1-methyl-1-phenylethyl peroxyneodecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, 473 and 476, GLP).

In a bacterial reverse gene mutation test conducted according to OECD 471, the target substance did not induce mutagenicity. In a mammalian cell HPRT mutation assay conducted according to OECD 476, the target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was also tested negative for inducing mutagenic effects. Moreover, the target substance was tested negative in an in vitro cytogenicity study in human lymphocytes conducted according to OECD 473. Based on the lack of mutagenicity/cytogenicity in all in vitro assays, 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000-07-25 to 2000-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 July 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST MATERIAL
- Name: Cumyl peroxyneodecanoate
- Brand name: LUPEROX 188M70
- CAS-No.: 26748-47-0
- Purity: 70% (in isododecane)
- Appearance: brown liquid

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Source: ATOFINA. Cours Michelet, La Défense 10, 92091 Paris-la-Défense CEDEX, France; Batch no.: E-660 0005-001MXL
- Expiration date of the lot/batch: May 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At -20 °C in the dark
- Solubility: 0.2 mg/L in water, miscible with organic solvents
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was dissolved in the vehicle at a concentration of 100 mg/mL (expressed as active material) for the preliminary toxicity test and both mutagenicity experiments. The preparations were made immediately before use.

Target gene:

histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: B.N. Ames' Laboratory (University of California, Berkeley, USA)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stored in a cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethyl sulfoxide) in a liquid nitrogen container.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Since the test substance was freely soluble and sometimes toxic, the highest dose-level was either 5000 µg/plate or based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:
- 312.5, 625, 1250, 2500 and 5000 µg/plate, for TA1537, TA98, TA100 and TA102 (Experiment 1)
- 156.25, 312.5, 625, 1250 and 2500 µg/plate, for TA1535 (Experiment 1)
- 78.125, 156.25, 312.5, 625, 1250 and 2500 µg/plate, for all (Experiment 2)

Experiments with S9 mix:
- 312.5, 625, 1250, 2500 and 5000 µg/plate, for TA1537, TA98, TA100 and TA102 (Experiment 1) and for TA1535, TA98 and TA102 (Experiment 2)
- 156.25, 312.5, 625, 1250 and 2500 µg/plate, for TA1535 (Experiment 1)
- 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate, for TA1537 and TA100 (Experiment 2)
- 1250, 1875, 2500, 3750 and 5000 µg/plate, for TA1537 (Experiment 3)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol; Batch no.: V8M 084248 M (Carlo Erba, 27106 Val de Reuil, France)
- Justification for choice of solvent/vehicle: The test substance was freely soluble in the vehicle

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthraminev(2AM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine (2AM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; experiment 1); pre-incubation (experiment 2)

EXPERIMENTAL PERFORMANCE:
In two independent experiments, five dose-levels of CUMYL PEROXYNEODECANOATE (three plates/dose-level) were tested on each strain, with and without S9 mix. In a complementary experiment, five dose-levels of the test substance (three plates/dose-level) were tested on the TA 1537 strain, with S9 mix.
Direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first and third experiment with S9 mix):
- 0.05 mL test substance solution
- 0.5 mL S9 mix
- 0.1 mL bacterial suspension
- mixed with 2 mL of overlay agar (containing traces of relevant amino acid and biotin and maintained at 45 °C)
After rapid homogenization, mixture was overlaid onto Petri plate containing minimum medium.

Preincubation method (second experiment with S9):
- 0.05 mL test substance solution
- 0.5 mL S9 mix
- 0.1 mL bacterial suspension
- mixed with agar after 60 minutes incubation time at 37 °C

After 48 to 72 hours of incubation at 37 °C revertants were scored with an automatic counter

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Preliminary toxicity test: Six dose levels (one plate/dose-level) were tested in the TA98, TA100 and TA102 strains with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn

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. See Table 1 in box 'Any other information on materials & methods incl. tables' for historical data.

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with our historical data (appendix 2)
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

Statistics:

not applicable

Key result

Species / strain:

other: TA1535, TA100, TA102, TA 1537, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

EXPERIMENTS WITHOUT S9 MIX:
In the first experiment, a slight to moderate toxicity was induced in the TA1535 and TA98 strains at dose-level ≥1250 µg/plate. In the TA100 strain, a slight thinning of the bacterial lawn was noted at 5000 µg/plate.
In the second experiment, except for a slight toxicity observed in the TA1537 at 2500 µg/plate, no noteworthy toxicity was induced.
In the first experiment, a 2-fold increase in the number of revertants was noted at 312.5 µg/plate in the TA1537 strain. However, this slight increase was not considered as biologically relevant since it was neither induced at higher dose-level nor reproduced in the second experiment. No noteworthy increase in the number of revertant was observed in the remaining tester strains.

EXPERIMENTS WITH S9 MIX:
No toxicity was noted towards all the strains used, in the first experiment.
In the second experiment (preincubation), using the preincubation method, up to 2.8-fold increase in the number of revertants was noted with this tester strain, without any clear evidence of a dose relationship.
Even though the individual and the mean values obtained in these two experiments remained clearly within the vehicle control historical range (3-25), in order to check the reliability of the increase in revertants mainly noted in the first experiment, it was decided to perform a third experiment, using a closer range of dose-levels.
In this third experiment, no noteworthy increase in the number of revertants was induced.
Therefore, the more than two-fold increases previously noted were considered as sporadic and were attributed to the low vehicle control values.
The test substance did not induce any noteworthy increase in the number of revertants, in the remaining tester strains.

For individual results see Table 2 in 'Any other information on results incl. tables'.

Table 2: First experiment without S9 mix

strains	doses* (µg/plate)	T	E	revertants per plate			mean	SD	ratio
TA 1535	0	0	0	8	7	5	7	2	-
	156.25	0	0	9	9	3	7	3	1.05
	312.5	0	0	15	6	7	9	5	1.40
	625	0	0	9	6	4	6	3	0.95
	1250	1	1	9	7	10	9	2	1.30
	2500	2	1	6	2	3	4	2	0.55
	NaN3(l)	-	1	372	408	357	379	26	56.85
TA 1537	0	0	0	7	3	3	4	2	-
	312.5	0	0	9	9	8	9	1	2.00
	625	0	1	3	9	5	6	3	1.31
	1250	0	1	6	4	8	6	2	1.38
	2500	0	1	9	8	8	8	1	1.92
	5000	0	2	8	12	2	7	5	1.69
	9AA(50)	-	-	204	166	183	184	19	42.54
TA 98	0	0	0	22	15	18	18	4	-
	312.5	0	0	10	17	16	14	4	0.78
	625	0	1	16	23	16	18	4	1.00
	1250	1	1	20	14	17	17	3	0.93
	2500	1	1	21	27	13	20	7	1.11
	5000	2	2	22	9	11	14	7	0.76
	2NF(0.5)	-	-	162	160	182	168	12	9.16
TA 100	0	0	0	52	70	73	65	11	-
	312.5	0	0	70	59	55	61	8	0.94
	625	0	1	61	70	59	63	6	0.97
	1250	0	1	64	44	42	50	12	0.77
	2500	0	1	68	47	61	59	11	0.90
	5000	1	1	54	70	69	64	9	0.99
	NaN3(l)	-	-	278	237	296	270	30	4.16
TA 102	0	0	0	145	119	131	132	13	-
	312.5	0	0	99	89	79	89	10	0.68
	625	0	1	138	132	143	138	6	1.05
	1250	0	1	146	99	80	108	34	0.82
	2500	0	1	145	165	119	143	23	1.09
	5000	0	2	110	107	81	99	16	0.75
	MMC(0.5)	-	-	850	663	735	749	94	5.69

0: vehicle control (ethanol)

*: doses expressed as active material

T: toxicity

E: emulsion

ratio: (number of revertants with the test substance)/(number of revertants with vehicle)

Table 3: Second experiment without S9 mix

strains	doses* (µg/plate)	T	E	revertants per plate			mean	SD	ratio
TA 1535	0	0	0	6	12	7	8	3	-
	78.125	0	0	8	8	12	9	2	1.12
	156.25	0	0	10	8	9	9	1	1.08
	312.5	0	0	8	10	3	7	4	0.84
	625	0	1	11	7	13	10	3	1.24
	1250	0	1	4	9	13	9	5	1.04
	2500	0	1	18	11	8	12	5	1.48
	NaN3(l)	-	-	420	372	344	379	38	45.44
TA 1537	0	0	0	9	6	2	6	4	-
	78.125	0	0	8	8	3	6	3	1.12
	156.25	0	0	2	5	4	4	2	0.65
	312.5	0	0	6	4	3	4	2	0.76
	625	0	1	6	7	11	8	3	1.41
	1250	0	1	7	2	11	7	5	1.18
	2500	1	1	7	7	6	7	1	1.18
	9AA(50)	-	-	173	217	181	190	23	33.59
TA 98	0	0	0	31	27	23	27	4	-
	78.125	0	0	19	26	18	21	4	0.78
	156.25	0	0	34	28	19	27	8	1.00
	312.5	0	0	29	30	15	25	8	0.91
	625	0	1	24	26	22	24	2	0.89
	1250	0	1	31	25	19	25	6	0.93
	2500	0	1	27	26	28	27	1	1.00
	2NF(0.5)	-	-	147	177	175	166	17	6.16
TA 100	0	0	0	72	76	56	68	11	-
	78.125	0	0	55	64	54	58	6	0.85
	156.25	0	0	67	61	68	65	4	0.96
	312.5	0	0	64	60	56	60	4	0.88
	625	0	1	76	61	54	64	11	0.94
	1250	0	1	70	66	67	68	2	1.00
	2500	0	1	100	68	90	86	16	1.26
	NaN3(l)	-	-	473	461	414	449	31	6.61
TA 102	0	0	0	198	189	197	195	5	-
	78.125	0	0	123	111	100	111	12	0.57
	156.25	0	0	172	194	131	166	32	0.87
	312.5	0	0	154	114	107	125	25	0.64
	625	0	1	192	180	197	190	9	0.97
	1250	0	1	176	131	126	144	28	0.74
	2500	0	1	187	162	149	166	19	0.85
	MMC(0.5)	-	-	990	1079	1001	1023	49	5.26

Table 4: First experiment with S9 mix

strains	doses* (µg/plate)	T	E	revertants per plate			mean	SD	ratio
TA 1535	0	0	0	15	8	24	16	8	-
	156.25	0	0	16	16	15	16	1	1.00
	312.5	0	0	20	23	16	20	4	1.26
	625	0	1	13	12	19	15	4	0.94
	1250	0	1	20	16	19	18	2	1.17
	2500	0	1	27	19	25	24	4	1.51
	2AM(2)	-	-	138	143	103	128	22	8.17
TA 1537	0	0	0	5	7	5	6	1	-
	312.5	0	0	11	6	2	6	5	1.12
	625	0	0	6	12	7	8	3	1.47
	1250	0	1	9	13	6	9	4	1.65
	2500	0	1	16	13	10	13	3	2.29
	5000	0	2	17	13	12	14	3	2.47
	2AM(2)	-	-	62	66	70	66	4	11.65
TA 98	0	0	0	22	36	28	29	7	-
	312.5	0	0	17	7	21	15	7	0.52
	625	0	0	19	26	21	22	4	0.77
	1250	0	1	26	16	23	22	5	0.76
	2500	0	1	17	23	15	18	4	0.64
	5000	0	1	27	24	21	24	3	0.84
	2AM(2)	-	-	397	386	371	385	13	13.42
TA 100	0	0	0	87	69	83	80	9	-
	312.5	0	0	99	85	105	96	10	1.21
	625	0	0	100	94	108	101	7	1.26
	1250	0	1	152	128	114	131	19	1.65
	2500	0	1	141	154	135	143	10	1.80
	5000	0	2	172	166	131	156	22	1.96
	2AM(2)	-	-	936	987	897	940	45	11.80
TA 102	0	0	0	194	197	177	189	11	-
	312.5	0	0	177	116	111	135	37	0.71
	625	0	0	169	191	201	187	16	0.99
	1250	0	1	176	136	133	148	24	0.78
	2500	0	1	223	152	216	197	39	1.04
	5000	0	1	223	184	154	187	35	0.99
	2AM(2)	-	-	821	700	719	747	65	3.94

Table 5: Second experiment with S9 mix: Preincubation method

strains	doses* (µg/plate)	T	E	revertants per plate			mean	SD	ratio
TA 1535	0	0	0	13	6	12	10	4	-
	312.5	0	0	13	9	8	10	3	0.97
	625	0	0	8	15	17	13	5	1.29
	1250	0	1	10	13	12	12	2	1.13
	2500	0	1	14	16	13	14	2	1.39
	5000	0	2	16	17	13	15	2	1.48
	2AM(2)	-	-	81	89	90	87	5	8.39
TA 1537	0	0	0	3	5	2	3	2	-
	156.25	0	0	5	13	10	9	4	2.80
	312.5	0	0	14	2	2	6	7	1.80
	625	1	0	5	11	7	8	3	2.30
	1250	1	0	11	6	11	9	3	2.80
	2500	2	1	11	11	6	9	3	2.80
	5000	2	1	8	4	5	6	2	1.70
	2AM(2)	-	-	63	49	63	58	8	17.50
TA 98	0	0	0	25	20	20	22	3	-
	312.5	0	0	17	23	23	21	3	0.97
	625	0	0	20	17	25	21	4	0.95
	1250	0	0	25	23	26	25	2	1.14
	2500	0	1	21	21	23	22	1	1.00
	5000	0	2	35	32	31	33	2	1.51
	2AM(2)	-	-	489	399	465	451	47	20.82
TA 100	0	0	0	118	89	101	103	15	-
	156.25	0	0	100	103	95	99	4	0.97
	312.5	0	0	90	115	95	100	13	0.97
	625	0	0	113	103	82	99	16	0.97
	1250	0	1	94	82	83	86	7	0.84
	2500	1	1	113	111	92	105	12	1.03
	5000	1	2	127	120	123	123	4	1.20
	2AM(2)	-	-	689	609	558	619	66	6.03
TA 102	0	0	0	205	253	196	218	31	-
	312.5	0	0	187	135	175	166	27	0.76
	625	0	0	227	187	209	208	20	0.95
	1250	0	0	201	216	176	198	20	0.91
	2500	0	1	277	319	309	302	22	1.38
	5000	0	2	328	243	219	263	57	1.21
	2AM(2)	-	-	1012	741	740	831	157	3.81

Conclusions:

In conclusion, the test substance does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in the presence or absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA1537, TA1535, TA102, TA100 and TA98) were exposed to Cumyl Peroxyneodecanoate (1 -Methyl-1 -phenylethyl peroxyneodecanoate; 70% purity in isododecane) in ethanol at concentrations of 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/ plate in the presence and absence of metabolic activation. The positive controls did induce the appropriate response in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-01-14 to 2003-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21st July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Supplier: ATOFINA
- Chemical Name: Alpha-CUMYL PEROXYNEODECANOATE
- Batch number: 0210840414
- Description: colourless liquid
- Container: one plastic flask
- Storage conditions: at -20 °C and protected from light
- Purity: 75% in isododecane

The test item was dissolved in the vehicle at a concentration of 612.9 mg/mL for the first experiment and 306.45 mg/mL for the second experiment.
The preparations were made immediately before use.

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Cells used: Human lymphocytes
- Modal number of chromosomes: 46
- Cell cycle time: 12 - 14
- Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.

MEDIA USED
RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, toxicity indicated by the reduction of mitotic index (MI) in the first experiment, if any, was also taken into account.
- Experiment I (with and without S9): 0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 mM
- Experiment II (with S9): 0.16, 0.31, 0.63, 1.25, 2.5 and 5 mM
- Experiment II (without S9): 0.04, 0.08, 0.16, 0.31, 0.63 and 1.25 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol, batch no. V8M084248 M (Carlo Erba, 27106 Val de Reuil, France)

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9: 3 µg / mL ( 3h) or 0.2 µg / mL (continuous experiment)

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9: 12.5, 25 or 50 µg / mL

Details on test system and experimental conditions:

Treatment
In two independent experiments, using duplicate cultures, the cells were tested, with and without S9 mix, with:
- at least five dose-levels of the test item,
- the vehicle control,
- the appropriate positive control.
To prepare each culture, 0.5 mL of heparinised whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide). The cultures were then placed at 37°C for 48 hours.

In the first experiment, lymphocyte cultures were then exposed to the test or control items, both in the absence and presence of S9 mix, for 3 hours then rinsed. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:
• without S9 mix, cells were exposed continuously to the test or control items, until harvest,
• with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.

Preparation of microscope slides
After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded, so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

Determination of cytotoxicity
The cytotoxicity of the test item was evaluated using the mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether a item induces mitotic inhibition. Mitotic index was determined without blind scoring. Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberrations were observed. All metaphase analyses were performed blind. The following structural aberrations were recorded for each metaphase (c, d): gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition, the following numerical aberrations were recorded when encountered: polyploidy and endoreduplication. The metaphase analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford.

Evaluation criteria:

A reproducible 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:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ2 test, in which p= 0.05 was used as the lowest level of significance.

Species / strain:

lymphocytes: human

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: pH was approximately 7.7 (7.4 for the vehicle control) and the osmolality equal to 313 mOsm/kg H2O (377 for the vehicle control).
- Other confounding effects: No emulsion was observed in the culture medium at the end of the treatment period.

Experiments without S9 mix:

Cytotoxicity:
Following the 3-hour treatment, a moderate to marked decrease in mitotic index (56-81% decrease) was noted at dose-levels ≥ 1.25 mM. Following the 20-hour treatment, a moderate decrease in mitotic index (41% decrease) was noted at 1.25 mM. Following the 44-hour treatment, a slight to marked decrease in mitotic index (31-70% decrease) was noted at dose-levels ≥ 0.63 mM.

Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 0.31, 0.63 and 1.25 mM for the 3-hour and the 20-hour treatments, the latter inducing 56 and 41% decreases in mitotic index, respectively,
- 1.25 mM, for the 44-hour treatment, this dose-level inducing 70% decrease in mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44-hour treatments.


Experiments with S9 mix:

Cytotoxicity:
A slight to marked decrease in mitotic index (29-78% decrease) was noted at dose-levels ≥ 0.63 mM at the 20-hour harvest time.
At the 44-hour harvest time, a moderate to strong decrease in mitotic index was noted at dose-levels ≥ 1.25 mM (59-89% decrease).

Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 1.25, 2.5 and 5 mM, for the 20-hour harvest time in the first experiment, the latter inducing 59% decrease in mitotic index,
- 0.31, 0.63 and 1.25 mM, for the 20-hour harvest time in the second experiment, the latter inducing 68% decrease in mitotic index,
- 1.25 mM, for the 44-hour harvest time, this dose-level inducing 59% decrease in mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Table 1: Experiment 1 without S9 mix: chromosome aberration (3 -hour treatment, 20 -hour harvest)

Doses mM	Slide Nb.	Nb. Of cells scored	NA	Structural chromosome aberrations (type and number)									Cells with structural chromosome aberrations
				G	Chromatid		Chromosome		MA	PU	Total +G	Total -G	Nb. +G	%	Nb. -G	%
					D	Exch	D	Exch
0	14M	100	0	0	1	0	0	0	0	0	5	3	1	2.0	1	1.5
	23F	100	1	2	0	0	1	1	0	0			3		2
0.31	22M	100	1	2	1	0	2	2	0	0	9	7	6	4.0	4	3.0
	6F	100	0	0	1	1	0	0	0	0			2		2
0.63	5M	100	0	2	0	0	0	0	0	0	6	2	2	3.0	0	1.0
	15F	100	1	2	2	0	0	0	0	0			4		2
1.25	39M	100	2	3	1	0	1	1	0	0	10	5	6	5.0	3	2.5
	12F	100	0	2	2	0	0	0	0	0			4		2
MMC	9M	50	0	4	18	11	3	0	0	0	89	78	21	52.0	19	48.0 ***
3µg/mL	18F	50	0	7	28	14	4	0	0	0			31		29

Table 2: Second experiment without S9 mix: chromosome aberration (20 -hour treatment, 20 -hour harvest)

Doses mM	Slide Nb.	Nb. of cells scored	NA	Structural chromosome aberrations (type and number)									Cells with structural chromosome aberrations
				G	Chromatid		Chromosome		MA	PU	Total +G	Total -G	Nb. +G	%	Nb. -G	%
					D	Exch	D	Exch
0	48M	100	0	2	2	0	0	0	0	0	5	3	4	2.5	1	1.5
	57F	100	2	0	0	0	1	0	0	0			1		2
0.31	51M	100	0	1	0	0	0	0	0	0	3	1	1	1.5	4	0.5
	75F	100	0	1	1	0	0	0	0	0			2		2
0.63	64M	100	1	1	1	0	0	0	0	0	6	1	2	3.0	0	0.5
	44F	100	0	4	0	0	0	0	0	0			4		2
1.25	60M	100	0	0	0	0	0	0	0	0	3	2	0	1.5	3	1.0
	71F	100	1	1	2	0	0	0	0	0			3		2
MMC	54M	50	0	3	5	4	1	0	0	0	30	23	10	24.0	19	21.0 ***
3 µg/mL	49F	50	0	4	6	5	2	0	0	0			14		29

Table 3: Experiment 2 without S9 mix: chromosome aberration (44 -hour treatment, 44 -hour harvest)

Doses mM	Slide Nb.	Nb. of cells scored	NA	Structural chromosome aberrations (type and number)									Cells with structural chromosome aberrations
				G	Chromatid		Chromosome		MA	PU	Total +G	Total -G	Nb. +G	%	Nb. -G	%
					D	Exch	D	Exch
0	84M	100	0	2	0	0	2	0	0	0	4	2	4	2.0	2	1.0
	97F	100	1	0	0	0	0	0	0	0			0		0
Jan 25	81M	52	0	1	2	0	0	0	0	0	15	11	3	5.5	2	3.5
	87F	148	3	3	2	0	7	0	0	0			8		5

Table 4: Experiment 1 with S9 mix: chromosome aberration (3 -hour treatment, 20 -hour harvest)

Doses mM	Slide Nb.	Nb. of cells scored	NA	Structural chromosome aberrations (type and number)									Cells with structural chromosome aberrations
				G	Chromatid		Chromosome		MA	PU	Total +G	Total -G	Nb. +G	%	Nb. -G	%
					D	Exch	D	Exch
0	32M	100	0	1	1	0	0	0	0	0	5	3	2	2.5	1	1.5
	8F	100	0	1	0	0	2	0	0	0			3		2
1.25	2M	100	1	4	0	0	1	0	0	0	9	2	5	4.5	1	1.0
	33F	100	1	3	0	0	0	1	0	0			4		1
2.5	38;	100	2	3	1	0	1	1	0	0	13	9	6	5.5	3	3.5
	26F	100	1	1	3	0	3	0	0	0			5		4
5	16M	100	0	7	0	0	0	0	0	0	10	1	7	5.0	0	0.5
	4F	100	3	2	0	0	1	0	0	0			3		1
CPA	27M	50	0	6	26	5	1	0	0	0	91	78	22	52.0	21	48.0 ***
50 µg/mL	17F	50	0	7	35	6	4	0	1	0			30		27

Table 5: Experiment 2 with S9 mix: chromosome aberration (4 -hour treatment 20 -hour harvest)

Doses mM	Slide Nb.	Nb. Of cells scored	NA	Structural chromosome aberrations (type and number)								Cells with structural chromosome aberrations
				Chromatid		Chromosome		MA	PU	Total +G	Total -G	Nb. +G	%	Nb. -G	%
				D	Exch	D	Exch
0	43M	100	0	1	0	0	0	0	0	5	4	1	2.5	1	2.0
	53F	100	0	2	0	1	0	0	0			4		3
0.31	52M	100	0	0	0	0	0	0	0	3	1	0	1.5	0	0.5
	69F	100	2	1	0	0	0	0	0			3		1
0.63	66M	100	1	1	0	0	0	0	0	5	4	1	2.0	1	1.5
	47F	100	0	1	0	1	1	0	0			3		2
Jan 25	56M	100	0	1	0	0	0	0	0	6	4	3	3.0	1	2.0
	72F	100	0	1	0	1	1	0	0			3		3
CPA	73M	50	0	10	3	3	0	0	0	38	31	16	28.0	13	23.0 ***
12.5 µg/mL	45F	50	0	12	2	1	0	0	0			12		10

Table 6: Experiment 2 with S9 mix: chomosome aberration (3 -hour treatment 44 -hour harvest)

Doses mM	Slide Nb.	Nb. Of cells scored	NA	Structural chromosome aberrations (type and number)								Cells with structural chromosome aberrations
				Chromatid		Chromosome		MA	PU	Total +G	Total -G	Nb. +G	%	Nb. -G	%
				D	Exch	D	Exch
0	77M	100	0	1	1	0	0	0	0	3	1	2	1.5	1	0.5
	91F	100	0	0	0	0	0	0	0			1		0
1.25	83M	100	0	2	1	0	0	0	0	5	2	3	2.5	1	1.0
	94F	100	1	1	1	0	0	0	0			2		1

NA: numerical aberrations, G: gap, D: deletion, Exch: exchange, MA: multiple aberrations, PU: pulverization.

M: male

F: female

0: vehicle control (DMSO)

MMC: mitomycin C

Statistical analysis: Chi-Quadrat test ***: p < 0.001 (performed only for cells with structural aberrations excluding gaps)

Nb.: number

(1): expressed as active

Conclusions:

In this study, the test substance did not induce chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, the test item is not considered to be clastogenic in this test system.

Executive summary:

In an in-vitro mammalian chromosome aberration test conducted in accordance with OECD 473, the potential of the test item (75% in isododecane) to induce chromosome aberrations in cultured human lymphocytes was evaluated in the presence and absence of a liver metabolizing system (S9). In the first experiment, lymphocytes were exposed to 0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 mM with and without S9 mix for 3 hours, then rinsed. Cells were harvested 20 hours after beginning of treatment. For the second experiment, the cells were treated with 0.16, 0.31, 0.63, 1.25, 2.5 and 5 mM with S9 mix for 3 hours, then rinsed and with 0.04, 0.08, 0.16, 0.31, 0.63 and 1.25 mM without S9 mix until harvest. Cells were harvested 20 and 44 hours after the beginning of treatment. For all tested treatment groups, no dose-response relationship could be observed. The positive controls did induce the appropriate response. There was no significant increase in the frequency of cells with structural chromosomal aberrations in both experiments and at both harvest times. Based on these results, the test item did not induce chromosome aberrations in cultured human lymphocytes. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-03-24 to 2017-08-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TEST MATERIAL
- Name: 1-methyl-1-phenylethyl peroxyneodecanoate
- Product name: Peroxan CND
- CAS No.: 26748-47-0
- Physical state: fluid
- Colour: colourless/yellowish
- Density: 0.962 g/cm³
- Active component: 69.6% peroxide

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ; Batch no.: 1207870-01
- Purity test date: 2017-02-21


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤ -20 °C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test THF was most suitable. The test item was dissolved in THF and diluted prior to treatment. The different standard solutions were added to cell culture medium (MEM + 0% FBS 4 h treatment) with a final concentration of 0.5% (v/v). The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration in experiment 1 (250 µg/mL) was 373 mOsmol/kg.

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus of V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELL SOURCE
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH
- Suitability of cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Complete Culture Medium (MEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B)
Treatment Medium (MEM medium supplemented with 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B)
Selective Medium (MEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 11 µg/mL thioguanine (TG))
- Periodically checked for Mycoplasma contamination: yes (each cell batch)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-experiment: 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476.

Experiment 1:
without metabolic activation: 0.5, 1, 2, 5, 10, 30, 50, 100 and 250 µg/mL
with metabolic activation: 1, 2, 5, 10, 20, 30, 40, 50, 100 and 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles. Based on the results, tetrahydrofuran was most suitable.

Untreated negative controls:

yes

Remarks:

treatment medium, duplicate cultures

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran, Applichem Lot No. I864407

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; final concentration: 300 µg/mL

Untreated negative controls:

yes

Remarks:

treatment medium, duplicate cultures

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran, Applichem Lot No. I864407

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; final concentrations: 1.0 and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5 x 10^6 cells per negative, solvent and positive controls were seeded in complete culture medium (with and without metabolic activation) and in total 10 x 10^6 cells for each test item concentration.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 h
- Expression time (cells in growth medium): 6-7 days
- Selection time (if incubation with a selection agent): 7-9 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: approx. 200

DETERMINATION OF CYTOTOXICITY
- Method: Relative survival, based on the cloning efficiency

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data.

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was noted at concentrations of 100 µg/mL and higher (with metabolic activation).

RANGE-FINDING/SCREENING STUDIES
The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476.
In experiment 1 250 µg/mL (without metabolic activation) and 100 µg/mL (with metabolic activation) were selected as the highest concentrations due to cytotoxic effects and precipitation, respectively. Experiment 1 with and without metabolic activation was performed as a 4 h short-term exposure assay.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive controls, DMBA (1 and 1.5 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.
- Negative (solvent/vehicle) historical control data: The mean mutant value of the negative and solvent controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.

ADDITIONAL INFORMATION ON CYTOTOXICITY
A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment 1 without metabolic activation.
In experiment 1 without metabolic activation the relative survival was 17% for the highest concentration (250 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 100 µg/mL due to precipitation with a relative survival of 88%.

For individual results see Table 2 to 5 in box 'Any other information on results incl. tables'.

Table 2: Experiment 1 - Toxicity, without metabolic activation

Dose group	Concentration µg/mL	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10,000,000	10,931,000	147	149	148	74	81	122
NC2		10,000,000	11,509,000	159	139	149	75	86	129
S1	0	10,000,000	8,942,000	163	166	165	82	74	100
S2		10,000,000	8,619,000	136	140	138	69	59
1	0.5	20,000,000	17,374,000	151	133	142	71	62	93
2	1	20,000,000	16,592,000	166	172	169	85	70	105
3	2	20,000,000	15,402,000	144	143	144	72	55	83
4	5	20,000,000	16,558,000	127	127	127	64	53	79
5	10	20,000,000	12,512,000	128	137	133	66	41	62
6	30	20,000,000	13,736,000	141	169	155	78	53	80
7	50	20,000,000	15,062,000	161	155	158	79	59	89
8	100	20,000,000	12,444,000	163	169	166	83	52	78
9	250	20,000,000	4,148,000	102	116	109	55	11	17
PC1	300	10,000,000	11,220,000	171	181	176	88	99	148
PC2	300	10,000,000	10,693,000	153	173	163	82	87	131

PC: positive control (ethylmethanesulfonate)

NC: negative control

S: solvent control (tetrahydrofuran)

CE: cloning efficiency

Table 3: Experiment 1 - Mutagenicity, without metabolic activation

Dose group	Concentration µg/mL	CE in non-selective medium				CE in selective medium							Mutant frequency per 10^6cells
		Number of colonies per flask			CE [%]	Number of colonies per flask
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	154	146	150	75	9	15	8	13	14	11.8	2.8	39.3
NC2		140	126	133	67	8	7	10	4	4	6.6	2.3	24.8
S1	0	173	157	165	83	7	9	3	11	6	7.2	2.7	21.8
S2		133	143	138	69	10	9	8	8	9	8.8	0.7	31.9
1	0.5	143	174	159	79	8	11	8	8	9	8.8	1.2	27.8
2	1	160	157	159	79	12	11	13	14	16	13.2	1.7	41.6
3	2	150	157	154	77	9	9	9	14	15	11.2	1.7	36.5
4	5	176	148	162	81	11	9	8	16	15	11.8	3.2	36.4
5	10	120	150	135	68	3	8	8	5	4	5.6	2.1	20.7
6	30	120	122	121	61	8	13	9	10	16	11.2	2.9	46.3
7	50	124	132	128	64	8	9	10	5	4	7.2	2.3	28.1
8	100	148	142	145	73	12	8	9	4	8	8.2	2.6	28.3
9	250	150	145	148	74	4	10	10	9	10	8.6	2.3	29.2
PC1	300 µg/mL	131	149	140	70	62	72	80	74	86	74.8	8.1	267.1
PC2	300 µg/mL	160	135	148	74	76	68	68	80	77	73.8	4.9	250.2

PC: positive control (ethylmethanesulfonate)

NC: negative control

S: solvent control (tetrahydrofuran)

CE: cloning efficiency

SD: standard deviation

Table 4: Experiment 1 - Toxicity, with metabolic activation

Dose group	Concentration µg/mL	Number of cells at the		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10,000,00	14,671,000	175	192	184	92	135	124
NC2		10,000,00	14,195,000	195	185	190	95	135	125
S1	0	10,000,00	12,546,000	163	156	160	80	100	100
S2		10,000,00	12,240,000	204	176	190	95	116
1	1	10,000,00	12,376,000	198	178	188	94	116	108
2	2	10,000,00	12,546,000	195	234	215	107	135	124
3	5	10,000,00	13,855,000	175	159	167	84	116	107
4	10	10,000,00	12,376,000	172	192	182	91	113	104
5	20	10,000,00	11,883,000	208	196	202	101	120	111
6	30	10,000,00	11,220,000	189	174	182	91	102	94
7	40	10,000,00	12,733,000	153	164	159	79	101	93
8	50	10,000,00	12,053,000	208	205	207	103	124	115
9 P	100	10,000,00	11,016,000	183	162	173	86	95	88
10 P	250	10,000,00	10,472,000	187	168	178	89	93	86
PC 1	1	10,000,00	13,311,000	153	162	158	79	105	97
PC 2	1.5	10,000,00	14,246,000	101	104	103	51	73	67

PC: positive control (ethylmethanesulfonate)

NC: negative control

S: solvent control (tetrahydrofuran)

CE: cloning efficiency

P: precipitation at the end of treatment

SD: standard deviation

Table 5: Experiment 1 - Mutagenicity, with metabolic activation

Dose group	Concentration µg/mL	CE in non-selective medium				CE in selective medium							Mutant frequency per 10^6cells
		Number of colonies per flask			CE [%]	Number of colonies per flask
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	179	166	173	86	16	11	11	16	14.0	14.0	2.4	40.6
NC2		147	160	154	77	11	7	9	11	10.2	10.2	2.0	33.2
S1	0	144	155	150	75	7	10	10	5	7.8	7.8	1.9	26.1
S2		162	154	158	79	7	7	4	4	5.6	5.6	1.4	17.7
4	10	165	160	163	81	5	7	5	7	6.0	6.0	0.9	18.5
5	20	171	137	154	77	10	13	5	16	11.4	11.4	3.7	37.0
6	30	160	180	170	85	8	15	9	13	10.8	10.8	2.7	31.8
7	40	160	172	166	83	21	15	17	10	15.6	15.6	3.6	47.0
8	50	149	170	160	80	14	11	17	12	13.0	13.0	2.3	40.8
9 P	100	153	160	157	78	6	5	11	6	7.8	7.8	2.6	24.9
PC1	1	156	137	147	73	146	153	159	142	151.6	151.6	6.7	517.4
PC2	1.5	152	131	142	71	205	192	208	179	192.8	192.8	12.1	681.3

Conclusions:

Under the experimental conditions reported, the test item 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell HPRT gene mutation assay according to guideline OECD 476, strains of Chinese hamster lung fibroblasts (V79) were exposed to 1 -Methyl-1 -phenylethyl peroxyneodecanoate (69.6% Peroxide) in tetrahydrofuran at concentrations of 0.5, 1, 2, 5, 10, 30, 50, 100 and 250 µg/mL (without metabolic activation) and (with metabolic activation) 1, 2, 5, 10, 20, 30, 40, 50, 100 and 250 µg/mL. The positive controls did induce the appropriate response in the corresponding strains. There was no evidence of induced mutant colonies over background. According to the clear negative results observed in the first experiment (with and without metabolic activation), the second experiment with long time exposure (without metabolic activation) was not considered necessary.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mammalian cell transformation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, 473 and 476, GLP).

In a bacterial reverse gene mutation test conducted according to OECD 471, the target substance did not induce mutagenicity. In a mammalian cell HPRT mutation assay conducted according to OECD 476, the target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was also tested negative for inducing mutagenic effects. Moreover, the target substance was tested negative in an in vitro cytogenicity study in human lymphocytes conducted according to OECD 473. Based on the lack of mutagenicity/cytogenicity in all in vitro assays, 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-genotoxic.

Justification for classification or non-classification

Based on the lack of mutagenicity/cytogenicity in all conducted in vitro assays with the target substance, 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.