2-benzyl-4,4,6-trimethyl-1,3-dioxane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): not mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 April 2016 and 12 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

In accordance with GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine or tryptophan locus in
the genome of five strains of bacteria

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. 5000 μg/plate was the maximum recommended dose level.

Vehicle / solvent:

The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in
dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

Controls performed in triplicate

Details on test system and experimental conditions:

Sterility controls as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular
only).

Bacteria
The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitution
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

All of the Salmonella strains are histidine dependent by virtue of a mutation through the
histidine operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty
lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an
inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced
mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In
addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This
deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and
Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on
17 August 1987.
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen
freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in
nutrient broth (Oxoid Limited; lot number 1712138 07/20) and incubated at 37 °C for
approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity
with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in
dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Dimethyl sulphoxide was therefore selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in
dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No
correction was made for purity. Prior to use, the solvent was dried to remove water using
molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of
4 x 10^-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable
for this period. Analysis for concentration, homogeneity and stability of the test item
formulations is not a requirement of the test guidelines and was, therefore, not determined.
This is an exception with regard to GLP and has been reflected in the GLP compliance
statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was
5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each
tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive
control was added to 2 mL of molten, trace amino-acid supplemented media containing
0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were
then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls
were also performed on the same day as the mutation test. Each concentration of the test
item, appropriate positive, vehicle and negative controls, and each bacterial strain, was
assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the
addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the
molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the
presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the
pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and
was 5 to 5000 μg/plate. In the initial second experiment the toxicity of the test item yielded
results that differed slightly from the Experiment 1 and consequently an insufficient number
of non-toxic dose levels were attained for TA1537 (presence of S9-mix only). Therefore,
TA1537, dosed in the presence of S9-mix was repeated employing an amended test item dose
range of 0.5 to 1500 μg/plate.
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a
minimum of four non-toxic dose levels and the toxic limit of the test item following the
change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of
the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were
incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten,
trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
Negative (untreated) controls were also performed on the same day as the mutation test
employing the plate incorporation method. All testing for this experiment was performed in
triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the
addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added
to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with
shaking) and addition of molten, trace amino-acid supplemented media. All testing for this
experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the
presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity).

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by
their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and
Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per
plate in the vehicle and untreated controls (negative controls). Acceptable ranges are
presented as follows:

TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the
intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the
frequency of revertant colonies, which are at least the minimum positive control value over
the previous two years, both with or without metabolic activation.

There should be a minimum of four non-toxic test item dose levels.

There should be no evidence of excessive contamination.

Evaluation criteria:

Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following
can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above
criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the
data generated will prohibit making a definite judgment about test item activity. Results of
this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05)
for those values that indicate statistically significant increases in the frequency of revertant
colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous
reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and
the S9-mix used in both experiments was shown to be sterile. The test item formulation was
also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The maximum dose level of the test item in the first experiment was selected as the maximum
recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation
method) the test item induced a weakening of the bacterial background lawn of bacterial
strain TA100 in the absence of S9-mix at 5000 μg/plate. There was no visible reduction in
the growth of the bacterial background lawns noted for any of the remaining tester strains at
any dose level, either in the presence or absence of metabolic activation (S9-mix).

Consequently the same maximum dose level was used in the second mutation test for all of
the tester strains except TA1537 dosed in the presence of S9-mix. The test item was noted to
induce a much stronger toxic response in the second mutation test after employing the
pre-incubation method with a visible reduction in the growth of the bacterial background
lawns noted to all of the tester strains dosed in the absence of S9-mix from 500 μg/plate. In
the presence of S9-mix, weakened bacterial background lawns were noted to all of the
Salmonella strains, initially from 500 μg/plate (TA100 and TA1535) and 1500 μg/plate
(TA98 and TA1537). No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the
presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test
item varied slightly between strain type, exposures with or without S9-mix and experimental
methodology. A test item precipitate (globular in appearance) was noted under a
low-power microscope at 5000 μg/plate, this observation did not prevent the scoring of
revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of
the bacterial strains, with any dose of the test item, either with or without metabolic
activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no
toxicologically significant increases in the frequency of revertant colonies were recorded for
any of the bacterial strains, with any dose of the test item, either with or without metabolic
activation (S9-mix) in Experiment 2 (pre-incubation method). Statistically significant
increases in TA1535 revertant colony frequency were observed in the presence of S9-mix at
1500 and 5000 μg/plate in the second mutation test. These increases were considered to have
no biological relevance because weakened bacterial background lawns were also noted at the
same dose levels. These responses are, therefore, considered to be due to additional histidine
being available to His- bacteria allowing these cells to undergo several additional cell
divisions and presenting as non-revertant colonies.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the
normal range. All of the positive control chemicals used in the test induced marked increases
in the frequency of revertant colonies in excess of the minimum positive control values over
the previous two years, both with or without metabolic activation. Thus, the sensitivity of the
assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: No mutagenic potential

Conclusions:

Under the conditions of this study, Reseda body was considered to be non-mutagenic under the conditions of this test.

Executive summary:

A study was performed according to the OECD Guidelines for "Bacterial Reverse Mutation Test" (OECD TG 471). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Reseda body was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A study was performed according to the OECD Guidelines for "Bacterial Reverse Mutation Test" (OECD TG 471). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Reseda body was considered to be non-mutagenic under the conditions of this test.

Justification for classification or non-classification

Based on the results of the Ames test, Reseda body does not need to be classified for mutagenicity in accordance with the criteria outlined in EU CLP (1272/2008/EC and its amendments).