Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2016 to 28 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test" (Adopted July 21, 1997).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

EC Number:

701-003-6

Cas Number:

1454803-04-3

Molecular formula:

C20H39NO3 to C26H51NO3

IUPAC Name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Test material form:

liquid

Details on test material:

Identification: MLA-3202
Appearance: Clear amber-red liquid
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 17 February 2019 (expiry date)
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)
CAS Number: 1454803-04-3
Test item handling No specific handling conditions required

Specific details on test material used for the study:

Batch: RC-1045Study specific test item informationPurity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3Test item handling: No specific handling conditions required

Method

Target gene:

The objective of this study was to evaluate MLA-3202 for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of MLA-3202 used in the subsequent mutation assay was 5000 μg/plate.

Vehicle / solvent:

No correction was made for the purity/composition of the test item. A solubility test was performed. MLA-3202 was dissolved in dimethyl sulfoxide.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

Cell culture Preparation of bacterial cultures Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for testing.Agar plates Agar plates (ø 9 cm) containing 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma). Top agar Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C. Environmental conditions All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.9 – 39.8°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity. Metabolic activation system Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch is characterised with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.Preparation of S9-mix S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml Milli-Q water (first experiment) and 5.0 ml Milli-Q water (second and third experiment) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment. Study design Dose range finding test Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of MLA-3202 used in the subsequent mutation assay was 5000 μg/plate. Mutation assay At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains. An additional experiment was performed with tester strain TA98 in the absence of S9-mix, and TA100 in the absence and presence of 10% (v/v) S9-mix. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted. Colony counting The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP2uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair substitution mutagens.

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range. A test item is considered negative (not mutagenic) in the test if: a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control. b) The negative response should be reproducible in at least one follow-up experiment. A test item is considered positive (mutagenic) in the test if: a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control. b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test/first mutation experiment MLA-3202 was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate. Precipitate Precipitation of MLA-3202 on the plates was observed at the start of the incubation period at concentrations of 1600 μg/plate and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period. Toxicity To determine the toxicity of MLA-3202, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence and presence of S9-mix. In the tester strains TA1535 (absence of S9-mix), TA1537 (absence and presence of S9-mix) and TA98 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies. MutagenicityNo increase in the number of revertants was observed upon treatment with MLA-3202 under all conditions tested.Experiment 2 To obtain more information about the possible mutagenicity of MLA-3202, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitate Precipitation of MLA-3202 on the plates was observed at the start of the incubation period at concentrations of 1568 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period. Toxicity In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants was observed at any of the concentrations was observed in the absence and presence of S9-mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1537 and TA98 in the absence of S9-mix, in tester strain TA1535 in the presence of S9-mix and in tester strain TA100 in the absence and presence of S9-mix. In strain WP2uvrA and TA1535, both in the absence of S9-mix, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies. Mutagenicity In the second mutation assay, no increase in the number of revertants was observed upon treatment with MLA-3202 under all conditions tested..Experiment 3 Since in the second mutation test, not enough non-toxic dose levels were present in the tester strains TA98 (absence of S9-mix) and TA100 (absence and presence of S9-mix), an additional experiment was performed. In this third mutation experiment, the following lower dose range was tested: 17, 52, 164, 512, 1600 and 5000 μg/plate. Precipitate Precipitation of MLA-3202 on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period. Toxicity Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain TA98, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants was observed at any of the concentrations tested. Mutagenicity In the third mutation assay, no increase in the number of revertants was observed upon treatment with MLA-3202 under all conditions tested.

Any other information on results incl. tables

Dose range finding test: Mutagenic response of MLA-3202 in the

Salmonella typhimuriumreverse mutation assay and in the

Escherichia colireverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
	TA100		WP2uvrA
	Without S9-mix
Positive control Solvent control 1.7 5.4 17 52 164 512 1600 5000	1228± 127 ± 114 ± 109 ± 117 ± 111 ± 72 ± 37 ± 49 ± 35 ±	56 5 17 28 1 9 4 22 10^NP 3^n SP	1672± 23 ± 24 ± 22 ± 22 ± 27 ± 28 ± 25 ± 32 ± 21 ±	106 8 4 8 7 3 5 1 7^NP 9^n SP
	With S9-mix^1
Positive control Solvent control 1.7 5.4 17 52 164 512 1600 5000	1530± 109 ± 124 ± 111 ± 118 ± 129 ± 121 ± 65 ± 52 ± 44 ±	90 4 5 2 9 6 11 11 9^NP 6^n SP	461± 28 ± 25 ± 24 ± 22 ± 28 ± 27 ± 27 ± 26 ± 31 ±	51 4 10 6 10 1 6 6 3^NP 8^n SP

¹             Plate incorporation assay (5% S9)

NP         No Precipitate

SP           Slight Precipitate

n             Normal bacterial background lawn

 

Experiment 1: Mutagenic response of MLA-3202 in theSalmonella typhimurium

reverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA1535		TA1537		TA98
	Without S9-mix
Positive control Solvent control 5.4 17 52 164 512 1600 5000	859± 8 ± 5 ± 10 ± 4 ± 2 ± 7 ± 3 ± 5 ±	39 5 3 7 4 2 3 4^NP 3^n SP	572± 5 ± 2 ± 4 ± 2 ± 4 ± 4 ± 1 ± 3 ±	70 2 3 1 2 1 4 1^NP 4^n SP	1519± 11 ± 15 ± 11 ± 12 ± 11 ± 7 ± 11 ± 14 ±	87 1 4 6 7 1 5 6^NP 4^n SP
	With S9-mix^1
Positive control Solvent control 5.4 17 52 164 512 1600 5000	297± 8 ± 9 ± 10 ± 10 ± 8 ± 3 ± 5 ± 6 ±	50 3 3 2 4 4 4 2^NP 4^n SP	436± 4 ± 8 ± 4 ± 1 ± 5 ± 3 ± 3 ± 4 ±	36 3 3 1 1 2 1 2^NP 1^n SP	1429± 19 ± 16 ± 16 ± 18 ± 21 ± 14 ± 12 ± 9 ±	93 7 2 5 4 8 10 8^NP 8^n SP

¹              Plate incorporation assay (5% S9)

NP          No precipitate

SP           Slight Precipitate

n             Normal bacterial background lawn

 

Experiment 2: Mutagenic response of MLA-3202 in theSalmonella typhimuriumreverse mutation assay and in the

Escherichia colireverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plate (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535		TA1537		TA98		TA100		WP2uvrA
	Without S9-mix
Positive control Solvent control 492 878 1568 2800 5000	946± 6 ± 3 ± 4 ± 3 ± 3 ± 3 ±	34 4 1 5 2 2^NP 2^n SP	990± 5 ± 4 ± 3 ± 2 ± 2 ± 2 ±	80 2 1 2 2 2^NP 3^n SP	1873± 14 ± 7 ± 7 ± 5 ± 5 ± 4 ±	107 2 2 6 3 4^NP 1^n SP	866± 88 ± 36 ± 23 ± 20 ± 23 ± 19 ±	45 28 8 1 9 4^NP 7^n SP	1585± 18 ± 12 ± 19 ± 16 ± 14 ± 16 ±	34 9 6 1 2 5^NP 7^n SP
	With S9-mix^1
Positive control Solvent control 492 878 1568 2800 5000	151± 8 ± 4 ± 4 ± 6 ± 4 ± 2 ±	27 2 3 1 3 3^NP 1^n SP	590± 11 ± 7 ± 7 ± 5 ± 8 ± 7 ±	28 5 2 4 1 1^NP 3^n SP	712± 18 ± 20 ± 17 ± 18 ± 17 ± 15 ±	20 7 5 1 6 3^NP 10^n SP	1447± 103 ± 68 ± 44 ± 40 ± 49 ± 32 ±	123 18 6 4 10 10^NP 11^n SP	512± 25 ± 26 ± 25 ± 18 ± 15 ± 15 ±	15 9 3 9 4 7^NP 5^n SP

¹              Plate incorporation assay (10% S9)

NP          No precipitate

SP           Slight Precipitate

n             Normal bacterial background lawn

 

Experiment 3: Mutagenic response of MLA-3202 in the

Salmonella typhimuriumreverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA98		TA100
	Without S9-mix
Positive control Solvent control 17 52 164 512 1600 5000	1979± 15 ± 15 ± 20 ± 15 ± 17 ± 13 ± 10 ±	104 9 4 2 2 3 7^NP 5^n SP	1207± 99 ± 98 ± 106 ± 82 ± 55 ± 42 ± 37 ±	39 6 7 6 9 13 4^NP 10^n SP
	With S9-mix^1
Positive control Solvent control 17 52 164 512 1600 5000			1684± 104 ± 101 ± 92 ± 124 ± 78 ± 43 ± 36 ±	61 6 9 5 24 12 8^NP 5^n SP

¹              Plate incorporation assay (10% S9)

NP          No precipitate

SP           Slight Precipitate

n             Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that MLA-3202 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of MLA-3202 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

 

MLA-3202 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). An additional experiment was performed with tester strain TA98 in the absence of S9-mix, and TA100 in the absence and presence of S9-mix.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. The test item was dissolved in dimethyl sulfoxide.

 

In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. MLA-3202 precipitated on the plates at the dose level of 5000 μg/plate. In tester strain TA100, toxicity was observed in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first mutation assay.

 

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in several tester strains, TA1535 (presence of S9-mix), TA1537 (absence of S9-mix), TA98 (absence of S9-mix) and TA100 (absence and presence of S9-mix).

 

In the second mutation test, not enough non-toxic dose levels were present in the tester strains TA98 (absence of S9-mix) and TA100 (absence and presence of S9-mix), therefore an additional experiment was performed. In this mutation experiment, the test item was tested at a concentration range of 17 to 5000 μg/plate in the absence of S9-mix in both tester strains, and in the presence of 10% (v/v) S9-mix in tester strain TA100. The test item precipitated on the plates at dose level of 5000 μg/plate. In tester strain TA100, toxicity was observed in the absence and presence of S9-mix. In tester strain TA98, no toxicity was observed at any of the dose levels tested.

 

MLA-3202 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that MLA-3202 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.