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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:
1994-10-18 until 1994-10-25 (plate incorporation test) and 1994-12-14 until 1994-12-20 (preincubation test)
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
Test material form:
liquid
Details on test material:
Test was performed with dibenzyltoluene, new name: dibenzylbenzene, ar-methyl derivative

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
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 / 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, 1538; Sodium azide (2.5 µg/plate) for TA 100, 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 resultsopen allclose all
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
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: at a concentration 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: no signs of cytotoxicity both with and without metabolic activation observed
Remarks on result:
other: strain/cell type: Salmonella typhimurium
Remarks:
Migrated from field 'Test system'.

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*

 32±8

 51±4

 no

 117±5

 123±6

 no

 12±5

15±1 

 no

8

 28±1

 60±4

 no

 105±5

 149±3

 no

 7±2

 12±1

 no

40

 34±5

 49±10

 no

 103±4

 146±10

 no

 10±2

 12±6

 no

200

 29±8**

 58±2**

 no

 97±9**

 134±8**

 no

 9±1**

 15±2**

 no

1000

 36±5**

 55±4**

no

 101±16**

 114±3**

 no

 11±6**

 9±2**

 no

5000

 33±4**

 48±7**

 no

 113±9**

 138±21**

 no

 10±1**

 10±3**

 no

Positive control

 204±3

 1657±189

 no

 414±14

 2043±69

 no

 412±29

 191±41

 no

*solvent control with DMSO ** precipitation  

Table #1 continued: 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*

 16±4

 14±5

 no

 21±4

 33±7

 no

 

 

 

8

 18±3

 16±3

 no

 26±4

 33±5

 no

 

 

 

40

 19±3

 14±6

 no

 26±1

 33±8

 no

 

 

 

200

 18±6**

 16±8**

 no

 32±2**

 35±8**

 no

 

 

 

1000

 20±1**

 19±6**

 no

 25±2**

 31±6**

 no

 

 

 

5000

 17±6**

 20±8**

 no

 22±1**

 34±5**

 no

 

 

 

Positive control

 79±19

 195±60

 no

 239±16

 1839±100

 no

 

 

 

*solvent control with DMSO ** precipitation  

Table #2: 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*

 34±11

 50±4

 no

 136±6

 128±4

 no

 10±3

 14±4

 no

8

 30±4

 50±8

 no

 112±7

 146±22

 no

 9±2

 12±3

 no

40

 37±3

 54±4

 no

 121±4

 138±5

 no

 13±1

 17±0

 no

200

 28±4**

 46±12**

 no

 128±27**

 124±19**

 no

 10±1**

 13±5**

 no

1000

 34±4**

 45±3**

 no

 110±4**

 119±3**

 no

 13±2**

 12±5**

 no

5000

 30±7**

 54±3**

 no

 128±21**

 136±14**

 no

 13±4**

 12±2**

 no

Positive control

 16012

 1945±100

 no

 385±29

 2150±66

 no

 361±10

 217±9

 no

*solvent control with DMSO ** presipitation  

Table #2 continued: 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*

 37±4

 41±8

 no

 29±6

 37±6

 no

 

 

 

8

 35±3

 41±7

 no

 22±9

 35±8

 no

 

 

 

40

 32±7

 47±12

 no

 22±5

 45±1

 no

 

 

 

200

 29±8**

 41±7**

 no

 20±3**

 36±2**

 no

 

 

 

1000

 35±6**

 46±6**

 no

 18±4**

 32±6**

 no

 

 

 

5000

 28±1**

 45±14**

 no

 24±5**

 38±9**

 no

 

 

 

Positive control

 218±49

 362±10

 no

 175±2

 953±90

 no

 

 

 

*solvent control with DMSO ** precipitation  

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was found to be not mutagenic in a standard bacterial reverse mutation assay (Ames test).
Executive summary:

The test material was found to be not mutagenic in a standard bacterial reverse mutation assay (Ames test) in concentrations up to 5000 µg/plate in the presence and absence of metabolic activation system.