Dilauroyl peroxide

Genetic toxicity: in vitro

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Apparently well conducted GLP study; only one positive control with S9; no viable cell concentration provided; marker verification was not reported (if conducted)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dilauroyl peroxide

EC Number:

203-326-3

EC Name:

Dilauroyl peroxide

Cas Number:

105-74-8

Molecular formula:

C24H46O4

IUPAC Name:

dodecanoyl dodecaneperoxoate

Details on test material:

The test substance, DILAUROYL PEROXIDE, used in the study was supplied by Elf Atochem Deutschland.
Documentation supplied by the Sponsor identified the test substance as follows:
denomination:
- protocol:DILAUROYL PEROXIDE

batch number:
- protocol and labelling: 110-9502-34
description: whitish powder
quantity and container: two plastic bags each containing 0.1 kg
date of receipt: 18.5.95
storage conditions: at room temperature, protected from light
purity: 99.7%.

Method

Target gene:

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the
histidine operon, resulting in a requirement for histidine.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: See "Any other information on materials and methods incl. tables"

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: See "Any other information on materials and methods incl. tables"

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

The top dose-level was selected according to the criteria specified in the international
regulations. Since the test substance was non-toxic, poorly soluble, the top dose-level was
the lowest precipitating dose: approximately 1500 ug/plate.
The selected dose-levels were for the first experiment:
· 15, 50, 150, 500 and 1500 ug/plate;
as precipitation again interfered with the scoring at 1500 ug/plate, the dose-treatments were
decreased for the second experiment as follows:
5, 15, 50, 150 and 500 ug/plate.
The dose-levels selected for the third experiment with the TA 1537 with S9 mix were: 15,
50, 150, 500 and 1500 ug/plate.

The dose-levels of the positive controls were as follows:
without S9 mix:
1 ug/plate of sodium azide (NaN3): TAl535 and TA 100 strains,
50 ug/plate of 9-Aminoacridine (9AA): TA 1537 strain,
0.5 ug/plate of 2-Nitrofluorene (2NF): TA 98 strain,
0.5 ug/plate of Mitomycin C (MMC): TAl02 strain.

with S9 mix:
2 ug/plate of2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,
10 ug/plate of 2-Anthramine (2AM): TA 102 strain.

Vehicle / solvent:

tetrahydrofuran

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

Positive controls:

yes

Remarks:

-S9

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: TA 1535, TA 100

Controls 2

Positive controls:

yes

Remarks:

-S9

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: TA 1537

Controls 3

Positive controls:

yes

Remarks:

-S9

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: TA 98

Controls 4

Positive controls:

yes

Remarks:

-S9

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: TA 102

Controls 5

Positive controls:

yes

Remarks:

+S9

Positive control substance:

other: 2-anthramine

Details on test system and experimental conditions:

The experiments were performed according to:
Direct plate incorporation method (preliminary toxicity tests, both experiments without
S9 mix, first and third experiments with S9 mix): test substance solution (0.05 to 0.1 ml),
S9 mix (0.5 ml) when required and bacterial suspension (0.1 ml) were mixed with 2 ml
of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at
45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing
minimum medium.

Preincubation method (second experiment with S9 mix): test substance solution (0.05 to
0.1 ml), S9 mix (0.5 ml) and bacterial suspension (0.1 ml) were incubated for 60 minutes
at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar
plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic
counter (Artek counter, model 880, O.S.I., 75015 Paris, France).

Evaluation criteria:

Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. ,

Acceptance criteria
This study was considered valid since the following criteria were fully met:
· the number of revertants in the vehicle controls was within the range of our historical data

· the number of revertants in the positive controls was higher than that of the vehicle
controls and was within the range of our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle
controls, in any strain at any dose-level and/or evidence of a dose-relationship was
considered as a positive result. Reference to historical data, or other considerations of
biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

NA

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The number of revertants of the vehicle and positive controls was as specified in the
acceptance criteria and within the range of the historical data.

The top dose-level was selected according to the criteria specified in the international
regulations. Since the test substance was non-toxic, poorly soluble, the top dose-level was
the lowest precipitating dose: approximately 1500 ug/plate.

The selected dose-levels were for the first experiment:
. 15, 50, 150, 500 and 1500 ug/plate;
as precipitation again interfered with the scoring at 1500 ug/plate, the dose-treatments were
decreased for the second experiment as follows:
. 5, 15, 50, 150 and 500 ug/plate.
The dose-levels selected for the third experiment with the TA 1537 with S9 mix were: 15,
50, 150, 500 and 1500 ug/plate.

The test substance did not induce any significant increase in the number of revertants, with
and without S9 mix, in any of the five strains. Indeed, the slight 2.4 fold increase observed
at 500 and 1500 ug/plate in the first experiment with S9 mix in the TA 1537 strain was
attributed to the low value of revertants in the concurrent vehicle controls and was not
taken into account since it was not reproduced in the second and third experiments and
since the value obtained remained in our historical data.

Any other information on results incl. tables

See attachment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, the test substance DILAUROYL
PEROXIDE did not show mutagenic activity in this bacterial reverse mutation test on
Salmonella typhimurium.

Executive summary:

The objective of this study was to evaluate the potential of the test substance DILAUROYL PEROXIDE to induce reverse mutation in Salmonella typhimurium.

Preliminary toxicity tests were performed to define the dose-levels of DILAUROYL PEROXIDE to be used for the mutagenicity study. DILAUROYL PEROXIDE was then tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. A third experiment with S9 mix was also performed in the TA 1537 strain. The experiments were performed according to the direct plate incorporation method except the second with S9 mix, which was performed according to the preincubation method (60 minutes, 37 deg C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37 deg C, the revertant colonies were scored. The test substance DILAUROYL PEROXIDE was dissolved in tetrahydrofuran.

The top dose-level was selected according to the criteria specified in the international regulations. Since the test substance was non-toxic, poorly soluble, the top dose-level was the lowest precipitating dose: approximately 1500 ug/plate. The selected dose-levels were for the first experiment: 15, 50, 150,500 and 1500 ug/plate; as precipitation again interfered with the scoring at 1500 ug/plate, the dose-treatments were decreased for the second experiment as follows: 5, 15, 50, 150 and 500 ug/plate. The dose-levels selected for the third experiment with the TA 1537 with S9 mix were: 15, 50, 150, 500 and 1500 ug/plate.

The test substance did not induce any significant increase in the number of revertants, with and without S9 mix, in any of the five strains. Indeed, the slight 2.4 fold increase observed at 500 and 1500 ug/plate in the first experiment with S9 mix in the TA 1537 strain was attributed to the low value of revertants in the vehicle controls and was not taken into account since it was not reproduced in the second and third experiments.

Under the experimental conditions, the test substance DILAUROYL PEROXIDE did not show mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.