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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Supplier: Arkema France
- Name of test material (as cited in study report): Hexylene glycol
- Physical state: liquid
- Analytical purity: 99.95%
- Impurities (identity and concentrations): water <= 0.05%
- Purity test date: 22nd July 2008
- Lot/batch No.: A1TX6K010101
- Expiration date of the lot/batch: July 2010

Method

Target gene:
TK Locus (Trifluorothymidine Resistance)
Species / strain
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
Controls
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.

Results and discussion

Test results
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).

Applicant's summary and conclusion

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.