Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
17 Oct 1989 - 30 Jan 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions. Analytical purity of the test substance not specified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
analytical purity of test substance not specified
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
analytical purity of test substance not specified
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trade name, fatty acid C18 and C18 unsaturated, epoxidized, Ester with ethylene glycol
- Physical state: white powder
- Analytical purity: no data
- Lot/batch No.: No. 41, dated February 24, 1988, with the designation 041/8/055
- Storage condition of test material: at room temperature

Test animals

Species:
mouse
Strain:
other: outbred albino mice, strain CFW 1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 20.7 - 32.8 g (males) and 19.5-24.8 g (females)
- Fasting period before study: animals were fasted overnight prior to dosing and until approximately 3-4 h after administration of test substance and control material.
- Housing: male mice were housed individually in Macrolon cages type I, female mice were housed in groups up to three in Macrolon cages type II.
- Diet: standard animal diet Altromin No. 1314 with 10 mm pellet diameter (Altrogge Spezialfutter), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days (dose range finding study) and at least 4 - 6 days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Concentration of test material in vehicle: 300, 400 and 500 mg/mL (dose range finding study) and 500 mg/mL (main study)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in arachis oil (80°C and applied after cooling at a temperature of approx. 35°C) at an application volume of 10 mL/kg. The test substance concentrations were prepared immediately before use. Homogeneity was maintained during application using a magnetic stirrer.
Duration of treatment / exposure:
24, 48 and 72 h
Frequency of treatment:
single treatment
Post exposure period:
24, 48 and 72 h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3000, 4000 and 5000 mg/kg bw/day
Basis:
actual ingested
dose range finding study
Remarks:
Doses / Concentrations:
5000 mg/kg bw/day
Basis:
actual ingested
main study
No. of animals per sex per dose:
2 (dose range finding study)
6 (main study)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 10 mg/kg bw in water, application volume 10 mL/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
As the acute oral toxicity (LD50 mouse) was determined to be higher than 5000 mg/kg bw according to acute toxicity studies, the following doses were chosen initially to determine the maximum tolerated dose: 3000, 4000 and 5000 mg/kg bw. According to the results of the dose range finding study, a dose level of 5000 mg/kg was chosen for the main study, because it is generally recommended to use the maximum tolerated dose for the micronucleus test.

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed 24, 48 or 72 h after treatment.

DETAILS OF SLIDE PREPARATION:
The slides were fixed with methanol, air dried at least overnight and stained with Giemsa according to modification of Gollapudi and Kamra.

METHOD OF ANALYSIS:
Three slides per animal were prepared, one slide was randomly chosen and analysed. The slides of 5 males and 5 females per treatment group were analysed and the ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time.

Evaluation criteria:
The test is considered acceptable if the positive control substance induced statistically significant increase in polychromatic erythrocytes and the incidence of micronuclei should reasonably fall within the historical control data range of the laboratory. The test is considered positive if the test substance induced biologically as well as statistically significant (p < 0.05) increase in the frequency of micronuclei at any dose either in the male or in the female groups. The test is considered negative if the test substance did not induce any biologically as well as statistically significant (p < 0.05) increase in micronuclei at any dose either in the male or in the female groups.
Statistics:
Statistical significance of test substance values versus negative controls were calculated with the aid of tables of Kastenbaum and Bowman.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 3000, 4000 and 5000 mg/kg bw
- Solubility: soluble
- Harvest times: 24 h
- Clinical signs of toxicity in test animals: no findings were observed in any animals up to 72 h after administration.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant induction
- Clinical signs of toxicity in test animals: signs at clinical examination were not noticed
- Ratio of PCE/NCE (for Micronucleus assay): no statistically significant changes were observed
- Statistical evaluation: the investigated sample did not induce a statistically significant (time dependent) increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of male or female mice.

Any other information on results incl. tables

Table 1: Mean values per group of PCE and PCE/NCE

Treatment group (sampling time

Species and sex

Dose

Mean of

Micronucleated cells/ 1000 PCE

Ratio of PCE/NCE

Negative control (vehicle)

(24 h)

male mice

10 mL/kg

1.6

1.0

female mice

1.8

0.9

Positive control

(24 h)

male mice

10 mg/kg

9.8

1.1

female mice

7.4

1.0

Test substance

 

24 h

male mice

5000 mg/kg

2.4

1.1

female mice

1.6

1.0

48 h

male mice

5000 mg/kg

1.2

1.0

female mice

1.4

1.0

72 h

male mice

5000 mg/kg

1.2

1.3

female mice

1.4

1.2

PCE: Polychromatic erythrocytes

NCE: Nonchromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative