Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative for mutagenicity in vitro in the Ames test, a Mouse Lymphoma Assay and in vitro micronucleus. Therefore no further in vivo cytogenicity and/or mutagenicity testing is required.

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: see 'Remark'
Remarks:
With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have. No independent repeat was performed to confirm the negative result.
Qualifier:
no guideline available
Principles of method if other than guideline:
The procedures used in this mutagenic assay are described in detail by Ames et al (1975).
GLP compliance:
no
Remarks:
pre-GLP
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 0.012, 0.04, 0.11, 0.33, 1.0 mg test product per 0.1 ml acetone per plate
0, 12, 40, 110, 330, 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: None
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: colonies (revertants which are histidine-independent) are counted, and the background lawn of bacterial growth examined.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
No data
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:
cytotoxicity
Remarks:
With strain TA 1538 the backgound lawn of bacterial growth, was slightly less dense at 1mg per plate than in the concomitant control plates.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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
Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that the test substance did not reveal mutagenic activity in the Salmonella/microsome mutagenicity test.
Executive summary:

The mutagnic activity. Of the test subsatnce was examined in the Salmonella/micosome mutagenicity test, using a set of five histidine

requiring mutants of S. typhimurium (TA 1535, TA 1537, TA1538, TA 98 and TA 100) and liver homogenate of Aroclor-induced rats.

Incorporation of the test product with the bacteria up to non-inhibitory levels (i.e. 1.0 mg per plate) did not increase the numbers of his revertants in any of the five tester strains either in the presence or in the absence of the liver microsome activation system.

It was concluded that the present results did not reveal any mutagenic activity of the test sample in the Salmonella/microsome mutagenicity test.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-19 to 2012-10-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD Guideline 487 and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
OECD Guideline for the Testing of Chemicals, adopted July 22, 2010, Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test”.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 0.2, 0.3, 0.7, 1.4, 2.7, 5.5, 10.9, 21.9, 43.8, 87.5, 175.0, 350.0, 700.0, 1400.0 µg/mL
Experiment II: 0.7, 1.4, 2.7, 5.5, 10.9, 21.9, 43.8 µg/mL

Without metabolic activation:
Experiment I: 0.2, 0.3, 0.7, 1.4, 2.7, 5.5, 10.9, 21.9, 43.8, 87.5, 175.0, 350.0, 700.0, 1400.0 µg/mL
Experiment II: 0.7, 1.4, 2.7, 5.5, 10.9, 21.9, 43.8, 87.5, 175.0, 350.0, 700.0, 1400.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 487
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: demecolcin
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 20 hours without S9 mix. The chromosomes were prepared 40 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 20 hours (- S9 mix)
- Recovery after 4 hours treatment: 16 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours


CYTOKINESIS BLOCK (cytogenetic assays): Cytochalasin B 20 hours
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF BINUCLEATED CELLS EVALUATED: 1000 per culture


DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)




Evaluation criteria:
Evaluation of the slides will be performed using NIKON microscopes with 40 x objectives. The micronuclei will be counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells will be reported as % micronucleated cells. To describe a cytotoxic effect the CBPI is determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CBPI=(MONCx1)+(BINCx2)+(MUNCx3)/n

CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BINC Binucleate cells
MUNC Multinucleate cells

Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]

T Test item
C Solvent control
Statistics:
Statistical significance can be confirmed by means of the Chi square test.
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Dihexadecyl peroxodicarbonate (CAS 26322-14-5), dissolved in THF, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 1400.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 10.9 µg/mL and above in the absence of S9 mix and at 5.5 µg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II at 5.5 µg/mL and above in the absence and presence of S9 mix at the end of treatment. No relevant influence on osmolarity or pH value was observed.
In this study, at both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity could be observed.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.30 - 1.20 % micronucleated cells) were close to the range of the solvent control values (0.40 - 0.95 % micronucleated cells) and within the range of the laboratory historical control data.
In both experiments, either Demecolcin (100.0 ng/mL), MMC (2.0 µg/mL) or CPA (7.5 or 12.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. Demecolcin showed increased micronucleus rates in mono- and binucleate cells.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Summary of results of thein vitro micronucleus test in human lymphocytes with
Dihexadecyl peroxodicarbonate (CAS 26322-14-5)

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Negative control

2.03

 

0.40

 

 

Solvent control1

1.98

 

0.95

 

 

Positive control2

1.71

31.1

10.70S

 

 

2.7

1.95

3.4

0.70

 

 

5.5

1.95

4.0

0.75

 

 

10.9P

1.94

4.3

1.00

Exposure period 20 hrs without S9 mix

II

40 hrs

Negative control

1.72

 

0.80/0.55***

 

 

Solvent control1

1.67

 

0.40

 

 

Positive control3

1.50

29.8

2.10S/2.60S***

 

 

1.4

1.66

1.2

0.45

 

 

2.7

1.69

n.c.

0.55

 

 

5.5P

1.71

n.c.

0.30

*    For the positive control groups, the relative values are related to the negative controls;
for the test item treatment groups the values are related to the solvent controls

**  The number of micronucleated cells was determined in a sample of 2000 binucleated cells

*** The number of micronucleated cells was determined in a sample of 2000 mononucleated cells

P     Precipitation occurred at the end of treatment

S     The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1     THF                0.5 % (v/v)
2
         MMC              2.0 µg/mL
3
         Demecolcin  100.0 ng/mL


Summary of results of thein vitro micronucleus test in human lymphocytes with Dihexadecyl peroxodicarbonate (CAS 26322-14-5) (continued)

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs with S9 mix

I

40 hrs

Negative control

2.07

 

0.45

 

 

Solvent control1

2.08

 

0.70

 

 

Positive control2

1.83

22.7

 2.45S

 

 

1.4

2.04

4.3

0.45

 

 

2.7

2.09

n.c.

1.00

 

 

5.5P

2.02

6.0

0.65

II

40 hrs

Negative control

1.87

 

1.05

 

 

Solvent control1

1.85

 

0.80

 

 

Positive control3

1.65

25.1

 2.95S

 

 

1.4

1.78

8.1

1.10

 

 

2.7

1.81

4.4

1.20

 

 

5.5P

1.86

n.c.

0.60

*    For the positive control groups, the relative values are related to the negative controls;
for the test item treatment groups the values are related to the solvent controls

**  The number of micronucleated cells was determined in a sample of 2000 binucleated cells

P     Precipitation occurred at the end of treatment

S     The number of micronucleated cells is statistically significantly higher than corresponding control values

1     THF    0.5 % (v/v)
2
         CPA    7.5 µg/mL

3         CPA  12.5 µg/mL

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Dihexadecyl peroxodicarbonate (CAS 26322-14-5) is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.
Executive summary:

The test item Dihexadecyl peroxodicarbonate (CAS 26322-14-5), dissolved in THF, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

Without S9-Mix

With S9-Mix

 

Exp. I

Exp. II

Exp. I and II

Exposure period

 4 hrs

20 hrs

 4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Preparation interval

40 hrs

40 hrs

40 hrs

Total culture period

88 hrs

88 hrs

88 hrs

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

The highest applied concentration in this study (1400.0 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

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: see 'Remark'
Remarks:
according to OECD 476 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation
GLP compliance:
yes (incl. certificate)
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: 10.0; 21.9; 43.8; 87.5; 175.0 (p); and 350.0 (p) µg/mL
with S9 mix: 10.0; 21.9; 43.8; 87.5; 175.0 (p); and 350.0 (p) µg/mL
Experiment II:
without S9 mix: 10.0; 21.9; 43.8 (p); 87.5 (p); 175.0 (p); and 350.0 (p) µg/mL
with S9 mix: 10.0; 21.9; 43.8; 87.5 (p); 175.0 (p); and 350.0 (p) µg/mL
Following the expression phase of 48 hours the cultures (printed in bold letters) at the lowest concentration of 10.9 µg/mL with and without metabolic activation in experiment I and II were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- 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:
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 (pH 7.48 in the solvent control versus pH 7.52 at 1400 µg test item/mL)
- Effects of osmolality: Not increased (341 in the solvent control versus 329 at 1400 test item/mL)
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation:
Main experiments:
Precipitation was noted at 175 and 350 µg/mL following 4 hours treatment with and without metabolic activation in both experiments. At the end of the 24h treatment period precipitation occurred at 43.8 µg/mL and above in the second experiment without metabolic activation.

- 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 10.9 µg/mL and 1400 µg/mL were used with respect to the current guidelines.
Toxic effects leading to RSG values below 50% were solely observed at 350 µg/mL and above in the absence of metabolic activation following 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of the treatment period (4 hours) before the test item was removed. Precipitation was observed at 175.0 µg/mL and above after 4 hours treatment with and without metabolic activation, and at 87.5 µg/mL and above following 24 hours treatment.
Both, pH value and osmolarity was determined in the pre-experiment at the highest concentration of the test item and in the solvent control without metabolic activation. There was no relevant shift of both parameters.
The dose range of the main experiments was set according to the solubility data of the test item. In both main experiments the individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity and solubility the main experiments were started with more than four concentrations.


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 THF - 100.0 119 245 100.0  90 216
Pos. control with MMS  19.5 -  90.3 440 245  53.1 292 216
Test item  10.9 - culture was not continued# culture was not continued#
Test item  21.9 -  92.4 137 245 132.0 105 216
Test item  43.8 - 111.5  87 245 108.8  90 216
Test item  87.5 - 124.7  94 245 121.8  86 216
Test item 175.0 (p) - 130.5 144 245 103.9  82 216
Test item 350.0 (p) -  73.2  97 245 113.1  76 216
       
Solv. control with THF + 100.0 102 228 100.0 106 232
Pos. control with CPA   3.0 +  58.9 365 228  35.9 374 232
Pos. control with CPA   4.5  +   28.9 609 228  15.7 708 232
Test item  10.9  +  culture was not continued# culture was not continued#
Test item  21.9  +  125.1 105 228 102.1  69 232
Test item  43.8  +  120.7 109 228  89.0 107 232
Test item  87.5  +  118.3 122 228  75.7  89 232
Test item 175.0 (p)  +   91.1  80 228  81.7  92 232
Test item 350.0 (p)  +  103.2  99 228  52.2 130 232
Experiment II / 24 h treatment   culture I culture II
Solv. control with THF - 100.0 107 233 100.0  99 225
Pos. control with MMS  13.0 -  24.4 370 233  41.0 409 225
Test item  10.9 - culture was not continued# culture was not continued#
Test item  21.9 -  87.7  49 233  96.2 106 225
Test item 43.8 (p) -  78.0  50 233 158.4  76 225
Test item 87.5 (p) -  51.7  67 233 149.3 146 225
Test item 175.0 (p) -  73.1  84 233 168.7  56 225
Test item 350.0 (p) -  69.6  64 233 105.7 111 225
Experiment II / 4 h treatment   culture I culture II
Solv. control with THF + 100.0 109 235 100.0  99 225
Pos. control with CPA   3.0 +  69.6 241 235  34.3 263 225
Pos. control with CPA   4.5 +  53.4 294 235  36.1 507 225
Test item  10.9 + culture was not continued# culture was not continued#
Test item  21.9 + 109.8 100 235  93.3  84 225
Test item  43.8 +  87.7 134 235  80.9 104 225
Test item 87.5 (p) + 105.7  95 235  71.2 113 225
Test item 175.0 (p) + 108.9  87 235  70.6  98 225
Test item 350.0 (p) +  94.8  78 235  76.5  90 225

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

#   culture was not continued since a minimum of only four analysable concentrations is required
p
   precipitation at the end of treatment visible to the naked eye

 

Conclusions:
Interpretation of results (migrated information):
negative

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 of Dihexadecyl peroxodicarbonate (CAS 26322-14-5) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

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 hours. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations with and without metabolic activation:

Experiment I

without S9 mix:                              21.9; 43.8; 87.5; 175.0; 350.0 µg/mL
with S9 mix:                                   21.9; 43.8; 87.5; 175.0; 350.0 µg/mL

Experiment II

without S9 mix:                              21.9; 43.8; 87.5; 175.0; 350.0 µg/mL
with S9 mix:                                   21.9; 43.8; 87.5; 175.0; 350.0 µg/mL

Precipitation of the test item at the end of treatment was evaluated by the naked eye. Precipitation was noted at 175 and 350 µg/mL following 4 hours treatment with and without metabolic activation in both experiments. At the end of the 24h treatment period precipitation occurred at 43.8 µg/mL and above in the second experiment without metabolic activation.

No relevant cytotoxic 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.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 90 up to 119 mutant colonies per 106cells; the range of the groups treated with the test item was from 49 up to 146 mutant colonies per 106cells.

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 with at least one of the concentrations.

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.

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

Genetic toxicity in vivo

Description of key information

The substance is negative for mutagenicity in vitro in the Ames test, a Mouse Lymphoma Assay and in vitro micronucleus. Therefore no further in vivo cytogenicity and/or mutagenicity testing is required.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
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 selection of genetic toxicity endpoint

Ames + MLA+ Micronucleus, K1: The study was performed according to OECD guidelines and GLP.

Short description of key information:

The substance is negative for mutagenicity in vitro in the Ames test, a Mouse Lymphoma Assay and in vitro micronucleus. Therefore no further in vivo cytogenicity and/or mutagenicity testing is required.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Available data shows a negative outcome for genotoxicity based on these results, the data is conclusive but not sufficient for classification.