Di-tert-butyl 1,1,4,4-tetramethylbut-2-yn-1,4-ylene diperoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1987-06-29 to 1987-07-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-butyl 1,1,4,4-tetramethylbut-2-yn-1,4-ylene diperoxide

EC Number:

213-944-5

EC Name:

Di-tert-butyl 1,1,4,4-tetramethylbut-2-yn-1,4-ylene diperoxide

Cas Number:

1068-27-5

Molecular formula:

C16H30O4

IUPAC Name:

2,5-bis(tert-butylperoxy)-2,5-dimethylhex-3-yne

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Remarks:

and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: With metabolic activation: 2-aminoanthracene (all strains); Without metabolic activation: 4-nitro-o-phenylene-diamine (TA1538), daunomycine (TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

MUTATION ASSAY:
Five different doses of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain.
Top agar in top agar tubes is melted and heated to 45 °C. The following solutions are successively added to 3 mL of top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in dimethylsulphoxide (DMSO) of spectroscopic quality (Merck), and in the case of activation assays 0.5 mL of S9-mix. The ingredients are mixed on a Vortex and the contents of the top agar tube are poured onto a selective agar plate. After solidification of the top agar, the plates are turned and incubated in the dark at 37 °C for 48h. After this period revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.

DETERMINATION OF CYTOTOXICITY
- Method: The percentage survival of the TA 100 culture is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the Ames test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. If the test substance shows in the first test only a positive response at one or two concentrations, the assay is repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

, but tested up to limit concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

, but tested up to limit concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

, but tested up to limit concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

, but tested up to limit concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

, but tested up to limit concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Mutagenic Response of the Test Substance in the Ames Salmonella/Microsome Plate Test

Dose (µg/plate)	Mean number of revertant (His+) colonies/ 3 replicate plates (± S.D.) with different strains of S. typhimurium
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
Without S9-mix
100	6 ± 4	7 ± 4	8 ± 2	27 ± 11	57 ± 9
333	9 ± 3	12 ± 3	8 ± 3	31 ± 6	70 ± 3
1000	8 ± 2	15; 8^b)	8 ± 3	30 ± 5	54 ± 5
3330	8 ± 2	13 ± 3	11 ± 6	30 ± 9^c)	57 ± 10
5000	7 ± 4	9 ± 4	8 ± 3	7 ± 7^d)	56 ± 5
Solvent control	8 ± 6	12 ± 5	5 ± 3	23 ± 5	54 ± 12
Positive control	286 ± 27	1329 ± 79	544 ± 160	659; 175^f)	890 ± 40
With S9-mix
100	8 ±	6 ± 3	13 ± 3	19 ± 2	54 ± 8
333	13 ±	3 ± 3	13 ± 3	22 ± 8	61 ± 4
1000	12 ±	3 ± 1	12 ± 3	20 ± 5	61 ± 6
3330	8 ±	4 ± 2	13 ± 4	19 ± 4	57 ± 6
5000	8 ±	4 ± 3^c)	9 ± 5	23 ± 10	61 ± 12
Solvent control	11 ±	6 ± 0	15 ± 2	28 ± 3	91 ± 27
Positive control	132 ±	67 ± 7	744 ± 99	693 ± 28	1036 ± 123

a) 0.1 mL DMSO.

b) One plate infected with other bacteria.

c) Bacterial background lawn slightly reduced.

d) Bacterial background lawn moderately reduced.

e) Test substance precipitated in the top agar.

f) Inadvertently no bacteria plated on one plate; value of 175 is outside historical range.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the test substance can be considered as non mutagenic in the Ames Salmonella/microsome assay.

Executive summary:

The test item was tested in the Ames Salmonella/microsome test up to 5000 µg/plate. The test substance induced no dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535; TA1537; TA1538; TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as nonmutagenic in this test system.