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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:
1990-03-14 until 1991-04-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited number of values for assessment due to high cytotoxicity; poor documentation; strain to detect oxidising mutagens, cross-linking agents and hydrazines is missing; nevertheless considered sufficient for evaluation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

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

Constituent 1
Reference substance name:
2-tert.-butylphenol
IUPAC Name:
2-tert.-butylphenol
Constituent 2
Chemical structure
Reference substance name:
2-tert-butylphenol
EC Number:
201-807-2
EC Name:
2-tert-butylphenol
Cas Number:
88-18-6
Molecular formula:
C10H14O
IUPAC Name:
2-tert-butylphenol
Constituent 3
Reference substance name:
o.-tert.-butylphenol
IUPAC Name:
o.-tert.-butylphenol
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 2-tert.-butylphenol
- Physical state: liquid
- Analytical purity: 99.97%
- Purity test date: 2014-05-07
- Lot/batch No.: 1419
- Expiration date of the lot/batch: 05/2015
- Stability under test conditions: stable
- Storage condition of test material: under N2 in tightly closed container at a cool, well ventilated place
- Colour: clear

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:
Arochlor 1254 induced liver S9 mix
Test concentrations with justification for top dose:
Plate incorporation test: 8 / 40 / 200 / 1000 / 5000 µg/plate
Pre-incubation test: 32 / 63 / 125 / 250 / 500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 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 and TA 1538; Sodium azide (2.5 µg/plate) for TA 100 and TA 1535; Aminoacredine (50 µg/plate) for TA 1537, Aminoanthracen (10 µg/plate) for TA 100
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Aminoanthracene (10 µg/plate) with strain TA 100
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 causes no mutagenic effects at a concentration of 5000 µg/plate can be considered as non-mutagenic.
Statistics:
No statistics reported.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Tables 1-4 below for concentrations with cytotoxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Tables 1-4 below for concentrations with 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:
cytotoxicity
Remarks:
See Tables 1-4 below for concentrations with cytotoxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Tables 1-4 below for concentrations with cytotoxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Tables 1-4 below for concentrations with 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: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: not performed

COMPARISON WITH HISTORICAL CONTROL DATA: not performed

ADDITIONAL INFORMATION ON CYTOTOXICITY: See Tables 1-4 below for concentrations with cytotoxicity.

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]

- S9

+ S9

Cytotoxic

(yes/no)

- S9

+ S9

Cytotoxic
(yes/no)

- S9

+ S9

Cytotoxic
(yes/no)

0*

22 ± 10

49 ± 4

no

266 ± 23

165 ± 17

no

4 ± 4

15 ± 5

no

8

23 ± 2

52 ± 6

no

276 ± 9

181 ± 8

no

2 ± 1

10 ± 4

no

40

15 ± 4

46 ± 7

no

224 ± 30

170 ± 23

no

0

20 ± 4

yes

200

8 ± 3

24 ± 1

no

237 ± 6

143 ± 5

no

0

14 ± 5

yes

1000

0

0

yes

0

0

yes

0

0

yes

5000

0

0

yes

0

0

yes

0

0

yes

Positive control

334 ± 79

 not tested

no

302 ± 7

564 ± 167

no

 250 ± 18

not tested

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]

- S9

+ S9

Cytotoxic

(yes/no)

- S9

+ S9

Cytotoxic

(yes/no)

0*

3 ± 3

18 ± 4

no

47 ± 31

52 ± 5

no

8

0

20 ± 7

yes

39 ± 11

54 ± 10

no

40

0

25 ± 10

yes

32 ± 2

48 ± 14

no

200

0

7 ± 4

yes

30 ± 9

54 ± 12

no

1000

0

0

yes

0

0

yes

5000

0

0

yes

0

0

yes

Positive control

408 ± 233

not tested

no

177 ± 15

not tested

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]

- S9

+ S9

Cytotoxic
(yes/no)

- S9

+ S9

Cytotoxic (yes/no)

- S9

+ S9

Cytotoxic

(yes/no)

0*

34 ± 4

49 ± 11

no

120 ± 23

115 ± 16

no

15 ± 4

19 ± 2

no

32

40 ± 8

56 ± 2

no

114 ± 15

129 ± 3

no

18 ± 4

18 ± 4

no

63

33 ± 4

54 ± 4

no

130 ± 15

120 ± 31

no

13 ± 4

19 ± 6

no

125

0

54 ± 4

yes

0

120 ± 14

yes

0

14 ± 8

yes

250

0

51 ± 2

yes

0

93 ± 12

yes

0

0

yes

500

0

0

yes

0

0

yes

0

0

yes

Positive control

325 ± 24

not tested

no

578 ± 16

2531 ± 234

no

487 ± 39

not tested

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]

- S9

+ S9

Cytotoxic (yes/no)

- S9

+ S9

Cytotoxic

(yes/no)

0*

12 ± 1

16 ± 2

no

43 ± 9

42 ± 4

no

32

12 ± 3

17 ± 6

no

35 ± 5

47 ± 9

no

63

9 ± 4

23 ± 5

no

36 ± 10

45 ± 5

no

125

0

19 ± 7

yes

31 ± 4

61 ± 9

no

250

0

3 ± 6

yes

0

50 ± 6

yes

500

0

0

yes

0

0

yes

Positive control

633 ± 106

 not tested

no

155 ± 15

not tested

no

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Increase of revertant colonies was observed neither at non-cytotoxic concentrations nor at the limit concentration. Therefore, based on the results from this Ames-test the test substance can be considered as non-mutagenic.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA 1535, TA 1537 and TA 1538 ofS. typhimurium were exposed to o-tert-butylphenol in DMSO at concentrations of 8, 40, 200, 1000, and 5000 µg/plate (plate incubation assay) and 32, 63, 125, 250, 500 µg/plate (pre-incubation assay) in the presence and absence of mammalian metabolic activation.

O-tert-butylphenol was tested up to cytotoxic concentrations and the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is considered to be acceptable. This study satisfies the requirements for test guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data, except that a strain to detect oxidising mutagens, cross-linking agents and hydrazines is missing (e.g. TA102 or E. coli WP2 uvrA).