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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 10, 1992 to March 25, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD)

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: EC Commission directive 84/449/EEC of 25 . April 1984, Part B : Methods for the determination of Toxicity ; Sub-acute Toxicity (oral) ; Official Journal of the European Communities No . L 251, p . 118 - 121, 1984
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5,6-trimethylcyclohex-2-en-1-one
EC Number:
243-473-0
EC Name:
2,5,6-trimethylcyclohex-2-en-1-one
Cas Number:
20030-30-2
Molecular formula:
C9H14O
IUPAC Name:
2,5,6-trimethylcyclohex-2-en-1-one
Details on test material:
- Name of test material (as cited in study report): 2,5,6-Trimethyl-2-cyclohexen-1-on
- Test substance number : 90/73 1
- Lot/batch No.: production, Kopf K 204 from November 13, 1990, 11.00 am
- Physical state: clear colorless and homogeneous liquid
- Storage condition of test material: room temperature
- Stability under test conditions: the degree of purity of the test substance was 95.7% before the start of the study and 94.3% after the end of
the study . Thus the test substance was stable for the period of the study

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Chbb :THOM (SPF)
- Source: Dr . KARL THOMAE GmbH, Biberach/Riss, Germany
- Identification: ear tattoo
- Age at study initiation: 42 days old
- Weight at study initiation: 185 (180 - 194) g for the male, 148 (139 - 158) g for the females .
- Housing: housed singly in type DK III stainless steel wire cages supplied by BECKER & Co., Castrop-Rauxel, Germany (floor area about 800 cm^2) .
- Diet (e.g. ad libitum): ad libitum, ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingentalmühle AG, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): ad libitum, drinking water
- Acclimation period: 8 days


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

OTHER
The animal room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin-ammoniabased terminal disinfector) before the start of the study . The floor was cleaned twice a week and the walls were cleaned once a week . The cleansing liquid used in each case was water containing about 0 .1% Incidin perfekt® (supplied by HENKEL, Düsseldorf, FRG) .

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- 2,5,6-Trimethyl-2-cyclohexen-1-on was weighed depending on the dose group, then olive oil DAB 9 was added and the mixture was
subsequently dissolved by shaking .
- The test substance preparations were made in intervals for which the stability of the test substance preparations was proven .


VEHICLE
- Concentration in vehicle: 0.3, 1.2, 6 g/100 ml
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): olive oil DAB 9
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each concentration taken from the first test substance preparation were sent to the analytical laboratory for determination of the correctness of the concentrations of the test substance preparations .
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily (except Saturdays and Sundays)
Doses / concentrations
Remarks:
Doses / Concentrations:
15; 60; 300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: due to results obtained from 14 day study (7.5.1 BASFAG11S0731/9009 6.Repeated dose toxicity: oral 14 days rat)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice (Mondays to Fridays) or once a day (Saturdays, Sundays and on public holidays)


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


BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period; during the administration period the body weight of the animals was determined once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5 per sex per group
- Parameters checked in table were examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: No
- How many animals: 5 per sex per dose
- Parameters were examined:
1. Enzymes: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-glutamyltransferase
2. Blood chemistry: sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
3 . Immunoglobulins: immunoglobulin G (IgG), immunoglobulin M (IgM)


URINALYSIS: Yes
- Parameters were examined: volume, color, turbidity,nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
In the statistical evaluation of the study, means and standard deviation were calculated for the variables food consumption, body weight and body weight change for the animals in each test group, and printed out together with the individual values in the form of tables (except body weight change) .
The statistical significance of the clinical data (body weight and body weight change) was determined using KRUSKAL-WALLIS and MANN-WHITNEY - U-Test. If the results of this test were significant, p-markers (* for p< 0 .05, ** for p< 0 .02, *** for p< 0 .002) were printed together with the group means in the tables.
For some urinalisys parameters a pairwise comparison of each dose group with the control was carried out using Fisher's exact test for the hypothesis of equal proportions . If the results of this test were significant, labels (* for p < 0 .05 , ** for p< 0 .01) were printed in the tables .

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- During the first week of administration all male and female animals of the highest dose group showed reduced activity after application for about one hour.
- From the 2nd application onwards the males and females of the 300 mg/kg bw dose group showed salivation after application for about 30 min.
- In one male rat of the lowest dose group a tail injury was noted (animal No . 6) . This injury resulted from
mechanical influence and is not to be considered as a treatment - related effect .
- In all other male and female animals of the dose groups with 15 and 60 mg/kg bw/day no clinically observable signs of toxicity were detected

BODY WEIGHT AND WEIGHT GAIN
- No statistically significant differences were obtained in any of the dose groups
- Body weight change: In the highest dose group, body weight change was impaired about 12 to 28% during the first 1 to 2 weeks of the study being statistically significant in males only .

FOOD CONSUMPTION
On day 7 of the study in the test group with 300 mg/kg bw/day, food consumption was decreased in males (about 18%) and females (about 12% lower than the control values) . In test group 1 and 2, no substance-related effects were observed .

ORGAN WEIGHTS
- statistically significant increase of the absolute and relative liver weights of females in the 300 mg/kg dose group
HAEMATOLOGY
The results of the hematological examinations did not reveal any substance-related changes in the treatment groups of either sex.

CLINICAL CHEMISTRY
The results of the blood chemistry examinations did not reveal any substance-related changes in the treatment groups of either sex.
The results of the enzyme examinations did not reveal any substance-related changes in the treatment groups of either sex

URINALYSIS
In the results of the urinalyses no statistically significant differences were detected in both sexes. However, in the sediment of two males of the 300 mg/kg bw/day group (animals number 17 and 19) increased renal tubular and transitional epithelial cells and granular casts (animal number 19) were found at the end of the study. These findings are assessed as being treatmentrelated and are signs of slight kidney damage .

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical signs: liver and kidney

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Substance-related effects were obtained at 300 mg/kg bw,
target organs were liver and kidneys. The clinical signs
observed were assessed as being the consequence of local
irritation but not due to permanent morphological altera-
tions of the nervous system; no signs of immunotoxicity
were recorded. The increased renal tubular and transitional
epithelial cells in the sediment of two males and increased
granular casts in the urine of one male is probably
associated with the problem of alpha-2-microglobuline. There
is also evidence of this due to the precence of
hyalin-droplets in the cytoplasm of proximal tubular
epithelial cells in the kidneys of males.
Alpha-2-microglobuline occurs only in the male rat and is
not relevant to humans.

Applicant's summary and conclusion

Conclusions:
Due to clinical signs regarding liver and kidney a NOEL of 60 mg/kg bw/day and a LOAEL of 300 mg/kg bw/day were calculated.

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