at terminal sacrifice. This was considered to be unrelated to treatment, as histomorphology of their reproductive organs indicated physiological sexual cycling.

As a conclusion, no test item-related pathological lesions were noted in male and female reproductive organs in this study. The NOAEL (No Observed Adverse Effect Level) for pathology of reproductive organs is therefore considered to be 1000 mg/kg/day under the conditions of this study.

Macroscopic colour changes of kidney and mesenteric lymph node were considered to be directly caused by the test item, but were not evaluated histologically.

Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 109.2%, 107.5% and 90.5% of the nominal concentration, respectively.

Stability of formulation samples was investigated in study week 1 for LD and HD dose groups. After 6 hours storage at -20°C recovery compared to starting value

was 112.3 and 98.9%.

Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group

was 113.9 and 105.8% of the nominal value, and 89.5 and 106.5% for HD dose group. The coefficients of variation of the different sampling locations

(top, middle, bottom) were 3.2 and 1.7% in LD dose group, and 2.5 and 2.2% in HD dose group. 

Conclusion

In conclusion, the repeated dose administration ofFAT 40821/A in sterile waterto the male (28 days) and female (maximum 54 days) Wistar rats at dosages

of 100, 300 and 1000 mg/kg body weightrevealedneither mortalities nor findings of toxicological relevance.

Based on the data generated from this reproduction/ developmental toxicity screening test withFAT 40821/A, the no observed adverse effect level (NOAEL)

is considered to be 1000 mg/kg body weight forreproduction/ developmental toxicity screening in males and females.

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 compliant guideline study, klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The aim of this study was to assess the possible effects of the test substance on male and female fertility and embryofetal development after repeated dose administration in Wistar rats (BSL Bioservice 2013). The test item dissolved in water, was administered daily to 3 groups of 10 animals/sex (0, 100, 300, 1000 mg/kg bw). Animals were treated during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. No mortality was observed and no test item related clinical signs were observed. No toxicologically relevant effects were observed on body weight development and food consumption. No test item-related findings were noted in the male and female reproductive organs. Reproductive organs of most females showed typical post-partum histomorphology. One control female, one female treated at 100 mg/kg/day and three females treated at 1000 mg/kg/day did not show any indication of recent pregnancy at terminal sacrifice. This was considered to be unrelated to treatment, as histomorphology of their reproductive organs indicated physiological sexual cycling.

At necropsy of male and females, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in mid dose and high dose male animals were yellow spots on epididymides, dark discolouration of organs like kidneys and mesenteric lymph nodes. In males, statistically significant decrease in absolute prostate (with seminal vesicles and coagulating gland) weight in mid dose group and relative prostate weight in high dose group when compared with the control was observed. In females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls. Macroscopic colour changes of kidney and mesenteric lymph node were considered to be directly caused by the test item.

No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4. Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with corresponding controls. No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births. Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls. No treatment related effect on copulation index, delivery index and viability index was observed when compared with controls. Reduced fertility index was observed in high dose group (70 %) dose group as compared to control (90%), low dose (90%) and mid dose groups (100 %). No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls. No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings like hematoma at head in one pup from control group female, redness on the nose in one pup from LD group female and dark spot at the neck in one pup from HD group was observed on PND 0. All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

As a conclusion, no test item-related pathological lesions were noted in male and female reproductive organs in this study. The NOAEL (No Observed Adverse Effect Level) for fertility and developmental toxicity is therefore considered to be 1000 mg/kg/day under the conditions of this study.


Short description of key information:
Under the conditions of the reproduction/ developmental toxicity screening test (OECD 421, GLP), the NOAEL for fertility and developmental toxicity was determined to be 1000 mg/kg bw.

Justification for selection of Effect on fertility via oral route:
Only 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 absence of adverse effect on reproductive organs or tissues in the reproduction/developmental toxicity screening test, classification is not necessary for toxicity to fertility in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Based on the absence of developmental effects in the reproduction/developmental toxicity screening test classification is not necessary for developmental toxicity in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.