Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-04-23 to 1992-05-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 12.05.1981
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
19.09.1984
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Versuchstierzucht Winkelmann, Borchen
- Other: Animals form the Wistar strain (Bor:WISW (SPF Cpb)) were used for toxicological tests at the Bayer AG for years. Historic data regarding pysiology and spontaneous alterations were available. The health was spot-tested for important specific infectious agents.
- Age at study initiation: ca. 7 weeks
- Mean weight at study initiation: males: 146 g (136-155 g); females: 122 g (114-134 g).
- Housing: before randomization: Makrolon cages Type III, groups of 5 animals; during the study: Makrolon cages Type II, dust-free wooden granulate, singl-housed.
- Identification of the animals was performed based on their cage carts which lists test substance anme, animal number, dose, gender, study number and tattoo (ear).
- Nutrition: The animals received fixed-formula standard diet Altromin® 1324 pellets and tap water ad libitum.
- Other: Females were nulliparous and not pregnant. The condition of the animals' health was checked before the start of experiment. Only healthy animals, without any clinical signs, were included in the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 220 +/- 2 °C
- Humidity: 50 +/- 10 %
- Air changes: ca. 10 times per hour
- Photoperiod: 12 h light (from 6 am to 6 pm MEZ), artificial illumination

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1,2-Propandiol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulation of test substance in vehicle


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Formulation and analysis:
For the oral application the test substance was formulated daily with the vehicle (1,2-Propandiol) in accordance with the dose scheme. For the calculation it was assumed that the test item has a purity of 100 %. These application formulations were prepared at room tempertaure and were administered immediately.
Once during the experimental period the content of the test substance in the application formulations was controlled and analytically determined, respectively. To make sure, that the test substance is stable over the study period and is homogenously distributed, samples were also analytically checked.
Duration of treatment / exposure:
29 days
Frequency of treatment:
- once a day, 7 days/week, in total 29 applications
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40 mg/kg bw
Basis:
other: nominal in 1,2-Propanediol
Remarks:
Doses / Concentrations:
200 mg/kg bw
Basis:
other: nominal in 1,2-Propanediol
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
other: nominal in 1,2-Propanediol
No. of animals per sex per dose:
5 animals per dose per sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Appication duration, doses and animal groups:
Volume of application formulation: 2 mL/kg bw. Control animals were administered the vehicle (1,2-Propanediol) only. The application valume was adjusted individually to the animals after weighing them.
Before application the animals were randomized and were assigned to the animal groups. The animals were separated gender-specific and were put into a tray. Thereafter the animals were randomly assigned to the animal groups using the software "Scientific Subroutine Package", IBM Institute für Biometrie, Bayer AG.

- Dose selection rationale:
The doses were selected after the performance of a dose-range-finding test using 500 and 1000 mg/kg (7 days, each 3 male and female Wistar rats). The inveatigation of the acute oral toxicity showed that a dose of 2000 mg/kg caused no symptoms.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: Once per week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Once per week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of treatment (week 4)
- Anaesthetic used for blood collection: Yes, diethylether
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of treatment (week 4)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of treatment (week 4)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked in table [No.4] were examined.
Sacrifice and pathology:
Autopsy was performed on day 30 ca. 24 hours after the last application.
GROSS PATHOLOGY: Yes, see table No. 5
HISTOPATHOLOGY: Yes, see table No. 6
Other examinations:
No
Statistics:
From results coming from individual animals, exanmination of animal weights and organ weights as well as from uptakes of food and water arithmetic averagegroup values and standard deviations were caluculated. Test of significance was performed using the Mann´s U-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality. Ruffled fur in the 1000 mg/kg bw/day dose group.
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality. Ruffled fur in the 1000 mg/kg bw/day dose group.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Increased food consumption of female animals of the 1000 mg/kg bw/day dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increased water consumption in male and female animals of the 1000 mg/kg bw/day dose group.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Significant reduction of the hemoglobin concentration, the hematocrit value and the reticulocyte count in male animals. Increased thrombocyte count in male animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Significant decreased ALT activity and increased albumin concentration in male animals. Significant increased AST activity, total protein and albumin concentration in female animals.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day group: Increased absolute and relative liver weights in male and female animals. In male animals adrenal gland weights were also significantly increased. Significantly increased kindey weights were noted in female animals.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the entire study. Animals of the 1000 mg/kg bw/day group showed ruffled fur during week 3 and 4 of the treatment.

BODY WEIGHT AND WEIGHT GAIN
No test item related effects were observed.

FOOD CONSUMPTION
The mean food consumption of female animals of the 1000 mg/kg bw/day group was 18 % higher than in the control group. No treatment related impact on food consumption was observed in male animals of the same dose group or in na y other treatment group.

WATER CONSUMPTION
The mean water consumption was significantly increased in male and female animals of the 1000 mg/kg bw/day dose group.

HAEMATOLOGY
In hematological and histopathological analysis, no test item related effects could be observed on blood parameters and hematopoietic organs up to 200 mg of the test item/kg bw/day. There was a significant decrease in hemoglobin concentration and hematocrit value and a lower reticulocyte count compared to control group in male animals of the 1000 mg/kg bw/day dose group. In addition, a significant increase of the platelet count compared to control group were noted. Blood coagulation was not affected in all treatment groups. No test-item related effect on leukocyte count were noted.

CLINICAL CHEMISTRY
No treatment related effects on clinical chemistry parameter were found up to a dose level of 200 mg/kg bw/day in male and female animals. ALT activity was significantly decreased in male animals of the 1000 mg/kg bw/day group. Albumin and total protein levels were significantly increased in male animals of the same dose group. In female animals of the 1000 mg/kg bw/day dose group, AST activity, total protein level and albumin concentration were significantly increased in comparison to the control group.

URINALYSIS
There were no test item related effects on the volume and specific gravity of the urin in all treatment groups. The pH value and levels of blood, glucose, ketons, bilirubin and urobilinogene were in the range of the control animals in all treated animals. A reduced protein excretion in males of the 1000 mg/kg bw/day group was observed, however without any (histo-)pathological correlate in the kidney.

ORGAN WEIGHTS
Absolute and relative organ weights of animals of the 40 mg/kg bw/day dose group were in comparison with the control group. In male animals of the 1000 mg/kg bw/day group a significant increase of absolute and relative liver weights and adrenal gland weights were noted in comparison to the control group. In female animals of the same dose group absolute and relative liver and kidney weights were significantly increased compared to the control group. Liver and kidney weights relative to body weight were also significantly increased in female animals of the 200 mg/kg bw/day dose group compared to control group.

GROSS PATHOLOGY
Necropsy observation did not reveal any test item related macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histological examination did not reveal any toxic or test item related lesions in the investigated organs.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: haematology; clinical chemistry; organ weights

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The results of the 28-Day Repeated Dose Oral Toxicity Study of Incozol EH in rats showed no significant toxicological changes related to administration of test substance up to 200 mg/kg bw/day. Therefore the NOAEL was determined to be 200 mg/kg bw/day under the conditions of this study.
Executive summary:

The 28-Day Repeated Dose Oral Toxicity Study with Incozol EH in Rats was conducted according the OECD TG 407 (12 May 1981) and Directive 84/449/EEC, B.7 (19 September 1984).

Incozol EH was repeatedly administered by oral gavage at dose levels of 0 (control group), 40 (low dose), 200 (mid dose), and 1000 mg/kg bw/day (high dose) to male and female Wistar rats (Bor:WISW) for 29 consecutive days to assess the toxicological effects of Incozol EH. The control group was given 1,2-propandiol only. Each group consisted of 5 males and 5 females.

There was no mortality during the entire study period. No clinical signs were observed in all test item treated animals. Male and female animals of the high dose group (1000 mg/kg bw/day) showed ruffled fur from the 3rdweek of administration on. An increased water consumption compared to control group was observed in male and female animals of the high dose group. Food consumption was only increased in female animals of the 1000 mg/kg bw/day dose group.

In hematological and histopathological analysis, no test item related effects could be observed on blood parameters and hematopoietic organs up to 200 mg test item/kg bw/day in male animals and up to 1000 mg/kg bw/day in female animals. There was a significant decrease in hemoglobin concentration and hematocrit value and a lower reticulocyte count compared to control group in male animals of the 1000 mg/kg bw/day dose group. In addition a significant increase of the platelet count compared to control group was noted. Blood coagulation was not affected in all treatment groups.

As a result of the blood biochemical analysis and macroscopic and microscopic pathology, there was no treatment related effect on the intermediary metabolism and organ morphology in male and female animals up to a dose level of 200 mg/kg bw/day. Administration of 1000 mg/kg bw/day revealed an increased AST activity in female animals and a reduced ALT activity in male animals compared to control group. Albumin and total protein level were significantly increased in both sexes of the 1000 mg/kg bw/day dose group.

There was a significant increase of liver weights in male and female animals of the 1000 mg/kg bw/day dose group. In the same dose group, an increased adrenal gland and kidney weight was observed in male and female animals, respectively. As there was no macroscopic or microscopic pathological correlation a test item related kidney damage is not considered. Organ weights were not affected up to 200 mg test item/kg bw/day in male and female animals.

There was no test substance related change in urinalysis. In male animals of the high dose group protein concentration in urine was significantly increased compared to control. This observation was not considered to be test item related as there was no pathological correlation and female animals were not affected.

As a result, there was no test substance related change following 28-day repeated oral administration of Incozol EH up to a dose level of 200 mg/kg bw/day. Based on these results, a no-observed-adverse-effect-level (NOAEL) of Incozol EH was determined to be 200 mg/kg bw/day under the conditions of this study.