3-((C12-18)-acylamino)-N-(2-((2-hydroxyethyl)amino)-2-oxoethyl)-N,N-dimethyl-1-propanaminium chloride

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

427-370-1

EC Name:

-

Cas Number:

164288-56-6

Molecular formula:

C21H44N3O3Cl

IUPAC Name:

{[(2-hydroxyethyl)carbamoyl]methyl}dimethyl(3-octadecanamidopropyl)azanium chloride

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sufficient albino Crl:CD-1TM(ICR)BR strain mice were supplied by Charles River (UK) Limited, Margate, Kent. At the start of the main study the mice weighed 26 to 30g and were approximately five to eight weeks old. After a minimum acclimatisation period of seven days the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.

The animals were housed in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding. Free access to mains drinking water and food (Certified Rat and Mouse Diet 5LF2, IPS Product Supplies Ltd, P.O. Box 6655, Weliingborough, Northants) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

distilled water
The vehicle was supplied by IVAX Healthcare Ltd, as follows:
Supplier's identification : water for irrigation
Supplier's lot number : A2044
Safepharm serial number : V-2443
Date received : 15 March 2002
Description : clear colourless liquid
Storage conditions : room temperature

Duration of treatment / exposure:

once

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
2000 mg/kg

Doses / concentrations 2

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
1500 mg/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate

Examinations

Tissues and cell types examined:

Bone Marrow:
- Polychromatic erythrocytes (PCE)
- Normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

Immediately following termination (je. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grûnwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

Evaluation criteria:

Stained bone marrow smears were coded and examined blind using light microscopy at xl000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Premature deaths were observed at 1500 mg/kg in the 48-hour group. Clinical signs were observed in animals dosed with the test material at 1500 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: diarrhoea, hunched posture, pilo-erection and distended abdomen. It was considered that the loss of the two animals due to premature death did not affect the integrity of the study, because at least five analysable animals were availabie per group, as recommended in the OECD guidelines.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

Introduction.

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the revised OECD Guidelines for Testing of Chemicals No.474 “Micronucleus Test”, Method B12 of the EEC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods.

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 1500 mg/kg with 750 and 375 mg/kg as the two lower dose levels.

Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single oral dose of distilled water (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectiveiy. Vehicle control animals were killed 24 or 48 hours later, and positive control animais were killed after 24 hours.

Results.

Marked decreases in PCE/NCE ratios were observed in both the 24 and 48-hour 1500 mg/kg test material dose groups, and a clear dose-related response was seen within the 24- hour test material dose groups. Although decreases in the PCE/NCE ratio at 1500 mg/kg, and a dose-related response within the 24-hour groups were observed this did not result in any statistically significance. It was considered that the relatively high standard deviation value of the 24-hour exposure resulted in the absence of a statistical significance. However, the reductions in PCE/NCE ratios, the occurrence of premature deaths and clinical signs was taken to indicate that systemic absorption had occurred. Whilst premature deaths were recorded in the 48-hour1500 mg/kg test material group, the minimum number of animals required for analysis stated in the OECD Guideline was achieved. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

Conclusion.

The test material was considered to be non-genotoxic under the conditions of the test.