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Genetic toxicity in vitro

Description of key information

The genotoxic potential of hexylene glycol has been assessed in four GLP in vitro studies, including a bacterial reverse mutation assay (Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and Escherichia coli WP2 uvr A pKM 101; equivalent to OECD test guideline 471), a mammalian gene mutation assay (Mouse lymphoma assay according to OECD test guideline 476), a yeast gene mutation assay (Saccharomyces cerevisiae; equivalent to OECD test guideline 480) and a mammalian chromosome aberration test (Chinese hamster ovary cells; equivalent to OECD test guideline 473). In all four studies negative results were reported in the presence and absence of metabolic activation.

Gene mutation assays

In a GLP bacterial reverse mutation assays equivalent to OECD test guideline 471 (Meyeret al.,1985; Brookset al.,1988), hexylene glycol (HG) was tested at doses of 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, or 4000 µg/plate inSalmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and inEscherichia coliWP2 uvr A pKM 101. Incubations at each concentration were done in triplicate and the experiment was conducted twice, both times in the presence and absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9). Sterile distilled water was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed and no increase in the reverse mutation rate was observed at any HG concentration in any of the tester strains in the presence or absence of metabolic activation. Incubation with positive control substances in the presence of metabolic activation resulted in anticipated increases in the reverse mutation rate but did not always do so in the absence of metabolic activation. This study is therefore considered reliable with restrictions.
The potential of HG to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according to the international guidelines OECD No. 476 and Good Laboratory Practice (Simar, 2010). After a preliminary toxicity test, HG was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 5 x 10e6 (3-hour treatment) or 1.25 x 10e6 (24-hour treatment) cells/mL in 10 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 5%), at 37°C. Cytotoxicity was measured by assessment of plating efficiency, Relative Survival Growth (RSG), and Relative Total Growth (RTG), after treatment (T0) and 48 hours after treatment (PE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. HG was dissolved in water and the positive controls were methylmethane sulfonate (without S9 mix) and Cyclophosphamide (with S9 mix). In the culture medium, the concentration of 10 mM was freely soluble. At this dose-level, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The selected concentrations were 10, 5, 2.5, 1.25 and 0.63 mM for both experiments, with and without S9 mix. Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no toxicity was induced at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the Adj. RTG. Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no noteworthy increase in the mutation frequency, in comparison to the vehicle control was noted. HG did not show any mutagenic activity in the mouse lymphoma assay.
In a GLP yeast gene mutation assay equivalent to OECD test guideline 480 (Meyeret al.,1985; Brookset al.,1988), HG was tested at doses of 0, 0.01, 0.1, 0.5, 1.0, or 5.0 mg/mL inSaccharomyces cerevisiae. Incubations at each concentration were done at least in triplicate and the experiment was conducted twice, both times in the presence and absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9). Sterile distilled water was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed and no increase in the rate of mitotic gene conversion was observed at any HG concentration in the presence or absence of metabolic activation. Incubation with positive control substances in the presence (cyclophosphamide) or absence (4-Nitroquinoline-N-oxide) of metabolic activation resulted in anticipated increases in the rate of mitotic gene conversion. As the post-treatment period was 3 days as opposed to the recommended 4 to 7 days, this study is considered reliable with restrictions.


Chromosomal aberrations assay

In a GLP mammalian chromosome aberration test equivalent to OECD test guideline 473 (Meyeret al.,1985; Brookset al.,1988), HG was tested at doses of 0, 1250, 2500, or 5000 µg/mL in Chinese hamster ovary (CHO) cells. Incubations at each concentration were done in triplicate and the experiment was conducted twice, both times in the presence and absence of exogenous metabolic activation (Aroclor 1254 induced rat liver S9). Sterile distilled water was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed and no chromosome damage was observed at any HG concentration in the presence or absence of metabolic activation. Incubation with positive control substances in the presence (cyclophosphamide) or absence (ethyl methane-sulphonate) of metabolic activation resulted in anticipated increases in chromatid damage.

Link to relevant study records

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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: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK Locus (Trifluorothymidine Resistance)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat induced by Aroclor 1254
Test concentrations with justification for top dose:
10 – 5 – 2.5 – 1.25 – 0.63 – 0.31 – 0.16 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9-mix : methyl methanesulfonate. With S9-mix : cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration:
Without S9-mix : 3 hours (short treatment) and 24 hours (continuous treatment)
With S9-mix : 3 hours
- Expression time (cells in growth medium): 2 days after treatment
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
Evaluation criteria:
Under the experimental conditions and when the criteria for validity are fulfilled, a test item is considered as mutagenic in this system if the following conditions are fulfilled:
1. The induced mutation frequency for at least one tested concentration is higher than the mutation frequency in the vehicle control cultures by at least the global evaluation factor of 126 x10-6 (Moore et al., 2006).
2. A statistical trend test demonstrates a positive dose related increase in the mutation frequency (Moore et al., 2006).
3. The results have to be reproducible in an independent study, at least from a qualitative point of view.
If none of the three criteria mentioned above is fulfilled, the tested test item is considered as not mutagenic in this study system.
In all other cases, the results are discussed case by case, and the results obtained on other study systems are taken into account.
All these criteria are not absolute: however, they give help when a decision has to be taken, making a conclusion possible in the majority of the cases.
Statistics:
Statistical evaluation of data for the total number of mutants and for small colony mutants is performed using the method proposed by Robinson et al. (1990).
Briefly, the statistical analysis procedure includes the following steps:
• Test for consistency of duplicate cultures at each dose level for a single experiment. The limit is 10.8 times the current heterogeneity factor (H) (10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinite degrees of freedom. If the current heterogeneity factor is higher than 10.8 for survival heterogeneity factor (Hs) or mutation heterogeneity factor (Hm), this dose level is excluded from further consideration.
• The new heterogeneity factor (Hexp) is calculated for doses not excluded. If it exceeds the value for that number of degrees of freedom in the experiment, the experiment should be discarded
• For each dose level, mutant frequency (MF), log mutant frequency (LMF), the variance (V) of LMF and the weight (W) of MF are calculated for non-excluded dose levels (LMF could be slightly different in statistically evaluation because LMF is calculated on pooled independent cultures).
• Each treatment is compared with the control by means of one-sided Dunnett's test for multiple comparisons with the same control.
• Test for linear trend of mutant frequency with dose is performed. The slope b and its variance var (b) are calculated to form the test statistic b2/var (b) that should be compared with tabulated critical values of chi2 with 1 degree of freedom.
• Consistency across experiments is checked if possible (particularly if the same doses are used in both experiments) and if consistency is acceptable, each treatment (series A and B combined) is compared with the control.
Species / strain:
mouse lymphoma L5178Y cells
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
Additional information on results:
No toxicity was noted either with or without (3 or 24-hour treatment) metabolic activation (see attache Table 1). The concentration of 10 mM was thus retained as the highest concentration to be tested in the mutagenicity assays.
HEXYLENE GLYCOL induced no biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation, in the two independent assays (see attached Tables 2 & 3).
Conclusions:
Interpretation of results (migrated information):
negative

In the both independent assays with and without metabolic activation, no statistical or biological significant increase in the mutation frequency of total induced mutants (small and large colonies) or in the mean number of small colonies and in the mutation frequency of small colony mutants was noted at any concentrations tested in the presence of HEXYLENE GLYCOL.
Executive summary:

The potential of Hexylene glycol to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according to the international guidelines OECD No. 476 and Good Laboratory Practice.

After a preliminary toxicity test, Hexylene glycol was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 5 x 10e6 (3-hour treatment) or 1.25 x 10e6 (24-hour treatment) cells/mL in 10 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 5%), at 37°C.

Cytotoxicity was measured by assessment of plating efficiency, Relative Survival Growth (RSG), and Relative Total Growth (RTG), after treatment (T0) and 48 hours after treatment (PE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype.

The Hexylene glycol was dissolved in water and the positive controls were methylmethane sulfonate (without S9 mix) and Cyclophosphamide (with S9 mix). In the culture medium, the concentration of 10 mM was freely soluble. At this dose-level, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

The selected concentrations were 10, 5, 2.5, 1.25 and 0.63 mM for both experiments, with and without S9 mix.

Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no toxicity was induced at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the Adj. RTG.

Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no noteworthy increase in the mutation frequency, in comparison to the vehicle control was noted.

Hexylene glycol did not show any mutagenic activity in the mouse lymphoma assay.

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:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
- source/origin and conditions of cultivation or maintenance of the test system was not provided in the study report
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenates (S9)
Test concentrations with justification for top dose:
0, 1250, 2500, or 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methane-sulphonate (- S9) or cyclophosphamide (+ S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours (+ S9) or 24 hours (- S9)

NUMBER OF REPLICATIONS: Each concentration and control were tested in triplicate and the experiment was repeated.

NUMBER OF CELLS EVALUATED: 100 cells/culture (and, therefore, 300 cells/concentration).
Evaluation criteria:
Comparison of the frequency of aberrations.
Statistics:
Not reported.
Species / strain:
Chinese hamster Ovary (CHO)
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 applicable
Positive controls validity:
valid
Additional information on results:
There was no significant dose-related increase in the frequency of chromosome damage in CHO cultures exposed to hexylene glycol concentrations up to 5000µg/ml either in the presence or in the absence of the S9 mix (see attached tables).
Conclusions:
Interpretation of results (migrated information):
negative

Hexylene glycol did not induce chromosome damage in cultured Chinese hamster (CHO) cells under the experimental conditions described.
Executive summary:

In a GLP mammalian chromosome aberration test equivalent to OECD test guideline 473 (Meyer et al., 1985; Brooks et al., 1988), hexylene glycol was tested at doses of 0, 1250, 2500, or 5000µg/mL in Chinese hamster ovary (CHO) cells. Incubations at each concentration were done in triplicate and the experiment was conducted twice, both times in the presence and absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9). Sterile distilled water was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed and no chromosome damage was observed at any hexylene glycol concentration in the presence or absence of metabolic activation. Incubation with positive control substances in the presence (cyclophosphamide) or absence (ethyl methane-sulphonate) of metabolic activation resulted in anticipated increases in chromatid damage.

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:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (Positive control results for strains TA 98, TA 100, TA 1537 and TA 1538 in the absence of S9 fraction did not produce anticipated result)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
reliability scoring based on 1997 guideline.
Deviations:
yes
Remarks:
Not tested up to limit or cytotoxic dose levels
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see Table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37°C for 48-72 hrs

NUMBER OF REPLICATIONS: 3 plates per test; two independent experiments performed.

Evaluation criteria:
Reproducible values of 2.5 x control value or greater are considered to indicate a mutagenic response.
Statistics:
Revertant colony numbers were calculated as average and standard deviation. No statistical analysis test was performed.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
> 4000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
other: Positive control results for strains TA 98, TA 100, TA 1537 and TA 1538 in the absence of S9 fraction did not produce anticipated results.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
> 4000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Hexylene glycol did not induce a cytotoxic effect up to a concentration of 4000 µg/plate either in the presence or in the absence of rat liver S9 fraction (see attached table).
Conclusions:
Interpretation of results (migrated information):
negative

Applications of hexylene glycol up to 4000 µg/plate did not increase the reverse gene mutation rate in Escherichia coli WP2 uvrA pKM 101 or in Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100, either in the presence or in the absence of rat liver S9 fraction.
Executive summary:

In a GLP bacterial reverse mutation assay equivalent to OECD test guideline 471 (Meyer et al., 1985; Brooks et al., 1988), hexylene glycol was tested at doses of 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, or 4000 µg/plate in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and in Escherichia coli WP2 uvr A pKM 101. Incubations at each concentration were done in triplicate and the experiment was conducted twice, both times in the presence and absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9). Sterile distilled water was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed and no increase in the reverse mutation rate was observed at any hexylene glycol concentration in any of the tester strains in the presence or absence of metabolic activation. Incubation with positive control substances in the presence of metabolic activation resulted in anticipated increases in the reverse mutation rate but did not always do so in the absence of metabolic activation. This study is therefore considered reliable with restrictions.

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)
Version / remarks:
Reliability scoring based on 1986 guideline
Deviations:
yes
Remarks:
- source/origin and conditions of cultivation or maintenance of the test system not provided and post-treatment incubation period was 3 days instead of 4 to 7 days
GLP compliance:
yes
Type of assay:
gene mutation assay in fungi
Species / strain / cell type:
Saccharomyces cerevisiae
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenates (S9)
Test concentrations with justification for top dose:
0, 0.01, 0.1, 0.5, 1.0, or 5.0 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-N-oxide (-S9) or cyclophosphamide (+ S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 hours at 30 ºC (-S9) or 2 hours at 37 ºC followed by 16 hours at 30 ºC (+ S9)

NUMBER OF REPLICATIONS: Each concentration and control were at least tested in triplicate and the experiment was repeated.
Evaluation criteria:
Values of greater than twice the control value are considered to indicate a mutagenic response.
Statistics:
None performed (number of revertant colonies counted).
Species / strain:
Saccharomyces cerevisiae
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 applicable
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

Hexylene glycol did not induce conversion in the yeast Saccharomyces cerevisiae JD1 under the experimental conditions described.
Executive summary:

In a GLP yeast gene mutation assay equivalent to OECD test guideline 480 (Meyer et al., 1985; Brooks et al., 1988), hexylene glycol was tested at doses of 0, 0.01, 0.1, 0.5, 1.0, or 5.0 mg/mL in Saccharomyces cerevisiae. Incubations at each concentration were done at least in triplicate and the experiment was conducted twice, both times inthe presence and absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9). Sterile distilled water was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed and no increase in the rate of mitotic gene conversion was observed at any hexylene glycol concentration in the presence or absence of metabolic activation. Incubation with positive control substances in the presence (cyclophosphamide) or absence (4-Nitroquinoline-N-oxide) of metabolic activation resulted in anticipated increases in the rate of mitotic gene conversion. As the post-treatment period was 3 days as opposed to the recommended 4 to 7 days, this study is considered reliable with restrictions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Not classification is warranted according to CLP (EC 1272/2008).