Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqeuous solution
Details on test material:
Cocamidopropyl PG-dimonium chloride
CAS# 136920-08-6
Lot# CJ9157
% active: 35%
date: 11/19/2003

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system (10% liver S9 in standard co-factors)
Vehicle / solvent:
sterile distilled water
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With S9 activation system
Positive controls:
yes
Positive control substance:
other: 1,8-dihydroxyanthraquinone
Remarks:
with S9 activation system
Details on test system and experimental conditions:
Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate. The test was performed by mixing 0.1 mL of bacterial culture (TA100), 2mL of molten, trace histidine supplemented, top agar, 0.1mL of test material, 0.5mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30mL/plate). Ten doses of the test material and a vehicle control (sterile distilled water) were tested. In addition, 0.1mL of the maximum concentration of the test material and 2mL of a molten, trace histidine supplemented, top agar was overaid onto sterile nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hrs incubation at 37C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of bacterial background lawn.

Mutation test - Experiment 1
Seven concentrations of the test material (1.5, 5, 15, 50, 150, 500 and 1500 microg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.Additional dose levels were included to allow for test material induced toxicity, ensuring that a minimum of four non-toxic doses were achieved.
Measured aliquots (0.1mL) of one of the bacterial cultures were dispended into sets of test tubes followed by 2mL of molten, trace histidine supplemented, top agar, 0.1mL of the test material formulation, vehicle or positive control and either 0.5mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate) This procedure was repeated, in triplicate for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37C for approximately 48 hrs and the frequency of revertant colonies assessed using Domino colony counter.

Mutation test - Experiment 2
Te second experiment was perfromed using methodology described for Experiment 1, using fresh bacterial cultures test material and control solutions. The test material dose range was amended slightly following the results obtained from experiment ! and was as follows:
all tester strains without S9-mix: 1.5 to 1500 microg/plate
All tester strains with S9-mix: 5 to 1500 microg/plate
Evaluation criteria:
Validation criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strains cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method.

The approprite characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.

All tester strains cultures should be in the approximate range of 1 to 9.9x10^9 bacteria per mL.

Each mean positive value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutaenic exposure and the integrity of the S9-mix. The historical control range for 2002 and 2003 are presented in Apendix 1 in full report attached.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria
The test material may be considered positive in this test system if the following criteria are met:
the test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Additional information on results:
Preliminary toxicity test
the test matrial was toxic at and above 5000 microg/plate to the strain of Salmonella used (TA100) The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile.

Mutation test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate. All were found to be satisfactory. These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in table 1, and were considered to be acceptable. These data for concurrent untreated control plates performed on the same day as the mutation test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation are presented in table 2 to 5 in the attached full report.

The test material caused a visible reduction in growth of the bacterial background lawn to all of the tester strains initially at 150 and 500 microg/plate, without and with S9 respectively. The test material was therefore tested up to the toxic limit. No test material precipitate was observed on the plates at any doses tested in either the presence or absence of S9-mix.
No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either with or without metabolic activation.

A small but statisclly significant increase in revertant colony frequency was observed in TA98 (without S9-mix) at 15 microg/plate in Experiment 1 only. However, all the plate counts were within the specified range of the tester strain, there was no evidence of a dose-response relationship, the fold increase was only 1.37 times that of the concurrent solvent control and finally, the response was not reproduced in the second experiment. Therefore, this small response was considered to be of no toxicological or biological significance.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of S9-mix and the sensitivity of the bacterial strains

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

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
The test material supplied contained the actove ingredient cocopropyl dimethyl dihydroxypropyl ammonium chloride (35%). All results given are based on the test material as whole and has not been corrective for active ingredient content.
Executive summary:

The method was designed to meet the requirements of the OECD guidelines No 471.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of the rat liver homogenate metabilising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 1500 microg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The test material dose range for Experiment 2 was amended slightly following the results obtained from Eperiment 1.

Additional dose levels were included in both experiment to allow for test material induced toxicity, ensuring that a minimum of four non toxic levels were achieved.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metaolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn to all of the testes strains initially at 150 and 500 microg/plate without and with activation system respectively. The test material was therefore tested up to the toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of test material, either with or without metabolic activation.

A small but statisticallly significant increase in revertant colony frequency was observed in TA98 (without S9-mix) at 15 microg/plate in Experiment 1 only. However, all the plate counts were within the specified range of the test strain, there was no evidence of a dose-response relationship, the fold increase was only 1.37 times that of the concurrent solvent control and, finally the response was not reproduced in the second experiment. Therefore this small response was considered to be of no toxicological or biological significance.

The test material was therefore considered to be non-mutagenic under the conditions of this test.

The test material supplied contained the actove ingredient cocopropyl dimethyl dihydroxypropyl ammonium chloride (35%). All results given are based on the test material as whole and has not been corrective for active ingredient content.