Registration Dossier

Administrative data

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:
2012-08-16 to 2013-09-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: FAT 40224/H TE
Batch No.: TZ 2382-BB-503C81
Physical State: powder
Colour: red
Density: 1.46 g/cm3 (20°C)
pH: 6.5 to 7.5 conc.(% w/w): 1%
Melting Point: > 200°C
Purity / Active Components: sum of all coloured substances: 85.4%,
main constituents: 50.6%
Date of Analysis: 23.07.2012
Storage Conditions: at room temperature
Expiry Date: 28.01.2017

Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.



Test animals

Species:
rat
Strain:
Wistar
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:
272 - 303 g (mean: 288.10 g, ± 20% = 230.48 – 345.72 g), females: 184 - 217 g (mean: 199.48 g, ± 20% = 159.58 – 239.37 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 [7] 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. 0856)
-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. 030512)
-Certificates of food, water and bedding are filed at BSL BIOSERVICE
-Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. Females with unsuccessful mating will be allowed to mate with other male of the same group.
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 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).


Samples for the nominal concentration verification was 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 was 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 was taken in the first week of the study, 0 hours after the preparation (at room temperature), from high and low dose
formulations (2 samples).

The dose formulation analysis was performed at BSL Bioservice Scientific Laboratories GmbH.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle for a 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.
Frequency of treatment:
7 days/ week
Details on study schedule:
Arrival of the Test Item: 12 June 2012

Study Initiation Date: 16 August 2012

1st Amendment to Study Plan: 09 October 2012

2nd Amendment to Study Plan: 21 March 2013

Experimental Starting Date: 29 August 2012

Experimental Completion Date: 12 October 2012

Completion Date of Delegated
Phase (Histopathology): 09 September 2013

Completion Date of Delegated
Phase (Formulation Analysis): 07 May 2013






Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Number and sex of the animals
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Preparation of the animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange. 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.

Dosage
Iin consultation with the sponsor the following doses (Table 1) 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 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.
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 injectionem using the same
volume as used for the high dose group.


Administration of doses
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.


Mating
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. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. Females with unsuccessful mating will be allowed to mate with other male of the same group.
The cages were arranged in such a way that possible effects due to cage placement were minimised.



Clinical observation
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.
Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a
thorough macroscopic examination.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations will be made in all animals outside the home cage
in a standard arena. 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.


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.


Pathology
Gross necropsy
Males were sacrificed after the completion of mating period (total dosing of 28 days), pregnant females were sacrificed on respective post natal day 4 and non pregnant females sacrificed on their respective day 26 after the evidence of mating or completion of mating period by using
Ketamine/Xylazine (2:1) at the dose volume of 1.4 ml/kg body weight. At the time of sacrifice the adult animals were examined macroscopically for
any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. The pups were killed on post natal
day 4 by decapitation.
Dead pups and pups killed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.
The ovaries, uterus with oviduct and 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. Testes and
epididymides were fixed in modified Davidson’s Solution for 24 hours and then transferred to 10 % neutral buffered formalin.


Organ weight
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.


Histopathology
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in Control and HD animals. All organs showing gross lesions were examined in all groups.
All non-pregnant female animals were examined histopathologically.


For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The 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 was performed according to the corresponding SOP’s of the test sites.




Examinations

Parental animals: Observations and examinations:
Body weight, food consumption, clinical signs, pathology, organ weight (reproductive organs), histopathology (reproductive organs)
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not Examined
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):
yes
Postmortem examinations (offspring):
not examined
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical
biochemistry and 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.5.01 software
(p<0.05 was considered as statistically significant).
Reproductive indices:
Copulation, fertility, delivery indices
Offspring viability indices:
yes

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
male: none; female: slight piloerection (9/10 HD; 1/10 C ), moderate piloerection (4/10 HD ; 0/10 C ), slight salivation (4/10 HD ; 0/10 C ), and moving the bedding (5/10 HD ; 0/10 C ), indicating discomfort, assumed to be test-item related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
in male and female, but no adversity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
in male and female, but no adversity.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
but no adversitiy
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
discoloured yellow testes (2/10 HD; 0/10 C) and discoloured yellow oviduct and cervix (2/10 HD; 0/10 C)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
minor, non adverse.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
assumed to be incidental

Details on results (P0)

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

Clinical observation
No relevant differences were observed concerning functional and behavioural examinations in male treated groups when compared with male
controls.
For female animals, more clinical symptoms were found in HD group when compared to C group. In particular, slight piloerection (9/10 HD animals; 1/10 C animals), moderate piloerection (4/10 HD animals; 0/10 C animals), slight salivation (4/10 HD animals; 0/10 C animals), and moving the
bedding (5/10 HD animals; 0/10 C animals) were observed. These symptoms in female animals indicate discomfort and are assumed to be test item
related.


Body weight and body weight change
In male animals, a slight tendency towards a decreased body weight development was observed for HD animals during the whole treatment period.
In this regard, a significantly decreased body weight gain in HD group was found during treatment days 1-7.
In female animals, no test item related influence on body weight development during the pre-mating period was measured. A significantly decreased (p<0.05) body weight gain during pre-mating days 1-7 (C: +3.8 g; HD: -2.4 g) is considered to be not of toxicological relevance since it recovered
during the second week of the pre-mating period. During gestation and lactation, no significant influence on body weight development of female
animals was observed.
For both – male and female animals – a test item relation is possible. However, due to the mild characteristics as well as the partially reversibility an
adverse toxicological relevance cannot be assumed.


Food consumption
In male animals, food consumption was not different in treatment groups when compared to C group.
In female animals, a significantly increased (p<0.05) food consumption was recognized for HD group during premating days 7-14. A consumption of 107.6 g (C) and 120.3 g (HD) was measured. This increased food consumption is not assumed to be of toxicological relevance. During the rest of the application period, there was no further change in food consumption of treatment groups when compared to C group.


Precoital interval and duration of gestation
No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group.
The values were comparable between the groups. All pregnancies resulted in normal births.
Successful mating resulted in 6 pregnancies in the control, 9 in low, and 10 in medium and high dose group animals. The reduced number of
pregnancies in C and LD group is considered to be incidental.





Gross pathology
Few specific gross pathological changes were recorded for the male and female animals. The most common were discoloured yellow testes (2/10 HD; 0/10 C) and discoloured yellow oviduct and cervix (2/10 HD; 0/10 C). Other findings were just seen individually.



Organ weight
In male animals, the relative total testes weight (to body weight) was significantly increased (p<0.05) in HD group (1.0325 %) when compared to C
group (0.9484 %). In this regard, also the relative weight (to body weight) of the left testis was significantly (p<0.05) increased in HD group
(0.5203 %) when compared to C group (0.4751 %).
Since no finding was present in histopathology, this weight increase is not considered to be test item related and of toxicological relevance.


Histopathology
Histologically, in the males, yellow-brown pigment in interstitial macrophages was seen at a minimal or mild degree in the testis and at a minimal
degree in the epididymis in all rats treated at 1000 mg/kg/day. This change confirmed the macroscopic observation of yellow discoloration in
some of these organs and was considered to represent test item deposition in macrophages. As there was no indication of other structural changes or functional impairment of these organs, the pigmentation was considered non-adverse.
No test item-related histological findings were noted in the other male and in the female reproductive organs.
Reproductive organs of those control and high dose females having undergone pregnancy showed typical post-partum histomorphology. Four
control females and one female treated at 100 mg/kg/day did not show any indication of recent pregnancy. As there was no dose relationship this
was considered to be unrelated to treatment. Histomorphology of the non-pregnant control females indicated physiological sexual cycling, the
female treated at 100 mg/kg/day was not evaluated histologically.
As a conclusion, only minor test item-related pathological findings were noted in this study and were considered to be directly caused by test item
deposition in macrophages and to be non-adverse.



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 96.0%, 111.3% and 114.6% 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 95.8% and 102.1%. 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
100.1 and 97.7% of the nominal value and 113.0 and 107.6% for HD dose group. The coefficients of variation of the different sampling locations
(top, middle, bottom) were 1.1 and 5.6% in LD dose group and 2.6 and 4.6% in HD dose group..


Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
1 000 mg/kg bw/day
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.

Results: F1 generation

General toxicity (F1)

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:
effects observed, treatment-related
Description (incidence and severity):
minor, incidental
Histopathological findings:
not examined

Details on results (F1)

Litter weight data
No difference was found for mean litter weights at PND 0 and PND 4 of treatment groups when compared to C group. The total litter weight was
slightly (p>0.05) reduced in HD group. Furthermore, the male litter weight was slightly (p>0.05) decreased, too, at PND0 and PND4 in HD group.
This belongs mainly to the reduced litter size and a reduced number of male animals which hence leads to a reduced total litter weight.

Pre and post natal data
A slightly reduced number of implantation sites was counted for HD group (p>0.05). In particular, 9.3 (HD) and 11.67 (C) were counted/calculated. Due to a high variance and a missing significance, this is not assumed to be test item related.
Furthermore, an increased percentage of pre-implantation loss was detected in HD group: 23.26 % (HD) and 7.83 (C) were calculated. This was not
statistical significant (combined with a high variance). Furthermore, when comparing the total number of corpora lutea and implantation sites
these values were no significantly different among the groups. Hence, a test item relation is not assumed.


Litter data
A slightly decreased total number of pups born was observed for HD animals. In particular, 9.1 (HD) and 11.17 (C) were counted/calculated.
This correlates with a slightly decreased number of male pups (3.6 (HD) in comparison to 5.83 (C)). However, this was not statistical significant
(combined with a high variance) and the degree of decrease was rather mild.
One still birth was detected in LD group (animal No. 59) as well as MD group (animal No. 61). However, since no other still births were detected
(especially in HD group) this is not assumed to be test item related but incidental.
In correlation with PND 0, the total number of living pups was still slightly (p>0.05) decreased in HD group at PND 4: 9.1 (HD) and 11.17 (C) were
counted.


Reproductive indices
A reduced fertility index (number of pregnant females/ number of copulated females X 100) was observed in the C (60 %) and medium dose group
(90 %). MD, and HD groups exhibited 100 %. The copulation index ((No. of rats copulated / No. of pairs) X 100) as well as delivery index (No. of dams with live newborns / No.of pregnant dams) X 100) were 100 % for each group.
The viablity index was 100 % for C, LD, MD, and HD animals.
The reduced fertility index is assumed to be incidental.



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.

Pup external findings
No treatment-related gross external findings were observed in any of the treated groups. Few incidences of external findings were observed but
none of them was considered to be test item related.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of this reproduction/ developmental toxicity screening test with FAT 40224/H 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:
In HD group, increased clinical symptoms (piloerection, salivation, moving the bedding) were found when compared to C group.
Furthermore, a slightly attenuated body weight gain could be found as well as a slight weight increase in testes. However, although these symptoms could be test item related they are not assumed to display adverse effects. Hence, the NOAEL for maternal/paternal toxicity is 1000 mg/kg BW within this study.
In HD animals, the total number of pubs born was decreased for HD animals. This correlated with a decreased number of male pubs born.
However, this was not statistical significant (combined with a high variance) and the degree of decrease was rather mild. An increased percentage of pre-implantation loss in HD group was not statistical significant (combined with a high variance), too. Furthermore, when comparing the total
number of corpora lutea and implantation sites these values were similar among the groups. Hence, the NOAEL for foetal toxicity is 1000 mg/kg within this study.

Executive summary:

The aim of this study was to assess the possible effects of FAT 40224/H 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 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 receivedaqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistar 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 days 29 and 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 postnatalday 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.

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 in aqua 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 28-30 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight. Summary Results

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

No relevant differences were observed concerning functional and behavioural examinations in male treated groups when compared with male controls.

For female animals, more clinical symptoms were found in HD group when compare to C group. In particular, slight piloerection (9/10 HD animals; 1/10 C animals), moderate piloerection (4/10 HD animals; 0/10 C animals), slight salivation (4/10 HD animals; 0/10 C animals), and moving the bedding (5/10 HD animals; 0/10 C animals) were observed.

In male animals, a slight tendency towards a decreased body weight development was observed for HD animals during the whole treatment period. In this regard, a significantly decreased body weight gain in HD group was found during treatment days 1-7.

In female animals, no test item related influence on body weight development during the pre-mating period was measured.

A significantly decreased (p<0.05) body weight gain during pre-mating days 1-7

In male animals, food consumption was not different in treatment groups when compared to C group.

In female animals, a significantly increased (p<0.05) food consumption was recognized for HD group during premating days 7-14.

A slightly decreased total number of pubs born was observed for HD animals. In particular, 9.1 (HD) and 11.17 (C) were counted/calculated. In correlation with PND 0, the total number of living pubs was still slightly (p>0.05) decreased in HD group at PND 4.

No difference was found for mean litter weights at PND 0 and PND 4 of treatment groups when compared to C group. The total litter weight was slightly (p>0.05) reduced in HD group. Furthermore, the male litter weight was slightly (p>0.05) decreased, too, at PND0 and PND4 in HD group.

No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group

Successful mating resulted in 6 pregnancies in the control, 9 in low, and 10 in medium and high dose group animals.

A slightly reduced number of implantation sites was counted for HD group (p>0.05).

In this regard, an increased percentage of pre-implantation loss was detected in HD group: 23.26 % (HD) and 7.83 (C) were calculated.

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 any of the treated groups.

Few specific gross pathological changes were recorded for the male and female animals. The most common were discoloured yellow testes (2/10 HD; 0/10 C) and discoloured yellow oviduct and cervix (2/10 HD; 0/10 C).

In male animals, the relative weight of testes (to body weight) was significantly increased (p<0.05) in HD group. In this regard, also the relative weight (to body weight) of the left testis was significantly (p<0.05) increased in HD group when compared to C group.

Histologically, in the males, yellow-brown pigment in interstitial macrophages was seen at a minimal or mild degree in the testis and

at a minimal degree in the epididymis in all rats treated at 1000 mg/kg/day.

No test item-related histological findings were noted in the other male and in the female reproductive organs.

Reproductive organs of those control and high dose females having undergone pregnancy showed typical post-partum histomorphology.