Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol]and propylidynetrimethanol and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Genetic toxicity: in vitro

Administrative data

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:

3rd May 2012 to 17th August 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with GLP and OECD Guideline 471, with no deficiencies to affect the validity of the study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not applicable

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-diethyl-2,6-bis(hydroxymethyl)heptane-1,3,5,7-tetrol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

EC Number:

904-155-3

Molecular formula:

C12H28O6 C12H26O5 C6H14O3

IUPAC Name:

2,6-diethyl-2,6-bis(hydroxymethyl)heptane-1,3,5,7-tetrol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): HB TMP
- Analytical purity: 69%
- Composition of test material, percentage of components: 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] (CAS: 23235-61-2, EC No.: 245-509-0), Propylidynetrimethanol (CAS: 77-99-6, EC No.: 201- 074-9) and 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] (CAS: 93983-16-5, EC No.: 301-304-9).
- Lot/batch No.: 7LTQA002
- Expiration date of the lot/batch: 8th May 2014
- Storage condition of test material: Stored at ambient temperature in the dark when not in use.

Method

Target gene:

Histidine and tryptophan.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The S. typhimurium strains contain mutations in the histidine operon, thereby imposing the essential requirement for histidine in the growth medium. Three mutations in the histidine operon are involved his G 46 in TA 1535 and TA 100, his C 3076 in TA 1537 and his D 3052 in TA 98. All 4 strains also contain the deep rough (rfa) mutation.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

The strain contains an ochre mutation in the trpE locus and can be mutated to tryptophan-independence either by a base-pair reversion of an A-T base-pair in the trpE locus, or more commonly, by a base-pair substitution within a number of transfer RNA loci elsewhere in the chromosome.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 16.6, 49.8, 166, 498, 1659 and 4978 μg per plate
First mutation test: 16.6, 49.8, 166, 498, 1659 and 4978 μg per plate
Second mutation test: 17, 50, 167, 500, 1667 and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

2 μg per plate with S typhimurium TA 1535 and TA 1537, 0.5 μg per plate with S. typhimurium TA 98 and TA 100 and 20 μg per plate with E. coli WP2uvrA

Positive control substance:

other: 2-aminoanthracene

Remarks:

Prepared in DMSO ; With S9 mix

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

1 μg per plate with S. typhimurium TA 1535 and TA 100

Positive control substance:

sodium azide

Remarks:

Prepared in water ; Without S9 mix

Controls 3

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

80 μg per plate with S. typhimurium TA 1537

Positive control substance:

9-aminoacridine

Remarks:

Prepared in DMSO ; Without S9 mix

Controls 4

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

1 μg per plate with S. typhimurium TA 98

Positive control substance:

2-nitrofluorene

Remarks:

Prepared in DMSO ; Without S9 mix

Controls 5

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

2 μg per plate with E. coli WP2uvrA

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Prepared in DMSO ; Without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation method.

The Direct Plate Incorporation Method was used in the initial toxicity testing to determine suitable exposure levels to be used for the subsequent mutation tests. In the initial toxicity testing, a single strain of bacteria, S. typhimurium TA 100 was used, and one plate per exposure level of test item was prepared.

In the mutation tests, Diluted agar (Difco Bacto-agar (0.6%, w/v), NaCl (0.6%, w/v)) was autoclaved and, just before use, were supplemented with sterile L-histidine.HCl (1.0 mM)/biotin (1.0 mM) was added at 50 mL per litre of soft agar for S. typhimurium. For E. coli, sterile L-tryptophan (1.35 mM) was added at 10 mL per litre of soft agar. These soft agars were thoroughly mixed and kept in a water bath set to maintain a temperature of 45°C.

For the Direct Plate Incorporation Method, soft agar (2 mL) was dispensed into a small plastic sterile tube. S9 mix or phosphate buffer (0.05 M, pH 7.4) (0.5 mL) was added, followed by the bacteria (0.1 mL) and, finally, the test solution. The tube contents were mixed, then poured on to minimal medium plates prepared in-house. The plates contained BBL Purified Agar (1.5%, 20 mL) in Vogel-Bonner Medium E (Vogel and Bonner, 1956) with glucose (2%, w/v).

For the Pre-incubation Method, S9 mix or phosphate buffer (0.05 M, pH 7.4) (0.5 mL) was dispensed into a small plastic sterile tube followed by the bacteria (0.1 mL) and, finally, the test solution. The tubes were then placed for 20 min in a shaking incubator set to maintain a temperature of 37°C. After incubation, soft agar (2 mL) was added to each tube. The tube contents were then mixed and poured onto minimal medium plates. When the soft agar had set, the plates were inverted and placed in an incubator set to maintain a temperature of 37°C for 3 days and then examined. The numbers of mutant colonies on each plate were determined and captured electronically in a validated software system. The plates were also examined microscopically for precipitates and for microcolony growth.

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: All test items were tested in triplicate, (100 μL per plate), both in the absence and in the presence of S9 mix.

DETERMINATION OF CYTOTOXICITY
- Method: Colony count

Evaluation criteria:

For S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli WP2uvrA, at least a doubling of the mean concurrent vehicle control value is required before mutagenic activity is suspected. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value is required before mutagenic activity is suspected.

If the mean colony count on the vehicle control plates is less than 10, then a minimum count of 20 (representing a 2-fold increase over 10) is required before a response is registered. Rationale: As the vehicle control counts for strains with low spontaneous mutation rates (such as TA 1535, TA 1537 and particularly E. coli WP2uvrA) often range from 0 to 30, it is normal to have occasions where the vehicle mean is <10 while random treatment means are in the range 10 to 20. The use of the ‘minimum of 20’ rule avoids unnecessary repeat work due to strict application of the 2-fold rule.

A concentration-related response is also required for identification of a mutagenic effect. At high concentrations this relationship may be reversed because of, for example, toxicity of the test item to the bacteria, specific toxicity of the test item to the mutants, or inhibition of S9 enzymes (where a mutagen requires metabolic activation by the S9 mix). A response should be reproducible in an independent test.

Statistics:

Not performed in this study.

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of HB TMP was observed at any concentration in either mutation test.

RANGE-FINDING/SCREENING STUDIES:
In the first test, conducted by the Direct Plate Incorporation Method, no toxicity to the bacteria was observed at any concentration of HB TMP. In the second test, conducted by the Pre-incubation Method, toxicity to all 5 strains was observed as a slight thinning of the background lawn of microcolonies. This observation was made at the highest concentration of 5000 μg per plate, only in the absence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle and positive control groups were all within the normal/historical ranges for each bacterial strain and activation condition.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation.

Under the conditions of this study, HB TMP is not considered mutagenic when tested on Salmonella typhimurium and Escherichia coli in the presence and absence of metabolic activation at concentrations up to the predetermined maximum of 5000 μg per plate.

Executive summary:

In a study to determine the mutagenic potential of HB TMP, a bacterial reverse mutation assay was conducted in accordance with OECD guideline 471. The test strains used were Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2uvrA. DMSO was used as the vehicle and the positive controls included 2 -Aminoanthracene in the presence of S9 mix and Sodium azide, 9-Aminoacridine, 2-Nitrofluorene and N-Ethyl-N-nitro-N-nitrosoguanidine in the absence of S9 mix.

Initial toxicity testing was conducted to determine the concentrations to use in the 2 subsequent tests. In the first mutagenicity test conducted by the Direct Plate Incorporation method, the concentrations used were 16.6, 49.8, 166, 498, 1659 and 4978 μg per plate while in the second mutagenicity test conducted by the Pre-incubation method, concentrations of 17, 50, 167, 500, 1667 and 5000 μg- per plate were used. Triplicate plates were used at each exposure level.

In the initial toxicity test, no toxicity to the bacteria was observed at any concentration in either the absence or the presence of S9 mix and no precipitation of HB TMP was observed at any tested concentration. In the subsequent mutation tests, in the direct plate incorporation method, no toxicity to the bacteria was observed at any concentration of HB TMP. In the second test, conducted by the Pre-incubation Method, toxicity to all 5 strains was observed as a slight thinning of the background lawn of microcolonies. This observation was made at the highest concentration of 5000 μg per plate, only in the absence of S9 mix. No precipitation of HB TMP was observed at any concentration in either mutation test.

Under the conditions of this study, HB TMP is not considered mutagenic when tested on Salmonella typhimurium and Escherichia coli in the presence and absence of metabolic activation at concentrations up to the predetermined maximum of 5000 μg per plate. Consequently, HB TMP does not require classification according to CLP.