Multi constituent substance

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD guideline 471 in compliance to GLP.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Reference 2

Reference Type:

other: Published secondary source

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Multi constituent substance

EC Number:

916-603-5

IUPAC Name:

Multi constituent substance

Test material form:

solid: particulate/powder

Details on test material:

Test substance : Basic Red 76 (COLIPA number C008)
Batch number : 0057891101
Methylsulphate anion : 15.9%
Chloride ion : 2.7%
Water : 4.1%
Purity : 98.6% (HPLC)

Method

Target gene:

Histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Pre-experiment : 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate without and with metabolic activation
Experiment I : 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate without and with metabolic activation
Experiment II : 33, 100, 333, 1000, 2500 and 5000 ug/plate without and with metabolic activation

To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in the pre-experiment were the same as described for the experiment I (plate incorporation test). The pre-experiment is reported as part of main experiment I since the following criteria are met : evaluable plates (>0 colonies) at five concentrations or more in all strains used. In the pre-experiment the concentration range of the test item was 3-5000 ug/plate. The pre-experiment is reported as part of experiment I. Since thecriteria mentioned above were met 5000 ug/plate was chosen as the maximum concentration.

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine (TA1537, TA 98 (without metabolic activation)), 2-aminoanthracene (TA 1535, TA 1537, TA 98, TA 100, TA 102 (with metabolic activation))

Details on test system and experimental conditions:

Treatment
Experiment I : Direct plate incorporation with 48 hours incubation without and with metabolic activation
Experiment II : Pre-incubation method (60 minutes) and at least 48 hours incubation without and with metabolic activation

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (TA 98, TA 100, TA 102) or three times (TA 1535, TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of metabolic activation in both experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Reduced background growth was observed with and without metabolic activation in strains TA 98 and TA 100 in experiment I. In experiment II, reduced background growth was observed with and without metabolic activation in all strains used.

Toxic effects, evident as a reduction in the number of revertants were observed at the concentrations detailed below.

Strain	Experiment I and IA		Experiment II
	Without metabolic activation	With metabolic activation	Without metabolic activation	With metabolic activation
TA 1535	5000	no toxic effects observed	no toxic effects observed	no toxic effects observed
TA 1537	5000	5000	5000	5000
TA98	5000 (I), no toxic effect (IA)	5000	1000 -5000	5000
TA 100	2500, 5000	5000	1000 -5000	5000
TA 102	2500, 5000	5000	2500, 5000	5000

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generalyy acknowledged border of biological relevance with the exception of strain TA 98 in the absence of metabolic activation in experiment I. This strain showed a minor increase in revertant colony numbers at 1000 ug/plate. However, the required threshold of two times the number of the corresponding solvent control only slightly exceeded (indication factor of 2.2). To verify the results of this experiment an independent repeat experiment was performed under identical conditions with strain TA 98 in the absence of metabolic activation. No increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the first experiment was judged as biologically irrelevant. The results of the repeat experiment are reported as experiment IA. Also in experiment II strain TA 98 showed a minor increase in the absence of metabolic activation. The number of colonies reached the threshold of twice the number of the corresponding solvent control at concentrations as low as 100 ug/plate. Since the factor is just reached and did not show a dose dependent increase this effect is judged as biological irrelevant.

Appropriate reference mutagens were used as positive controls. Distinct increases in induced revertant colonies were observed.

The number of colonies exceeded the historical control range of the laboratory in the solvent control of strain TA 102 without metabolic activation in experiment I. Since this deviation is rather small this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the experimental conditions employed the test item (Basic Red 76) was not mutagenic in this bacterial gene mutation test both in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations : pre-experiment : 3, 10, 3, 100, 333, 1000, 2500, 5000 ug/plate ; experiment I : 10, 33, 100, 333, 1000, 2500, 5000 ug/plate ; experiment II : 33, 100, 333, 1000, 2500, 5000 ug/plate. Reduced background growth was observed with and without metabolic activation in strains TA 98 and TA 100 in experiment I. In experiment II, reduced background growth was observed with and without metabolic activation in all strains used. Toxic effects evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.