Registration Dossier

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:
14 April 2008 to 20 May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 21 August 2007 Date of Signature: 15 October 2007
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Not reported.
- Storage condition of test material: room temperature in the dark
- Other: Not reported.

Test animals

Species:
mouse
Strain:
other: Crl:CD-1™(ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, UK
- Age at study initiation: 5 to 8 weeks of age.
- Weight at study initiation: 22 - 30 g.
- Fasting period before study: Overnight fasting immediately before dosing and for approximately three to four hours after dosing.
- Housing: The animals were housed in groups of up to 7 in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): Certified Rat and Mouse Diet Code 5LF2, IPS Ltd., London, UK ad libitum throughout the study.
- Water (e.g. ad libitum): Mains drinking water ad libitum throughout the study.
- Acclimation period: At least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 deg C to 25 deg C.
- Humidity (%): 30 to 70%.
- Air changes (per hr): Approximately 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

IN-LIFE DATES: From: Day 0 To: Day 2

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Water for irrigation
- Concentration of test material in vehicle: 25, 50 and 100 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no. (if required): 300290909/V-4100
Details on exposure:
All animals were dosed by gavage using a metal cannula attached to a graduated syringe.
Duration of treatment / exposure:
Not applicable (treatment was once only).
Frequency of treatment:
Once only.
Post exposure period:
24, 48 hours.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg
Basis:
nominal in water
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal in water
No. of animals per sex per dose:
7 animals in each group as in Table 1.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test. Therefore it was used to confirm the sensitivity of the system under the conditions of the test.
- Route of administration: Oral by gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Erythrocytes in bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg in the range-finding toxicity study. (As a result, a premature death occurred at 1200 mg/kg, and clinical signs were observed at and above 1000 mg/kg. The maximum tolerated dose (MTD) of the test material, selected for use in the main test was 1000 mg/kg, with 500 and 250 mg/kg as the lower dose levels.)

DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 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, stained in May-Grünwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.

METHOD OF ANALYSIS: Examination of bone marrow slides microscopically.

Evaluation criteria:
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 was 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.

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 and 1200 mg/kg
- Clinical signs of toxicity in test animals: The following clinical signs were observed at and above 1000 mg/kg: Hunched posture, ptosis, pilo-erection, lethargy, ataxia, splayed gait, decreased respiratory rate, laboured respiration and hypothermia.
- Other:
Mortality: A premature death occurred at 1200 mg/kg.
Justification of dose setting for the main test: The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated via the oral route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, selected for use in the main test was 1000 mg/kg, with 500 and 250 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
A summary of the results of the micronucleus test is given in Table 2. A modest, but statistically significant, decrease in the PCE/NCE ratio was observed in the 24-hour 1000 mg/kg test material dose group when compared to the concurrent control group. The reduction in PCE/NCE ratio, together with the observation of clinical signs at and above 500 mg/kg and a premature death in the 48-hour 1000 mg/kg dose group, was taken to indicate that systemic absorption had occurred and exposure to the target tissue achieved. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

MORTALITY DATA AND CLINICAL OBSERVATIONS
A premature death was seen in the 48-hour 1000 mg/kg dose group. Clinical signs were observed in animals dosed with the test material at and above 500 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ptosis, diarrhoea, noisy respiration, lethargy, ataxia and splayed gait.

Any other information on results incl. tables

Table 2 Micronucleus Test - Summary of Group Mean Data

Treatment Group

Number of PCE with Micronuclei per 2000 PCE

PCE/NCE Ratio

Group Mean

SD

Group Mean

SD

1.       Vehicle Control (Distilled water)
10 ml/kg
48-hour Sampling Time

1.1

1.5

1.33

0.19

2.       Vehicle Control (Distilled water)
10 ml/kg
24-hour Sampling Time

1.9

0.7

1.18

0.12

3.       Positive Control (Cyclophosphamide)
50 mg/kg
24-hour Sampling Time

58.0***

19.6

1.11

0.31

4.       3-Octadecyloxypropyl-N,N, N-trimethylammonium chloride
1000 mg/kg
48-hour Sampling Time

1.2

1.9

0.98

0.48

5.       3-Octadecyloxypropyl-N,N, N-trimethylammonium chloride
1000 mg/kg
24-hour Sampling Time

1.0

1.2

0.91**

0.19

6.       3-Octadecyloxypropyl-N,N, N-trimethylammonium chloride
500 mg/kg
24-hour Sampling Time

1.1

1.1

1.05

0.26

7.       3-Octadecyloxypropyl-N,N, N-trimethylammonium chloride
250 mg/kg
24-hour Sampling Time

0.4

0.5

1.06

0.21

PCE   = Polychromatic erythrocytes

NCE  = Normochromatic erythrocytes

SD     = Standard deviation

**     = P <0.01

***   = P < 0.001

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 and does not meet the criteria for classification according to EU classification system.
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 1997 OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method B12 of the EC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

Methods. A range-finding test was performed to find suitable dose levels of the test material and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose 1000 mg/kg and with 500 and 250 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 respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Results. A modest, but statistically significant, decrease in the PCE/NCE ratio was observed in the 24-hour 1000 mg/kg test material dose group when compared to the concurrent control group. The reduction in PCE/NCE ratio, together with the observation of clinical signs at and above 500 mg/kg and a premature death in the 48-hour 1000 mg/kg dose group, was taken to indicate that systemic absorption had occurred and exposure to the target tissue 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 group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion. The test material was considered to be non-genotoxic under the conditions of the test. The test material does not meet the criteria for classification according to EU classification system.