Di-tert-butyl sec-butylidene 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:

The study was conducted between May 1978 and August 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The procedures followed in the present protocol mimic as closely as possible those described by Ames and coworkers, in Mutation Research 31 (1975), 347-365.

GLP compliance:

no

Remarks:

Study pre-dates GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-butyl sec-butylidene diperoxide

EC Number:

218-507-2

EC Name:

Di-tert-butyl sec-butylidene diperoxide

Cas Number:

2167-23-9

Molecular formula:

C12H26O4

IUPAC Name:

2,2-bis(tert-butylperoxy)butane

additive 1

Reference substance name:

Dibutyl phthalate

EC Number:

201-557-4

EC Name:

Dibutyl phthalate

Cas Number:

84-74-2

Molecular formula:

C16H22O4

IUPAC Name:

dibutyl phthalate

Constituent 2

Reference substance name:

Butanone

EC Number:

201-159-0

EC Name:

Butanone

Cas Number:

78-93-3

Molecular formula:

C4H8O

IUPAC Name:

butan-2-one

Test material form:

liquid

Specific details on test material used for the study:

The study report covers multiple substances. In this report the substance is referred to as Trigonox D-B50.

Method

Target gene:

the histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix using liver homogenate of Aroclor-induced rats

Test concentrations with justification for top dose:

0.08, 0.4, 2 and 10 µl test liquid/0.1ml acetone/plate
All dilutions/solutions were prepared immediately before use.

Vehicle / solvent:

acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

0.1, 0.25, 0.5 and 1.0 µg/0.1ml DMSO/plate for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

with and without S9-mix

Details on test system and experimental conditions:

Bacterial strains
Origin
The Salmonella typhimurium mutants used viz. s. typh. TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were provided by Dr. B.N. Ames, Berkeley, California, USA. They are stored as frozen cultures at -80ºC.

Cultures for testing
To obtain cultures for mutagenesis testing, nutrient broth is inoculated with a thawed aliquot of the appropriate bacterial culture (0.1 ml per 10 ml nutrient bouillon) and grown up overnight with shaking at 37°C for 16 hours. The optical density is used as a measure for the total number of bacteria per ml. The bacterial cultures are stored in a refrigerator at 5ºC for up to four days.

MUTAGENESIS ASSAYS
Plate incorporation assay - Solids and non-volatile liiquids
The procedure used in this assay has been described in detail by Ames et al. (1975). Briefly the procedure is as follows: to 2 ml molten top agar (56°C) are added in this sequence 0.1 ml of a fully grown culture of one of the tester strains containing about 10^9 cells/ml , 0.1 ml of the appropriate dilution/suspension of the test product and 0.5 ml of the S-9 mix if indicated. The ingredients are thoroughly mixed and immediately poured onto minimal glucose agar plates.
After the top agar has been allowed to harden, the plates are incubated at 37°C for three days. Then the colonies (revertants which are histidine independent) are counted and the background lawn of bacterial growth is examined microscopically. Routinely, four to five different doses and a solvent control are tested with 5 different strains, viz. Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 99 and TA 100 with and without the liver microsome activation system. All determinations are made in triplicate.

S-9 mix and test product are checked for sterility. The dose range used in the mutagenesis assay is based on a preliminary test performed to assess the toxicity of the compound for the bacteria. If possible, the lowest toxic dose is taken as the highest dose for mutagenesis assay. In the case of questionable results the plate incorporation assay is partly repeated and/or, a number of revertant colonies is checked for histidine requirement and for other strain characteristics if appropriate.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results obtained with the various peroxides did not provide evidence of a dose-related, reproducible increase in the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of S-9 mix.
Incorporation of 1 µl Triqonox 48 and of 0.01 µl Trigonox HM per plate appeared slightly toxic to the bacteria as revealed by a less dense background lawn of bacterial growth. Incorporation of the other four peroxides did not reveal any indication of an interference of chemical toxicity with mutagenicity testing.
From the present results it can be concluded that none of the six peroxides examined revealed mutagenic activity in the plate incorporation assay with s. typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Applicant's summary and conclusion

Conclusions:

From the present results it can be concluded that none of the six peroxides examined revealed mutagenic activity in the plate incorporation assay with s. typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Executive summary:

1. The mutagenic activity of six organic peroxides: Trigonox B, Laurox, Trigonox 48, Trigonox HM, Trigonox D-B50 and Perkadox SE 10 was examined in the Salmonella/microsome mutagenicity test, using a set of five histidine requiring mutants of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and liver homogenate of Aroclor-induced rats.

2. Incorporation of the peroxides up to non-inhibitory levels did not increase the number of his+ revertants in any of the five tester strains, either in the presence or in the absence of the liver microsome activation system.

3. It was concluded that the present results did not reveal mutagenic activity with any of the test products in the Salmonella/microsome mutagenicity test.