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Toxicological information

Genetic toxicity: in vitro

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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
Controlsopen allclose all
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
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
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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.