Glycine, N-methyl-N-(1-oxododecyl)-, 1-methylethyl ester

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21-07-1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Identity: ISOPROPYL N-LAUROYLSARCOSINATE
Description: pale yellow liquid
Batch number: 002013
Purity: 92.9 %
Stability of test item: stable under storage conditions
Expiry date: 31-03-2003

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRI
Source: RCC Ltd., Füllinsdorf / Switzerland
Number of animals: 72 (36 m / 36 f)
Age at start of acclimatization: 8 – 10 weeks
Body weight at start: 27.6 – 33.8 g

Acclimatization: About one week under test conditions after health examination.
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with target ranges for temperature 22 +/- 3°C and for relative humidity between 30-70 %. 12 hours fluorescent light/12 hours dark, music during the light period.
Accommodation: Individually in Makrolon type I cages
Diet: Pelleted standard Altromin 1324 ad libitum
Water: Community tap-water ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Dose Selection
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Study Procedure
Test Groups: 6 m / 6 f were assigned to each test group. Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h and 24 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Duration of treatment / exposure:

One oral dosing via gavage and sacrifice after 24 h or 48 h

Frequency of treatment:

One oral dosing via gavage and sacrifice after 24 h or 48 h

Post exposure period:

24 h or 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Remarks:

dosing for 24 h preparation interval

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

dosing for 24 h preparation interval

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

dosing for 24 h and 48 h preparation interval

No. of animals per sex per dose:

5 m / 5 f

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

Examinations

Tissues and cell types examined:

At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides and 10 animals (5 m / 5 f) per test group were evaluated.

Details of tissue and slide preparation:

According to guideline

Evaluation criteria:

Acceptance Criteria
The study was considered valid as the following criteria are met:
-the negative controls are in the range of the historical control data (0.01 - 0.15 %; mean = 0.066 ± 0.032 PCEs with micronuclei)
-the positive controls are in the range of the historical control data (0.91 - 2.975 %; mean = 1.644 ± 0.446 PCEs with micronuclei)
-at least 80 % of animals are evaluable

Evaluation of Results
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. 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

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kg: reduced spontaneous activity, eyelid closure, ruffled fur

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It can be stated that the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 and 48 hrs after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 m / 5 f) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic (PCE) and normochromatic (NCE) erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide used as positive control and administered orally showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.