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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Rose oxide 90
- Physical state: liquid/colorless, clear
- Analytical purity: 99.0 area-% on a DB-1 column; 98.1 area-% on a DB-wax column
- Batch No.: 01567829U0
- Homogeneity: given (visually)
- Storage condition of test material: room temperature
- Stability: The stability of the test substance under storage conditions over the test period was guaranteed

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Mean weight at study initiation: males 166.1-170.3 g; females 132.3-136.4 g. The weight variation of the animals used did not exceed 20% of the mean weight of each sex.
- Housing: 5 animals per cage; in polysulfonate cages (TECNIPLAST, Hohenpeißenberg, Germany; floor area about 2065 cm^2).
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced at least once a week. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The administration volume was 4 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean values of Rose oxide 90 in corn oil were found to be in the range of 90-110% of the nominal concentrations. These results demonstrated the correctness of the concentrations of Rose oxide 90 in corn oil
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the administration period and thereafter at weekly intervals

FOOD CONSUMPTION:
Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.

WATER CONSUMPTION :
- Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 31 in the morning
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes (16-20 hours)
- How many animals: 5 animals per test group and sex

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 31 in the morning
- Animals fasted: Yes (16-20 hours)
- How many animals: 5 animals per test group and sex

URINALYSIS: Yes
- Time schedule for collection of urine: on day 25
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on day 26 (male), on day 27 (female)
- Dose groups that were examined: all animals
- Battery of functions tested:
Home cage observations (Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities)
Open field observations (Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait abnormalities, Activity/ arousal level, Feces excreted within 2 minutes (number/ appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes)
Sensory motor tests/ reflexes (Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Auditory startle response),Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings)
Motor activity assessment: Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.
Sacrifice and pathology:
ORGAN WEIGHTS: YES
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Prostate
10. Seminal vesicles with coagulating glands
11. Spleen
12. Testes
13. Thymus
14. Thyroid glands
15. Uterus with cervix

GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The
exsanguinated animals were necropsied and assessed by gross pathology.

HISTOPATHOLOGY: Yes
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Eyes with optic nerve
12. Heart
13. Ileum
14. Jejunum
15. Kidneys
16. Liver
17. Lungs
18. Lymph nodes (mesenteric and axillary lymph nodes)
19. Ovaries
20. Peyer’s patches
21. Pituitary gland
22. Prostate
23. Rectum
24. Sciatic nerve
25. Seminal vesicles
26. Skeletal muscle
27. Spinal cord (cervical, thoracic and lumbar cord)
28. Spleen
29. Sternum with marrow
30. Stomach (forestomach and glandular stomach)
31. Testis, left
32. Thymus
33. Thyroid glands
34. Trachea
35. Urinary bladder
36. Uterus
37. Vagina
Other examinations:
Sperm parameters (see Chapter 7.8.1)
Estrous cycle determination (see Chapter 7.8.1)
Statistics:
DUNNETT's test: Body weight, body weight change
KRUSKALWALLIS; post test WILCOXON test: Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity, Blood
parameters, Urine pH, volume, specific gravity, color and turbidity, organ weight parameters
WILCOXON-test: Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)
WILCOXON-test; post test Bonferroni-Holm:Spermanalysis parameters

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study. No signs of general systemic toxicity in Wistar rats of either sex were observed. Slight to moderate salivation after treatment was observed in animals of both sexes of test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) on several days of the study as well as in two male and one female animals of test group 1 (100 mg/kg bw/d). From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related changes in body weight data were observed.

FOOD CONSUMPTION
No treatment-related changes in food consumption were observed. Deviating food consumption values in comparison to the control group were assessed as being incidental and of no biological relevance as body weights were not affected in the treated animals.

WATER CONSUMPTION
No overt changes in water consumption were observed.

HAEMATOLOGY
- Test group 3 (1000 mg/kg bw/d):
Both sexes: red blood cell counts (not significantly in females) and hemoglobin concentrations were decreased, whereas relative reticulocyte counts were increased.
Males: hematokrit values were decreased
Females: mean corpuscular hemoglobin concentration (MCHC) as well as platelet counts were lower compared to controls
No other treatment-related changes observed.

- Test group 2 (300 mg/kg bw/d)
Females: hemoglobin values were statisically significant lower compared to controls, but this value was marginally below the historical control range and the only altered red blood cell parameter in rats of this test group.
Overall, no treatment-related changes observed.

- Test group 1 (100 mg/kg bw/d)
No treatment-related changes observed.

CLINICAL CHEMISTRY
- Test group 3 (1000 mg/kg bw/d):
- Females: alanine aminotransferase (ALT) activities as well as triglyceride and cholesterol levels were increased.
No other treatment-related changes observed.
- Test group 2 (300 mg/kg bw/d) + Test group 1 (100 mg/kg bw/d)
No treatment-related changes observed.

URINALYSIS
- Both sexes: In test group 3 (1000 mg/kg bw/d) urine volume was higher compared to controls. This finding was not correlated with any other change in the urine or among kidney parameters and therefore, it was regarded as treatment-related, but not adverse.
No other treatment-related changes observed.

NEUROBEHAVIOUR
- Functional observational battery
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No treatment-related changes were observed.

- Motor activity measurement
Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female rats.

ORGAN WEIGHTS
- Test group 3 (1000 mg/kg bw/d):
Epididymides: Significantly increased absolute (26%) and relative (33%) mean weights
Kidney: weight increase (abs.: +22/24% ♂/♀); (rel.: +28/24% ♂/♀)
Liver: weight increase (abs.: +36/52% ♂/♀); (rel.: +42/52% ♂/♀)
Spleen: weight increase (abs.: +30/20% ♂/♀); (rel.: +36 /19% ♂/♀)
No other treatment-related changes observed.

- Test group 2 (300 mg/kg bw/d)
Kidney: weight increase (abs.: +13/17% ♂/♀); (rel.: +13/18% ♂/♀)
Liver: weight increase (abs.: +20 /23% ♂/♀); (rel.: +20/23% ♂/♀)
No other treatment-related changes observed.

Test group 1 (100 mg/kg bw/d)
No treatment-related changes observed.

GROSS PATHOLOGY
All findings occurred individually. They were considered to be incidental in nature and not related to treatment.

HISTOPATHOLOGY:
- Left epididymis:
In comparison to the mature epididymal ducts of the control group, the ducts of the distal portion of the corpus and the cauda epididymis were immature in males of test group 2 and 3 (300 and 1000 mg/kg bw/d). The immature epididymal ducts were characterized by epithelial infolding or cribiform pattern, lack of distension and narrow ductal lumina. They were totally or partially devoid of spermatozoa. In contrast to the mature epididymal ducts, immature ducts showed undifferentiated epithelial cells. An interstitial edema with minimal infiltration of mixed-cellular type was more frequently observed in those segments affected by highly immature ducts as seen in test group 3 (1000 mg/kg bw/d). Intraductal infiltration with granulocytes was found in single distended ducts of the cauda containing densely condensed spermatozoa, most likely revealing sperm stasis.

- Spleen:
Extramedullary hematopoiesis was observed in the males and females of test group 3 (1000 mg/kg bw/d).

- Kidneys:
Special stains performed in the kidneys of all males (Mallory-Heidenhain’s stain for eosinophilic droplets and immunohistochemical staining for the detection of anti-alpha2uglobuline) showed that minimal intracytoplasmic eosinophilic droplets in the epithelial cells of proximal convoluted tubules were consistent with alpha2u-globuline. Eosinophilic droplets were equally distributed between control and treated animals.

- Testis, left:
No histopathological changes were found in the left testes.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental in nature and not related to treatment.



ESTRUOS CYCLE + Sperm analysis
See Chapter 7.8.1

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: clinical pathology, pathology
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: clinical pathology, pathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

With regard to clinical examinations, no signs of general systemic toxicity in Wistar rats of either sex were observed. Slight to moderate salivation after treatment was observed. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Regarding clinical pathology in high dose rats of both sexes a slight anemia was present. Additionally, in females of this test group liver cell membranes as well as the liver cell metabolism was affected, indicated by increased alanine aminotransferase (ALT) activities and higher triglyceride and cholesterol values. Because the mentioned effects were due to functional liver cell alterations, a histopathological correlate could not be detected.

 

In mid/ high dose males sperm function and morphology in the cauda epididymidis were adversely affected.

 

Regarding pathology, target organs were the epididymides in males as well as the liver, kidneys and spleen in males and females.

Histopathology revealed in the left epididymis the presence of immature ducts in the distal corpus and/or cauda epididymis in all mid/ high dose males. Immature ducts increased in severity dose dependently. With increasing severity the immature ducts were accompanied by increasing interstitial edema, which correlated with the significant weight increase found in the high dose group. In only two of five high dose males, additional intraductal granulocytic infiltration was observed in single distended ducts of the cauda with apparent sperm stasis. All of these findings were attributed to treatment and were regarded as adverse.

 

In the spleen; slight extramedullary hematopoiesis was seen in high dose males and females. This finding correlated with the relative weight increase of the spleen in males and females. The extramedullary hematopoiesis was considered

to be secondary to the anemia detected at hematology and was regarded as an adaptive response.

 

Significant absolute and relative liver weight increases occurred in males and females of mid/ high dose groups, without a histopathological correlate. Nevertheless, the liver weight increment was regarded as treatment-related but

not adverse in nature.

No histopathological findings were detected in the kidneys of males and females that could explain their weight increase observed, which was therefore regarded as treatment-related but not adverse.

Applicant's summary and conclusion