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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
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 peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
Hageman et al
Year:
1988
Bibliographic source:
Mutation Research

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Ames assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl-4-methoxyphenol
EC Number:
246-563-8
EC Name:
tert-butyl-4-methoxyphenol
Cas Number:
25013-16-5
Molecular formula:
C11H16O2
IUPAC Name:
(1,1-Dimethylethyl)-4-methoxyphenol
Details on test material:
- Name of test material: N Butylated hydroxyanisole
- Molecular formula : C11H16O2
- Molecular weight: 181.2533 g/mol
- Substance type: Organic

Method

Target gene:
his+ gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA104
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 fractions prepared from Aroclor-treated rats
Test concentrations with justification for top dose:
1, 10, 50, 100, 200, 500 or 1000 μg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: Without S9 mix Strain TA97: 4-nitro-o-phenylene-diamine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Witout S9: Strain TA100: sodium azide Strain
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: TA102 tert.-butylhydroperoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: Strain TA104: methylglyoxal
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Mutagenicity test consisting of the combination of the test compound, the bacterial tester strain, and S9 mix in soft agar. Positive and negative controls are usually also included in each assay. However, negative control are not included in the current study. After incubation at 37°C for 48 h, revertant colonies are counted. A liquid preincubation procedure was also applied for some of the experiments to provide with a more sensitive assessment of mutagenic activity.

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Triplicate replications

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: N/A

OTHER EXAMINATIONS: N/A
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for no. of revertants/plate
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 97
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:
valid
Species / strain:
S. typhimurium TA 100
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:
valid
Species / strain:
S. typhimurium TA 102
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:
valid
Species / strain:
S. typhimurium, other: TA104
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:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: Because BHA have antimicrobial properties a non-toxic dose range was established.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Mutagencity of the test chemical Salmonella Microsome Assay

                                            No. of his+ revertants per plate in strains

 

 

         TA97

       TA100

         TA102

         TA104

Compound

Dose

μg/plate

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

BHA

1

121 ±3

147± 9

83± 4

85± 8

243± 13

363 ±57

333 ±5

361 ±91

 

10

117 2

146 6

76 12

81 14

224 24

387 2

371 21

409 50

 

100

109 12

138 18

92 11

97 12

197 38

277 16

329 16

391 47

 

200

136 9

174 19

102 8

76 12

171 11

381 9

32811

47254

 

500

106 13

144 12

54 9

90 10

18 9

112 8

367 29

39268

 

1000

0

0

0

0

15 1

182 67

0

17 10

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
The test chemical dissolved in dimethylsulfoxide and given in the concentration of 1, 10, 100, 200, 500 or 1000 mg/plate, was not mutagenic in the Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions and hence it is not likely to be mutagenic as per the criteria mentioned in CLP regulation.
Executive summary:

In a Salmonella/microsome assay, the mutagenic activity of the test chemical was evaluated in Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions from Aroclor-induced rats. At doses of 100 mg/plate, the phenolic antioxidant BHA exhibited toxic effects. However, a modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Therefore, exposure of the test chemical in Salmonella typhimurium, at concentrations below 500 mg/plate with or without metabolic activation by S9 liver fractions, is not regarded to be mutagenic.