Benzyltoluene

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:

1994-10-20 to 1994-12-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Marlotherm L, neu

IUPAC Name:

Marlotherm L, neu

Constituent 2

Reference substance name:

Benzyltoluene

EC Number:

248-654-8

EC Name:

Benzyltoluene

Cas Number:

27776-01-8

Molecular formula:

C14H14

IUPAC Name:

1-benzyl-2-methylbenzene; 1-benzyl-3-methylbenzene; 1-benzyl-4-methylbenzene

Details on test material:

- Name of test material (as cited in study report): MARLOTHERM L, neu
- Substance type: product
- Physical state: liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver S9 mix

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: not mentioned

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Nitrofluorene (2.5 µg/plate) for the strains TA 98, TA 1538; Sodium azide (2.5 µg/plate) for TA 100, TA 1535; Aminoacredine (25 µg/plate) for TA 1537

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Aminoanthracene (2.5 µg/plate) with all strains

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation, performed in two independent tests


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 96 hours


NUMBER OF REPLICATIONS: 3 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: not mentioned


OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (DMSO), test substance concentrations and positive controls; determination of the titers of overnight cultures


OTHER: none

Evaluation criteria:

According to Ames a test article which caused no mutagenic effects at a concentration of 5000 µg/plate will be called non-mutagenic.

Statistics:

no statistics performed

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not mentioned
- Precipitation: yes, at concentrations of 200 µg/plate and higher
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: not performed


COMPARISON WITH HISTORICAL CONTROL DATA: not performed


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- With metabolic activation:
Plate incorporation test: background lawn reduced at 200 µg/plate and complete clearing of background lawn at 1000 and 5000 µg/plate (TA 1535); background lawn reduced at 1000 µg/plate and complete clearing of background lawn at 5000 µg/plate (TA 1537)
Incubation test: background lawn reduced at 200, 1000 and 5000 µg/plate (TA 1535); complete clearing of background lawn at 1000 and 5000 µg/plate (TA 1537)
- Without metabolic activation:
Plate incorporation test: background lawn reduced at 200, 1000 and 5000 µg/plate (TA 1535); background lawn reduced at 200 µg/plate and complete clearing of background lawn at 1000 and 5000 µg/plate (TA 1537)
Incubation test: background lawn reduced at 200, 1000 and 5000 µg/plate (TA 1535); complete clearing of background lawn at 200, 1000 and 5000 µg/plate (TA 1537)

Any other information on results incl. tables

Table #1: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 98]			[Strain TA 100]			[Strain TA 1535]
Conc. [unit]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	35	54	no	132	143	no	16	18	no
8	33	62	no	140	156	no	20	16	no
40	33	46	no	141	144	no	17	17	no
200	31	50	no	144	140	no	13	12	yes
1000	38	46	no	115	136	no	11	8	yes
5000	25	50	no	97	113	no	9	3	yes
Positive control	156	2153	no	405	1994	no	445	236	no

*solvent control with DMSO

Table #2: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 1537]			[Strain TA 1538]			[-]
Conc. [unit]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	15	17	no	27	34	no
8	18	14	no	28	32	no
40	12	11	no	23	36	no
200	2	11	yes	26	39	no
1000	0	5	yes	24	49	no
5000	0	0	yes	25	32	no
Positive control	901	264	no	178	1428	no

*solvent control with DMSO  

Table #3: Preincubation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 98]			[Strain TA 100]			[Strain TA 1535]
Conc. [unit]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	30	40	no	150	147	no	10	15	no
8	31	45	no	151	166	no	11	15	no
40	40	49	no	145	146	no	8	15	no
200	33	45	no	147	159	no	5	16	yes
1000	32	43	no	120	138	no	1	6	yes
5000	31	47	no	100	105	no	2	2	yes
Positive control	162	1669	no	432	2081	no	354	199	no

*solvent control with DMSO 

Table #4: Preincubation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 1537]			[Strain TA 1538]			[-]
Conc. [unit]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	22	20	no	21	28	no
8	13	21	no	24	38	no
40	15	20	no	19	43	no
200	0	22	yes	24	31	no
1000	0	5	yes	23	34	no
5000	0	0	yes	18	32	no
Positive control	68	272	no	169	864	no

*solvent control with DMSO 

Applicant's summary and conclusion

Conclusions:

Benzyltoluene did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella.

Executive summary:

The potential of benzyltoluene to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100) was evaluated in accordance with the international guidelines (OECD 471, ) and in compliance with the Principles of Good Laboratory Practice. Benzyltoluene was tested, with and without a metabolic activation system; bacterias were exposed to benzyltoluene at 8, 40, 200, 1000 and 5000 µg/plate. Nonoteworthy increase in the number of revertantswas observed for all doses with and without metabolic activation on the 5 tested strains. Under our experimental conditions, benzyltoluene did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella. typhimurium.