Decane-1,10-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All the three required in vitro genotoxicity studies were performed on 1,10 -decanediol. Negative results were obtained in the In vitro gene mutation study in bacteria (OECD 471), in the In vitro cytogenicity / micronucleus study (OECD 487) and in the In vitro gene mutation study in mammalian cells (OECD 490). Based on these results, 1,10 -decanediol is considered to be not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November - December 1995

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:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

n/a

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

30 - 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (all strains with S9)

Details on test system and experimental conditions:

Five dose levels of test item ranging up to a maximum of 5000 µg/plate in the absence and in the presence of metabolic activation were spaced at helf-log intervals.
In the test #1, the plate incorporation method was used. And in the test #2, the preincubation method was used.

Rationale for test conditions:

The following criteria must be met for the mutagenicity assay to be considered valid :
-in the solvent control, each tester strain culture must exhibit a characteristic mean number of spontaneous revertants
-to ensure that appropriate numbers of bacteria are plated, overnight culture titers must be in excess of 10^8 bacteria/ml
-the mean of each positive control must exhibit a significant increase on the number of revertants over the mean value of the respective vehicle control
-normally, at least four non-toxic dose levels are required to evaluate the assay data

Evaluation criteria:

For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reqroducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Excessive toxicity, incdicated by a reduced growth or total absence of the bacterial background lawn, was detectable with :
-TA100 at 5000 µg/plate (without S9, preincubation test)
-TA1535 at 5000 µg/plate (+/-S9, plate incorporation test) and at 3000 (without S9, preincubation test)
-TA1537 at 5000 µg/plate (+/-S9, plate incorporation test) and at 100, 3000 (without S9, preincubation test) and at 3000 (with S9, preincubation test)

All four bacterial strao,ns exhibited a positive mutagenic response with the positive controls test both with and without metabolic activation by S9 mix. Negative (sovent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable.
In both experiments, no indication of test compound induced mutagenicity was observed with either one of the four tester TA98, TA100, TA1535 and TA1537 with or without metabolic activation.
All criteria for a valid study were met as described.

Conclusions:

Decane-1.10-diol did not induce a mutagenic effect in S.typhimurium. It is therefore not considered to ba a bacterial mutagen.

Executive summary:

Decane-1,10 -diol was tested for its ability to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were treated with the test compound by the Ames test plate incorporation (test #1) as well as the preincubation method (test #2). Five dose levels covering the range between 30 and 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Aroclor 1254 induced rat liver S9 mix) were employed.

All four bacterial strains exhibited mutagenic responses to the appropriate positive control substances. Solvent controls also tested with each strain and the mean mumbers of spontaneous revertants were in an acceptable range.

Mutagenic activity of the test compound to one or more of the tester strains was not observed in either experiment with and without metabolic activation.

It is therefore concluded, taht decane-1,10 -diol is not a bacterial mutagen.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 September 2016 -19 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

Treatments without S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels selected for the treatments were as follows:
- 0.063, 0.125, 0.25, 0.5, 0.75, 1, 2 and 4 mM for the 3-hour treatment,
- 0.031, 0.063, 0.125, 0.25, 0.375, 0.5 and 1 mM for the 24-hour treatment.

Treatment with S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: selection based on available solubility data and according to recommendation of the OECD guideline No. 487.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary cytotoxicity test
Without S9 mix 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h recovery

Main cytogenetic experiment
Without S9 mix 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h recovery

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were dropped onto clean glass slides. The slides were air-dried before being stained for approximately 15 min in 5% Giemsa.

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per dose

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- micronuclei should be clearly surrounded by a nuclear membrane,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- non-refractility of the micronuclei,
- micronuclei should not be linked to- the main nucleus via nucleoplasmic bridges,
- micronuclei should be located within the cytoplasma of the cell,
- only mononucleated cells with a number of micronuclei = 5 should be scored to exclude apoptosis and nuclear fragmentation.

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

yes

Key result

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: none
- Effects of osmolality: none
- Evaporation from medium: not determined
- Precipitation: >= 2 mM
- Definition of acceptable cells for analysis:
Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995), according to the following criteria:
- micronuclei should be clearly surrounded by a nuclear membrane,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- non-refractility of the micronuclei,
- micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
- micronuclei should be located within the cytoplasma of the cell,
- only mononucleated cells with a number of micronuclei = 5 should be scored to exclude apoptosis and nuclear fragmentation.

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Using a test item solution at 174.29 mg/mL in the vehicle and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1742.9 µg/mL) was achievable. Thus, the dose-levels selected for the treatment of the preliminary test were 0.005, 0.014, 0.041, 0.123, 0.37, 1.11, 3.33 and 10 mM.
At the highest dose-level of 10 mM, the pH of the culture medium was approximately 7.1 (as for the vehicle control) and the osmolality was 434 mOsm/kg H2O (468 mOsm/kg for the vehicle control). Therefore, none of the tested dose-levels was considered to produce extreme culture conditions.

A precipitate was observed in the culture medium at the end of the treatment periods at dose-levels >= 3.33 mM.

Following the 3-hour treatment without S9 mix, a moderate to severe cytotoxicity was observed at dose levels >= 1.11 mM, as shown by a 42 to 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a slight to severe cytotoxicity was observed at dose-levels >= 0.37 mM, as shown by a 36 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a severe cytotoxicity was observed at the dose-level of 10 mM, as shown by a 100% decrease in the PD.

NUMBER OF CELLS WITH MICRONUCLEI
see attached document

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document
- Negative (solvent/vehicle) historical control data: see attached document

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: population doubling

Conclusions:

Under the experimental conditions of the study, the test item, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item, to induce an increase in the frequency of micronucleated cellsin the mouse lymphoma cell line L5178Y TK +/-.

 

Methods

 

After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide (DMSO), was tested in a single cytogenetic experiment, with a metabolic activation system prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254 (3 h treament + 24h recovery) and without metabolic activation (3h treatment + 24 h recovery, and 24h treatment + 0h recovery).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each condition of the cytogenetic experiment, micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Results

 

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

 

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

A precipitate was observed in the culture medium at the end of the 3-hour treatment period, at dose-levels superior or egal to 2 mM.

 

Treatments without S9 mix

With a treatment volume of 1% (v/v) in culture medium, the dose-levels selected for the treatments were as follows:

- 0.063, 0.125, 0.25, 0.5, 0.75, 1, 2 and 4 mM for the 3-hour treatment,

- 0.031, 0.063, 0.125, 0.25, 0.375, 0.5 and 1 mM for the 24-hour treatment.

Cytotoxicity

Following the 3-hour treatment, a slight to severe cytotoxicity was observed at dose-levels superior or egal to 0.5 mM, as shown by a 32 to 100% decrease in the PD.

Following the 24-hour treatment without S9 mix, a slight to moderate cytotoxicity was observed at dose-levels superior or egal to 0.25 mM, as shown by a 27 to 59% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for the micronucleus analysis were as follows:

- 0.125, 0.25 and 0.5 mM for the 3-hour treatment, the latter inducing a 32% decrease in the PD but higher dose-levels being considered as too cytotoxic,

- 0.125, 0.375 and 1 mM for the 24-hour treatment, the latter inducing a 59% decrease in the PD.

 

No statistically significant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed.

 

The recommended level of cytotoxicity was not reached after the 3-hour treatment. However, considering the narrow dose-levels spacing used in this experiment, the available results were considered to be suitable to allow a reliable interpretation.

In the absence of S9 mix, the results met the criteria of a negative response.

 

Treatment with S9 mix

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM.

 

Cytotoxicity

A slight to severe cytotoxicity was observed at dose-levels superior or egal to 1 mM, as shown by a 27 to 100% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for the micronucleus analysis were 0.5, 1 and 2 mM, the latter being the lowest dose-level showing precipitate in the culture medium at the end of the treatment period.

 

No increase in the frequency of micronucleated cells was noted after the 3-hour treatment experiment. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed. These results met the criteria of a negative response.

 

Conclusion

Under the experimental conditions of the study, the test item, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 September 2016 - 30 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines (i.e. decrease in Adj. RTG).

Experiments without S9 mix
The selected dose-levels were 0.156, 0.313, 0.625, 1.25, 2.5, 5, 7.5 and 10 mM.

Experiments with S9 mix
The selected dose-levels were as follows:
- 0.156, 0.313, 0.625, 1.25, 2.5, 5, 7.5 and 10 mM for the first experiment,
- 0.25, 0.5, 1, 2, 3, 4, 5 and 7.5 mM for the second experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: selection based on available solubility data and according to recommendation of the OECD guideline No. 490yes.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; Relative Total Growth and Relative Suspension Growth.

Evaluation criteria:

IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity up to dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there no culture showing an Adj. RTG value between 10 and 20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

yes

Key result

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: none
- Effects of osmolality: none
- Evaporation from medium: not determined
- Precipitation: >= 3 mM
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Using a test item solution at 174.29 mg/mL in the vehicle and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1742.9 µg/mL) was achievable.

Thus, the dose-levels selected for the treatment of the preliminary test were 0.005, 0.014, 0.041, 0.123, 0.37, 1.11, 3.33 and 10 mM.
At the highest dose-level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 426 mOsm/kg H2O (450 mOsm/kg for the vehicle control). Therefore, none of the tested dose-levels was considered to produce extreme culture conditions.

A precipitate was observed in the culture medium at the end of the treatment period at the dose-level of 10 mM.

Following the 3-hour treatment without S9 mix, a moderate to severe cytotoxicity was induced at dose levels = 3.33 mM, as shown by a 46 to 100% decrease in the Adj. RTG.
Following the 3-hour treatment with S9 mix, a severe cytotoxicity was induced at the dose-level of 10 mM, as shown by a 100% decrease in the Adj. RTG.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document
- Negative (solvent/vehicle) historical control data: see attached document

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

Conclusions:

Under the experimental conditions of this study, the test item, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells.

Methods

After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide (DMSO), was tested in two independent experiments, with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO2 humidified incubator.

 

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Results

The Cloning Efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

 

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines (i.e. decrease in Adj. RTG).

 

Experiments without S9 mix

The selected dose-levels were 0.156, 0.313, 0.625, 1.25, 2.5, 5, 7.5 and 10 mM.

A precipitate was observed in the culture medium at the end of the treatment period at dose-levels = 5 mM. This precipitate did not prevent any scoring.

Cytotoxicity

Following the 3-hour treatment, a severe cytotoxicity was induced at dose-levels = 5 mM, as shown by a 96 to 99% decrease in the Adj. RTG.

 

Mutagenicity

No noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression.

 

There was no culture showing an Adj. RTG value between 10 and 20%, however there was no evidence of mutagenicity in a series of data points between 100 and 25% and there was also a negative data point between 10 and 1% Adj. RTG. Thusthese results did not meet the criteria of a positive response.

Experiments with S9 mix

The selected dose-levels were as follows:

- 0.156, 0.313, 0.625, 1.25, 2.5, 5, 7.5 and 10 mM for the first experiment,

- 0.25, 0.5, 1, 2, 3, 4, 5 and 7.5 mM for the second experiment.

 

A precipitate was noted in the culture medium at the end of the treatment period, at dose-levels = 3 mM. This precipitate did not prevent any scoring.

 

Cytotoxicity

In the first experiment, a severe cytotoxicity was induced at dose-levels = 5 mM, as shown by a 100% decrease in the Adj. RTG.

In the second experiment, a moderate to severe cytotoxicity was induced at dose-levels = 4 mM, as shown by a 43 to 91% decrease in the Adj. RTG.

 

Mutagenicity

No noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, in either experiment, and no dose-response relationship was demonstrated by the linear regression.

These results did not meet the criteria of a positive response.

Conclusion

Under the experimental conditions of this study, the test item, 1,10-decanediol, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria (OECD 471, Erbert 1996):

Decane-1,10 -diol was tested for its ability to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were treated with the test compound by the Ames test plate incorporation (test #1) as well as the preincubation method (test #2). Five dose levels covering the range between 30 and 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Aroclor 1254 induced rat liver S9 mix) were employed.

All four bacterial strains exhibited mutagenic responses to the appropriate positive control substances. Solvent controls also tested with each strain and the mean mumbers of spontaneous revertants were in an acceptable range.

Mutagenic activity of the test compound to one or more of the tester strains was not observed in either experiment with and without metabolic activation.It is therefore concluded that decane-1,10-diol is not a bacterial mutagen.

In vitro cytogenicity / micronucleus study (OECD 487, Sire 2017a) :

The objective of this study was to evaluate the potential of the test item, to induce an increase in the frequency of micronucleated cells in the mouse lymphoma cell line L5178Y TK +/-.

 After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide (DMSO), was tested in a single cytogenetic experiment, with a metabolic activation system prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254 (3 h treament + 24h recovery) and without metabolic activation (3h treatment + 24 h recovery, and 24h treatment + 0h recovery).

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

 In the treatments without S9 mix, following the 3-hour treatment, a slight to severe cytotoxicity was observed at dose-levels superior or egal to 0.5 mM, as shown by a 32 to 100% decrease in the PD. Following the 24-hour treatment without S9 mix, a slight to moderate cytotoxicity was observed at dose-levels superior or egal to 0.25 mM, as shown by a 27 to 59% decrease in the PD. No statistically significant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed. The recommended level of cytotoxicity was not reached after the 3-hour treatment. However, considering the narrow dose-levels spacing used in this experiment, the available results were considered to be suitable to allow a reliable interpretation. In the absence of S9 mix, the results met the criteria of a negative response.

In the treatment with S9 mix, a slight to severe cytotoxicity was observed at dose-levels superior or egal to 1 mM, as shown by a 27 to 100% decrease in the PD. No increase in the frequency of micronucleated cells was noted after the 3-hour treatment experiment. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed. These results met the criteria of a negative response.

Under the experimental conditions of the study, the test item, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

In vitro gene mutation study in mammalian cells (OECD 490, Sire 2017b):

The objective of this study was to evaluate the potential of the test item, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells.

After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide (DMSO), was tested in two independent experiments, with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

In the experiments without S9 mix, following the 3-hour treatment, a severe cytotoxicity was induced at dose-levels = 5 mM, as shown by a 96 to 99% decrease in the Adj. RTG. No noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression. There was no culture showing an Adj. RTG value between 10 and 20%, however there was no evidence of mutagenicity in a series of data points between 100 and 25% and there was also a negative data point between 10 and 1% Adj. RTG. Thusthese results did not meet the criteria of a positive response.

With S9 mix, in the first experiment, a severe cytotoxicity was induced at dose-levels = 5 mM, as shown by a 100% decrease in the Adj. RTG. In the second experiment with S9 mix, a moderate to severe cytotoxicity was induced at dose-levels = 4 mM, as shown by a 43 to 91% decrease in the Adj. RTG. No noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, in either experiment, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria of a positive response.

Under the experimental conditions of this study, the test item, 1,10-decanediol, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Justification for classification or non-classification

Based on the weight of evidence, 1,10 -decanediol is considered to be not mutagenic. No classification is required according to the Regulation EC n°1272/2008.