Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 16 - April 16, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl.WI (Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: (P) Males 11 wks; Females 10 weeks (F1) 4 days
- Weight at study initiation: (P) Males: 313 ( 296 – 337) g; Females: 205 ( 186 – 232) g; (F1) Males: 5.89 - 6.35 g; Females: 5.65 - 6.05 g
- Fasting period before study: no
- Housing: single-housed in type III Makrolon® cages
- Diet: Provimi Kliba 3433.0 ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.5 – 23.0 ºC
- Humidity: 37.8 – 64.5 %
- Air changes (per hr): 10 times
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
Details on exposure:
- Justification for use and choice of vehicle: standard vehicle used at testing facility, without toxic properties
- Concentration in vehicle: 0, 3, 9, and 27 mg/mL
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until gestation day (GD) 0, max 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as GD 0
- Further matings after two unsuccessful attempts: n.a.
- After successful mating each pregnant female was caged (how): single
- Any other deviations from standard protocol: no

- Further details: At the start of mating the male rats were approximately 14 weeks old and the female rats 13 weeks. First, the males and females were treated for 2 weeks (pre-mating phase), afterwards, mating took place according to the following scheme: one male is caged with one female. The animals were staying together day and night, and allocated pairs were recorded. Daily vaginal smears were taken from the females, their cycles were monitored and the presence of sperm was examined. The day of positive sperm finding is defined as gestation day (GD) 0. From GD 0 onwards, males and females were separated, and the animals were kept individually. Final confirmation of pregnancy was either the delivery of pups or resorptions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The quantification of the test material in the dosing formulations was performed using a HPLC method with UV detection. During the course of the study each dosing formulation (including control) of two preparation periods was sampled and analyzed at the beginning and the end of usage, resulting in 4 time points of formulation analysis.
Duration of treatment / exposure:
males: 6 weeksfemales: up to 7 weeks
Frequency of treatment:
daily (7d / week)
Details on study schedule:
Males: The males were treated altogether for 42 days: 2 weeks during premating, max. 2 weeks during mating and further on until day 42.
Females: The females were treated first for 2 weeks during premating, then for 2 weeks (maximum) during the mating period and later until day 4 post partum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
45 mg/kg bw/day (actual dose received)
Dose / conc.:
135 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 (12 m / 12 f)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The test material has been investigated in a subacute 4-week toxicity study with daily oral administration of 40, 200, and 1000 mg/kg. A control group was treated similarly with the vehicle, corn oil. Recovery animals for 2 weeks were kept in the control and 200 mg/kg groups. All animals treated with 1000 mg/kg were killed in extremis after 6 days of treatment and 2 more days without treatment. One female treated with 200 mg/kg died spontaneously on treatment day 21, one hour after application without any previous symptoms. The cause of death could no be established. Another female of this group and 2 control females died on the day of scheduled necropsy after blood sampling. Death was attributed to the blood sampling procedure. One female treated with 200 mg/kg died on the first day of the recovery period, cause of death could no be established. Treatment-related findings were generally restricted to reduced food consumption and reduced body weight and body weight gain in all animals treated with 200 or 1000 mg/kg. Decreased hematocrit and hemaglobin in males and females treated with 200 mg/kg and increased hemaglobin concentration distribution width in females of the 200 mg/kg group were present after 4 weeks of treatment and also after recovery. Decreased erythrocyte numbers in both sexes treated with 200 mg/kg were also present after recovery, however, males were within internal laboratory range.In clinical chemistry, main changes were increased creatinine, decreased phospholipids and increased alanine aminotransferase with alanine aminotransferase showing a tendency of recovery. Other affected parameters included increased aspartate aminotransferase indicating changes in the liver and kidneys. A slightly increased number of leucocytes in urine was seen after 4 weeks of treatment in females at 200 or 40 mg/kg with a tendency of reversibility during recovery (200 mg/kg). In the absence of macroscopic or microscopic correlating findings, it is considered test-item related but not adverse. Histologically, alterations in the liver, adrenal glands, and testes were found in animals of the 200 mg/kg group. Hepatocellular hypertrophy correlated with increased liver weights and enlarged livers. It was not present after recovery and was considered to be of metabolic nature due to the absence of any further lesion. Cortical atrophy in adrenal glands often combined with focal/multifocal subacute inflammation was recorded in both sexes treated with 200 mg/kg. Cortical atrophy with focal vacuolar degeneration was recorded in one female treated with 200 mg/kg. These findings were also present after the recovery period. Cortical atrophy corresponds to macroscopically reduced size of adrenal glands and decreased adrenal weights. Findings in the adrenals are considered to be test-item related. Focal/multifocal multinucleated spermatid giant cells at minimal severity were observed in 2 animals of the 200 mg/kg group. This finding was not persistent after recovery. The presence of focal/multifocal multinucleated spermatid giant cells is often associated with early stages of atrophic seminiferous tubules. Based on the results of this study, 40 mg/kg could be established as NOAEL (no observed adverse effect level).

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: checked twice daily at working days and once at off days, at the same time (s) each day

Functional observational battery: Yes
- Time schedule: pre-dose ,day 7, day 29 (m) or day 4 post partum (f)

Motor Activity: yes
- Time schedule: day 29 (m) or day 4 post partum (f)

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment and thereafter once a week. From the date of mating the females will be weighed daily, within 24 hours of parturition (day 0 post-partum), and on day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE: yes, once a week before mating of all animals. No food consumption was measured for the males further on. Food consumption measurements of the females were started again after the positive vaginal smears, then on days 7, 14, 21 post coitum and again on day 4 post-partum

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER: Hematology and Clinical Chemistry undertaken on day 13
- parameters hematology: Red blood cells (erythrocytes), Hemoglobin, Hematocrit, Mean cell volume, Mean hemoglobin content, Mean hemoglobin concentration, Platelets, Reticulocytes, Absolute number of reticulocytes, White blood cells (leukocytes), Absolute number of neutrophilic granulocytes, Absolute number of lymphocytes, Absolute number of eosinophilic granulocytes, Absolute number of basophilic granulocytes, Absolute number of monocytes, Absolute number of large unstained cells, Neutrophilic granulocytes, Lymphocytes, Eosinophilic granulocytes, Basophilic granulocytes, Monocytes, Large unstained cells, Prothrombin time, Partial thromboplastin time
- parameters clinical chemistry: Sodium, Potassium, Calcium, Chloride, Inorganic phosphate, Glucose, Urea, Creatinine, Total bilirubin, Cholesterol, Triglycerides, Bile acids, Total protein, Albumin, A/G ratio, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Urinalysis, pH value, Protein, Glucose, Bilirubin, Blood, Urobilinogen, Ketone, Sediment, Specific gravity
Oestrous cyclicity (parental animals):
assessed
Sperm parameters (parental animals):
not assessed
Litter observations:
Live pups/dam at birth, Live pups/dam on day 4 pp, Sex ratio (m/f) at birth, Sex ratio (m/f) on day 4 pp, Pup weight males on day 0 pp (live), Pup weight males on day 4 pp (live), Pup weight females on day 0 pp (live), Pup weight females on day 4 pp (live), Litter weight on day 0, Litter weight on day 4 pp, Gross abnormalities (day 0 – 4 pp), Gross abnormalities at evisceration, Behavioral abnormalities day 0-4 pp, Runts
Postmortem examinations (parental animals):
SACRIFICE
The male animals were sacrificed on day 43, the pregnant females after day 4 post-partum of the study.

GROSS NECROPSY
All adult rats were necropsied and examined for gross pathological alterations. The rats were killed by anesthesia with a carbon dioxide air mixture and exsanguination after opening of the abdominal vessels.

ORGAN WEIGHTS
Terminal body weight (after exsanguination), Heart, Liver, Kidneys (together), Spleen, Thymus, Testes (together), Prostate, Ovaries (together), Uterus, Adrenals (together after fixation), Thyroids with Parathyroids (together after fixation), Brain (after fixation), Seminal vesicles, Epididymides (together)

HISTOPATHOLOGY (Groups 1 and 4 examined, intermediates as required)
Adrenal (2), Bone with bone marrow (sternum, femur), Brain (cerebrum, cerebellum, pons, brain stem), Eye (2), Heart, Intestine, large Cecum, Colon, Rectum, Intestine, small Duodenum, Jejunum, Ileum, Kidney (2), Liver (left lateral and right medial lobe), Lung (with mainstem bronchi), Lymph nodes mandibular (2) and mesenteric, Mammary gland (inguinal), Muscle, skeletal (thigh), Nerve, sciaticPeyer’s Patches, Reproductive organs, female: Ovary (2), Oviduct (2), Uterus (cornu/corpus/cervix), Vagina, Reproductive organs, male Epididymis (2), Prostate, Seminal vesicle, Testis (2), Spinal cord (cervical, thoracal, lumbal), Spleen, Stomach (proventricular, fundic, pyloric), Thymus, Thyroid (2), Trachea, Urinary bladder, All tissues showing abnormality
Postmortem examinations (offspring):
SACRIFICE
Dead pups and pups killed on day 4 post-partum (day 4 pp), will be carefully examined externally for gross abnormalities. On day 4 pp, pups will be killed in the laboratory, by a pain-free method (overdose of pentobarbital i.p or s.c). Macroscopic examination will be performed afterwards.

GROSS NECROPSY
Each pup was eviscerated, and examined for gross abnormalities. The inner sexual organs was compared with the sex determined by the anogenital distance. In case of discrepancies, the inner sex overrules the external sex. For each pup the following minimum evaluation were performed: External examination of body, Sex external, Eye (2), Palatine with tongue, Sex internal / reproductive organs, Urinary bladder, Ureter (2), Pancreas, Spleen, Kidney (2), Adrenal (2), Liver, Gastrointestinal tract, Diaphragm, Heart, Lung, Thymus
Statistics:
Statistical test
- Body weight and body weight gain compared to day 0 (males and females during premating period): DT, 2-sided
- Body weight and body weight gain compared to gestation day 0 (pregnant females during gestation period): DT, 2-sided
- Food consumption (males and females during premating and pregnant females): DT, 2-sided
- FOB numerical parameters (males and females before and week 1, males on day 29 + pregnant females on day 4 pp): Kruskal Wallis (2-sided) + Wilcoxon
- Motor activity (males on day 29, pregnant females on day 4 pp): Kruskal Wallis (2-sided) + Wilcoxon
- Organ weights (males + pregnant females): DT, 2-sided
- Hematological and clinical chemistry parameters: Wilcoxon (2-sided) + Bonferroni-Holm
- Pup weight males on day 0 pp (live): Dunnett-test 2-sided
- Pup weight males on day 4 pp (live): Dunnett-test 2-sided
- Pup weight females on day 0pp (live): Dunnett-test 2-sided
- Pup weight females on day 4pp (live): Dunnett-test 2-sided
- Litter weight on day 0: Dunnett-test 2-sided
- Litter weight on day 4 pp: Dunnett-test 2-sided
Reproductive indices:
Pairs started, Conceiving days, Pregnancy duration, Females showing evidence of copulation, Females achieving pregnancy, Live young born, Live young on day 4 pp, Sex ratio at birth (on day 0 pp), Sex ratio on day 4 pp, Corpora lutea, Implantations, Resorptions, Gross abnormalities (day 0 – 4 pp), Gross abnormalities at evisceration, Behavioral abnormalities day 0-4 pp,

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals survived the treatment period. No treatment-related clinical signs were observed in males at any time point. In the females, no treatment-related signs were observed during the pre-mating period, during pregnancy, but increased incidences of hair loss was observed in group 4 (135 mg/kg) females.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain was not affected in males treated daily orally for a period of 42 days at doses of up to 135 mg/kg at any time point (including pre-mating, end of mating or end of treatment period). During the pre-mating period, the females did not show statistically significant differences of body weight between dose- and control group, however, body weight gain was decreased in group 4 (135 mg/kg) females during the 2nd week of treatment (day 7-14)compared to control. During pregnancy, body weight was decreased (without statistical significance) in group 3 (45 mg/kg) from day 6 onwards, and with statistical significance in group 4 (135 mg/kg) females from study day 8 onwards (with the exception of day 22+23) compared to control. Body weight gain was decreased until gestation day 20, not always statistically significant. No treatment-related effects on food consumption were 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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant alterations were seen in some clinical chemistry parameters but were all within the known internal reference interval, low in degree and therefore considered incidental and not treatment-related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
During the functional observational battery no treatment-related relevant changes were observation days 0, 7, 29 (males only), or day 4 p.p. (females only) in the autonomous and senso-motor domains. In the neuromuscular domain, hind limb foot splay showed a slight trend of reduction in all treatment group males (15 to 135 mg/kg) on study day 29 compared to control. Group 4females (135 mg/kg) on day 4 p.p. showed decreased hind limb foot splay as well. However, the observation was without statistical significance. In the central nervous domain a trend of reduction of the raising number in group 4 females (135 mg/kg) on day 7, and in group 3 (45mg/kg) and 4 (135 mg/kg) on day 4 p.p. was noted. Body temperature was slightly reduced (statistically significant) in group 4 (135 mg/kg) females on day 7 (pre-mating period) and day 4p.p. compared to control.

Motor activity (number of counts) measured on day 29 in males and on day 4 p.p. in females was unaffected at all dose levels (up to 135 mg/kg). In group 4 (135 mg/kg) females on day 4 p.p. a trend towards slightly reduced rearing numbers and rearing time was noted.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histopathology, group 4 (135 mg/kg) showed pronounced cortical atrophy of the adrenalgland combined with endothelial cell activation and mononuclear infiltrates in both sexes. In the liver, increased centrilobular single cell necrosis was observed in eight females whereas males were not affected. In group 3 (45 mg/kg), cortical atrophy of the adrenal gland was slightly less pronounced than in group 4 (135 mg/kg) in both sexes. Endothelial activation in females was comparable to group 4 (135 mg/kg) whereas in males only three animals showed endothelial activation. Mononuclear infiltrates were observed in 4/24 rats. In the liver, increased amounts of single cell necrosis weredetected in one male and three females. In group 2 (15 mg/kg), three females showed a minimal to slight degree of endothelial activation, and four females and one male showed mononuclear infiltrates. These findings are not considered to be adverse due to the slight degree and small animal number affected. In the liver three female rats had a minimal degree of increased centrilobular single cell necrosis which is also not considered to represent an adverse effect. Extramedullary hematopoiesis was observed in the adrenal gland of females of all dose-groups including controls. This finding is considered to be related to the former pregnancy of those rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
All females in all groups including control paired, showed evidence of conception, and achieved pregnancy. Conceiving days 1-5 were evident in all females (n=12) of the 3 dose groups (15 to 135 mg/kg), and in 11 (of 12) control females. One control female conceived later (day 12).
Pregnancy duration varied between 21 and 24 days, with most females delivering after 22 pregnancy days. Controls and group 3 (45 mg/kg) had either 22 (10/12) or 23 (2/12) days of pregnancy each, group 2 (15 mg/kg) had 21 days (2/12), 22 days (9/12) or 23 days (1/12) of pregnancy, and group 4 (135 mg/kg) had either 21 days (1/10), 22 days (6/10), 23 days (2/10), or 24 days (1/10) of pregnancy. Therefore, most variation of pregnancy duration was observed in group 4 (135 mg/kg) females.

Pre-implantation loss (corpora lutea minus implantations) was observed in all groups including control. No treatment-relationship could be established.

Pre-natal loss (implantations minus live births) was observed in all groups including control. The number of females with more than three losses was dose-dependently increased in groups 3 (45 mg/kg) and 4 (135 mg/kg).

Post-natal loss (live births minus alive at post-natal day 4) was observed in groups 3 (45 mg/kg) and 4 (135 mg/kg). Females with one post-natal loss each, were seen in groups 3 (45 mg/kg) and 4 (135 mg/kg). One female with two post-natal losses was seen in group 3 (45 mg/kg) only.

Details on results (P0)

Please refer to result tables attached.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
45 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
45 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Abnormal live pups were only observed in one female of group 4 (135 mg/kg), having one abnormal pup diagnosed with unilateral hydronephosis (left) during evisceration. All other females had no abnormal pups. Hydronephrosis is a common spontaneous observation in pups. The finding is considered incidental. No runts were observed in any group.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no treatment-related effect on the number of dead pups at birth. The number of dead pups on day 4 p.p. was slighlty higher in groups 3 (45 mg/kg bw/day) and 4 (135 mg/kg bw/day) compared to control, however, no dose-relationship could be established.
The number of live pups on days 0 p.p. and 4 p.p. was reduced in groups 3 (45 mg/kg) and 4 (135 mg/kg). However, this effect is related to an increased number of resorptions in groups 3 (45 mg/kg bw/day) and 4 (135 mg/kg bw/day).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean pup weight of male and female pups at birth and on day 4 p.p. did not show any statistically significant differences between treatment groups and control. A trend towards slightly decreased pup weights on day 0 in group 4 (135 mg/kg) was observed (-7% in both genders). On day 4 p.p., no dose-relationship of weight changes could be established.
Litter weight at birth and on day 4 p.p. was statistically significantly decreased due to a reduced number of live pups in groups 3 (45 mg/kg) and 4 (135 mg/kg) by 26% and 57%, respectively.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Description (incidence and severity):
135
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio on day 0 p.p. in control was shifted towards females with a ratio of 0.61 (4.4/7.2). In groups 2 (15 mg/kg) and 3 (45 mg/kg), the ratio was approximately 1, 1.02 and 1.07, respectively. In group 4 (135 mg/kg) the sex ratio was slightly shifted towards females with a ratio of 0.78 (1.8/2.3). Therefore, it is concluded that no treatment-related change can be observed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Please refer to result tables attached.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
135 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects were seen

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
45 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
In conclusion, the no adverse effect level (NOAEL) regarding systemic toxicity and reproduction parameters is considered to be 15 mg/kg bw/day.
Executive summary:

The test material was administered orally by gavage, once daily, 7 times a week to 3 groups of male and female Crl:WI (Han) rats at doses of 15, 45 or 135 mg/kg. Males received the test item for 6 weeks, whereas females had a treatment period of up to 7 weeks.

A similarly constituted control group received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), and served to generate contemporary control data. All dose groups consisted of 12 male and 12 female rats each.

Males and females were first treated for 14 days separately, thereafter, a mating period of maximally 2 weeks started. As soon as the females showed a positive sperm result, animals were separated again. The males were treated until day 42 (6 weeks treatment duration) and the females were treated throughout birth of pups until necropsy (after day 4 post partum, approximately 7 weeks treatment duration). One control female had a late conception and was vehicle-treated for approximately 8 weeks.

The rats were single housed, except for the mating period, under conventional conditions. During the mating period of maximally 2 weeks, animals were paired (1 male and 1 female per cage) within the dose groups.

Group Dose [mg/kg] Males Females
No. of animals Animal Nos. No. of animals Animal Nos.
1 0 12 1-12 12 49-60
2 15 12 13-24 12 61-72
3 45 12 25-36 12 73-84
4 135 12 37-48 12 85-96
Total 48 48


Survey of inlife investigations: Observations/Measurements Time schedule


Adults (males and females)
Appearance and behavior: Daily
Mortality: Daily
Motor activity: Day 29 males / Day 4 p.p. females
Functional observational battery: Predose (day 0), day 7, day 29 males / day 4 p.p. females
Body weight males: Once a week
Body weight females: Once a week, from mating daily, then day 0 p.p. and day 4 p.p.
Food consumption males: Once a week before mating
Food consumption females: Once a week before mating, after positive vaginal smears on days 7, 14, 21 and day 4 p.p.
Hematology: Day 13
Clinical chemistry: Day 13
Urinalysis: Day 13
Formulation analysis: Weeks 1 + 4

Reproductive parameters (pregnant females)
Conceiving day: Daily vaginal smears during mating period
Delivery day: Once between day 20 - 24
Uteri staining, resorptions, implantation sites: After day 4 p.p.
Ovaries: number of corpora lutea: After day 4 p.p.

All adult rats were subjected to macroscopic and histopathological examinations. Selected organs were weighed from each surviving rat at the end of the treatment period.

The offspring were weighed, sexed, and observed for mortality, clinical signs, appearance and behavior. All surviving pups were subjected to external macroscopy, evisceration with internal sexing of animals and examination for gross pathology changes.

The concentration of the test material in the dosing formulations was within the predefined acceptance limits (±15 % of the nominal concentration). No test material was detected in the control formulations.
All animals survived the treatment period.
No treatment-related clinical signs were observed in males at any time point. In the females, no treatment-related signs were observed during the pre-mating period, during pregnancy, but increased incidences of hair loss was observed in group 4 (135 mg/kg) females.
Body weight and body weight gain was not affected in males treated daily orally for a period of 42 days at doses of up to 135 mg/kg at any time point (including pre-mating, end of mating or end of treatment period). During the pre-mating period, the females did not show statistically significant differences of body weight between dose- and control group, however, body weight gain was decreased in group 4 (135 mg/kg) females during the 2nd week of treatment (day 7-14) compared to control. During pregnancy, body weight was decreased (without statistical significance) in group 3 (45 mg/kg) from day 6 onwards, and with statistical significance in group 4 (135 mg/kg) females from study day 8 onwards (with the exception of day 22+23) compared to control. Body weight gain was decreased until gestation day 20, not always statistically significant. No treatment-related effects on food consumption were observed.
During the functional observational battery no treatment-related relevant changes were observed on days 0, 7, 29 (males only), or day 4 p.p. (females only) in the autonomous and sensomotoric domains. In the neuromuscular domain, hind limb foot splay showed a slight trend of reduction in all treatment group males (15 to 135 mg/kg) on study day 29 compared to control. Group 4 females (135 mg/kg) on day 4 p.p. showed decreased hind limb foot splay as well. However, the observation was without statistical significance. In the central nervous domain a trend of reduction of the raising number in group 4 females (135 mg/kg) on day 7, and in group 3 (45 mg/kg) and 4 (135 mg/kg) on day 4 p.p. was noted. Body temperature was slightly reduced (statistically significant) in group 4 (135 mg/kg) females on day 7 (pre-mating period) and day 4 p.p. compared to control.
Motor activity (number of counts) measured on day 29 in males and on day 4 p.p. in females was unaffected at all dose levels (up to 135 mg/kg). In group 4 (135 mg/kg) females on day 4 p.p. a trend towards slightly reduced rearing numbers and rearing time was noted. Overall, only single behavioral parameters within the different domains were slightly affected without statistical significance. The effect on rearing numbers and time together with the reduced body weight is probably a treatment-related clinical effect on the general condition of these females.
Hematology, coagulation, clinical chemistry, and urinary parameters were measured on study day 13. Statistically significant alterations were seen in some clinical chemistry parameters but were all within the known internal reference interval, low in degree and therefore considered incidental and not treatment-related.
At necropsy of the adult animals only spontaneous findings were observed. Female high dose rats (135 mg/kg) exhibited a terminal body weight reduction of approximately 8 %. Adrenal weights of both sexes were absolutely and relatively reduced at 45 mg/kg and 135 mg/kg. At histopathology, group 4 (135 mg/kg) showed pronounced cortical atrophy of the adrenal gland combined with endothelial cell activation and mononuclear infiltrates in both sexes. In the
liver, increased centrilobular single cell necrosis was observed in eight females whereas males were not affected.
In group 3 (45 mg/kg), cortical atrophy of the adrenal gland was slightly less pronounced than in group 4 (135 mg/kg) in both sexes. Endothelial activation in females was comparable to group 4 (135 mg/kg) whereas in males only three animals showed endothelial activation. Mononuclear infiltrates were observed in 4/24 rats. In the liver, increased amounts of single cell necrosis were detected in one male and three females.
In group 2 (15 mg/kg), three females exhibited slight degree of endothelial activation, and four females and one male showed mononuclear infiltrates. These findings are not considered to be adverse due to the slight degree and small animal number affected. In the liver three female rats had a minimal degree of increased centrilobular single cell necrosis which is also not considered to represent an adverse effect.
Extramedullary hematopoiesis was observed in the adrenal gland of females of all dose-groups including controls. This finding is considered to be related to the former pregnancy of those rats.

Reproduction parameters:
All females of all groups, including control, paired and showed evidence of conception, and achieved pregnancy. No treatment-related effects on male or female fertility were observed. Conceiving days 1-5 were evident in all females of the 3 dose groups (15 to 135 mg/kg), and in 11/12 control females. One control female conceived later (day 12). Pregnancy duration varied between 21 and 24 days, with most females delivering after 22 pregnancy days. Slightly higher
variation of pregnancy duration was observed in group 4 (135 mg/kg) females.
Live young on day 0 and 4 p.p. were observed in all females (n=12) of the control, group 2 (15 mg/kg) and group 3 (45 mg/kg). In group 4 (135 mg/kg), live young were observed in 9/12 females on day 0 and 4 p.p.. Abnormal pups were only observed in one female of group 4 (135 mg/kg) with one abnormal live pup (unilateral hydronephrosis). The other females had no abnormal pups. Hydronephrosis is a common spontaneous observation in pups. The finding is considered incidental. No runts were observed in any group.
Pre-implantation loss (corpora lutea minus implantations) was observed in all groups including control. No treatment-relationship could be established.
Pre-natal loss (implantations minus live births) was observed in all groups including control. The number of females with more than three losses was dose-dependently increased in groups 3 (45 mg/kg) and 4 (135 mg/kg).
Post-natal loss (live births minus alive at post natal day 4) was observed in groups 3 (45 mg/kg) and 4 (135 mg/kg). Females with one post-natal loss each, were seen in groups 3 (45 mg/kg) and group 4 (135 mg/kg). One female with two post-natal losses was seen in group 3 (45 mg/kg) only.
The number of corpora lutea and implants was slightly reduced in group 4 (135 mg/kg). In groups 3 (45 mg/kg) and 4 (135 mg/kg) the number of resorptions was increased whereas the number of live pups on days 0 and 4 p.p. was reduced.
There was no treatment-related effect on the number of dead pups at birth. The number of dead pups on day 4 p.p. was slightly higher in groups 3 (45 mg/kg) and 4 (135 mg/kg) compared to control, however, no dose-relationship could be established.
Sex ratio at birth did not show any treatment-related differences between treatment groups and control.
The mean pup weights of males and females at birth and on day 4 p.p. did not show any relevant differences between treatment groups and control.
Litter weight at birth and on day 4 p.p. was clearly decreased in group 3 (45 mg/kg) and 4 (135 mg/kg) females, by 26% and 57%, respectively.

Conclusions

Daily oral treatment by gavage of 15 mg/kg of the test item to rats according to the study design was tolerated whereas 45 or 135 mg/kg caused cortical atrophy of the adrenal glands was observed in both sexes. A statistical significant weight reduction of both absolute and relative weights correlates to this effect.
Clinically, all doses (15, 45, and 135 mg/kg) were tolerated over a treatment period of 42 days in males. The females, treated for approximately up to 7 weeks showed clinically an increased incidence of hair loss in group 4 (135 mg/kg) and statistically significant body weight decreases in group 4 (135 mg/kg) during the gestation period. No effects on clinical pathology parameters were noted in both genders of any dose group on study day 13. Behavioral parameters (FOB and
motor activity) showed some slight changes in females of group 4 (135 mg/kg) on days 7 and 4p.p. with a slight reduction of rearing time and number, plus a body temperature decrease, however, a treatment-relationship could not be defined equivocally.
The most prominent findings regarding reproduction parameters was an increased number of resorptions in groups 3 (45 mg/kg) and 4 (135 mg/kg). Related to this, the number of live pups and litter weights were decreased in these groups on days 0 and 4 p.p..
In addition, the following findings were noted: a slightly higher variation of pregnancy duration and a reduced number of females with live young was observed in group 4 (135 mg/kg). Prenatal loss (implantations minus live births) was increased in groups 3 (45 mg/kg) and 4 (135 mg/kg). Post-natal loss (live births minus alive at post natal day 4) was only noted in groups 3 (45 mg/kg) and 4 (135 mg/kg), however, only at a very low incidence. The number of corpora lutea and implants was slightly reduced in group 4 (135 mg/kg), whereas the number of resorptions was increased in groups 3 (45 mg/kg) and 4 (135 mg/kg). No treatment-related effects on male or female fertility were observed.

In conclusion, the no adverse effect level (NOAEL) regarding systemic toxicity and reproduction parameters is considered to be 15 mg/kg bw/d.