Bisdecanoyl peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint, three in vitro tests are available and gave negative findings. Therefore didecanoyl peroxide is not considered as genotoxic. No in vivo test is available and required.

In vitro gene mutation in bacterias

The potential of didecanoyl peroxide to induce reverse mutation in Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98, and TA 100) was evaluated in an Ames test (Willems, 1979). Didecanoyl peroxide was tested in two independent studies: with and without a metabolic activation system. It was tested using the direct plate incorporation. Bacteria were exposed to the substance at four dose-levels: 0, 16, 80, 400, 2000µg/plate. After three days of incubation at 37°C, the revertant colonies were scored. No noteworthy increase in the number of revertants was observed for all doses with and without metabolic activation on the 5 tested strains.

In vitro chromosomal aberration assay

Didecanoyl was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors in two independent experiments both in the absence and presence of metabolic activation (S9 mix), according to the OECD n° 473 Guideline (Haddouk, 2002). This substance was tested in two independent experiments, both with and without a metabolic activation system.

In the first experiment, lymphocyte cultures were exposed to the test or control items, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. As this first experiment was negative, a second experiment was performed as follows: without S9 mix, cells were exposed continuously until harvest to the test or control items; with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

There were no increase in frequencies of cells with structural aberrations compared to control and historical data. In conclusion, didecanoyl peroxide did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested in the absence and presence of S-9mix.

In vitro gene mutation assay in mammalian cells (Mouse Lymphoma Assay):

Didecanoyl peroxide was examined for mutagenic activity for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method, in an OECD 476 test (Salvador, 2012). In the first experiment without S9, the cells were exposed to the test item for a short treatment time (3 hours) to doses levels of 0.42, 0.84, 1.68, 3.36, 6.72 and 13.4 µg/mL. Mild toxicity was observed at the highest dose level selected reducing relative total growth (%RTG) to 53%. The second experiment without S9 was performed using a longer treatment time (24 hours), with dose levels of 1.88, 3.75, 7.5, 15.0, 22.5, 30.0 µg/mL. The highest dose level selected yielded marked toxicity reducing RTG to 22%. Dose-related toxicity was seen over the remaining concentrations. In the first experiment with S9, the cells were exposed to the test item 3 hours to doses levels of 13.4, 26.9, 53.8, 108, 215 and 430 µg/mL. Slight toxicity was seen at the three higher dose levels (430, 215 and 108 µg/mL) reducing RTG in a range of 66-80% of the concurrent negative control value. In the second experiment with S9, the cells were exposed to the test item 3 hours to dose levels of 113, 170, 255, 382, 573, 860 µg/mL. Moderate toxicity was noted at the highest dose level (860 µg/mL) reducing RTG to 39% of the concurrent negative control value. Slight toxicity covering a range from 52 to 70% of RTG was observed over the remaining concentrations tested.

In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time. In the second main assay, using the long treatment time in the absence of S9 metabolism, a linear trend was indicated (p<5%) and a statistically significant increase in mutant frequency was observed at the intermediate dose level of 15.0 µg/mL. The IMF value was lower than the global evaluation factor and the mutant frequency observed at this dose level was within the historical control range at the CRO. Hence, the observed linear trend was considered to be attributable to a chance event not related to the action of the test item and of no biological significance. In conclusion didecanoyl peroxide did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro with or without S9.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2002-02-25 to 2002-08-5

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

primary culture, other: human lymphocytes

Details on mammalian cell type (if applicable):

- they have stable karyotype with 46 chromosomes
- they have an average cell cycles time of 12-14 hours

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction and the cofactors necessary for their function. S9 fraction was obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg,intraperitoneal route)

Test concentrations with justification for top dose:

With and without S9:
- 3-hour treatment, 20-hour harvest: 0, 1.07; 2.13; 4.26; 8.52; 17.05; 34.09; 68.18; 136.36 µg/ml
- 20-hour treatment, 20-hour harvest and 44-hour treatment, 44-hour harvest: 0, 4.26; 8.52; 17.05; 34.09; 68.18; 136.36 µg/ml

Vehicle / solvent:

- Ethanol
- The substance was not soluble in water and in dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: mitomycine C, with S9: Cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 hours
- Cells were harvested 20 hours after beginning of treating, corresponding approximately 1.5 normal cell cycles, or for one test 44 hours after the beginning of the treatment.

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: microscopic evaluation
- Determination of endoreplication: microscopic evaluation

Evaluation criteria:

A reproductible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

If necessary, the comparison was performed with chi deux test, in which p = 0.05 was used at the lowest level of significance.

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- At 136.36 µg/ml Ph and osmolality values were equivalent to those of the vehicule control culture
- Water solubility: no
- Precipitation: a slight precipitation was observed at the end of the treatment, at dose-levels >=68.18µg/ml
- Cytotoxicity: both with and without S9 mix, except for some slight decreases in the mitotic index noted mainly at dose-levels = 68.18 µg/mL, no noteworthy toxicity was induced.
- All the experiment were performed in duplicate

Conclusions:

DIDECANOYL PEROXIDE did not induce chromosome aberrations in cultutred human lymphocytes.

Executive summary:

DIDECANOYL PEROXIDE was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors in two independent experiments both in the absence and presence of metabolic activation (S9 mix), according to the OECD n° 473 Guideline and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice.

This substance was tested in two independent experiments, both with and without a metabolic activation system.

In the first experiment, lymphocyte cultures were exposed to the test or control items, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

As this first experiment was negative, a second experiment was performed as follows:

. without S9 mix, cells were exposed continuously until harvest to the test or control items.

. with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

Mitomycine C and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

Cultures treated with DIDECANOYL PEROXYDE in the absence and presence of S-9 (Experiment 1 and 2) resulted in frequencies of cells with structural aberrations, which were similar to those seen in concurrent negative controls. All cultures receiving the test article had numbers of cells with structural aberrations (excluding gaps) that were within historical negative (normal) control ranges.

Under these experimental conditions, this substance did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in any experiment or at any harvest time. 

In conclusion, DIDECANOYL PEROXYDE did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested in the absence and presence of S-9mix.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January to 7 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; coherence between data, results and conclusions

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Minimal medium A

RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 IU/ml + Penicillin G 50.000 units/ml
F 68 Pluronic

Minimal medium B

RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 units/ml + Penicillin G 50.000 units/ml

Complete medium (5%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium (10%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium A (20%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium B (20%)

Minimal medium B
Horse serum (heat-inactivated)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix:
13.4, 6.72, 3.36, 1.68, 0.840 and 0.420 ug/mL for the first experiment (3-hour treatment)
30.0, 22.5, 15.0, 7.50, 3.75 and 1.88 ug/mL for the second experiment (24-hour treatment)

Experiments with S9 mix:
430, 215, 108, 53.8, 26.9 and 13.4 ug/mL for the first experiment (3-hour treatment)
860, 573, 382, 255, 170 and 113 ug/mL for the second experiment (3-hour treatment)

Vehicle / solvent:

Vehicle used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Acetone)

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

(with S9 mix)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Acetone)

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(without S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: fluctuation method in medium

DURATION
- Exposure duration: 3 hours in the absence and presence of S9 metabolism
24 hour hours in the absence of S9 metabolism
- Expression time (cells in growth medium): Two days after treatment (48 hours)
- Selection time (if incubation with a selection agent): 13 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF CELLS EVALUATED: 1.6 cells/well plated in each of 96-well plates (two)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

DETERMINATION OF MUTATION
- Method: Induced mutant frequency (IMF)

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:

(i) The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell
method (126 x 10-6) at one or more doses.

(ii) There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive
responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological
significance. Any increase in mutant frequency should lie outside the historical control range to have biological
relevance.

At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder
effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and
not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is
generally recommended that such data are treated with caution or excluded from consideration.

Statistics:

Dunnett test. Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No precipitate was noted upon addition of the test item to the cultures in all treatment series and by the end of treatment incubation period.
The addition of the test item solution did not have any obvious effect on the osmolality of the treatment medium. The addition of the test item solutions generated a slight dose-dependent decrease in pH. However, pH values measured at all dose levels were within the physiological range.

RANGE-FINDING/SCREENING STUDIES (Cytotoxicity test):
On the basisi of solubility results, a citotoxicity assay was performed in the absence and presence of S9 metabolic activation, at a maximum dose level of 860 µg/mL and at a wide range of lower dose levels: 430, 215, 108, 53.8, 26.9, 13.4, 6.72 and 3.36 µg/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, severe toxicity was observed at the six higher dose levels tested. At the next lower dose level (13.4 µg/mL) relative survival (RS) was reduced to 10% of the concurrent negative control value. Test item treatment at 6.72 and 3.36 µg/mL yielded mild toxicity reducing RS to 36% and 50%, respectively.
Using the 24 hour treatment time, no cells survived to treatment at the six higher dose levels. Test item treatment at 13.4 µg/mL yielded mild toxicity reducing RS to 37% and dose-related toxicity was observed over the remaining dose levels tested.
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), slight toxicity was seen at the two higher dose levels while no relevant toxicity was observed over the remaining dose levels tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive control treatments were included in the mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

ADDITIONAL INFORMATION ON MUTAGENICITY:
Based on the solubility and cytotoxicity results obtained in the preliminary assay, two independent assays for mutation to trifluorothymidine resistance were performed using the dose levels described in the following table:

Experiment No. S9 Treatment time (h) Dose levels (ug/mL)
1 - 3 13.4, 6.72, 3.36, 1.68, 0.840 and 0.420
1 + 3 430, 215, 108, 53.8, 26.9 and 13.4
2 - 24 30.0, 22.5, 15.0, 7.50, 3.75 and 1.88
2 + 3 860, 573, 382, 255, 170 and 113


In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time.
In the second main assay, using the long treatment time in the absence of S9 metabolism, a linear trend was indicated (p<5%) and a statistically significant increase in mutant frequency was observed at the intermediate dose level of 15.0 µg/mL. The IMF value was lower than the global evaluation factor and the mutant frequency observed at this dose level was within the historical control range at RTC. Hence, the observed linear trend was considered to be attributable to a chance event not related to the action of the test item and of no biological significance.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that BISDECANOYL PEROXIDE does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item, BISDECANOYL PEROXIDE, was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

The study was designed to comply with the OECD Guideline for the testing of chemicals No. 476 (adopted July 1997) and was in compliance with the Principles of Good Laboratory Practice.

The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.

The dose levels for the positive controls were:

-Without S9 mix:methylmethanesulphonate (MMS) 10.0 ug/mL

-With S9 mix:benzo(a)pyrene (B(a)P) 2.00 ug/mL

A cell suspension (1 x 10⁶ cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.

The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS). Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a longer treatment time (24 hours).

After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2 x 10⁵ cells/ml.The cultures were incubated at 37°C in a 5% CO₂ atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2 x 10⁵ cells/ml.

Plating for 5-trifluorothymidine resistance

After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/ml) and an estimated 2 x 10³ cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO₂ atmosphere (100% nominal relative humidity) for 13 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

Plating for viability

After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO₂ atmosphere (100% nominal relative humidity) for 13 days and wells containing clones were identified as above and counted.

Results

A main assay for mutation to trifluorothymidine resistance was performed using the dose levels described in the following table:

Experiment no		S9	Treatment time (h)	Dose levels (ug/mL)
1		-	3	13.4, 6.72, 3.36, 1.68, 0.840 and 0.420
1		+	3	430, 215, 108, 53.8, 26.9 and 13.4
2		-	24	30.0, 22.5, 15.0, 7.50, 3.75 and 1.88
2		+	3	860, 573, 382, 255, 170 and 113

Solvent and positive control cultures were included in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range (50-200 x 10^-6viable cells). The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). The cloning efficiencies at Day 2 in the negative control cultures and the control growth factor over 2 days fell within the range.The study was accepted as valid.

Survival after treatment

In the first experiment, in the absence of S9 metabolic activation, mild toxicity was observed at the highest dose level selected (13.4 µg/mL) reducing relative total growth (%RTG) to 53%. No toxicity was noted over the remaining dose levels tested. In the presence of S9 metabolic activation, slight toxicity was seen at the three higher dose levels (430, 215 and 108 µg/mL) reducing RTG in a range of 66-80% of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested.

In the second experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (30.0 µg/mL) yielded marked toxicity reducing RTG to 22%. Dose-related toxicity was seen over the remaining concentrations. In the presence of S9 metabolism, moderate toxicity was noted at the highest dose level (860 µg/mL) reducing RTG to 39% of the concurrent negative control value. Slight toxicity covering a range from 52 to 70% of RTG was observed over the remaining concentrations tested.

Mutation results

In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time.

In the second main assay, using the long treatment time in the absence of S9 metabolism, a linear trend was indicated (p<5%) and a statistically significant increase in mutant frequency was observed at the intermediate dose level of 15.0 µg/mL. The IMF value was lower than the global evaluation factor and the mutant frequency observed at this dose level was within the historical control range at RTC. Hence, the observed linear trend was considered to be attributable to a chance event not related to the action of the test item and of no biological significance. Conclusion It is concluded that BISDECANOYL PEROXIDE does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 1535. TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate of aroclor-induced rats

Test concentrations with justification for top dose:

0; 16; 80; 400; 2000 µg/ml

Vehicle / solvent:

- Vehicule used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), test performed in triplicate

NUMBER OF CELLS EVALUATED:
TA 1535: 5 x 10^9
TA 1537: 4 x 10^9
TA 1538: 2 x 10^9
TA 98: 9 x 10^9
TA 100: 5 x 10^9

Evaluation criteria:

Positive results: if it's provided evidence of dose-related, and reproductible increase in the number of HIS+ revertants

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table: Evaluation of test item in the salmonella/ microsome mutagenicity test

µg test item/ 0.1 ml acetone plate	S-9 mix  added	Number of his revertants (mean of 3 plates +/- SD) with
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
0	No	41 +/- 16	10 +/- 3	13 +/-2	19 +/-4	124 +/- 7
16	No	23 +/- 2	14 +/- 6	12 +/- 6	21 +/- 4	139 +/- 6
80	No	27 +/- 1	8 +/-1	10 +/- 2	24 +/- 10	137 +/- 32
400	No	32 +/- 9	7 +/-2	11 +/- 2	16 +/- 2	142 +/- 12
2000	No	24 +/- 5	9 +/- 3	7 +/- 3	16 +/- 6	139 +/- 21
0	Yes	18 +/- 2	11 +/- 5	56 +/- 11	74 +/- 17	155 +/- 13
16	Yes	31 +/- 10	9 +/- 4	55 +/- 6	72 +/- 8	108 +/- 34
80	Yes	21 +/- 6	10 +/- 4	47 +/- 4	84 +/- 19	118 +/- 17
400	Yes	32 +/- 7	13 +/- 2	42 +/- 9	88 +/- 19	131 +/- 10
2000	Yes	18 +/- 9	12 +/- 8	54 +/- 3	62 +/- 10	138 +/- 15
Number of bacteria/ml		5 x 10^9	4 x 10^9	2 x 10^9	9 x 10^9	5 x 10^9

--

Table: Dose-response relationship of 2 -aminoanthracene (2 -AA) in the salmonella/ microsome mutagenicity test (POSITIVE CONTROL)

2 - AA (µg/0.1ml DMSO plate)	Numbers of his^+evertants (mean of 3 plates +/- SD) with
	Without S-9 mix
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
0	12; 13; 19	3; 3; 7	13; 16; 19	26; 27; 38	108; 117; 134
0.1	7; 8; 20	3; 4; 7	11; 15; 20	24; 26; 29	103; 103; 117
0.25	11; 13; 14	2; 3; 8	11; 14; 23	22; 26; 36	115; 115; 131
0.5	16; 17; 17	3; 4; 6	8; 15; 23	22; 25; 32	105; 105; 128
1.0	7; 8; 14	8; 14; 17	11; 16; 23	26; 28; 29	101; 103; 158
	With S-9 mix (0.5 ml/plate)
0	7; 12; 15	8; 8; 12	31; 46; 46	47; 50; 67	100; 137; 159
0.1	24; 28; 40	13; 19; 19	62; 64; x	67; 68; 82	162; 198; 204
0.25	39; 51; 86	18; 18; 19	138; 173; 176	126; 128; 174	244; 279; 298
0.5	68; 100; 145	17; 34; 36	257; 308; 345	298; 302; 347	436; 522; 543
1.0	175; 198; 208	59; 72; 77	533; 621; 769	578; 621; 637	869; 938; 983

Conclusions:

Under the experimental conditions, DIDECANOYL PEROXYDE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The potential of DIDECANOYL PEROXYDE to induce reverse mutation in Salmonella typhimurium (5 strains: TA 1535, TA 1537, TA 1538, TA 98, and TA 100) was evaluated.

This substance was tested by two independent studies: with and without a metabolic activation system. It was tested using the direct plate incorporation.

Bacteria were exposed to DIDECANOYL PEROXYDE at four dose-levels: 0, 16, 80, 400, 2000 µg/plate. After three days of incubation at 37°C, the revertant colonies were scored.

No noteworthy increase in the number of revertants was observed for all doses with and without metabolic activation on the 5 tested strains.

Under our experimental conditions, DIDECANOYL PEROXYDE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), the substance is not classified for genotoxicity.