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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an apparently well conducted OECD 471 GLP in vitro bacterial reverse mutation study dilauroyl peroxide was not mutagenic in Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98, TA 100, or TA 102 with or without metabolic activation (Molinier, 1996). In several other less well documented bacterial reverse mutation assays, dilauroyl peroxide was not mutagenic in the various S. typhimurium strains tested (Seifried, 2006; Willems, 1979; Yamaguchi, 1980).

In an apparently well conducted OECD 476 GLP in vitro mouse lymphoma study, dilauroyl peroxide did not induce mutations in the mouse lymphoma thymidine kinase locus assay (Wollny, 2010). In a less well documented study, dilauroyl peroxide was considered non-mutagenic when evaluated under a doubling criteria and inconclusive under a "harmonized" criteria.

In an apparently well conducted OECD 473 in vitro mammalian chromosome aberration assay dilauroyl peroxide did not induce chromosomal aberrations in cultured human lymphocytes (Haddouk, 2002).

Based on the above in vitro assays, dilauroyl peroxide is not considered mutagenic and does not induce structural chromosomal damage. Therefore an in vivo study is scientifically unjustified.

Short description of key information:

One key and several supporting bacterial mutation studies are available.  All studies report dilauroyl peroxide was not mutagenic.  One key and one supporting in vitro mammalian mutation studies, with mouse lymphoma L5178Y cells, are available. Dilauroyl peroxide was not mutagenic to mouse lymphoma cells.  In an in vitro mammalian chromosome aberration assay, dilauroyl peroxide did not induce chromosomal aberration in cultured human lymphocytes.  

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

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)
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
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 / 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 / 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
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
Positive controls:
yes
Remarks:
-S9
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: TA 1537
Positive controls:
yes
Remarks:
-S9
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: TA 98
Positive controls:
yes
Remarks:
-S9
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: TA 102
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
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
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.

See attachment

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Apparently well conducted GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 hours. Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.

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.
Metabolic activation:
with and without
Metabolic activation system:
rat liver post-mitochondrial fraction
Test concentrations with justification for top dose:
6.39, 12.78, 25.57, 51.14, 102.3, 204.5, 409.1 and 818.2 μg/mL, for the first experiment with and without S9 mix,
25.57, 51.14, 102.3, 204.5, 409.1 and 818.2 μg/mL, for the second experiment with and without S9 mix.
Vehicle / solvent:
The vehicle was Tetrahydrofuran (THF), batch No. E 001261B (Carlo Erba, 27106 Val de Reuil, France).
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: (test without S9 mix 3 ug/ml or 0.2 ug/ml)
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: test with S9 (50 ug/ml or 25 ug/ml)
Details on test system and experimental conditions:
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.

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 hours 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. As this experiment
gave negative results, a 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.

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).

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 analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP.
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.
Key result
Species / strain:
lymphocytes: human
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 test item was soluble in the vehicle (THF), the limit of solubility being approximately 300 mg/mL. In the culture medium, the dose-level of 818.2
μg/mL (corresponding to 2.05 mM) showed a strong precipitate. At this dose-level, the pH was about 7.4 (7.4 for the vehicle control) and the
osmolality equal to 307 mOsm/kg H2O (312 for the vehicle control).

A slight to moderate precipitate was observed at the end of the treatment period, generally at dose-levels ≥ 102.3 μg/mL.

Experiments without S9 mix:
Cytotoxicity: No noteworthy toxicity was induced, in both experiments.
Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
204.5, 409.1 and 818.2 μg/mL, for the 3-hour and the 20-hour treatments,
818.2 μg/mL, for the 44-hour treatment.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44 hours treatments.

Experiments with S9 mix:
Cytotoxicity: No noteworthy toxicity was induced, in both experiments.
Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
204.5, 409.1 and 818.2 μg/mL, for the 20-hour harvest time in both experiments,
818.2 μg/mL, for the 44-hour harvest time.
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.
Conclusions:
Interpretation of results (migrated information):
negative

Under experimental conditions, the test item LUPEROX LP (Dilauroyl Peroxide, batch No. 9732-110-111-029, purity 99.3%) did not induce chromosome aberrations in cultured human lymphocytes.
Executive summary:

The study was conducted to evaluate the potential of Luperox LP to induce chromosome aberrations in cultured human lymphocytes in accordance with OECD 473. The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254. 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.

No noteworthy toxicity was induced, in both experiments. In the first experiment no significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44 hours treatments. In the second experiment 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.

Under experimental conditions, the test item LUPEROX LP (Dilauroyl Peroxide, batch No. 9732-110-111-029, purity 99.3%) did not induce chromosome aberrations in cultured human lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 476
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
Principles of method if other than guideline:
first experiment: 4 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I
without S9 mix: 8.1, 16.3, 32.5, 65.0, 130.0 (p), 260.0 (p) µg/mL
with S9 mix: 8.1, 16.3, 32.5, 65.0, 130.0, 260.0 µg/mL
Experiment II
without S9 mix: 8.1, 16.3, 32.5, 65.0 (p), 130.0 (p), 260.0 (p) µg/mL
with S9 mix: 32.5, 65.5 (p), 130.0 (p), 260.0 (p), 520.0 (p), 1040.0 (p) µg/mL
p = precipitation
Following the expression phase of 48 hours the cultures at 8.1 µg/mL in experiment I with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. In experiment II the cultures of the maximum concentrations of 260 µg/mL without and 1040 µg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
Species / strain:
mouse lymphoma L5178Y cells
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Water solubility: --
- Precipitation:
In the main experiments precipitation was determined at the following evaluated concentrations:
Experiment I, 4 hours treatment, without S9 mix: 130.0; 260.0 µg/mL
Experiment II, 24 hours treatment, without S9 mix: 65.0; 130.0 µ/mL
Experiment II, 4 hours treatment, with S9 mix: 65.5, 130.0, 260.0, 520.0 µ/mL


- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 32.8 µg/mL and 4200 µg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the purity (99.8 %) and the molecular weight (398.6 g/mol) of the test item.
Relevant toxic effects were observed at 2100 µg/mL and above in the absence (4 h treatment) and at 4200 µg/mL in the presence of metabolic activation (4 h treatment). Following continuous treatment (24 hours) toxicity was not observed up to the maximum concentration.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed at 131.3 µg/mL and above after both treatment periods with and without metabolic activation.
Both pH value and osmolarity were determined in the pre-experiment at the maximum concentration of the test item and in the solvent control without metabolic activation. The osmolarity at 4200 µg/mL (295 mOsm) remained at the physiological value of around 300 mOsm.
Based on the occurrence of toxicity and precipitation of the test item in the pre-experiment the dose range of the main experiments was chosen. The individual concentrations were spaced by a factor of 2.0.


COMPARISON WITH HISTORICAL CONTROL DATA: complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: none
Remarks on result:
other: strain/cell type: in vitro gene mutation assay with L5178Y cells
Remarks:
Migrated from field 'Test system'.
Summary Table
      relative mutant   relative mutant  
  conc. µg S9 total colonies/   total colonies/  
  per mL mix growth 106cells threshold growth 106cells threshold
Column 1 2 3 4 5 6 7 8
Experiment I / 4 h treatment   culture I culture II
Solv. control with medium - 100.0 100 226 100.0 148 274
Pos. control with MMS  19.5 -  25.4 340 226  15.4 541 274
Test item   8.1 - culture was not continued# culture was not continued#
Test item  16.3 - 108.3  98 226  54.2 218 274
Test item  32.5 - 139.8  88 226  55.4 206 274
Test item  65.0 -  90.6 107 226  65.3 204 274
Test item 130.0 (p) - 106.7  95 226  82.4 209 274
Test item 260.0 (p) - 129.7  86 226  59.0 186 274
       
Solv. control with medium + 100.0  82 208 100.0 185 311
Pos. control with CPA   3.0 +  43.5 149 208  57.2 290 311
Pos. control with CPA   4.5  +   25.1 341 208  28.8 496 311
Test item   8.1  +  culture was not continued# culture was not continued#
Test item  16.3  +   98.8 116 208 106.1 177 311
Test item  32.5  +   70.0 138 208  92.0 165 311
Test item  65.0  +   76.2 137 208  81.1 161 311
Test item  130.0  +   72.4 110 208  96.1 187 311
Test item  260.0  +   64.1 124 208  85.8 173 311
Experiment II / 24 h treatment   culture I culture II
Solv. control with medium - 100.0 113 239 100.0 145 271
Pos. control with MMS  13.0 -  14.5 475 239  21.2 406 271
Test item   8.1 - 100.8 126 239  45.1 168 271
Test item  16.3 -  76.3 219 239  78.5 186 271
Test item  32.5 -  48.6 173 239  63.5 236 271
Test item 65.5 (p) -  42.5 166 239  73.9 172 271
Test item 130.0 (p) -  41.6 170 239  88.2 142 271
Test item 260.0 (p) - culture was not continued## culture was not continued##
Experiment II / 4 h treatment   culture I culture II
Solv. control with water + 100.0 130 256 100.0 180 306
Pos. control with CPA   3.0 +  93.8 174 256  55.9 258 306
Pos. control with CPA   4.5 +  56.4 351 256  47.5 337 306
Test item  32.5 +  69.4 201 256 139.9 123 306
Test item 65.5 (p) + 109.9 129 256  68.6 271 306
Test item 130.0 (p) +  57.6 217 256  54.9 215 306
Test item 260.0 (p) +  87.7  91 256  64.4 206 306
Test item 520.0 (p) +  73.7 153 256  64.7 273 306
Test item 1040.0 (p) + culture was not continued## culture was not continued##

Threshold = number of mutant colonies per 106cells of each solvent control plus 126

#    culture was not continued since a minimum of four concentrations is required by the guidelines

##   culture was not continued due to heavy

p    precipitation visible to the unaided eye

 

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential ofDilauroyl Peroxide (CAS 105-74-8) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

This study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations:

Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998,
adopted, Guideline No. 476 “In vitro Mammalian Cell Gene Mutation Test”.

Commission Regulation (EC) No. 440/2008, B17: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated.

The assay was performed in two independent experiments, using two parallel cultures each.The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 4 h with and 24 h without metabolic activation. The maximum tested concentration in the pre-experiment was equal to about 10 mM. The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium.

Both main experiments were evaluated at the following concentrations:

Experiment I

without S9 mix: 16.3; 32.5; 65.0; 130; and 260 µg/mL
with S9 mix:      16.3; 32.5; 65.0; 130; and 260 µg/mL

Experiment II

without S9 mix: 8.1; 16.3; 32.5; 65.0; and 130 µg/mL
with S9 mix:      32.5; 65.5; 130; 260; and 520 µg/mL

No relevant toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

The isolated minor reduction of the relative total growth to 48.6, 42.5, and 41.6 % in the first culture of the second experiment without S9 mix was not considered a real cytotoxic effect since no comparable reduction was observed in the parallel culture performed under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. The threshold of 126 plus each solvent control count was not exceeded in any of the experimental parts.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In this study the range of the solvent controls was from 82 up to 185 mutant colonies per 106cells; the range of the groups treated with the test item was from 86 up to 273 mutant colonies per 106cells.The highest solvent control values (185 and 180 colonies per 106cells) exceeded the recommended acceptability data of 50 – 170 x 106control range. The data are acceptable however, since the spontaneous mutation frequency remained within the acceptable limits in the parallel cultures.The cloning efficiency exceeded the upper limit in the first culture of the first and second experiment without metabolic activation. Again, the cloning efficiency of the parallel culture remained in the acceptable range.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No study required as all in vitro results are negative.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In vitro mutation and genotoxicity studies demonstrate that dilauroyl peroxide does not meet the criteria for classification.