Reaction mass of sodium hydrogen N,N-dibutyl-10-(sulphonatooxy)octadecanamidate and sodium sulphooctadecanoate

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:

1999-03-29 to 1999-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 21st, July 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:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dibutyloleamide and oleic acid, sulphated, sodium salts

EC Number:

915-926-9

Molecular formula:

C18H34Na2O6S + C26H52NNaO5S

IUPAC Name:

N,N-dibutyloleamide and oleic acid, sulphated, sodium salts

Test material form:

liquid

Details on test material:

The water content of the actual test item was 57.6% (w/w); the water content of the REACH registration substance was analytically determined to be 3.8% (w/w).

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: males 37.6 g (33 – 42); females 28.2 g (25 – 31)
- Assigned to test groups randomly: yes
- Housing: macrolon cage type 3, 5 per cage, separatd by sex
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Photoperiod: 12 hours daily

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: water
- Concentration of test material in vehicle: 44.4 % (v/v)
- Amount of vehicle: 10 mL/kg

Frequency of treatment:

The test item was administered twice at an intervals of 24 hours.

Post exposure period:

24 hours

Doses / concentrations

Dose / conc.:

4 504.5 mg/kg bw/day

Remarks:

Dose of test substance received. Corresponds to ca. 2082 mg/kg bw of the REACH registration substance.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Justification for choice of positive control: Standard positive control substance
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg bw
- frequency: once

Examinations

Tissues and cell types examined:

bone marrow from femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: limited mortality in (2/6 animals) in a preliminary test at 4504.5 mg/kg bw of test substance (2000 mg/kg bw calculated based on water free content) combined with no systemic toxicity at lower dose

DETAILS OF SLIDE PREPARATION:
Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grünwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Enteltan®

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes. A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 3378.4 - 4504.5 mg/kg bw test substance (1500 - 2000 mg/kg bw calculated based on water free content)
- Solubility: aqueous solution
- Clinical signs of toxicity in test animals: motor activity decreased, squatting posture, coat bristling, palpebral fissure closed, respiratory sounds

Applicant's summary and conclusion

Conclusions:

The test item did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described according to OECD 474.

Executive summary:

The micronucleus test was carried out with the test item according to OECD 474. The test item was dissolved in deionized water and was given twice at an interval of 24 hours as an orally dose of 4504.5 mg/kg bw test substance (corresponding to 2082 mg/kg body weight of the REACH registration substance) to male and female mice, based on the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours after administration. Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg/kg body weight. The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was not less than 20 % of the control value. Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent. Under the conditions of the present study the results indicate that the test item is not mutagenic in the micronucleus test.