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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:
End of the study period: 30 April 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of male Sprague-Dawley rats liver induced with peritoneal Aroclor 1254 (= S9)
Test concentrations with justification for top dose:
- without S9: 31.25, 62.5, 125, 250 and 500 µg/plate for the 1st assay - 15.625, 31.25, 62.5, 125, and 250 µg/plate for the 2nd assay.
- with S9: 62.5, 125, 250, 500 and 1000 µg/plate for both assays.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (0.5 µg/pl): for TA 98 and TA 1538 without S9; sodium azide (1 µg/pl): for TA 100 and TA 1535 without S9; 9-amino-acridine (50 µg/pl): for TA 1537 without S9; 2-anthramine (1 or 2 µg/pl): for all strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 to 72 hours at 37°C
NUMBER OF REPLICATIONS: 3








Evaluation criteria:
- a test substance is considered as mutagenic if, for each test, it induces a doubling in the number of revertants when compared to that in the negative and/or solvent controls, for at least one of the tested strains and at one or more of the tested concentrations. In this case, a statistically significant dose relationship is investigated, using a linear regression analysis, and considered as significant if p ≤ 0.05 (for n values, the correlation coefficient r must be ≥ 0.47).
- a test substance is considered as non-mutagenic if the above two criteria are not fully met.
Statistics:
linear regression analysis

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: TA 98 and TA 100 strains: ≥ 1000 µg/plate without S9, ≥ 2500 µg/plate with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Toxicity:
In the preliminary assay, TBC was toxic to bacterial strains TA98 and TA100 stains at 1000 µg/plate or greater without S9 and 2500 µg/plate or greater with S9.
At lower concentrations, no toxicity was observed.

Mutagenic activity:
Without S9, TBC was toxic at 500 µg/plate and toxicity was also observed in the first test without S9 for the TA100 strain at some concentrations and varied according to the triplicate.
The solvent control results were equivalent to those usually obtained in the laboratory.
The number of revertants induced by the positive controls were higher than the controls, which demonstrated the sensitivity of the test.
The number of revertants obtained in the presence of TBC with and without S9 for the 5 strains was equivalent to that of the negative controls.

Any other information on results incl. tables

First assay(number of revertants/plate)

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

-S9

Cytotoxic

0*

10

no

1

no

2

no

31.25

8

no

3

no

2

no

62.5

4

no

3

no

7

no

125

9

no

5

no

6

no

250

6

no

3

no

3

no

500 (1)

t

yes

1

yes

4

yes

Positive control

10

no

35

no

22

no

 

TA 98

TA 100

 

 

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

 

0*

2

no

8

no

 

31.25

4

no

27

yes

 

62.5

7

no

6

yes

 

125

7

no

19

no

 

250

2

no

15

yes

 

500 (1)

3

yes

25

yes

 

Positive control

8

no

15

no

 

*solvent control with DMSO

t: toxicity

(1): yellow colouration

Second assay(number of revertants/plate)  

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

-S9

Cytotoxic

0*

14

no

10

no

23

no

15.625

16

no

13

no

18

no

31.25

17

no

13

no

15

no

62.5

17

no

9

no

24

no

125

10

no

13

no

25

no

250

11

no

8

no

25

no

Positive control

232

no

120

no

292

no

 

TA 98

TA 100

 

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

 

0*

24

no

118

no

 

15.625

29

no

112

no

 

31.25

27

no

117

no

 

62.5

21

no

93

no

 

125

27

no

120

no

 

250

19

no

61

no

 

Positive control

255

no

384

no

 

*solvent control with DMSO

First assay(number of revertants/plate)

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

+S9

Cytotoxic

0*

15

no

1

no

5

no

62.5

11

no

3

no

0

no

125

10

no

2

no

4

no

250

9

no

3

no

1

no

500

7

no

1

no

2

no

1000

6

no

1

no

2

no

Positive control

231

no

12

no

48

no

 

TA 98

TA 100

 

 

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

 

0*

5

no

26

no

 

62.5

2

no

22

no

 

125

4

no

7

no

 

250

7

no

10

no

 

500

4

no

16

no

 

1000

3

no

5

no

 

Positive control

27

no

106

no

 

*solvent control with DMSO

Second assay(number of revertants/plate)

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

+S9

Cytotoxic

0*

17

no

13

no

25

no

62.5

21

no

11

no

23

no

125

22

no

15

no

24

no

250

18

no

14

no

31

no

500

16

no

13

no

22

no

1000

13

no

12

no

29

no

Positive control

178

no

225

no

688

no

 

TA 98

TA 100

 

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

 

0*

34

no

112

no

 

62.5

32

no

126

no

 

125

29

no

112

no

 

250

34

no

95

no

 

500

23

no

100

no

 

1000

25

no

90

no

 

Positive control

1250

no

1283

no

 

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:

Negative without metabolic activation
Negative with metabolic activation

Under the experimental conditions, 4-tert-butylpyrocatechol did not show mutagenic activity in the Ames test up to 1000 µg/plate with metabolic activation and up to 5250 µg/plate without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria (CIT study 7682 MMO, 1992), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to 4 -tert-butylpyrocatechol (TBC) (purity: 99%), in DMSO at concentrations of 31.25, 62.5, 125, 250 and 500 µg/plate (first assay) or at concentrations of 15.625, 31.25, 62.5, 125 and 250 µg/plate (second assay) in the absence of mammalian metabolic activation (rat liver S9 fraction) and at concentrations of 62.5, 125, 250, 500 and 1000 µg/plate (both assays) in the presence of mammalian metabolic activation (rat liver S9 fraction) (plate incorporation method).

4 -tert-butylpyrocatechol was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Therefore, TBC was considered not mutagenic in the Ames test up to 1000 µg/plate with metabolic activation and up to 250 µg/plate without metabolic activation (the 500 µg/plate concentration was cytotoxic).

This study is classified as acceptable. This study satisfies the requirements of the test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation).