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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames test (OECD471) - negative for mutagenicity to 5 strains of Salmonella typhimurium in the presence and absence of metabolising enzymes. Mammalian cell gene mutation assay at the HPRT locus (OECD476) - negative for mutagenicity to V79 cells in the presence and absence of metabolising enzymes. Micronucleus assay in human lymphocytes (OECD487) - negative for induction of micronuclei by the test item.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 July 2015 to 20 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with guideline OECD 487 and to GLP.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline No. 487 'In vitro Mammalian Cell Micronucleus Test' (2014)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
other: human lymphocytes
Details on mammalian cell type (if applicable):
HUMAN LYMPHOCYTES:
- Peripheral blood lymphocytes from healthy non-smoking donors (Experiment I: male 23-year old; Experiment II: male 21-year old). All donors had previously established low incidence of micronuclei.
- Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.

CULTURE CONDITIONS
- Type and identity of media: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) supplemented with 200 m MGlutaMAX™, penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- All incubations were done at 37°C with 5.5% CO2 in humidified air.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
11.5, 20.1, 35.2, 61.6, 107.8, 188.7, 330.3, 578.0, 1011.4 and 1770.0 ug/mL. The highest concentration was selected in accordance of the OECD guideline at the time which was 10 mM and based on the molecular weight and 98.46% purity. Cytotoxicity and phase separation were observed in the presence and absence of S9 at 578.0 ug/mL and above.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, final concentration in the culture medium 0.5%
- Justification for choice of solvent/vehicle: DMSO was chosen as a solvent due to its solubility properties and its relatively non-toxicity to the cell cultures
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Pre-incubation period: yes, cells were stimulated for 48 hours by addition of phytohemeagglutinine (PHA)
- Exposure duration: 4 hours (Experiment I), 20 hours (Experiment II)
- Expression time (cells in growth medium): 16 hours (Experiment I), 0 hours (Experiment II)
- Selection time (if incubation with a selection agent): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B (4 ug/mL)
STAIN (for cytogenetic assays): The cells were stained with Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: cytogenetic damage: at least 1000 binucleated cells per culture, cytotoxic effect: CBPI (cytokinesis-block proliferation index) determined in 500 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: CBPI (cytokinesis-block proliferation index)
Evaluation criteria:
EVALUATION
Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. Evaluation of micronuclei was performed according to Countryman and Heddle (1976). At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

INTERPRETATION
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered to be unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or grain or loss in the test system.
Statistics:
Statistical significance will be confirmed by using the Chi-squared test (ɑ <0.05) using the validated R Script CH12.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none observed
- Effects of osmolality: none observed

RANGE-FINDING/SCREENING STUDIES: yes, 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Table 1. Summary of results

Experiment

Preparation interval

Test item concentration (μg/mL)

Proliferation index (CBPI)

Cytostasis (%)*

Micronucleated cells (%)**

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

2.08

 

0.15

 

 

Positive control2

1.33

69.4

22.30S

 

 

61.6

1.96

10.7

0.40

 

 

107.8

1.93

13.4

0.55S

 

 

188.7

1.81

25.1

0.85S

 

 

330.3

n.e.

n.e.

n.e.

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.85

 

0.55

 

 

Positive control3

1.36

58.0

3.35S

 

 

35.2

1.64

24.6

0.25

 

 

61.6

1.62

27.0

0.20

 

 

107.8

1.42

50.4

0.45

 

 

188.7

1.21

75.2

n.d.

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

1.80

 

0.50

 

 

Positive control4

1.56

29.6

5.45S

 

 

107.8

1.73

7.8

0.80

 

 

188.7

1.77

3.4

0.25

 

 

330.3

1.64

19.3

0.45

 

 

578.0

n.e.

n.e.

n.e.

* For the positive control groups and 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

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

n.e. Not evaluable due to cell debris

n.d. Not determined due to strong cytotoxicity

1 DMSO 0.5 % (v/v) 2 MMC 1.0 μg/mL

3 Demecolcin 100.0 ng/mL

4 CPA 15.0 μg/mL

Individual results and raw data are provided as background information.

Conclusions:
Interpretation of results (migrated information):
negative

Decan-1,2-diol, is considered to be non-mutagenic in the in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentrations.
Executive summary:

The test item decan-1,2-diol, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix, in two independent experiments according to OECD 487 guideline.

The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes under the experimental conditions reported. Therefore, decan-1,2 -diol is considered to be non-mutagenic in the in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentrations.

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

Additional information

Additional information from genetic toxicity in vitro:

The test item decan-1,2-diol, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments according to OECD 487 guideline. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes under the experimental conditions reported. Therefore, decan-1,2 -diol is considered to be non-mutagenic in the in vitro micronucleus test.

Decan-1,2-diol did not show any evidence of genotoxicity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA 100 and TA 102, in the presence and absence of a metabolising system. Furthermore, the test item did not induce gene mutations at the HPRT locus in V79 cells, in the presence and absence of a metabolising system.

Therefore, the test item is considered to be non-mutagenic.


Justification for selection of genetic toxicity endpoint
Of the three key in vitro genetic toxicity studies that have been conducted with decan-1,2-diol, it is the cytogenicity study in human lymphocytes that is considered to be the most relevant for this endpoint principally as a result of the use of human cells for the assay and the fact that the endpoint is the most severe (chromosome damage/disruption). The other two studies examine the potential for a mutagenic response either in bacterial cells or in non-human mammalian cells.

Justification for classification or non-classification

Negative results were produced in an in vitro cytogenicity study in mammalian cells, in a gene mutation study in mammalian cells and also in a gene mutation study in bacterial cells. Therefore, no classification is required for decan-1,2 -diol for this endpoint.