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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
(BASF AG, Department of Experimental Toxicology and Ecology)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Cyclohexane, oxidized, aquous extract
IUPAC Name:
Cyclohexane, oxidized, aquous extract
Constituent 2
Reference substance name:
Cyclohexane, oxidized, aq. ext.
EC Number:
272-810-4
EC Name:
Cyclohexane, oxidized, aq. ext.
Cas Number:
68915-38-8
Molecular formula:
not available
IUPAC Name:
Cyclohexane, oxidized, aq. ext.
Details on test material:
- Name of test material (as cited in study report): EP306/Acid Water
- Physical state: liquid, brownish
- Composition of test material, percentage of components:
The following results were obtained for the test item:
Formic acid 1.8 g/100g
Succinic acid 0.37 g/100g
Glutaric acid 1.3 g/100g
Adipic acid 14.3 g/100g
Acetic acid 0.30 g/100g
Propionic acid 0.23 g/100g
Butyric acid 0.65 g/100g
Valeric acid 0.76 g/100g
Hexane acid <0.1 g/100g
6-Hydroxy caproic acid 7.6 g/100g

The following results were obtained for the saponificated test item:
Formic acid 1.8 g/100g
Succinic acid 0.50 g/100g
Glutaric acid 1.7 g/100g (increase)
Adipic acid 18.1 g/100g (strong increase)
Acetic acid 0.50 g/100g (increase)
Propionic acid 0.17 g/100g
Butyric acid 0.09 g/100g
Valeric acid 0.76 g/100g
Hexane acid <0.1 g/100g
6-Hydroxy caproic acid 14.1 g/100g (strong increase)
- Stability under test conditions: storage stability are guaranteed throughout the study period
- Storage condition of test material: room temperature



Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5-8 weeks
- Mean weight at study initiation: 29.3 g
- Housing: single
- Diet (e.g. ad libitum): standardized pelleted feed, Kaiseraugst
- Water: ad libitum
- Acclimation period: 5 days


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


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: due to the good emulsifying of the test substance in corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The substance to be administered per kg body weight was emulsified in corn oil. To achieve an emulsion of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.


Duration of treatment / exposure:
2 applications with a 24 h interval inbetween.
Frequency of treatment:
2
Post exposure period:
24 h after last application
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide (CPP), 20 mg/kg bw once orally in a volume of 10 ml/kg bw
- Vincristine sulfate (VCR), 0.15 mg/kg bw, ip

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a pretest for the determination of the acute oral toxicity, following twice administration with a 24-hour interval 2000 mg/kg bw, the recommended highest dose according to the OECD Guideline was tolerated by all animals (male and female) without any signs or symptoms. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg bw were administered as further doses.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals of the vehicle control group and the dose groups were treated twice orally (gavage) with a volume of 10 ml/kg body weight of the test substance with a 24-hour interval between both administrations. Animals of the positive control groups were treated only once. All animals were sacrificed 24 hours after the last treatment, respectively.


Evaluation criteria:
Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i.e. = 2000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical negative control data both for PCEs and for NCEs.
- The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
- Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical negative control data.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical negative control data.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: The limit dose of 2000 mg/kg body weight was tolerated by all animals (male and female) without any signs or symptoms.

RESULTS OF DEFINITIVE STUDY
-The two oral administrations of the vehicle corn oil in a volume of 10 m/kg body weight led to the ratio of 1.1 (polychromatic erythrocytes containing micronuclei).
-After two administrations of the highest dose of 2000 mg/kg bw, the ratio of 0.8 (polychromatic erythrocytes containing micronuclei) was found.
- In the two lower dose groups, ratio (polychromatic erythrocytes containing micronuclei) of 1.4 each (500 and 1 000 mg/kg bw group) were detected.
- In all dose groups the number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) was close to the concurrent vehicle control value and was within the historical negative control data range.
- The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (a ratio of 13.4) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected. Vincristine sulfate, a spindle poison agent, produced a clear statistically significant increase (a ratio of 38.4) in the number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. a ratio of 7.6.
-The number of normochromatic erythrocytes containing micronuclei did not distinctly differ to any appreciable extent in the vehicle control group or in the various dose groups.
- No inhibition of erythropoiesis induced by the treatment of mice with EP306/Acid Water was detected, the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the vehicle control values in all dose groups.

Any other information on results incl. tables

 

Dose groups

Total numbers of

MN in

PCE

NCE

PCE

NCE

Vehicle

10000

3408

1.1

0.3

500 mg/kg bw

10000

3822

1.4

0.8

1000 mg/kg bw

10000

3889

1.4

0.0

2000 mg/kg bw

10000

3995

0.8

0.5

CPP 20 mg/kg bw

10000

4024

13.4*

0.0

VCR 0.15 mg/kg bw

10000

6004

38.4*

0.3

PCE = polychromatic erythrocytes
NCE = normochromatic erythrocytes

MN = Micronuclei

* p =0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative