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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 05 JUN 2012 to 04 AUG 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 422), GLP compliant
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]
EC Number:
239-898-6
EC Name:
4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]
Cas Number:
15793-73-4
Molecular formula:
C34H28Cl2N8O2
IUPAC Name:
4,4'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[5-methyl-2-(4-methylphenyl)-2,4-dihydro-3H-pyrazol-3-one]
Test material form:
solid: nanoform

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system 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: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups: males: 232 - 275 g
(mean: 253.78 g, +/- 20% = 203.02 – 304.53 g) females: 166 - 196 g
(mean: 178.45 g, +/- 20% = 142.76 – 214.14 g)
Number of animials: 10 males and 10 females per group
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. 1530)
- 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. 261111)
- 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:
corn oil
Details on exposure:
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 test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5mL/kg body
weight.
For each animals the individual dosing volume was calculated on the basis of the most recently measured body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration was analyzed for nominal concentration. Stability and homogeneity of the test item in the vehicle were
analyzed for the LD, MD and HD dosing formulation.
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) of control and all treatment groups (16 samples in total).
Samples for homogeneity were taken from the top, middle and bottom of HD, MD, and LD preparation in study week 1 and 5 (18
samples in total).
All formulation samples were analyzed on the day of sample collection at BSL BIOSERVICE Scientific Laboratories GmbH.
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.
Duration of treatment / exposure:
Males: 28 days; Females: Approx. 54 days
Frequency of treatment:
7 days/ week
Duration of test:
From 05 JUN 2012 to 04 AUG 2012
No. of animals per sex per dose:
10 animals/sex/ group
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. 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, respectively.

EXPERIMENTAL GROUPS AND DOSES:
According to the results of the dose range finding study and in consultation with the sponsor the following doses were selected for
the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
The animals were treated with the test item formulation or vehicle on 7 days per week for a 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 doses were
slected on the basis of data from a Dose Range Finding Study.
The animals in the control group were handled in an identical manner to the test group subjects and received corn oil using the
same volume as used for the 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 (PARENTAL ANIMALS):
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.
The cages were arranged in such a way that possible effects due to cage placement were minimised.







Examinations

Maternal examinations:
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and
weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days
(GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any
animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the
dose administration. Food consumption was not measured during the mating period in males and females and the post-mating
period in males.

CLINICAL OBSERVATION (PARENTAL ANIMALS):
General clinical observations were made at least once a day, at the same time each day.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were 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.

FUNCTIONAL OBSERVATIONS (PARENTAL ANIMALS):
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the
treatment in 5 randomly selected males and on day 3 of the lactation period in 5 randomly selected females (only lactating females
will be evaluated) outside the home cage using a functional observational battery of tests.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well
as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity,
visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex,
righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye
and fundus of eye).

HAEMATOLOGY (PARENTAL ANIMALS):
Haematological parameters from randomly selected males and females of each group were examined at the end of the treatment
prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.

BLOOD COAGULATION (PARENTAL ANIMALS):
Coagulation parameters from randomly selected males and females of each group were examined at the end of the treatment prior
to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in citrate tubes.

CLINICAL BIOCHEMISTRY (PARENTAL ANIMALS):
Parameters of clinical biochemistry from randomly selected males and females of each group were examined at the end of the
treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in serum separator tubes.

PATHOLOGY:
Gross necropsy-
Female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 2:1, Bremer Pharma GmbH and
Riemser Arzneimittel AG) was used.
Females showing no evidence of copulation up to 14 days of the mating period were sacrificed 24 to 26 days after the last day of
the 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.

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

ORGAN WEIGHTS (PARENTAL ANIAMALS):
The wet weight of the organs of 5 sacrificed adult males and females randomly selected from each group was recorded as soon as
possible. Paired organs were weighed separately. In addition reproductive organs of all animals were weighed.

liver
uterus with cervix
kidneys
thymus
adrenals
thyroid/parathyroid glands
testes
spleen
epididymides
brain
prostate, seminal vesicles and coagulating glands
pituitary gland
ovaries
heart

The following tissues of the from each group were preserved in 10% neutral buffered formalin except for testes and epididymides
that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 10% neutral buffered
formalin.

brain (cerebrum, cerebellum and pons)
ovaries (females)
spinal cord
uterus with cervix (females)
liver
vagina (females)
kidneys
testes (males)
adrenal glands
epididymides (males)
stomach
prostate and seminal vesicles with coagulating glands as a whole (males)
small and large intestines (including Peyer´s patches)
urinary bladder
thymus
lymphnodes (mesentric and axillary)
thyroid
peripheral nerve (e.g. sciatic nerve) with skeletal muscle
spleen
bone with bone marrow (sternum)
lung and trachea
pituitary gland
mammary glands
oesophagus
heart
all gross lesions

HISTOPATHOLOGY (PARENTAL ANIMALS):
A full histopathology was carried out on the preserved organs and tissues of 5 randomly selected male and 3 female animals of the
control group and 5 randomly selected male and 4 female animals of the high dose group which were sacrificed at the end of the
treatment period.
All organs and tissues listed in were evaluated from randomly selected males and females of the control and high dose group.
According to the study plan, five males and females per control and high dose should be evaluated. However, as some selected
females were found to be non-pregnant, less females were submitted to full histopathological evaluation, resulting in the following
list:

Males Nos.: 3, 4, 6, 8, 9, 31, 34, 37, 38, 39;
Females Nos.: 43, 44, 45, 71, 73, 76, 77.
Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and
macroscopic changes not considered to be related to test item color were evaluated in all study animals.
For paired organs, both sides were examined.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for
the 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 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.
Ovaries and uterine content:
macroscopic and micropscoic examination
Fetal examinations:
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 tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring
was recorded.
All surviving pups were killed by decapitation.
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 haematology, blood coagulation and
clinical biochemistry and absolute and relative organ weights were performed for each gender and in addition the values of litter
parameters 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).
Indices:
copulation, fertility, delivery and viability indices

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY:
There was no mortality observed in the study during the entire study period.

CLINICAL OBSERVATIONS:
There were no clinical signs considered to be of toxicological relevance observed in male and female treated group animals.
However, there were few clinical signs observed which were assumed to be incidental.
Males: C- Alopecia on both forelimbs (1/10 animals); LD- Alopecia on both forelimbs (1/10 animals); MD- aggressive (2/10 animals),
eschar on neck (1/10 animals).
Females: C- Alopecia on forelimb, hindlimb, abdomen, dorsal region, pilerection (1/10 animals), alopecia on flank (2/10 animals);
LD- alopecia on dorsal region (1/10 animals), small wound on right shoulder (1/10 animals); MD- moderate salivation (1/10
animals), slight piloerection (1/10 animals), aggressive (1/10 animals).
During the weekly detailed clinical observation no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

FUNCTIONAL OBSERVATIONS:
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the
treatment period. There were no biologically relevant differences in body temperature between the groups.

BODY WEIGHT DEVELOPMENT:
In both males and females, no treatment related changes were recorded for body weight and body weight change during the study
period. Statistical analysis revealed no significant differences between treated and corresponding control group.

FOOD CONSUMPTION:
No treatment related changes were observed on food consumption in any of the treated groups of male and female animals during This finding had no toxicological relevance.

HAEMATOLOGY AND COAGULATION:
In males and females, no treatment related changes were considered for measured haematological parameters (WBC, RBC, HGB,
HCT, MCV, MCH, MCHC, PLT, Neut, Lymph, Mono, Eos and Baso). However, there was statistically significant increase in MCH
value in male MD group. In the absence of dose response pattern this finding was not related to treatment.
There were no treatment related changes noted for blood coagulation parameters (PT and aPTT) in both males and females in
treated groups when compared to corresponding control. The statistical analysis revealed no significant differences between
treated and control groups.

CLINICAL BIOCHEMISTRY:
In males and females, the statistical analysis of clinical biochemistry data revealed no significant difference between the treated and
corresponding control groups. However, there was slight decrease noted for ASAT values in male LD and MD groups and female
HD group, substantial increase in mean ALP value in male and female LD group, considerable decrease in mean ALP value in
female MD group, slight decrease in mean TBA values in male LD, MD and HD groups; slight increase in mean CREA and CHOL
values in female MD and HD group. Histopathologically no treatment related changes were noted in liver or kidneys in treated
group animals. Hence, the above changes were considered to have no toxicological relevance.

PATHOLOGY:
Red discolorations of ileum and/or lymph nodes were observed in a number of test item treated animals, without dose-relationship.
Based on the histopathological evaluation, most of these macroscopic changes could be correlated to mucosal hemorrhage (ileum)
or intrasinusoidal blood resorption (lymph nodes), and they were therefore considered to be rather related to the technical
procedures of euthanasia and terminal blood sampling than to be test item-related.
Other macroscopic organ findings noted were very few. All of them were considered to be incidental and not to be test item-related.

ORGAN WEIGHT:
In males and females, no treatment related changes were considered for absolute and relative (to terminal body weight) organ
weights of the treated groups when compared to control group. However, there was statistically significant decrease in absolute and
relative weight of pituitary gland in female LD, MD and HD group and LD and MD groups, respectively. There was slight decrease
in absolute pituitary weight in male MD group, slight decrease in absolute adrenal gland weights in female LD, MD and HD groups,
slight decrease in relative weight of spleen in female LD, MD and HD groups, slight decrease in relative kidney weight of female MD
group, slight decrease in relative adrenal weight of female LD, MD and HD groups, slight decrease in relative thymus weight of
female HD group, slight decrease in relative thyroid/parathyroid weight of female MD and HD groups without attaining statistical
significance.
The above changes did not corroborate with the histopathological findings and any differences in organ weight in treated groups
when compared to controls were of no toxicological relevance.

HISTOPATHOLOGY:
Reproductive organs
There was no indication of test item-related histopathological findings in reproductive organs of male or female rats of this study.
Reproductive organs of most female study animals showed typical post-partum histomorphology without any relevant inter-group
difference.
The reproductive organs of the females found not to be pregnant at terminal sacrifice showed normal reproductive sexual cycle
(Nos. 42 and 75) or the sexual cycle could not be determined, together with a moderate unilateral luminal distension of the uterus
(No. 50). No test item relationship was established.
Other organs
No test item-related histopathological findings were noted in the other organs evaluated in this study.
In the kidney, mild basophilic tubules were noted in the cortex of 3/4 females treated at
1000 mg/kg/day, versus none of the controls. However, this finding is frequently observed in untreated rats of this strain and age
and was therefore not considered test item-related.
Moreover, in the kidney of males, minimal cortical basophilic tubules were seen equally in control males and males of HD group.
In the Peyer's patch of one female of HD group, a minimal amount of goldenbrown pigment was noted, but, in the view of its
isolated occurrence, was not considered sufficient evidence for a test item-related effect.
Other histopathological changes seen at terminal sacrifice were considered to be incidental in origin and/or within the range of
expected changes for rats of this age and strain kept under laboratory conditions.

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 104.6%, 101.1%, and 92.1% of the nominal concentration, respectively.
Samples for investigation of stability of formulation samples arrived at the Department Analytics for analysis in study week 1 for LD
and HD dose groups. Samples “0 hours storage” were analysed directly after arrival. The other samples were analysed after 6
hours storage at room temperature. For evaluation recovery after 6 hours storage was determined referring to starting value.
Recovery after 6 hours storage was between 96.3% and 100.9% at the dose groups investigated.
Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for
LD group were 97.2% and 104.0%, and for HD group 99.0% and 88.5% of the nominal value. The coefficients of variation of the
different sampling locations (top, middle, bottom) were in LD group 1.5% and 5.9%, and in HD group 1.7% and 0.3%.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
other: content C.I.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
other: content C.I.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
soft tissue and skeletal examination not made

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
visceral malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of the test item to the male (28 days) and female (maximum 54 days) Wistar rats at
dosages of 0, 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance.
Based on the data generated from this “Combined Repeated Dose Oral Toxicity Study with the Reproduction/ Developmental
Toxicity Screening Test”, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg body weight/day.
Executive summary:

The aim of this study was to assess the possible effects of the test item on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. It further provides information on the possible general health hazards likely to arise from repeated exposure over a relatively limited period of time.

The test item was administered daily in graduated doses to 3 groups of test animals. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. Each group comprised 10 male and 10 female Wistar rats. 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 was completed.

During the period of administration, the animals were observed each day for signs of toxicity. At termination 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.

Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from males and females randomly selected from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations were performed in randomly selected males in the week before the treatment and at the end of the study and on day 3 of the lactation period in randomly selected females.

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.

Pups sacrificed on post natal day 4, 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/d Medium Dose: 300 mg/kg body weight/d High Dose: 1000 mg/kg body weight/d. Dose calculation was adjusted to content C.I., i.e. dose values stand for mg Pigment Orange 34 per kg body weight. The test item formulation was prepared freshly on each day of administration. The test item was suspended in corn oil and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28-30 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration 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. There were no clinical signs considered to be of toxicological relevance observed in male and female treated group animals. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

In both males and female, no treatment related changes were recorded for body weight, body weight change and food consumption during the study period.

No treatment-related effect was noted for total number of pups born, still birth, runts on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4.

No treatment related effect was observed for precoital interval and duration of gestation in treated groups when compared with the control group. All pregnancies resulted in normal births.

Successful mating resulted pregnancy rate as follows, C 80%, LD 100%, MD 100% and HD 90%. The group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0, percentage of preimplantation loss and post-implantation loss remained unaffected due to the treatment with test item when compared to control group.

There were no treatment related changes considered for copulation index (%), fertility index (%) and viablity index (%) between treated and control groups.

No treatment related changes on survival of the pups from PND 0 to PND 4 was observed in any of the treated groups when compared with control. No treatment-related gross external findings were observed in any of the treated group animals. In males and females, no treatment related changes were considered for measured haematological parameters (WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, Neut, Lymph, Mono, Eos and Baso) and coagulation parameters (PT and aPTT). There were no treatment related changes noted for clinical biochemistry parameters measured in both males and females. Epididymal sperm motility and testicular sperm count indicated no changes considered to be of toxicological relevance in treated groups when compared to control.

The urinalysis performed in male animals revealed no test- item related effect in any of the treated groups when compared to control group.

Red discolorations of ileum and/or lymph nodes were observed in a number of test item treated animals, without dose-relationship. Based on the histopathological evaluation, most of these macroscopic changes could be correlated to mucosal hemorrhage (ileum) or intrasinusoidal blood resorption (lymph nodes), and they were therefore considered to be rather related to the technical procedures of euthanasia and terminal blood sampling than to be test item-related.

Other macroscopic organ findings noted were very few. All of them were considered to be incidental and not to be test item-related. In males and females, no treatment related changes were considered for absolute and relative (to terminal body weight) organ weights of the treatment groups when compared to control group.

No test item-related histopathological findings were noted in the reproductive organs and in the other organs evaluated in this study.

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 104.6%, 101.1%, and 92.1% of the nominal concentration, respectively. Samples for investigation of stability of formulation samples arrived at the Department Analytics for analysis in study week 1 for LD and HD dose groups. Samples “0 hours storage” were analysed directly after arrival. The other samples were analysed after 6 hours storage at room temperature. For evaluation recovery after 6 hours storage was determined referring to starting value. Recovery after 6 hours storage was between 96.3% and 100.9% at the dose groups investigated. Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 97.2% and 104.0%, and for HD group 99.0% and 88.5% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, and bottom) were in LD group 1.5% and 5.9%, and in HD group 1.7% and 0.3%..