Quaternary ammonium compounds, benzyl C12-C16 (even numbered)-alkyldimethyl chlorides

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:

1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

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: EEC Directive 79/831, Annex V, Method No. 431

Deviations:

no

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N-benzyl-N,N-dimethyltetradecan-1-aminium chloride

EC Number:

939-253-5

Cas Number:

68424-85-1

Molecular formula:

C12-14H25-29-(CH3)2-C6H5-N.Cl

IUPAC Name:

N-benzyl-N,N-dimethyltetradecan-1-aminium chloride

Test material form:

liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Breeders were purchased from G1. Bomholtgard Ltd., but the mice used were born in the Scantox Laboratories
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 25-30g
- Assigned to test groups randomly: Yes, in groups of 5
- Fasting period before study: No
- Housing: 5 animals/cage, males and females separately in type III Macrolone cages, bedding used was special softwood sawdust "Spanvall Special White " from Spanvall Ltd., DK-4535 Vallekilde
- Diet: Complete rodent diet "Altromin 1314" from Chr. Petersen Ltd., DK-4100 Ringsted, ad libitum
- Water: Drinking water adjusted to pH 2.5 with hydrochloric acid, ad libitum
- Acclimation period:

Environmental conditions
- Temperature: 21 ±2°C
- Humidity: 55 ± 15%
- Air changes: 10/h
- Photoperiod: 12h dark /12h light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Distilled water
- Concentration of test material in vehicle: 400 mg/10 mL distilled water for the dose level of 400 mg/kg bw.
- Amount of vehicle: The vehicle was administered orally at a volume of 10 mL/kg bw.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dilution of the test substance in distilled water

Frequency of treatment:

Once

Post exposure period:

24, 48 and 72 h

Doses / concentrations

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (positive control):
- Route of administration: Oral
- Doses / concentrations: 30 mg/10 mL equivalent to 30 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

Dose selection:
Preliminary investigations:
- A few mice were treated orally with various concentrations of the test substance diluted with distilled water. Thereby the maximum tolerated dose was estimated at 400 mg/kg bw. At this dosage bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).

Details of slide preparations: Immediately after sacrifice, femurs of a mouse were dissected free of muscle, and by a 1 mL syringe with needle the bone marrow was flushed out into 5 mL of fetal calf serum. After thorough shaking, the mixture was centrifuged for 10 min. at about 1000 rpm. Thereafter, smears were made after removal of the supernatant. The specimens were fixed in methanol and stained with May-Grunwald/Giemsa.

Method of analysis: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind.
The following counts were made:
Number of normochromatic erythrocytes (NCE) per 1000 erythrocytes
Number of polychromatic erythrocytes (PCE) per 1000 erythrocytes
Number of micronuclei (MN) in 1000 normochromatic erythrocytes
Number of micronuclei (MN) in 1000 polychromatic erythrocytes.

Evaluation criteria:

Increase in the frequency of micronucleated polychromatic erythrocytes in treated animals as compared to controls

Statistics:

The statistical difference was analysed by one-way ANOVA. In the case of PCE (%) the test was performed on the values observed, and for the MN (per thousand) the test was done on computed rank values transformed to normal scores according to Blom's method (Blom, 1958).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

one mortality in the 72h test group on Day 3 after treatment

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not genotoxic

Additional information on results:

Results of the dose-range finding study
- Dose range: Up to 400 mg/kg bw
- Clinical signs of toxicity in test animals: Maximum tolerated dose was estimated at 400 mg/kg bw. At a dose exceeding 400 mg/kg bw, the mortality was too high.
- Evidence of cytotoxicity in tissue analyzed: At 400 mg/kg bw, bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).
Results of the main test
- Induction of micronuclei: No significant difference as compared to controls.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice.

Executive summary:

A study was conducted to determine the in vivo toxicity of the test substance, C12-16 ADBAC (80.2% active in ethanol) according to OECD Guideline 474, in compliance with GLP. This study was performed to evaluate the chromosome-damaging effect of the test substance in mice. The experimental animals were 50 NMRI mice, divided into 5 groups. Of the 5 groups, three were test groups, one negative control group and one positive control group. The test groups were treated with 400 mg test substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively after treatment. From bone marrow smears micronucleus counts were made per 1000 polychromatic erythrocytes. Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice (Kallesen, 1985).