Propanamide, N,N',N''-1,3,5-benzenetriyltris[2,2-dimethyl-

Genetic toxicity: in vivo

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

Study period:

2005-07-05 to 2005-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant; Guideline Study (OECD 474)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Harlan Winkelmann GmbH; D-33178 Borchen;
- Initial Age at Start of Acclimatisation: Males: 5 - 8 weeks; Females: 10-11 weeks;
- Acclimatisation: minimum 5 days;
- Initial Body Weight at Start of Treatment: males mean value 37.0 g; females mean value 34.4 g;
- Housing: single;
- Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen);
- Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen);
- Feed: pelleted standard diet, ad libitum (Harian Winkelmann GmbH, D-33178 Borchen);
- Water: tap water, ad libitum (Gemeindewerke; D-64380 RoBdorf);

- Environment: temperature 22 ± 3 °C; relative humidity 41 - 92 %; artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: the vehicle was chosen to its relative non-toxicity for the animals
- Amount of vehicle: 20 mL/kg body weight orally

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in corn oil.

Duration of treatment / exposure:

single oral gavage treatment

Frequency of treatment:

one treatment

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 100 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

6 males and 6 females per dose per group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide; 40 mg/kg bw;

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes (At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. The ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.)

Details of tissue and slide preparation:

SAMPLING TIMES: Sampling of the bone marrow was done 24 (500, 1000, and 2000 mg/kg b.w.) and 48 hours (2000 mg/kg b.w.) after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using microscopes with 100x oil immersion objectives.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. test item formulated in corn oil. The animals expressed toxic reactions, i.e. reduction of spontaneous activity and ruffled fur.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei / statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item.
- Ratio of PCE/NCE: Not different from control, indicating that the test item had no cytotoxic properties in the bone marrow.
- Signs of systemic toxicity: At 1000 and 2000 mg/kg b.w. signs of toxicity consisted of reduction of spontaneous activity and ruffled fur.