Fatty acids, C18 unsatd., mono- and diesters with triethanolamine, di-Me sulfate quaternized

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:

1989-10-17 to 1990-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

May 26, 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Sept. 19, 1984

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18(even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate-quaternized

Cas Number:

91995-81-2 / 157905-74-3 / 93334-15-7

Molecular formula:

n.a. (UVCB)

IUPAC Name:

Fatty acids, C16-18(even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate-quaternized

Constituent 2

Reference substance name:

Propan-2-ol

EC Number:

200-661-7

EC Name:

Propan-2-ol

Cas Number:

67-63-0

Molecular formula:

C3H8O

IUPAC Name:

propan-2-ol

Test animals

Species:

mouse

Strain:

other: outbred albino mouse, strain CFW 1

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: bidest. water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was dispersed in preheated warm bidest. water (50 °C and applicated at a temperature of 35 °C) at an application volume of 20 ml/kg. The test item concentrations were prepared immediately before use. Homogencity was maintained during application using a magnetic stirrer.

The test substance and negative control was administered by oral intubation. the positive control substance was administrated intraperitoneal.

Duration of treatment / exposure:

The animals received the test item once.
Sampling of the bone marrow was carried out on animals 24, 48 and 72 h after treatment.

Frequency of treatment:

single exposure

Post exposure period:

The animals were sacrificed 24, 48 and 72 hours after treatment.

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Dose range finding study: 2 males, 2 females per group (3 groups)
Main study: 6 males, 6 females per group (5 groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP, CAS-No. 50-18-0, Endoxan, Asta Werke, Germany)
- Route of administration: intraperitoneal
- Doses / concentrations: 10 mg/kg dissolved in bidest. water
- Application volume: 10 ml/kg
- Sampling time: 24 h

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
1. Dose range finding study:
LD50, rat determined to be > 5000 mg/kg bw. therefore the maximum tolerated doses: 3000, 4000, and 5000 mg/kg bw were chosen.
2. Main study:
5000 mg/kg bw, chosen because of the results of dose range finding study (it is the maximum tolerated dose for the test).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were sacrificed 24, 48 or 72 hours after treatment under saturated atmosphere of carbondioxide. For each animal both femurs were removed and freed of blood and muscles. The proximal part of the femur were opend and extracted with aid of a syringe in fetal calf serum. The suspension was collected in a siliconised centrifuge tube, filled with 5 ml fetal calf serum. The cell suspensions were centrifuged at 1000 minE-1 (approx 100 g) for 5 minutes, the supernatant was removed with a Pasteur pipette. A drop of serum was left on the cell pellet. The cells of the sediment were carefully mixed.

DETAILS OF SLIDE PREPARATION:
A drop of the cell suspension was placed on a clean and marked slide, previously degreased with alcohol, and immediately spread on the slide. Three slides were prepared per animal.
The slides were air-dried at least overnight and then stained with Giemsa according to modification of Gollapudi and Kamra (Mutation Research 1979, 64, p. 45-46). The slides were fixed in 100 % methanol for 5 minutes, then rinsed twice in bidistilled water and thereafter stained in Giemsa solution (1 vol Giemsa Solution, Merck Art. No. 9204 plus 1 vol glycerol, dissolved 1:6 in bidistilled water).
After air-drying the back side was cleaned, if necessary, with ethanol and then dipped for 3 minutes in xylol.

METHOD OF ANALYSIS:
From the three slides prepared per animal, one slide was chosen and randomly coded. The slides of 5 male and 5 female animals per treatment group were scored microscopically at a mignification of 1000. The number of micronucleated cells was counted in 1000 polychromatic erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time.
Averages and standard deviations were calculated after deconding the complete scoring results.

Evaluation criteria:

The micronucleus test is considered acceptable if it meets the following criteria:
- the positive control substance induced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
- the incidence of micronuclei should reasonably fall within the historical control data range of the laboratory.

Test substance is considered positive:
if it induced a biologically as well as a statistically significant increase (p < 0.05) in the frequency of micronuclei at any dose or at any sampling either in the male or in the female groups.
Test substance is considered negative:
if none of the tested doses or sampling times showed a statistically significant (p < 0.05) increase in the incidence of micronuclei, neither in male nor in female groups.

Statistics:

Statistical analysis of data was performed by calculating the statistical significance versus negative controls with the aid of the tables of Kastenbaum and Bowman (Mutation Research, 1970, 9, p. 527-549).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

A slight reduction in the ration of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 3000, 4000, 5000 mg/kg bw
- Solubility: in bidest. water
- Application volume: 20 ml/kg bw
- No animal up to the highest dose died within the first three days or showed signs of severe morbidity. Slight piloerection was observed at all animals and all doses up to 24 hours after administration.
- Based on the results of this range-finding study 5000 mg/kg bw was selected as appropriate dose for the test.

RESULTS OF DEFINITIVE STUDY
- 5000 mg/kg bw was tested as the maximum dose in the main experiment at 24, 48 and 72 h. The volume administered was 20 ml/kg bw.
- Toxicity: No mortality was was noted after administration of the test item in any of the animals evaluated. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow.
- Signs of clinical examination: slight piloerection in all animals including positive and negative control.

RESULTS OF NEGATIVE CONTROL GROUP
- no mortality within 24 h
- was in the range of historical control data

RESULTS OF POSITIV CONTROL GROUP
- No mortality within 24 h
- Cyclophoshamide induced a statistically significant increase in the number of micronucleated cells in both sexes.

Applicant's summary and conclusion

Conclusions:

It can be stated that under the experimental conditions reported, the test item “partially unsaturated TEA-Esterquat” did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item “partially unsaturated TEA-Esterquat” is considered to be negative in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

In a mouse bone marrow micronucleus assay according to OECD guideline No. 474, 1983, 6 male and 6 female albino mice (CFW1) per group were treated by oral intubation with partially unsaturated TEA-Esterquat at a dose of 5000 mg/kg bw. Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.

There were no signs of toxicity as indicated by an enhanced mortality rate. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes were determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow.

Partially unsaturated TEA-Esterquat was tested at an adequate dose, based on the results of the range-finding test. The positive control induced the appropriate response.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirements of Test Guideline OECD 474 for in-vivo cytogenetic mutagenicity data.