AERO 5100 PROMOTER

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jan - 25 Feb 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 Dec 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

carbamothioic acid, 2-propenyl-, O-(2-methylpropyl) ester

IUPAC Name:

carbamothioic acid, 2-propenyl-, O-(2-methylpropyl) ester

Details on test material:

- Name of test material (as cited in study report): AERO 5100 Promoter
- Physical state: pale brown, liquid
- Analytical purity: 98%
- Lot/batch No.: 95
- Expiration date of the lot/batch: 1 July 2000
- Storage condition: at room temperature, in the dark

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ldt, Margate, UK
- Weight at study initiation: 28 - 30 g (males), 22 - 24 g (females)
- Housing: in groups of same sex in disposable cages
- Diet: pelleted expanded rat and mouse No. 1 diet SQC grade (Special Diets Services Ltd, Witham, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: corn oil
- Lot/batch no.: W 9088

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

24 h after treatment (all groups), 48 h after treatment (negative control and high-dose group)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Range-finding study: 2 males and 2 females
Main study: 10 (negative control and high-dose group), 5 (positive control and low- and mid-dose group)

Control animals:

yes, concurrent vehicle

Positive control(s):

- mitomycin C
- Route of administration: gavage
- Concentration: 0.6 mg/mL in water
- Dose: 12 mg/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: A range-finding study performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES: Sampling was performed 24 h after treatment (all groups) and 48 h after treatment (negative control and high-dose group)

DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with 10% Giemsa-solution for 10 minutes.

METHOD OF ANALYSIS: The proportion of immature erythrocytes is determined for each animal by counting of at least 1000 erythrocytes. 2000 polychromatic erythrocytes per animal were scored for the incidence of micronucleated erythrocytes.

Evaluation criteria:

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (p < 0.01). Individual and/or group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (p > 0.01) and where these values fall within the historical control range.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Statistics:

The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximize the power of statistical analysis.
For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation). Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected; for individual intergroup comparisons this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100 - 750 mg/kg bw
- Clinical signs of toxicity in test animals: Deaths (1/4, 2/4, 4/4, 3/4 and 4/4, respectively) occurred in all groups dosed with test substance at concentrations of 175, 250, 500, 600 and 750 mg/kg bw, respectively, and severe clinical signs were observed in the remaining animals. At 100 mg/kg bw, all animals showed signs of hunched posture, piloerection and irregular respiration but survived to terminal sacrifice. Results showed that a dose level of 100 mg/kg bw was approximately the maximum tolerated dose. This dose level was considered to be an appropriate maximum for use in the main study. Therefore, animals were treated with 25, 50 and 100 mg/kg bw test substance in the main study.

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay (males and females combined)

			Proportion of immature erythrocytes at sampling time		Mean number of micronucleated cells per 2000 immature erythrocytes at sampling time		Number of micronucleated cells per 2000 mature erythrocytes at sampling time
Exp. group	Number of animals per sampling time	Dose (mg/kg bw)	24 h	48 h	24 h	48 h	24 h	48 h
Vehicle control (corn oil)	5	-	41	42	0.8	0.4	1.0	2.0
Positive control (mitomycin C)		12	35	n.d	38.2***	n.d	1.8	n.d
Test substance		25	39	n.d	1.1	n.d	1.0	n.d
		50	34	n.d	1.2	n.d	1.5	n.d
		100	41	43	0.9	0.6	0.0	1.7

n.d. = not determined; *** statistically significant (p < 0.001)

CLINICAL SIGNS AND MORTALITY

No mortality was observed during the main study.

Clinical signs observed at 100 mg/kg bw were consistent with the maximum tolerated dose. No adverse clinical signs were observed for the vehicle or positive control group during the study period.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this mammalian erythrocyte micronucleus test in mice the test substances was non-clastogenic at any concentration tested up to 100 mg/kg bw.