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]

Endpoint summary

Description of key information

In the “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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

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 [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. 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

Route of administration:

oral: gavage

Vehicle:

corn oil

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.

Duration of treatment / exposure:

Males: 28 days; Females: Approx. 54 days

Frequency of treatment:

7 days/ week

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
other: based on content C.I., i.e. dose values stand for mg Pigment Orange 34 per kg body weigh

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 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
selected 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 (PRENTAL 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.

Positive control:

none

Observations and examinations performed and frequency:

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 OBSERVATIONS (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 OBERVATIONS (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).

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 tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring
was recorded.

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.

URINALYSIS (PARENTAL MALES):
A urinalysis was performed with samples collected from randomly selected males prior to or as part of the sacrifice of the animals.
Additionally, urine colour/ appearance were recorded.

EXAMINATION OF SPERM MOTILITY AND SPERM COUNT:
At necropsy (one day after the last administration), one epididymis and one testis were separated and used for evaluation of sperm
parameters. Epididymal sperm motility and testicular sperm count were evaluated in all male animals of each group using Hamilton
Thorn Sperm Analyser (TOX IVOS Version 13.0C).

Sacrifice and pathology:

PATHOLOGY:
Gross necropsy-
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30,
while female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 2:1, Bremer Pharma GmbH and
Riemser Arzneimittel AG). All surviving pups were killed by decapitation.
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
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:
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:
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.

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).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY (PARENTAL ANIMALS):
There was no mortality observed in the study during the entire study period.

CLINICAL OBSERVATIONS (PARENTAL ANIMALS):
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 (PARENTAL ANIMALS):
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 (PARENTAL ANIMALS):
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 (PARENTAL ANIMALS):
No treatment related changes were observed on food consumption in any of the treated groups of male and female animals during
the study period. However, there was a statistically significant increase in food intake between GD 14-20 in female HD group.
This finding had no toxicological relevance.

HAEMATOLOGY AND COAGULATION (PARENTAL 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). 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 (PARENTAL ANIMALS):
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.

EXAMINATION OF SPERM MOTILITY AND SPERM COUNT (PARENTAL MALES):
Statistical analysis of epididymal sperm motility and testicular sperm count indicated no statistical significant difference between the
treated and control groups. However, there were slight decreases in mean testicular sperm count in LD and HD groups. This finding
did not show dose response pattern. Hence, the finding was not related to treatment.

URINALYSIS (PARENTAL MALES):
The urinalysis performed in male animals revealed no test- item related effect in any of the treatment groups compared to the
control group.

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 (PARENTAL ANIMALS):
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 (PARENTAL ANIMALS):
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%.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

other: content C.I.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest dose tested

Critical effects observed:

not specified

LITTER DATA:

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

pups on PND 0 and PND 4.

No treatment related changes were considered for number of still births and runts. However, there were runts recorded in control

and LD groups and this was considered to be an incidental finding.

LITTER WEIGHT DATA:

There were no treatment related changes recorded for litter mean weight and total litter weight and male- female litter weight on

PND 0 and 4. Statistical analysis revealed no significant difference between the treated and corresponding control groups.

PRECOITAL INTERVAL AND DURATION OF GESTATION:

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%.

PRE- AND POST-NATAL DATA:

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 with the

control group. Statistical analysis revealed to significant difference between treatment and corresponding control groups.

REPRODUCTIVA INDICES:

A reduced fertility index (number of pregnant females/ number of copulated females X 100) was observed in the control (80 %) and

HD group (90%) as compared to LD (100%) and MD groups (100%). This difference in C and HD groups were considered

incidental.

No treatment related changes were considered for viablity index between treated and control groups.

PUP SURVIVAL DATA:

No treatment related changes on survival of the pups from PND 0 to PND 4 was observed in any of the treated group when

compared to controls.

PUP EXTERNAL FINDINGS:

No treatment-related gross external findings were observed in any of the treated groups.

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%.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

No classification

No advers effets was found in the Combined Repeated Dose Oral Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test up to the limit dose of 1000 mg/kg bw.