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Genetic toxicity in vitro

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Negative in the complete in vitro testing battery according to OECD 471, 473 and 476

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13. March to 18. April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to EC Directive 92/69/EEC and Regulation EC/440/2008 guideline methods under GLP conditions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitrochondrial fraction (S-9)
Test concentrations with justification for top dose:
see also table in attached report on page -12- regarding "Materials"/Test article".
1.6 to 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene 5µl, sodium azide 2 µg, 9-aminoacridine 50 µg, 4-nitroquinoline-1-oxide 2µg, Benzo[a]pyrene 10µg or 2-aminoanthracene 5 µg
Details on test system and experimental conditions:
2,4,7,9-tetramethyldecane-4,7-diol was assayed for mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA), both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of test item at 1.6, 8, 40, 200, 1000, and 5000 mg/plate, plus negative (solvent) and positive controls. Evidence of toxicity was observed following the top dose treatment in the absence and presence of S-9. Strain TA100 treatments were repeated in Experiment 1 in order to investigate the reproducibility of a statistically significant increase in revertant numbers following Range-Finder Experiment treatments in the absence of S-9.

Experiment 1 treatment of all the test strains in the absence and presence of S-9 retained the same test doses as employed for the Range-Finder Experiment. Evidence of toxicity was observed following the top one or two dose treatments of all strains in the absence and presence of S-9, except strain WP2 uvrA in the absence of S-9 where no clear evidence of toxicity was observed.

Experiment 2 treatment of strain TA1535 in the presence of S-9 and strain WP2 uvrA in the absence and presence of S-9 were performed up to 5000 mg/plate. All other treatments in this experiment were performed up to 2000 mg/plate (an estimate of the lower limit of toxicity). In each case narrowed dose intervals were used, in order to more closely investigate those doses of test item approaching the limit dose level, and therefore considered most likely to provide evidence of any mutagenic activity. In addition, treatments in the presence of metabolic activation were further modified by the inclusion of a pre-incubation step, in this way the range of mutagenic chemicals that can be detected in this system was increased. Evidence of toxicity was observed following the top dose treatment of all strains in the absence of S-9 (except strain WP2 uvrA where no toxicity was observed), and following the top three dose treatments of all strains in the presence of S-9.

The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed.

Negative (solvent) and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

No dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any test item mutagenic activity.

Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: See details above in "Details in Test System and Conditions"

Conclusions:
Interpretation of results: negative

No dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any 2,4,7,9-tetramethyldecane-4,7-diol mutagenic activity.

It was concluded that 2,4,7,9-tetramethyldecane-4,7-diol did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) when tested under the conditions of this study. These conditions included treatments at concentrations either up to 5000 µg/plate, or the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

2,4,7,9-tetramethyldecane-4,7-diol was assayed for mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA), both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of the test item at 1.6, 8, 40, 200, 1000, and 5000 µg/plate, plus negative (solvent) and positive controls. Evidence of toxicity was observed following the top dose treatment in the absence and presence of S-9. Strain TA100 treatments were repeated in Experiment 1 in order to investigate the reproducibility of a statistically significant increase in revertant numbers following Range-Finder Experiment treatments in the absence of S-9.

Experiment 1 treatment of all the test strains in the absence and presence of S-9 retained the same test doses as employed for the Range-Finder Experiment. Evidence of toxicity was observed following the top one or two dose treatments of all strains in the absence and presence of S-9, except strain WP2 uvrA in the absence of S-9 where no clear evidence of toxicity was observed.

Experiment 2 treatment of strain TA1535 in the presence of S-9 and strain WP2 uvrA in the absence and presence of S-9 were performed up to 5000 µg/plate. All other treatments in this experiment were performed up to 2000 mg/plate (an estimate of the lower limit of toxicity). In each case narrowed dose intervals were used, in order to more closely investigate those doses of test item approaching the limit dose level, and therefore considered most likely to provide evidence of any mutagenic activity. In addition, treatments in the presence of metabolic activation were further modified by the inclusion of a pre-incubation step, in this way the range of mutagenic chemicals that can be detected in this system was increased. Evidence of toxicity was observed following the top dose treatment of all strains in the absence of S-9 (except strain WP2 uvrA where no toxicity was observed), and following the top three dose treatments of all strains in the presence of S-9.

The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed.

Negative (solvent) and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

No dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any test item mutagenic activity.

It was concluded that the test item did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) when tested under the conditions of this study. These conditions included treatments at concentrations either up to 5000 µg/plate, or the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9)

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:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to EC Directive 92/69/EEC and Regulation EC/440/2008 guideline methods under GLP conditions
Qualifier:
according to
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:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
27.02 to 1200 µg/mL
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide and Cyclophosphamide
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
2,4,7,9-tetramethyldecane-4,7-diol did not induce chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

Executive summary:

2,4,7,9-tetramethyldecane-4,7-diol was tested in an in vitro cytogenetics assay using duplicate cultures of Chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest dose level tested, 1200 µg/mL, was in excess of the solubility limit in culture medium.

In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test item on relative cell number. Chromosome aberrations were analyzed at three dose levels. The highest concentrations chosen for analysis, 314.6 µg/mL in the absence of S-9 and 364.9 µg/mL in the presence of S-9, induced approximately 59% and 56% reduction in cell number, respectively.

In Experiment 2, treatment in the absence of S-9 was continuous for 20 hours. Treatment in the presence of S-9 was for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17). Chromosome aberrations were analyzed at three dose levels. The highest concentrations chosen for analysis, 204.8 mg/mL in the absence of S-9 and 331.7 µg/mL in the presence of S-9, induced approximately 48% and 55% reduction in cell number respectively.

Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

Treatment of cultures with the test item in the absence and the presence of S-9 resulted in frequencies of cells with structural aberrations, which were generally similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in most test article treated cultures fell within historical negative control (normal) ranges for the majority of cultures analyzed. The exceptions to these were single cultures at the highest concentration (314.6 µg/mL) in the 3+17 hour, -S-9 (Experiment 1) and at the intermediate concentration (315.1 µg/mL in 3+17 hour, +S-9 (Experiment 2) treatment regimes. The structural aberration frequencies (excluding gaps) in these cultures were marginally outside the historical negative control (normal) ranges. However, in both cases, these increases were not observed in the replicate cultures, and all other test item treated cultures had structural aberration frequencies that were within the normal ranges. These observations were therefore considered of no biological significance.

No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control range, were observed in the majority of cultures treated with the test article in the absence of S-9. The only exception was a single culture analyzed from the highest concentration (204.8 mg/mL) in the 20+0 hour, -S-9 treatment regime. The numerical aberration frequency in this culture marginally exceeded the historical control range. However, this increase was not observed in the replicate culture nor in any other test article treated cultures in this treatment regime. This observation is therefore considered of no biological significance.

Treatment cultures with the test item in the presence of S-9 (in both experiments) resulted in frequencies of cells with numerical aberrations, which were generally similar to those observed in the concurrent solvent-treated cultures. The numerical aberration frequency of the majority of test article treated cultures fell within the historical control range. Exceptions to this were observed in Experiment 2, where both cultures analyzed at the intermediate concentration (315.1 µg/mL) and a single culture analyzed at the highest concentration (331.7 µg/mL), had numerical aberration frequencies that marginally exceeded the normal range. No such increases were observed in Experiment 1, where very similar concentrations were analyzed. As such, this observation was considered of little or no biological significance.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
- they are manufactured from similar precursors under similar conditions
- they share structural similarities with common functional groups: both substances start with an acetylene group as core structure, however, in the target substance this acetylene group has been fully hydrogenated during the manufacturing process; geminal hydroxyl groups on the alpha carbon atoms; distal to the geminal hydroxyl groups is an isobutyl group (methyl isopropyl)
- they have similar physicochemical properties and thus, show a similar toxicokinetic behaviour
- they are expected to undergo similar metabolism: oxidation of the terminal methyl groups to result in alcohol, aldehyde and finally the corresponding acid

Since the central acetylene group in the source substance is sterically shielded by the neighbouring functional groups, this structural difference does not lead to major differences in reactivity and/or toxicity, which is demonstrated based on the available toxicological data.

Therefore, read-across from the existing toxicity studies on the source substance is considered as an appropriate adaptation to the standard information requirements of REACH regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
8.83 to 2260 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: ethylmethanesulphonate in in the absence of of metabolic activation; cyclophosphamide in the presence of metabolic activation
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Executive summary:

Intoduction:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, >>The method used meets the requirements of the OECD (476) and the Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods:

Two independent experiments wre performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozyous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 -hour exposure groups both in the asence and presence of metabolic activation (2% S9). In experiment 2, the cells were treated with the test material at up to eight dose levels using a 4 -hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 280 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 210 µg/ml in the presence of metabolic activation.

Results:

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of the test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cel line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory perfomance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For the assessment of the genetic toxicity potential of 2,4,7,9-tetramethyldecane-4,7-diol a bacterial reverse mutation test and a chromosome aberration test is available for the target substance itself. Supporting data are available for the source substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, which was also negative in these tests.

A gene mutation study in mammalian cells is available for the source substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol. A detailed justification for read-across is attached to iuclid section 13.

 

Bacterial reverse mutation test

2,4,7,9-tetramethyldecane-4,7-diol was assayed for mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA), both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of the test item at 1.6, 8, 40, 200, 1000, and 5000 µg/plate, plus negative (solvent) and positive controls. Evidence of toxicity was observed following the top dose treatment in the absence and presence of S-9. Strain TA100 treatments were repeated in Experiment 1 in order to investigate the reproducibility of a statistically significant increase in revertant numbers following Range-Finder Experiment treatments in the absence of S-9. Experiment 1 treatment of all the test strains in the absence and presence of S-9 retained the same test doses as employed for the Range-Finder Experiment. Evidence of toxicity was observed following the top one or two dose treatments of all strains in the absence and presence of S-9, except strain WP2 uvrA in the absence of S-9 where no clear evidence of toxicity was observed. Experiment 2 treatment of strain TA1535 in the presence of S-9 and strain WP2 uvrA in the absence and presence of S-9 were performed up to 5000 µg/plate. All other treatments in this experiment were performed up to 2000 µg/plate (an estimate of the lower limit of toxicity). In each case narrowed dose intervals were used, in order to more closely investigate those doses of test item approaching the limit dose level, and therefore considered most likely to provide evidence of any mutagenic activity. In addition, treatments in the presence of metabolic activation were further modified by the inclusion of a pre-incubation step, in this way the range of mutagenic chemicals that can be detected in this system was increased. Evidence of toxicity was observed following the top dose treatment of all strains in the absence of S-9 (except strain WP2 uvrA where no toxicity was observed), and following the top three dose treatments of all strains in the presence of S-9. The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed. Negative (solvent) and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any test item mutagenic activity. It was concluded that 2,4,7,9-tetramethyldecane-4,7-diol did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) when tested under the conditions of this study. These conditions included treatments at concentrations either up to 5000 µg/plate, or the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9)

 

The source substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol was also not mutagenic in a plate incorporation procedure with S. typhimurium strains TA1535, TA1537, TA98, and TA100 and E. coli strain WP2 (uvrA) over a dose range of 10 to 5000 µg/plate in both the presence and absence of an Aroclor 1254-induced rat-liver metabolic activation system. The initial experiment used 5 percent (v/v) metabolic activation and the repeat experiment used 10 percent (v/v) metabolic activation. The 5000 ug/plate dose formulation appeared to be immiscible in the tubes and on the plates. Precipitate was also observed in the tubes and on the plates at a dose level of 5000 ug. However, after the 48-hour incubation period the precipitate was no longer seen on the plates. Cytotoxicity, indicated by thinning of the background bacterial lawn and the formation of pinpoint nonrevertant colonies, was observed for all strains generally at dose levels of 1000 and 5000 µg/plate. No 2,4,7,9-tetramethyl-5-decyne-4,7-diol treatments of the test strains resulted in an increase in revertant numbers that was considered indicative of any mutagenic activity. 

 

Chromosome aberration test

2,4,7,9-tetramethyldecane-4,7-diol was tested in an in vitro cytogenetics assay using duplicate cultures of Chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest dose level tested, 1200 µg/ml, was in excess of the solubility limit in culture medium. In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test item on relative cell number. Chromosome aberrations were analyzed at three dose levels. The highest concentrations chosen for analysis, 314.6 µg/ml in the absence of S-9 and 364.9 µg/ml in the presence of S-9, induced approximately 59% and 56% reduction in cell number, respectively. In Experiment 2, treatment in the absence of S-9 was continuous for 20 hours. Treatment in the presence of S-9 was for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17). Chromosome aberrations were analyzed at three dose levels. The highest concentrations chosen for analysis, 204.8 µg/ml in the absence of S-9 and 331.7 µg/ml in the presence of S-9, induced approximately 48% and 55% reduction in cell number respectively. Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Treatment of cultures with the test item in the absence and the presence of S-9 resulted in frequencies of cells with structural aberrations, which were generally similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in most test article treated cultures fell within historical negative control (normal) ranges for the majority of cultures analyzed. The exceptions to these were single cultures at the highest concentration (314.6 µg/ml) in the 3+17 hour, -S-9 (Experiment 1) and at the intermediate concentration (315.1 µg/ml l in 3+17 hour, +S-9 (Experiment 2) treatment regimes. The structural aberration frequencies (excluding gaps) in these cultures were marginally outside the historical negative control (normal) ranges. However, in both cases, these increases were not observed in the replicate cultures, and all other test item treated cultures had structural aberration frequencies that were within the normal ranges. These observations were therefore considered of no biological significance. No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control range, were observed in the majority of cultures treated with the test article in the absence of S-9. The only exception was a single culture analyzed from the highest concentration (204.8 µg/ml) in the 20+0 hour, -S-9 treatment regime. The numerical aberration frequency in this culture marginally exceeded the historical control range. However, this increase was not observed in the replicate culture nor in any other test article treated cultures in this treatment regime. This observation is therefore considered of no biological significance. Treatment cultures with 2,4,7,9-tetramethyldecane-4,7-diol in the presence of S-9 (in both experiments) resulted in frequencies of cells with numerical aberrations, which were generally similar to those observed in the concurrent solvent-treated cultures. The numerical aberration frequency of the majority of test article treated cultures fell within the historical control range. Exceptions to this were observed in Experiment 2, where both cultures analyzed at the intermediate concentration (315.1 µg/ml) and a single culture analyzed at the highest concentration (331.7 µg/ml), had numerical aberration frequencies that marginally exceeded the normal range. No such increases were observed in Experiment 1, where very similar concentrations were analyzed. As such, this observation was considered of little or no biological significance.

 

The source substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol was also negative for chromosomal aberrations at concentrations of 19.5, 78.3, 312.5, 1250, and 3500 µg/ml in the presence or absence of metabolic activation (MA). A high dose of 3500 ug/ul was used based on the limit of solubility of the test article in dimethylsulfoxide (DMSO). Cells were exposed to the test article in the absence of MA for 3 and 21 hr and in the presence of MA for 3 hr. At 21 hr after exposure initiation, cells were harvested and evaluated. All of the cultures from the top two dose levels exhibited a significant decrease in confluency (0 to 25 percent) and therefore were not harvested. For cultures exposed to the test article for 3 hr in the presence or absence of MA, no significant reduction in mitotic index was observed at dose levels of 312.5 µg/ml and below. Cultures exposed for 21 hr to the test article at 312.5 µg/ml showed a significant reduction in mitotic index. Based on the cytotoxicity results, the initial chromosome aberration study was performed by exposing CHO cells for 3 hr to 2,4,7,9-tetramethyl-5-decyne-4,7-diol at concentrations of 19.5, 39.1, 78.1, 156.3, and 312.5 µg/ml in both the absence and presence of MA. At 21 hr after initiation of exposure, cells were harvested and evaluated. Cytotoxicity was evident in cultures exposed to 312.5 µg/ml under both MA conditions, so the cells were not harvested for evaluation. In cultures at the three dose levels scored in both MA conditions (39.1, 78.1, and 156.3 µg/ml), there was no statistically significant increase in the number of cells with structural aberrations and the mitotic index was comparable to that for the control. No increases in polyploidy were observed in the presence or absence of MA. The dose levels for the replicate experiment were based on the results of the cytotoxicity experiment (-MA) and the initial experiment (+MA) which indicated cytotoxicity and a significant reduction in confluency at the 312.5 µg/ml dose level. The replicate experiment was performed by exposing CHO cells for 21 hr to the test article at concentrations of 9.8, 19.5, 39.1, 78.1, and 156.3 µg/ml in the absence of MA and for 3 hr at concentrations of 19.5, 39.1, 78.1, and 156.3 µg/ml in the presence of MA. At 21 hr after initiation of exposure, cells were harvested and evaluated. At the three dose levels scored in both MA conditions (39.1, 78.1, and 156.3 µg/ml), there was no statistically significant increase in the number of cells with structural aberrations and the mitotic index was comparable to that for the control. No increases in polyploidy were observed in the presence or absence of metabolic activation. 

 

Gene mutation study in mammalian cells

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material 2,4,7,9-Tetramethyl-5-decyne-4,7-diol on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and the Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 -hour exposure groups both in the absence and presence of metabolic activation (2% S9). In experiment 2, the cells were treated with the test material at up to eight dose levels using a 4 -hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 280 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 210 µg/ml in the presence of metabolic activation.

Results:

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of the test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

There are no data gaps for the endpoint genetic toxicity. Overall, 2,4,7,9-tetramethyldecane-4,7-diol is not mutagenic nor clastogenic.

Justification for classification or non-classification

The test material was considered to be non-mutagenic according to the test conditions as given in OCED guidelines 471, 473 and 476.