1-hydroxybenzotriazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test

Test substance is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence and absence of metabolic activation system in all five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.

in vitro mammalian chromosome aberration test

Test chemical was evaluated for its mutagenic potential in mammalian cell by in vitro mammalian chromosome aberration test. The test result was considered to be negative both in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from SSS study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Bacterial Reverse Mutation Test of test substance in Salmonella typhimurium Tester Strains by Plate incorporation method, was conducted as per OECD guideline No. 471.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of the test material: 1-hydroxybenzotriazol
- Molecular formula: C6H5N3O
- Molecular weight: 135.126 g/mol
- Substance type: Organic
- Smiles: On1nnc2ccccc12

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Tester Strains Genotypes Tester Strains his Mutation Additional Mutations Plasmid Repair LPS TA98 hisD3052 uvrB rfa pKM101 TA100 hisG46 uvrB rfa PKM101 TA1535 hisG46 uvrB rfa - TA1537 hisC3076 uvrB rfa - TA102 hisG428 - rfa pKM101 and pAQ1

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 - induced rat liver Microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Trial 1: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate
Trial 2: 0, 128, 320, 800, 2000 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the solubility test dimethyl sulfoxide (Make: Sigma, Lot no.: BCBR0695V for preliminary cytotoxicity assay and Fischer scientific, Lot no.: 1967260517 for Main assay) was selected as a vehicle for the study.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2- Aminoanthracene (TA1537, TA1535, TA102, TA100 and TA98; Without S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, bacterial cell growth was observed
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

The Bacterial reverse mutation assay is considered acceptable if the following criteria are met:
- Regular background growth in the solvent control
- The positive control substances must produce a significant increase in revertant colony frequencies
- The spontaneous reversion rates in the solvent control must be in the range of the historical data.

Evaluation criteria:

Criteria for a Positive response:
• Tester Strains TA98, TA100 and TA102 : For a test item to be considered positive, it must produce at least a 2–fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response (minimum of 2 to 3 concentrations) to increasing concentrations of the test item.

• Tester Strains TA1537 and TA1535 : For a test item to be considered positive, it must produce at least a 3–fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response (minimum of 2 to 3 concentrations) to increasing concentrations of the test item.

Criteria For a Negative Response:
A test item for which the results do not meet the above criteria is considered non-mutagenic in this test.

Statistics:

No formal hypothesis testing was performed to analyse the data.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity study, the experiment was performed using tester strains TA98 and TA100 in the absence and presence of metabolic activation (5% v/v S9 mix) using plate incorporation method. Eight concentrations of test item were tested in triplicate plates along with vehicle and positive controls. The preliminary cytotoxicity assay was performed at the test concentrations of 0, 39.0625, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate both in the presence (5 % v/v S9 mix) and absence of a metabolic activation system along with vehicle and positive controls.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Bacterial cell growth
- Other observations when applicable: normal growth was observed in the cytotoxicity assay

Remarks on result:

other: No mutagenic potential

Summary Tables

Table 1

Revertant Colonies - Cytotoxicity Test

Test Concentration (µg/plate)	TA 98				TA 100
	+ S9		- S9		+ S9		- S9
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	29.67	1.53	24.67	2.08	133.33	7.37	138.33	7.51
39.0625	28.33	2.08	24.33	1.53	129.00	4.36	137.33	7.09
78.125	27.33	2.52	23.67	1.53	131.67	6.66	134.00	2.00
156.25	26.33	2.52	23.00	1.00	130.00	6.56	133.33	6.66
312.5	27.00	4.36	21.33	3.06	128.33	3.51	131.33	2.52
625	26.00	5.57	23.33	3.06	125.33	9.45	130.00	7.81
1250	25.67	4.73	22.00	2.00	123.33	5.51	128.00	6.24
2500	25.33	4.04	21.67	3.51	123.00	14.73	127.00	6.08
5000	26.67	2.52	19.33	3.51	118.67	3.51	124.00	8.72
PC	450.67	42.72	394.67	11.68	891.67	10.50	875.67	22.19

Key: SD = Standard Deviation, µg = Microgram, VC = Vehicle Control, PC = Positive Control,

       + S9 = Presence of S9, - S9 = Absence of S9, S9 = Rat liver Homogenate at 9000g.     

Table 2

 Revertant Colonies – Trial I

Plate Incorporation Method [Absence of metabolic activation (-S9)]
Test Concentration (µg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	10.67	5.51	14.67	2.52	223.67	2.52	24.67	2.08	138.33	7.51
312.5	5.00	2.00	13.00	2.65	220.33	3.21	21.33	3.06	131.33	2.52
625	7.67	2.08	13.33	1.15	218.00	6.24	23.33	3.06	130.00	7.81
1250	7.00	1.00	11.33	4.16	218.33	6.03	22.00	2.00	128.00	6.24
2500	7.33	1.53	8.00	2.00	215.67	5.69	21.67	3.51	127.00	6.08
5000	6.33	2.31	12.67	2.52	213.33	5.51	19.33	3.51	124.00	8.72
PC	216.33	23.35	315.33	20.84	922.33	11.02	394.67	11.68	875.67	22.19
PC 2Aa	NA	NA	14.00	2.00	NA	NA	NA	NA	NA	NA

 

Plate Incorporation Method [Presence of metabolic activation (+S9 5% v/v S9 Mix)]
Test Concentration (µg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	6.33	1.53	15.33	2.08	228.00	4.58	29.67	1.53	133.33	7.37
312.5	6.00	2.65	14.00	2.00	225.33	3.21	27.00	4.36	128.33	3.51
625	5.33	0.58	13.00	2.00	224.33	8.33	26.00	5.57	125.33	9.45
1250	9.33	2.08	14.33	2.52	218.67	6.03	25.67	4.73	123.33	5.51
2500	9.33	1.53	13.67	2.08	222.00	7.81	25.33	4.04	123.00	14.73
5000	8.67	4.51	13.33	2.08	217.00	6.00	26.67	2.52	118.67	3.51
PC	254.00	11.79	369.67	16.26	968.67	15.95	450.67	42.72	891.67	10.50

 

Key: SD = Standard Deviation, µg = Microgram, VC = Vehicle Control, PC = Positive Control,

       + S9 = Presence of S9, - S9 = Absence of S9, S9 = Rat liver Homogenate at 9000g,PC 2Aa = S9     Efficiency check in absence of metabolic activation with 2 Aminoanthracene, NA = Not Applicable.

 

Table 3

 Revertant Colonies -Trial II

 

Plate Incorporation Method [Presence of metabolic activation (+S9 10% v/v S9 Mix)]
Test Concentration (µg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	9.33	0.58	13.67	0.58	254.67	25.74	28.33	2.52	135.33	11.68
128	7.00	1.73	12.33	1.53	253.67	25.01	27.33	3.21	131.67	2.08
320	6.33	1.53	11.67	2.08	250.33	12.86	26.33	4.51	134.67	8.08
800	7.67	1.15	10.00	1.00	248.67	7.02	27.33	1.15	131.67	6.51
2000	6.67	1.15	11.33	1.15	234.00	8.19	26.33	1.53	129.67	4.73
5000	6.00	1.00	10.33	1.15	231.33	11.68	25.00	1.00	125.33	7.02
PC	243.67	7.09	350.67	7.57	901.00	20.66	445.33	9.07	873.67	27.57
PC 2Aa	NA	NA	17.33	1.53	NA	NA	NA	NA	NA	NA

Key: SD = Standard Deviation, µg = Microgram, VC = Vehicle Control, PC = Positive Control,                      + S9 = Presence of S9, S9 = Rat liver Homogenate at 9000g,PC 2Aa = S9 Efficiency check in           absence of metabolic activation with 2 Aminoanthracene, NA = Not Applicable.  
                                                                     Table 4 Fold Increase

Trial I - Presence of metabolic activation (+S9 5% v/v S9 Mix)
Test Concentration (µg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
312.5	0.47	0.95	0.89	0.91	0.99	0.99	0.86	0.91	0.95	0.96
625	0.72	0.84	0.91	0.85	0.97	0.98	0.95	0.88	0.94	0.94
1250	0.66	1.47	0.77	0.93	0.98	0.96	0.89	0.87	0.93	0.93
2500	0.69	1.47	0.55	0.89	0.96	0.97	0.88	0.85	0.92	0.92
5000	0.59	1.37	0.86	0.87	0.95	0.95	0.78	0.90	0.90	0.89
PC	20.28	40.11	21.50	24.11	4.12	4.25	16.00	15.19	6.33	6.69

 

Trial II – Presence of metabolic activation (+S910% v/v S9 mix)

Test Concentration (µg/plate)	TA1537	TA1535	TA 100	TA 102	TA 98
128	0.75	0.90	0.97	1.00	0.96
320	0.68	0.85	1.00	0.98	0.93
800	0.82	0.73	0.97	0.98	0.96
2000	0.71	0.83	0.96	0.92	0.93
5000	0.64	0.76	0.93	0.91	0.88
PC	26.11	25.66	6.46	3.54	15.72

 

     Key: µg = Microgram, PC = Positive Control

Table 5

Tester Strain Genotype Confirmation

Selective Media Plates	Details of Observation in Tester Strains
	TA1537	TA1535	TA98	TA100	TA102
Crystal Violet (rfamarker)	Zone of Inhibition	Zone of Inhibition	Zone of Inhibition	Zone of Inhibition	Zone of Inhibition
Ampicillin (pKM101 Plasmid)	No Growth Observed	No Growth Observed	Growth Observed	Growth Observed	Growth Observed
Tetracycline (pAQ1 Plasmid)	No Growth Observed	No Growth Observed	No Growth Observed	No Growth Observed	Growth Observed
Histidine Dependence	No Growth Observed	No Growth Observed	No Growth Observed	No Growth Observed	No Growth Observed
Biotin Dependence	No Growth Observed	No Growth Observed	No Growth Observed	No Growth Observed	Growth Observed
Histidine-Biotin Dependence	Growth Observed	Growth Observed	Growth Observed	Growth Observed	Growth Observed
UvrB	No Growth Observed	No Growth Observed	No Growth Observed	No Growth Observed	Growth Observed

Conclusions:

Test substance is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence and absence of metabolic activation system in all five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.

Executive summary:

Bacterial Reverse Mutation Test of test substance in Salmonella typhimurium Tester Strains by Plate incorporation method, was conducted at sa-FORD (Sanctuary for Research and Development), Maharashtra, India. This study was performed as per OECD guideline No. 471. Based on the solubility test, dimethyl sulfoxide was selected as a vehicle for the test item in the study. The Study was performed to evaluate the mutagenic potential of1-hydroxybenzotriazole (CAS no. 2592-95-2) using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 in Trial I (with 5 % v/v S9 mix and without metabolic activation) and Trial II (10% v/v S9 mix) along with vehicle (DMSO) and positive control in triplicates. Following concentrations were used for the respective trials: Trial I: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate and Trial II: 0, 128, 320, 800, 2000 and 5000 µg/plate. Trial I was performed at five test concentrations (factor 2) both in the presence (5 % v/v S9 mix) and absence of metabolic activation system along with vehicle and the positive control. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate both in the presence (5% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control. Trial II was conducted to confirm the negative results observed in Trial I. For the negative confirmation, the test item concentration was modified with a spacing factor of 2.5 and concentration of metabolic activation (S9 fraction) was increased to 10% v/v. Trial II was conducted with all the tester strains along with vehicle and positive control only in the presence of metabolic activation system. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate in the presence (10 % v/v S9 mix) of metabolic activation, when compared to the vehicle control. The spontaneous revertant colonies of the vehicle control were within the acceptable range of historical control data of all the tester strains. The positive controls used in the study exhibited significant increase in the mean number of revertant colonies as compared to vehicle control respective to their strains, indicating the sensitivity of the test system to specific mutagens. On the basis of the results of this study, it is concluded that test substance is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence or absence of metabolic activation system in all the five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentiom below

Principles of method if other than guideline:

Weight of evidence prepared from variuus publication mention below
1,To evaluate the mutagenic potential of test chemical in hamster cells by in vitro mammalian chromosome aberration test.
2,To evaluate the mutagenic potential of test chemical Human embryonic lung fibroblasts
(WI-38)by in vitro DNA damage and repair assay.

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian chromosome aberration test 2,DNA damage and repair assay

Species / strain / cell type:

other: hamster cells

Species / strain / cell type:

other: Human embryonic lung fibroblasts (WI-38)

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from rat liver

Test concentrations with justification for top dose:

1:Not specified
2:0.8, 8, 40, 80 ug/plate

Vehicle / solvent:

Not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Evaluation criteria:

Not specified

Species / strain:

other: hamster cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Human embryonic lung fibroblasts (WI-38)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: No mutagenic potential were observed

Conclusions:

Test chemical was evaluated for its mutagenic potential in mammalian cell by in vitro mammalian chromosome aberration test. The test result was considered to be negative both in the presence and absence of metabolic activation.

Executive summary:

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed tohamster cells inthe presence and absence of metabolic activation S9. Chromosome aberration, Chromosome gaps and breaks were not observed in the presence or absence of metabolic activation. No mutagenic potential was observed .Therefore test chemical was considered to be non -mutagenic inhamster cells byin vitro mammalian chromosome aberration test. Hence the substance cannot be classified as mutagenic in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data for the various test chemicals was reviewed to determine the mutagenic nature of 1-hydroxybenzotriazole (2592-95-2). The studies are as mentioned below:

AMES test

Bacterial Reverse Mutation Test of test substance in Salmonella typhimurium Tester Strains by Plate incorporation method, was conducted at sa-FORD (Sanctuary for Research and Development), Maharashtra, India. This study was performed as per OECD guideline No. 471. Based on the solubility test, dimethyl sulfoxide was selected as a vehicle for the test item in the study. The Study was performed to evaluate the mutagenic potential of1-hydroxybenzotriazole (CAS no. 2592-95-2) using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 in Trial I (with 5 % v/v S9 mix and without metabolic activation) and Trial II (10% v/v S9 mix) along with vehicle (DMSO) and positive control in triplicates. Following concentrations were used for the respective trials: Trial I: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate and Trial II: 0, 128, 320, 800, 2000 and 5000 µg/plate. Trial I was performed at five test concentrations (factor 2) both in the presence (5 % v/v S9 mix) and absence of metabolic activation system along with vehicle and the positive control. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate both in the presence (5% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control. Trial II was conducted to confirm the negative results observed in Trial I. For the negative confirmation, the test item concentration was modified with a spacing factor of 2.5 and concentration of metabolic activation (S9 fraction) was increased to 10% v/v. Trial II was conducted with all the tester strains along with vehicle and positive control only in the presence of metabolic activation system. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate in the presence (10 % v/v S9 mix) of metabolic activation, when compared to the vehicle control. The spontaneous revertant colonies of the vehicle control were within the acceptable range of historical control data of all the tester strains. The positive controls used in the study exhibited significant increase in the mean number of revertant colonies as compared to vehicle control respective to their strains, indicating the sensitivity of the test system to specific mutagens. On the basis of the results of this study, it is concluded that test substance is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence or absence of metabolic activation system in all the five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.

in vitro mammalian chromosome aberration test

Genetic toxicity in vitro study was assessed for test chemical. For this purpose In vitro DNA damage and repair assay was performed .The test material was exposed to Human embryonic lung fibroblasts (WI-38)by at the concentration of 0.8, 8, 40, 80 ug/plate. No mutagenic potential was observed .Therefore test chemical was considered to be non -mutagenic in Human embryonic lung fibroblasts (WI-38)byin vitro DNA damage and repair assay. The test result was considered to be negative. Hence the substance cannot be classified as mutagenic in vitro.

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed to hamster cells in the presence and absence of metabolic activation S9. Chromosome aberration, Chromosome gaps and breaks were not observed in the presence or absence of metabolic activation. No mutagenic potential was observed .Therefore test chemical was considered to be non -mutagenic in hamster cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as mutagenic in vitro.

Based on the data summarized, 1-hydroxybenzotriazole (2592-95-2) did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria the test chemical 1-hydroxybenzotriazole (2592-95-2)  did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.