3,3'-[(4-{(E)-[2-bromo-4-nitro-6-(trifluoromethyl)phenyl]diazenyl}phenyl)imino]dipropanenitrile

Endpoint summary

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:

2013-08-16 to 2014-09-12

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000.
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München , Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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: males: 10-11 weeks old, females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 266 - 309 g (mean: 288.30 g, ±20% = 230.64 – 345.96 g)
females: 177 - 205 g (mean: 191.78 g, ±20% = 153.42 – 230.13 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. 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)
- The animals were kept individually in IVC cages (except during the mating period when one female was paired with one male), type III H,
polysulphone cages on Altromin saw fibre bedding (lot no. 240113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

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 precision balance. The required volume of aqua ad iniectabilia was added and vortexed.
The formulation samples were subjected to homogenization for 1 to 2 minute using a homogenizer. Homogeneity of the test item
in the vehicle was maintained by constantly agitating the sample manually.
The formulation was immediately used for dose administration to animals.
The vehicle selected was used as per the study monitor’s suggestion. The test item formulation was prepared freshly on each administration day
before the administration procedure.
The vehicle (aqua ad iniectabilia) was also used as control item.

The followig doses were evaluated
Control: 0 mg/kg Body weight
Low Dose: 100 mg/kg Body weight
Mid 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 aqua ad iniectabilia using the dose
volume 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 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 analyzed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle was analyzed 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 and another sample 6 hours after the preparation (at room temperature), from high and low dose formulations (4 samples).
All formulation samples required for analytical verification were collected on the specified days (see above) and were stored at -20 °C. These samples were
analyzed at the end of the study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 134433.

Duration of treatment / exposure:

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 were completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Arrival of the Test Item: 23 July 2013
Study Initiation Date: 16 August 2013
1st Amendment to Study Plan: 16 September 2013
2nd Amendment to Study Plan: 16 October 2013
Experimental Starting Date: 20 August 2013
Experimental Completion Date: 05 October 2013
Completion Date of Delegated Phase (Histopathology): 28 August 2014
Completion Date of Delegated Phase (Formulation Analysis): 08 September 2014

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg body weight
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).

Control animals:

yes, concurrent vehicle

Positive control:

Not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Time Schedule: Daily
General clinical observations were made once at the same time each day. The health condition of the animals was recorded. Twice daily all animals
were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea,
changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to
handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT:
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.

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.

Oestrous cyclicity (parental animals):

Not Evaluated

Sperm parameters (parental animals):

Not Evaluated

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 sex identified and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum.
Live pups were identified by tattoo mark on paw. 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 an anaesthesia (ketamine/xylazin, 3:1, Pharmanovo, lot no: 24432, expiry date: 01/2015 and Serumwerk,
lot no: 00512, expiry date: 07/2014) was used.
Non-pregnant females were sacrificed on day 26 after the evidence of mating or the last day of mating period.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
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 testes and epididymides which were preserved in modified Davidson’s Solution and then
transferred in 70% Ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.


Organ Weights
The testes, epididymides and prostate (with seminal vesicle and coagulating gland) 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.

 
Histopathology
Testes, epididymides, prostate, seminal vesicle with coagulating gland, ovaries, uterus with cervix, vagina and all organs showing gross lesions
were examined in Control and HD animals and in non pregnant female animals of the LD and MD animals.
Histological examination of the reproductive organs of the two non pregnant female animals of low dose was carried out (no. 54 and 56).
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). 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 tissue processing issued a detailed phase plan which was attached to the study plan per amendment. The principal
histopathological investigator 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 pups were killed on day 4 post-partum by decapitation and 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).

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects; see below for details

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

no adverse effects; see below for details

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

Clinical Observations:
There were no clinical signs considered to be of toxicological relevance in male and female animals of dose groups. However, there were clinical signs including nasal discharge, piloerection, slight diarrhea, alopecia, abnormal breathing, exophthalmos, aggressiveness and scab noted in a single or
few animals of control and/or treated groups. These findings were transient.
Alopecia was seen in a single isolated animal of control and MD groups and was not considered to be of toxicological relevance.

Body Weight Development:
In males and females, there were no adverse treatment related changes for body weight and body weight gain during the study period. However, in
males there was statistically significantly lower body weight gain after mating/ postmating days 7-14 in HD group. Most of the males in HD group did not gain weight from mating/ postmating day 7 through day 14, but there was a weight gain noticed the next consecutive day (terminal
sacrifice). There were no relevant clinical signs (except for slight diarrhea in single animal) that could explain lower weight gain. Therefore, the
finding was not considered to be an adverse effect of treatment.
In females, there were statistically significantly lower body weight on GD 14 in LD and HD groups and lower body weight gain after premating days 1-7 in HD group when compared to corresponding control group. The changes in LD and HD group did not show dose response relation. The
changes in weight gain after premating days 1-7 in HD group was transient and all animals in the dose groups gained body weight (except animal
64 in MD group) with the study progress. Hence, this finding was not considered to be an adverse.
Furthermore, in females there was lower body weight gain in LD, MD and HD groups between the lactation days 0-4. This finding correlated to the
changes in food consumption, but in the absence statistically significant differences and relevant clinical signs the finding was not considered to
be an adverse effect of treatment.

Food Consumption:
In males and females, there were no adverse treatment related changes in food consumption during the study period. However, in males there was a
slightly lower food consumption in HD group after premating days 7-14, but in the absence of statistically significant difference the finding was not
considered adverse. In females there was a statistically significantly lower food consumption after premating days 7-14 in HD group, but the finding
did not correlate with the body weight data. In addition, there was slightly lower food consumption during the gestation days in LD and HD groups
when compared to control, but without statistical significance or dose response relation.
Furthermore, there was also a lower food consumption after lactation days 0-4 in LD, MD and HD groups in a dose response relation, which
correlated to the lower body weight gain. In the absence of statistical significance the finding was not considered to be an adverse affect of treatment.

Pathology:
There were no macroscopic findings related to the treatment with the test item.
The few findings recorded, sparsely distributed among control and treated animals, were considered to be within the range of normal background
alterations.

Organ Weight:
In males and females, there were no toxicologically relevant changes in organ weight (absolute and relative to body weight) data in treated groups
when compared to control. There were no statistically significant differences in absolute and relative organ weight.
However, in females there was marginally higher mean absolute and relative ovary weight in MD group and relative uterus (with cervix) weight in HD
group when compared to control, but these changes were considered to be incidental due to absence of statistical significance, dose response
relation and histopathological changes.

Histopathology:
At the end of the study, all animals were sacrificed, necropsied and examined post mortem. Histological examinations were performed on a small
set of organs from animals of control and HD groups. Histological examination of the reproductive organs of the two non pregnant female animals of LD group was carried out (no. 54 and 56).
At the end of the treatment period there were neither macroscopic nor microscopic treatment-related changes noted. Mortality was not affected by
the treatment.
The examination of the ovaries did not reveal any morphological difference between control and treated animals. The qualitative assessment of the
testes did not reveal any treatment-related effect.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL in this study was considered to be 1000 mg/kg body weight/ day for adult animals (both males and females) including reproduction toxicity and offspring developmental toxicity.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects; see below for details

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:
There were no adverse treatment related effect for total number of pups born, still births, runts on PND 0 and number of male and females pups, sex
ratio, live pups on PND 0 and PND 4. However, there were slightly lower number of live pups and male and female pups on PND 0 and 4 in LD and HD
groups when compared to control, but without statistical significance and dose response relation. All mean and most individual values of litter data
were within the range of historical control data. Hence, the changes were considered not to be an adverse effect of treatment.


Litter Weight Data:
There were no adverse changes of toxicological relevance considered for litter mean weight, total litter weight, male and female litter weight measured on PND 0 and 4. However, there were slightly lower male and female litter weight and total litter weight in LD and HD groups when compared to
control, but without the statistical significance and/ or dose response relation. All mean and most individual values of litter weight data were within
the range of historical control data. Hence, the changes were considered not to be an adverse effect of treatment.


Precoital interval and duration of gestation:
There were no treatment-related changes on precoital interval and the duration of gestation in dose groups when compared to corresponding
control group. All pregnancies resulted in normal births.

Pre and Postnatal Data:
There were no adverse changes of toxicological relevance for number of corpora lutea, implantation sites and live pups (PND 0 and 4). However, there were slightly lower implantation sites and live pups in treated groups (LD, MD and HD groups) when compared to control group, but without the statistical significance and/ or dose response relation. All mean and most individual values were within the range of historical control data.
There were higher percent pre implantation loss in LD, MD and HD groups and post implantation loss in LD and HD groups when compared to control. But the mean and most individual percent pre and post implantation loss were within the historical control data range. Furthermore, in the absence of statistical significance and dose response relation the changes in percent pre and post implantation loss were considered not be an adverse.


Reproductive Indices:
There were no treatment related effects for reproductive indices (Copulation, fertility, delivery and viability indices) in treatment groups when
compared to the corresponding control group.
A reduced fertility index (number of pregnant females/ number of copulated females X 100) was observed in the control (70%) and LD group (80%)
as compared to MD (100%) and HD groups (100%). The low pregnancy in Control and LD groups did not affect the validity as in the median and
higher dose groups the pregnancy rate was 100%.


Pup Survival Data
There was no treatment related effect on the survival of the pups from PND 0 to PND 4 when compared with controls. There were no mortalities of
pups in any of the groups between PND 0 and PND 4.


Pup External Findings:
There were no treatment-related gross external findings in any of the treated groups. Few incidences of external findings were in the control -dark
left hindpaw of pup 11 (female 47), dark head of pup 12 (female 49); LD- dark snout of pup 8 (female 55); MD- hypothermic on PND 0- all pups
(female 61) and HD- dark nose of pup 9 (female 77) groups. These findings were in few isolated females and were considered to be spontaneous and not related to the test item treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL in this study was considered to be 1000 mg/kg body weight/ day for adult animals (both males and females) including reproduction toxicity and offspring developmental toxicity.

Reproductive effects observed:

not specified

Conclusions:

In conclusion, the repeated dose administration of FAT 41029/A in aqua ad iniectabilia (water for injection) to the male (minimum 28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight/day produced no adverse toxicological effects in the adult
males and females or significant reproductive and developmental effects at any administered dose.
Based on the data generated from this reproduction/developmental toxicity screening test with FAT 41029/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 of FAT 41029/ A 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 were completed. Animals of a control group were handled identically as the dose groups but received aqua ad iniectabilia (water for injection), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

The following doses were evaluated:

Control (C):                  0        mg/kg body weight/ day

Low Dose (LD):            100     mg/kg body weight/ day

Medium Dose (MD):     300     mg/kg body weight/ day

High Dose (HD):           1000   mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. Dose volumes were adjusted individually based on the 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. At the conclusion of the test, all 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 the delivery 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 after the evidence of mating or the last day of mating period.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on postnatalday 4, were carefully examined for gross external abnormalities.

 

Histological examinations were performed on a small set of organs from animals of control and HD groups. Histological examination of the reproductive organs of the two non pregnant female animals of low dose was carried out (no. 54 and 56).

Summary Results

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

There were no clinical signs considered to be of toxicological relevance in male and female animals of the dose groups.

No adverse treatment related changes in body weight, body weight gain and food consumption were in male and female dose groups during the study period.

There were no treatment related effects for the reproductive indices (Copulation, fertility, delivery and viability indices) in dose groups when compared to the corresponding control group. A reduced fertility index was observed in the control (70%) and LD (80%) groups as compared to MD (100%) and HD (100%) groups. The low pregnancy in Control and LD groups did not affect the validity as in the MD and HD groups the pregnancy rate was 100%.

There were no treatment related changes on precoital interval and the duration of gestation in dose groups when compared with the corresponding control group. All pregnancies resulted in normal births.

There were no adverse changes of toxicological relevance for the number of corpora lutea, implantation sites and live pups (PND 0 and 4) in dose groups when compared to the control.

There were higher percent pre implantation loss in LD, MD and HD groups and post implantation loss in LD and HD groups when compared to control. In the absence of statistical significance and dose response relation the changes were considered not be an adverse effect of treatment.

There were no adverse treatment related effect on total number of pups born, still births, runts on PND 0 and number of male and female pups, sex ratio, live pups, total litter weight, litter mean weight, male and female litter weight on PND 0 and PND 4 in dose groups when compared to corresponding control.

There was no treatment related effect on the survival of the pups from PND 0 to PND 4 in dose groups when compared to control. There were no mortalities of pups in any of the groups between PND 0 and PND 4.

No treatment-related gross external findings were in the pups of dose groups.

There were no macroscopic findings in the adult animals related to the treatment with the test item.

In males and females, there were no toxicologically relevant changes in absolute and relative (to terminal body weight) organ weights (testes, epididymides, prostate including seminal vesicle and coagulating glands, ovaries and uterus with cervix) in dose groups when compared to control.

Histopathological examination revealed no microscopic treatment-related changes.The examination of the ovaries did not reveal any morphological difference between control and dose animals. The qualitative assessment of the testes did not reveal any treatment-related effect.

Conclusion

In conclusion, the repeated dose administration of FAT 41029/A in aqua ad iniectabilia (water for injection) to the male (minimum 28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight/day produced no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects at any administered dose. Based on the data generated from this reproduction/developmental toxicity screening test with FAT 41029/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.

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A screening study for reproduction / developmental toxicity according to OECD 421 was performed under GLP for the reference substance.

The aim of this study was to assess the possible effects of FAT 41029/ A 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 were completed. Animals of a control group were handled identically as the dose groups but received aqua ad iniectabilia (water for injection), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

The following doses were evaluated:

Control (C): 0 mg/kg body weight/ day

Low Dose (LD): 100 mg/kg body weight/ day

Medium Dose (MD): 300 mg/kg body weight/ day

High Dose (HD): 1000 mg/kg body weight/ day

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, all animals were sacrificed and observed macroscopically.

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 the delivery 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 after the evidence of mating or the last day of mating period.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post natal day 4, were carefully examined for gross external abnormalities.

Histological examinations were performed on a small set of organs from animals of control and HD groups. Histological examination of the reproductive organs of the two non pregnant female animals of low dose was carried out (no. 54 and 56). 

Summary Results

No mortality occurred in the control or any of the dose groups during the treatment period of this study and there were no clinical signs considered to be of toxicological relevance in male and female animals of the dose groups.

No adverse treatment related changes in body weight, body weight gain and food consumption were in male and female dose groups during the study period.

There were no treatment related effects for the reproductive indices (copulation, fertility, delivery and viability indices) in dose groups when compared to the corresponding control group. A reduced fertility index was observed in the control (70%) and LD (80%) groups as compared to MD (100%) and HD (100%) groups. The low pregnancy in Control and LD groups did not affect the validity as in the MD and HD groups the pregnancy rate was 100%.

There were no treatment related changes on precoital interval and the duration of gestation in dose groups when compared with the corresponding control group. All pregnancies resulted in normal births.

There were no adverse changes of toxicological relevance for the number of corpora lutea, implantation sites and live pups (PND 0 and 4) in dose groups when compared to the control.

There were higher percent pre implantation loss in LD, MD and HD groups and post implantation loss in LD and HD groups when compared to control. In the absence of statistical significance and dose response relation the changes were considered not be an adverse effect of treatment.

There were no adverse treatment related effect on total number of pups born, still births, runts on PND 0 and number of male and female pups, sex ratio, live pups, total litter weight, litter mean weight, male and female litter weight on PND 0 and PND 4 in dose groups when compared to corresponding control.

There was no treatment related effect on the survival of the pups from PND 0 to PND 4 in dose groups when compared to control. There were no mortalities of pups in any of the groups between PND 0 and PND 4.

No treatment-related gross external findings were in the pups of dose groups.

There were no macroscopic findings in the adult animals related to the treatment with the test item.

In males and females, there were no toxicologically relevant changes in absolute and relative (to terminal body weight) organ weights (testes, epididymides, prostate including seminal vesicle and coagulating glands, ovaries and uterus with cervix) in dose groups when compared to control.

Histopathological examination revealed no microscopic treatment-related changes. The examination of the ovaries did not reveal any morphological difference between control and dose animals. The qualitative assessment of the testes did not reveal any treatment-related effect.

 

Conclusion

In conclusion, the repeated dose administration of FAT 41029/A in aqua ad iniectabilia (water for injection) to the male (minimum 28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight/day produced no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects at any administered dose. Based on the data generated from this reproduction/developmental toxicity screening test with FAT 41029/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.

Short description of key information:
No effects on fertility were seen in the reproduction/developmental toxicity screening study according to OECD 421.

Effects on developmental toxicity

Description of key information

No effects on developmental toxicity were seen in the reproduction/developmental toxicity screening study according to OECD 421.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Additional information

Please see discussion on fertility for results of reproduction/developmental toxicity screening study.

Justification for classification or non-classification

Based on the results on the reproduction/developmental toxicity screening study, in which no effects on fertility and developmental toxicity were seen, the substance does not require classification for reproductive toxicity according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC).