Reaction mass of 4-tert-butylphenol and 1,3- phenylenedimethanamine and 2-({[3-(aminomethyl) benzyl]amino}methyl)-4-tert-butylphenol

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

Study period:

21 June - 25 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 4-tert-butylphenol and 1,3- phenylenedimethanamine and 2-({[3-(aminomethyl) benzyl]amino}methyl)-4-tert-butylphenol

EC Number:

939-071-6

Molecular formula:

(C10 H14 O . C8 H12 N2 . C H2 O)x

IUPAC Name:

Reaction mass of 4-tert-butylphenol and 1,3- phenylenedimethanamine and 2-({[3-(aminomethyl) benzyl]amino}methyl)-4-tert-butylphenol

Test material form:

other: clear colorless liquid

Details on test material:

The test article, Paraformaldehyde, oligomeric reaction products with 4-tert-butylphenol, m phenylenebis(methylamine) (Mannich Base PTBP-MXDA), was received by BioReliance on 04 April 2012 (Lot # VH101996M1).. In GLP analysis, three major components were detected. 4-tert-Butylphenol (PTBP) was quantified by external standard at 45.2% w/w. The other two major components, m-xylenediamine (MXDA) and primary reaction product (PTBP-MXDA) were detected at 21.2% and 17.4% by area, respectively. Water was detected at 0.31% w/w. Purity was determined by liquid chromatography and coulometric Karl Fisher titration with identification by nuclear magnetic resonance and liquid chromatography/mass spectrometry.

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the initial toxicity mutagenicity assay, the concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate.

In the confirmatory mutagenicity assay, the concentrations tested were 0.50, 1.5, 5.0, 15, 50 and 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation, 1.5, 5.0, 15, 50, 150 and 500 µg per plate with tester strains TA1537 and WP2 uvrA in the absence of S9 activation and tester strain TA100 in the presence of S9 activation and 5.0, 15, 50, 150, 500 and 1500 µg per plate with the remaining test conditions.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2 aminoanthracene-all strains with S9, 2-nitrofluorene-TA98, sodium azide-TA100 and TA1535, 9-aminoacridine-TA1537 and methyl methanesulfonate-E. coli without S9

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Overnight cultures were prepared by inoculating from the appropriate master plate, appropriate frozen permanent stock or with a lyophilized pellet into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2 °C for 11 to 15 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 (Lot No. 2939, Exp. Date: 09 May 2014) was prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60 °C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose 6 phosphate, 4 mM ß nicotinamide adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Solubility Test
A solubility test was conducted to determine the vehicle. The test was conducted using deionized water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.

Initial Toxicity-Mutation Assay
The initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and a minimum of eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.

Confirmatory Mutagenicity Assay
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. A minimum of five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures
The test system was exposed to the test article via the preincubation methodology described by Yahagi et al. (1977).

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L histidine, D biotin and L tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of deionized water for each 100 mL of minimal top agar. Bottom agar was Vogel Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).
Each plate was labeled with a code system that identified the test article, test phase, concentration, tester strain and activation, as described in detail in BioReliance's Standard Operating Procedures.

One half (0.5) milliliter of S9 or sham mix, 100 µL of tester strain (cells seeded) and 50 µL of vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2 °C. After vortexing, these mixtures were incubated with shaking for 20±2 minutes at 37±2 °C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2 °C. Plates that were not counted immediately following the incubation period were stored at 2 8 C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

For the test article to be evaluated positive, it must cause a reproducible, concentration-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA98, TA1535, TA1537 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA100 were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Initial Toxicity-Mutation Assay
In experiment B1 (initial toxicity-mutation assay), the maximum concentration tested was 5000 µg per plate, which is the maximum concentration recommended by test guidelines (Tables 1 and 2). This concentration was achieved using a concentration of 100 mg/mL and a plating aliquot of 50 µL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 50, 150 or 500 µg per plate. Based on the findings of the initial toxicity‑mutation assay, the maximum concentrations plated in the confirmatory mutagenicity assay were 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation, 500 µg per plate with tester strains TA1537 and WP2uvrA in the absence of S9 activation and tester strain TA100 in the presence of S9 activation and 1500 µg per plate with the remaining test conditions.

Confirmatory Mutagenicity Assay
In experiment B2 (confirmatory mutagenicity assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation (Tables 3 and 4). The concentrations tested were 0.50, 1.5, 5.0, 15, 50 and 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation, 1.5, 5.0, 15, 50, 150 and 500 µg per plate with tester strains TA1537 and WP2uvrA in the absence of S9 activation and tester strain TA100 in the presence of S9 activation and 5.0, 15, 50, 150, 500 and 1500 µg per plate with the remaining test conditions. No precipitate was observed. Toxicity was observed beginning at 50, 150 or 500 µg per plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility Test

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the results of the solubility assessment conducted as part of the analytical method validation study for this test article (BioReliance Study No. AD48HT.GTCHEM.BTL) and compatibility with the target cells. The test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

Dosing Formulation Analysis

Dosing formulations were submitted to the analytical chemistry laboratory at BioReliance for analysis. C

oncentration analysis indicates that the actual mean concentrations of the analyzed dose levels were 134.0 and 114.6% of their respective targets with < 5% RSD (relative standard deviation). Although the actual concentration of the low dose formulation was higher than expected, the critical top concentration was within 85 to 115% of target. This indicates that the regulatory-required top dose level was achieved. Therefore, the Study Director has concluded that the observed difference from nominal concentration had no adverse impact on the assessment of the test article’s mutagenicity and the results support the validity of the study conclusion. No test article was detected in the vehicle control sample.

Stability analysis of the formulations found Paraformaldehyde, oligomeric reaction products with 4‑tert-butylphenol, m‑phenylenebis(methylamine)(Mannich Base PTBP-MXDA) to be stable in DMSO at a concentration of 0.0134 mg/mL for at least three hours when stored at room temperature. Stability analysis was also conducted as part of BioReliance Study No. AD48HT.140R.BTL; and the results of the analysis indicate thatParaformaldehyde, oligomeric reaction products with 4‑tert-butylphenol, m‑phenylenebis(methylamine)(Mannich Base PTBP-MXDA) in DMSO at a concentration of 103.6 mg/mL was also stable for at least three hours when stored at room temperature.

In order to determine the stability of PTBP and MXDA in DMSO, the analytical chemistry laboratory at BioReliance prepared a fresh formulation ofParaformaldehyde, oligomeric reaction products with 4‑tert-butylphenol, m‑phenylenebis(methylamine)(Mannich Base PTBP-MXDA) at a concentration of 0.030 mg/mL. The results of the stability analysis indicate that PTBP and MXDA in DMSO at concentrations of 0.0326 and 0.0342 mg/mL, respectively, were stable for at least 16 hours when stored at room temperature. Stability analysis for PTBP and MXDA was also conducted as part of BioReliance Study No. AD48HT.140R.BTL; and the results of the analysis indicate that PTBP and MXDA in DMSO at concentrations of 104 and 102 mg/mL, respectively, were also stable for at least three hours when stored at room temperature. 

Table 1

Initial Toxicity-Mutation Assay without S9 activation

Test	Dose Level	Strain
Material	per plate	TA98	TA100	TA1535	TA1537	WP2uvrA
Mannich Base PTBP-MXDA	5000 ug	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
	1500 ug	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
	500 ug	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
	150 ug	0 + 0	0 + 0	0 + 0	0 + 0	28 + 8
	50 ug	19 + 6	73 + 0	9 + 0	7 + 4	34 + 9
	15 ug	26 + 4	98 + 20	9 + 1	11 + 0	29 + 6
	5.0 ug	30 + 4	103 + 28	15 + 5	7 + 2	33 + 0
	1.5 ug	23 + 1	95 + 10	14 + 5	8 + 3	28 + 5
DMSO	50 uL	22 + 4	113 + 4	8 + 4	10 + 1	36 + 6
2-nitrofluorene	1.0 ug	491 + 77	ND	ND	ND	ND
sodium azide	1.0 ug	ND	491 + 4	453 + 52	ND	ND
9-Aminoacridine	75 ug	ND	ND	ND	417 + 124	ND
methyl methanesulfonate	1000 ug	ND	ND	ND	ND	628 + 16

ND = No Data

Table 2

Initial Toxicity-Mutation Assay with S9 activation

Test	Dose Level	Strains
Material	per plate	TA98	TA100	TA1535	TA1537	WP2uvrA
Mannich Base PTBP-MXDA	5000 ug	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
	1500 ug	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
	500 ug	0 + 0	0 + 0	0 + 0	0 + 0	20 + 3
	150 ug	34 + 4	95 + 16	14 + 0	9 + 8	33 + 6
	50 ug	37 + 4	104 + 10	12 + 3	5 + 2	31 + 10
	15 ug	32 + 6	114 + 1	16 + 3	12 + 1	27 + 0
	5.0 ug	30 + 2	111 + 12	11 + 1	7 + 4	34 + 1
	1.5 ug	27 + 6	108 + 1	19 + 6	8 + 0	43 + 4
DMSO	50 uL	28 + 2	104 + 11	15 + 5	9 + 4	27 + 6
2-aminoanthracene	1.0 ug	792 + 18	ND	87 + 7	70 + 1	ND
2-aminoanthracene	2.0 ug	ND	887 + 70	ND	ND	ND
2-aminoanthracene	15 ug	ND	ND	ND	ND	200 + 33

ND = No Data

Table 3

Confirmatory Mutagenicity Assay without S9 activation

Test	Dose Level	Strains
Material	per Plate	TA98	TA100	TA1535	TA1537	WP2uvrA
Mannich Base PTBP-MXDA	500 ug	ND	ND	ND	0 + 0	0 + 0
	150 ug	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
	50 ug	0 + 0	0 + 0	4 + 3	8 + 2	32 + 9
	15 ug	20 + 7	69 + 8	9 + 1	5 + 1	32 + 8
	5.0 ug	16 + 9	71 + 14	8 + 3	6 + 3	26 + 7
	1.5 ug	15 + 6	82 + 8	11 + 7	4 + 3	28 + 3
	0.50 ug	16 + 5	57 + 5	9 + 4	ND	ND
DMSO	50 uL	23 + 4	82 + 6	12 + 3	4 + 1	25 + 7
2-nitrofluorene	1.0 ug	483 + 37	ND	ND	ND	ND
sodium azide	1.0 ug	ND	528 + 36	389 + 40	ND	ND
9-Aminoacridine	75 ug	ND	ND	ND	277 + 192	ND
methyl methanesulfonate	1000 ug	ND	ND	ND	ND	661 + 117

ND = No Data

Table 4

Confirmatory Mutagenicity Assay with S9 activation

Test	Dose Level	Strain
Material	per Plate	TA98	TA100	TA1535	TA1537	WP2uvrA
Mannich Base PTBP-MXDA	1500 ug	0 + 0	ND	0 + 0	0 + 0	0 + 0
	500 ug	0 + 0	0 + 0	0 + 0	0 + 0	32 + 12
	150 ug	26 + 7	64 + 5	9 + 6	14 + 5	34 + 6
	50 ug	34 + 13	83 + 4	11 + 4	8 + 4	41 + 6
	15 ug	27 + 9	90 + 22	8 + 4	11 + 3	38 + 11
	5.0 ug	32 + 4	98 + 13	12 + 3	8 + 5	27 + 7
	1.5 ug	ND	89 + 5	ND	ND	ND
DMSO	50 uL	32 + 2	87 + 10	14 + 1	9 + 3	33 + 11
2-aminoanthracene	1.0 ug	525 + 74	ND	82 + 2	42 + 8	ND
2-aminoanthracene	2.0 ug	ND	723 + 18	ND	ND	ND
2-aminoanthracene	15 ug	ND	ND	ND	ND	199 + 53

 ND = No Data

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In the initial toxicity as well as the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Executive summary:

The test article, Paraformaldehyde, oligomeric reaction products with 4-tert-butylphenol, m‑phenylenebis(methylamine)(Mannich Base PTBP-MXDA), was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor‑induced rat liver S9. The assay was performed in two phases using the preincubation method. The first phase, the initial toxicity‑mutation assay, was used to establish the dose‑range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the results of the solubility assessment conducted as part of the analytical method validation study for this test article (BioReliance Study No. AD48HT.GTCHEM.BTL) and compatibility with the target cells. The test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

In the initial toxicity-mutation assay, the maximum concentration tested was 5000 µg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a plating aliquot of 50 µL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 50, 150 or 500 µg per plate. Based on the findings of the initial toxicity‑mutation assay, the maximum concentrations plated in the confirmatory mutagenicity assay were 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation, 500 µg per plate with tester strains TA1537 and WP2uvrA in the absence of S9 activation and tester strain TA100 in the presence of S9 activation and 1500 µg per plate with the remaining test conditions.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The concentrations tested were 0.50, 1.5, 5.0, 15, 50 and 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation, 1.5, 5.0, 15, 50, 150 and 500 µg per plate with tester strains TA1537 and WP2uvrA in the absence of S9 activation and tester strain TA100 in the presence of S9 activation and 5.0, 15, 50, 150, 500 and 1500 µg per plate with the remaining test conditions. No precipitate was observed. Toxicity was observed beginning at 50, 150 or 500 µg per plate.

Dosing formulations were analyzed by the analytical chemistry laboratory at BioReliance. The analytically-determined concentrations ofParaformaldehyde, oligomeric reaction products with 4-tert-butylphenol, m‑phenylenebis(methylamine)(Mannich Base PTBP-MXDA)in the confirmatory dose formulations were 134.0 and 114.6% of their respective targets with < 5% RSD. Although the actual concentration of the low dose formulation was higher than expected, the critical top concentration was within 85 to 115% of target. This indicates that the regulatory-required top dose level was achieved and the results support the validity of the study conclusion. Formulation stability analysis found Paraformaldehyde, oligomeric reaction

products with 4-tert-butylphenol, m‑phenylenebis(methylamine) (Mannich Base PTBP-MXDA) to be stable in DMSO at concentrations of 0.0134 and 103.6 mg/mL for at least three hours when stored at room temperature. The stability of PTBP and MXDA in DMSO was also determined. The results of the analysis indicate that PTBP and MXDA in DMSO at concentrations of 0.0326 and 0.0342 mg/mL, respectively, were stable for at least 16 hours when stored at room temperature. PTBP and MXDA in DMSO at concentrations of 104 and 102 mg/mL, respectively, were also stable for at least three hours when stored at room temperature.

All criteria for a valid study were met as described in the protocol. The vehicle controls and positive controls in the initial toxicity-mutation and confirmatory mutagenicity assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay. Under the conditions of this study, Paraformaldehyde, oligomeric reaction products with 4‑tert-butylphenol, m‑phenylenebis(methylamine)(Mannich Base PTBP-MXDA)was concluded to be negative (non‑mutagenic) in the bacterial reverse mutation assay.

 [m1]Updated