O,O-tert-butyl isopropyl monoperoxycarbonate

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:

Experimental start date 19 October 1999 , Experimental completion date 25 October 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O,O-tert-butyl isopropyl monoperoxycarbonate

EC Number:

219-143-7

EC Name:

O,O-tert-butyl isopropyl monoperoxycarbonate

Cas Number:

2372-21-6

Molecular formula:

C8H16O4

IUPAC Name:

tert-butoxy propan-2-yl carbonate

additive 1

Reference substance name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

EC Number:

297-629-8

EC Name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

Cas Number:

93685-81-5

Molecular formula:

not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

isododecane

Test material form:

liquid

Details on test material:

Trigonox BPIC-C75

Specific details on test material used for the study:

Identification: TRIGONOX BPIC·C75
Description: Colourless liquid
Batch: 0419908130562
Purity: 75.8%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Not indicated
Expiry date: 04 October 2000 (allocated at NOTOX, 1 year after receipt of the test substance)

Method

Target gene:

The histidine locus in Salmonella typhimurium.
The tryptophan locus in Escherichia coli

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 extracted from the livers of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range-finding test:
with and without S9-mix - 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate

Based on results from the range-finding study, the following concentrations were used in the mutagenicity test:
with and without S9-mix - 100, 333, 1000, 3330 and 5000 μg/plate

Vehicle / solvent:

dimethylsulphoxide

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

60 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxid

True negative controls:

no

Positive controls:

yes

Remarks:

4 µg/plate for strain TA98

Positive control substance:

other: daunomycine

Remarks:

without S9-mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

650 µg/plate for strain TA100

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strain WP2uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 µg/plate for strain TA1537, 1 µg/plate for strains TA1535, TA98 and TA100 and 5 µg/plate for strain WP2uvrA

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

DURATION
- Exposure duration: 37ºC for 48 h. (Inadvertently, the plates of the tester strains TA 1535, TA 1537 and T A98 were incubated for 72 hours)
- Selection time (if incubation with a selection agent): 37ºC for 48 h. (Inadvertently, the plates of the tester strains TA 1535, TA 1537 and T A98 were incubated for 72 hours.)
- Environmental conditions: All incubations were carried out in the dark at 37°C. The temperature was monitored during the experiment.

SELECTION AGENT (mutation assays): histidine or tryptophan deficient media

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
To determine the toxicity of TRIGONOX BPIC-C75, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the 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 test if:
It induces a number of revertant colonies, dose related, greater than two-times the number of revertants 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.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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

Genotoxicity:

positive

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 1537

Metabolic activation:

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 98

Metabolic activation:

with

Genotoxicity:

positive

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 98

Metabolic activation:

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 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Metabolic activation:

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 8

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

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:

DOSE RANGE FINDING TEST
TRIGONOX BPIC·C75 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix.
This dose range finding test is reported as a part of the mutation test.

Precipitate
The test substance did not precipitate in the top agar. Precipitation of TRIGONOX BPIC-C75 on the plates was not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.

Toxicity
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.

Mutagenicity
In the absence of S9-mix, TRIGONOX BPIC-C75 did not induce a dose·related, two·fold, increase in the number of revertant (His+) colonies in tester strain TA 100 and in the number of revertant (Trp+) colonies in tester strain WP2uvrA.
In the presence of S9-mix in tester strain TA 100, TRIGONOX BPIC-C75 induced an up to 5.0·fold, dose related, increase in the number of revertant colonies compared to the solvent control.
In tester strain WP2uvrA, TRIGONOX BPIC-C75 induced an up to 8.7-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

MUTATION ASSAY
Based on the results of the dose range finding study, TRIGONOX BPIC-C75 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the mutation experiment. The mutation experiment was performed with the strains TA 1535, TA 1537 and TA98.

Precipitate
TRIGONOX BPIC-C75 did not precipitate in the top agar. Precipitation of TRIGONOX BPIC-C75 on the plates was not observed at the start or at the end of the incubation period in all tester strains.

Toxicity of the test substance
The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

Mutagenicity
In the absence of S9-mix, TRIGONOX BPIC-C75 did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the three tester strains (TA 1535, TA1 537 and TA98).
In the presence of S9-mix in tester strain TA 1537, TAIGONOX BPIC-C75 induced an up to 3.0-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, TRIGONOX BPIC-C75 induced an up to 1.5-fofd, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA 1535, TRIGONOX BPIC-C75 did not induce a dose-related increase in the number of revertant colonies.

Any other information on results incl. tables

MUTAGENIC RESPONSE OF TRIGONOX BPIC-C75 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Dose µg/plate	Mean number of revertant colonies/3 replicate plates (±S.D) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	382 ± 49	226 ± 21	706 ± 78	686 ± 81	908 ± 133
Solvent control	16 ± 3	11 ± 3	35 ± 9	76 ± 6	12 ± 3
3				75 ± 16	9 ± 4
10				73 ± 6	15 ± 4
33				57 ± 5	10 ± 5
100	16 ± 8	8 ± 3	32 ± 4	81 ± 10	12 ± 2
333	18 ± 6	6 ± 2	31 ± 5	74 ± 16	9 ± 3
1000	22 ± 1	9 ± 2	37 ± 7	74 ± 17	8 ± 1
3330	19 ± 7	11 ± 5	40 ± 9	83 ± 13	9 ± 2
5000	21 ± 5	14 ± 1	37 ± 5	96 ± 5	12 ± 2
With S9-mix^1
Positive control	237 ± 18	699 ± 34	1154 ± 55	1141 ± 99	71 ± 4
Solvent control	21 ± 2	10 ± 2	48 ± 2	85 ± 5	11 ± 2
3				80 ± 18	18 ± 2
10				78 ± 17	13 ± 4
33				81 ± 18	11 ± 1
100	15 ± 2	10 ± 3	48 ± 4	73 ± 13	12 ± 1
333	17 ± 5	11 ± 2	57 ± 9	101 ± 8	13 ± 3
1000	24 ± 13	15 ± 5	57 ± 8	102 ± 10	18 ± 6
3330	16 ± 6	24 ± 2	71 ± 8	386 ± 30	87 ± 9
5000	17 ± 4	30 ± 3	64 ± 6	429 ± 9	96 ± 14

Solvent control: 0.1 ml dimethylsulphoxide

¹ The S9-mix contained 5% (v/v) S9 fraction

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that TRIGONOX BPIC-C75 is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

TRIGONOX BPIC-C75 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and T A98)

and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvr A.

TRIGONOX BPIC-C75 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix. TRIGONOX BPIC-C75 did-not precipitate on the plates at this dose level.

The bacterial background lawn was not reduced at all concentrations tested and no decrease in the n umber of revertants was observed.

In the absence of S9-mix, TRIGONOX BPIC-C75 did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA 1537, TA98 and TA 100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA. ·

In the presence of S9-mix in tester strain TA 1537, TRIGONOX BPIC-C75 induced an up to 3.0-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, TAIGONOX BPIC-C75 induced an up to 1.5-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA 100, TRIGONOX BPIC-C75 induced an up to 5.0-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain WP2uvrA, TRIGONOX BPIC-C75 induced an up to 8.7-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA 1535, TRIGONOX BPIC-C75 did not induce a dose-related increase in the number of revertant colonies.

Based on the results of this study it is concluded that TRIGONOX BPIC-C75 is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.