Reaction mass of propylidynetrimethanol and 2-ethylpropane-1,3-diol and 5-ethyl-1,3-dioxane-5-methanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-05-08 to 2008-09-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Noshan No. 8417, Guideline 2-1-19-1, Agricultural Production Bureau

Version / remarks:

24 November 2000

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Official notice of J MHLW, METI and ME

Version / remarks:

21 November 2003

Deviations:

no

Test guideline 6

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

Version / remarks:

1996

Deviations:

no

Test guideline 7

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 5

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Includes MHRA GLP compliance certificate.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(5-ethyl-1,3-dioxan-5-yl)methanol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethylpropane-1,3-diol

EC Number:

904-153-2

IUPAC Name:

(5-ethyl-1,3-dioxan-5-yl)methanol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethylpropane-1,3-diol

Specific details on test material used for the study:

- Name of test material (as cited in study report): Polyol TD
- Physical state: Colourless liquid
- Analytical purity: Hydroxyl number, 760 mg KOH/g
- Lot/batch No.: 3777937
- Expiration date of the lot/batch: Indefinite
- Storage condition of test material: Room temperature in the dark, dry

Method

Target gene:

his/trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, 5000 µg/plate, first test.
50, 150, 500, 1500, 5000 µg/plate, second test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water, purified in-house by reverse osmosis
- Justification for choice of solvent/vehicle: The Sponsor indicated that the test substance was miscible with water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix: 2 µg/plate for strains TA100 and TA 1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix: 50 µg/plate for strain TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

:purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix: 2 µg/plate for strain TA98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

In the absence of S9 mix: 2 µg/plate for strain WP2 uvrA (pKM101)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

In the presence of S9 mix: 5 µg/plate for strains TA 100 and TA 1535; 10 µg/plate for strain WP2 uvrA (pKM101)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9 mix: 5 µg/plate for strains TA98 and TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - first test; preincubation - second test

DURATION
- Preincubation period: 30 minutes at 37ºC (second test only).
- Exposure duration: Plates incubated for 72 hours
- Expression time (cells in growth medium): 10 hours


SELECTION AGENT (mutation assays): Histidine (Salmonella strains) and tryptophan (E Coli strain)

NUMBER OF REPLICATIONS: Three plates at each concentration in each test.

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent backgroud bacterial lawn.


OTHER: all strains/cell types requested by OECD 471 tested

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Results and discussion

Test results

Test results 1

Species / strain:

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

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First test:
No evidence of toxicity was obtained following exposure to Polyol TD. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Polyol TD at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Second test:
No evidence of toxicity was obtained following exposure to Polyol TD. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Polyol TD at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported.
- Effects of osmolality: Not reported.
- Evaporation from medium: Not reported.
- Water solubility: Not reported.
- Precipitation:Not reported.
- Other confounding effects: Not reported.


RANGE-FINDING/SCREENING STUDIES:
No evidence of toxicity was obtained following exposure to Polyol TD. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

Any other information on results incl. tables

For further information and details on results, please refer to result tables, attached.

Applicant's summary and conclusion

Conclusions:

Based on the results from this baterial reverse mutation assay (according to OECD Guideline 471), It is concluded that Polyol TD showed no evidence of mutagenic activity in all bacterial strains (S. typhimurium TA 98, TA 102, TA1535, TA 1537 and E. coli WP2 uvrA pkM 101) which were investigated under the test conditions.

Executive summary:

In a reverse gene mutation assay in bacteria performed according to OECD TG 471, strains TA 98, TA 102, TA1535 and TA 1537 of S. typhimurium and WP2 uvrA pkM 101 of E. coli were exposed to Polyol TD at concentrations of 5, 15, 50, 150, 500, 1500, 5000 µg/plate in the first test and 50, 150, 500, 1500, 5000 µg/plate in the second test in the presence and absence of mammalian metabolic activation by a rat liver homogenate fraction (S9 mix). In the first test, the plate co-incubation method was used, while in the second test, a pre-incubation with the test substance was performed.

Polyol TD was tested up to the limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related response of induced mutant colonies over background. Based on the results, the test item can be considered to be non-mutagenic.

This study is calssified as acceptable as it satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.