Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2010 -02-22 till 2010-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted GLP study performed according to established guideline OECD 471 with no deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): di-tert-butyl peroxide
Identity: di-tert-butyl peroxide
CAS No.: 110-05-4
Trade Name: Trigonox B
Batch-No.: 0904130207
Purity: 99.0%
Stability in solvent: Not indicated by the sponsor
Flash Point: 6 °C
Storage: In the freezer at approximately - 20 °C, stored in original closed container, protected from direct sunlight, confinement and temperatures below – 30 °C should be avoided
Expiration Date: May 01, 2011

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:THF
- Justification for choice of solvent/vehicle: chosen because of its solubility properties
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no relevant
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used in experiment I. In experiment II reduced background growth was observed at 2500 µg/plate and 5000 µg/plate in all strains with and without metabolic activation
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strain TA 98 at 5000 µg/plate in the absence of metabolic activation.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

Study Name: 1294201

Study Code: Harlan CCR 1294201

Experiment: 1294201 VV Plate

Date Plated: 24/02/2010

Assay Conditions:

Date Counted: 02/03/2010

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without Activation

THF

27 ± 3

13 ± 3

38 ± 4

141 ± 11

393 ± 23

Untreated

20 ± 6

11 ± 3

39 ± 1

140 ± 26

388 ± 21

di-tert-butyl

3 µg

23 ± 5

12 ± 3

36 ± 10

137 ± 12

392 ± 12

peroxide

10 µg

23 ± 4

12 ± 2

38 ± 8

134 ± 13

380 ± 15

33 µg

20 ± 7

15 ± 1

33 ± 6

132 ± 18

394 ± 23

100 µg

30 ± 5

15 ± 2

40 ± 5

148 ± 17

378 ± 42

333 µg

19 ± 10

12 ± 2

40 ± 3

126 ± 13

386 ± 27

1000 µg

16 ± 3

14 ± 5

40 ± 12

116 ± 5

388 ± 12

2500 µg

23 ± 4

10 ± 0

41 ± 1

119 ± 12

419 ± 7

5000 µg

19 ± 1

12 ± 2

28 ± 2

132 ± 3

416 ± 11

NaN3

10 µg

1779 ± 90

2242 ± 24

4-NOPD

10 µg

266 ± 26

4-NOPD

50 µg

103 ± 9

MMS

3.0 µL

3642 ± 772

With Activation

THF

27 ± 7

25 ± 4

52 ± 3

163 ± 5

575 ± 8

Untreated

23 ± 9

25 ± 2

49 ± 4

158 ± 24

620 ± 21

di-tert-butyl

3 µg

22 ± 1

21 ± 3

63 ± 6

168 ± 4

586 ± 21

peroxide

10 µg

23 ± 1

22 ± 1

56 ± 11

155 ± 11

581 ± 29

33 µg

25 ± 9

23 ± 1

50 ± 2

171 ± 19

602 ± 20

100 µg

19 ± 4

23 ± 7

58 ± 8

169 ± 14

611 ± 32

333 µg

27 ± 4

26 ± 3

57 ± 3

162 ± 16

590 ± 31

1000 µg

26 ± 3

25 ± 6

53 ± 7

173 ± 10

561 ± 33

2500 µg

28 ± 4

25 ± 3

54 ± 6

154 ± 5

557 ± 37

5000 µg

18 ± 4

25 ± 3

64 ± 4

154 ± 9

570 ± 19

2-AA

2.5 µg

464 ± 54

582 ± 14

2334 ± 141

3605 ± 355

2-AA

10.0 µg

2563 ± 222

Key to Positive Controls

NaN3

2-AA

MMS

4-NOPD

sodium azide

2-aminoanthracene

methyl methane sulfonate

4-nitro-o-phenylene-diamine

Summary of Results Experiment II

Study Name: 1294201

Study Code: Harlan CCR 1294201

Experiment: 1294201 HV2 pre

Date Plated: 01/04/2010

Assay Conditions:

Date Counted: 07/04/2010

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without Activation

THF

14 ± 10

11 ± 1

29 ± 4

119 ± 5

371 ± 22

Untreated

10 ± 4

15 ± 4

26 ± 2

109 ± 16

329 ± 33

di-tert-butyl

33 µg

15 ± 5

9 ± 2

27 ± 4

113 ± 9

354 ± 15

peroxide

100 µg

17 ± 5

13 ± 1

32 ± 2

115 ± 32

385 ± 28

333 µg

17 ± 5

11 ± 5

30 ± 4

111 ± 4

384 ± 19

1000 µg

13 ± 2

10 ± 5

23 ± 1

115 ± 9

354 ± 11

2500 µg

14 ± 11R

7 ± 1R

28 ± 3R

108 ± 22R

375 ± 33R

5000 µg

11 ± 3R

5 ± 4R

7 ± 2R M

89 ± 15R

345 ± 31R

NaN3

10 µg

2133 ± 59

2255 ± 104

4-NOPD

10 µg

309 ± 7

4-NOPD

50 µg

74 ± 11

MMS

3.0 µL

3077 ± 243

With Activation

THF

21 ± 4

23 ± 4

42 ± 7

132 ± 10

530 ± 28

Untreated

23 ± 2

18 ± 5

43 ± 8

126 ± 9

501 ± 24

di-tert-butyl

33 µg

21 ± 7

23 ± 4

49 ± 13

122 ± 26

514 ± 66

peroxide

100 µg

21 ± 3

22 ± 3

45 ± 14

124 ± 30

590 ± 21

333 µg

24 ± 5

25 ± 4

47 ± 2

124 ± 5

541 ± 45

1000 µg

23 ± 1

22 ± 7

39 ± 5

116 ± 8

511 ± 49

2500 µg

18 ± 3R

13 ± 3R

54 ± 2R

132 ± 2R

508 ± 31R

5000 µg

17 ± 4R

11 ± 3R

40 ± 8R

130 ± 7R

563 ± 27R

2-AA

2.5 µg

308 ± 25

332 ± 18

2623 ± 269

2681 ± 63

2-AA

10.0 µg

3533 ± 206

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

MMS

4-NOPD

sodium azide

2-aminoanthracene

methyl methane sulfonate

4-nitro-o-phenylene-diamine

R

M

Reduced background growth

Manual count

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test item di-tert-butyl peroxide (CAS No. 110-05-4) was assessed for its potential to induce gene mutations ac­cording to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:    3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                        33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used in experiment I. In experiment II reduced background growth was observed at 2500 µg/plate and 5000 µg/plate in all strains with and without metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strain TA 98 at 5000 µg/plate in the absence of metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with di-tert-butyl peroxide (CAS No. 110-05-4) at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.