Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
06 August 2020 - 14 August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
updated 26 June 2020
Deviations:
yes
Remarks:
An impurity allowance of 2.1% was inadvertently employed for the solubility check and Exp. 1. The error was noted on the day of testing and the experiment was abandoned. There was considered no impact to integrity of the test and the study was repeated.
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]
EC Number:
245-509-0
EC Name:
2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]
Cas Number:
23235-61-2
Molecular formula:
C12H26O5
IUPAC Name:
2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol
Test material form:
solid: flakes
Details on test material:
- Name of test material (as cited in study report): Di-Trimethylolpropane, Di-TMP
- Lot/batch No.: 200504133
- Purity: 97.9%
- Appearance: White flakes
- Storage condition of test material: At room temperature in the dark.
Specific details on test material used for the study:
- Name of test material: Di-Trimethylolpropane, Di-TMP
- Lot/batch No.: 200504133
- Purity: 97.9%
- Appearance: White flakes
- Storage condition of test material: At room temperature in the dark

Method

Target gene:
Tryptophan locus
Species / strain
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (Exp. 1: Plate Incorporation Method)
15, 50, 150, 500, 1500 and 5000 μg/plate (Exp. 2: Pre-Incubation Method)
Vehicle / solvent:
Vehicle: Sterile distilled water
Justification for vehicle/solvent: The Sponsor stated that test item is fully soluble in sterile distilled water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
10 μg/plate for WP2uvrApKM101 (Direct acting compound in the absence of S9-mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
0.5μg/plate for WP2uvrApKM101 (Indirect acting compound in the presence of S9-mix)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- First experiment: in agar (plate incorporation)
- Second experiment: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period (Exp. 2): incubated at 37 ± 3°C for 20 minutes, with shaking
- Exposure duration: Plates were incubated at 37 ± 3°C for between 48 and 72 hours

OTHER EXAMINATIONS:
- Other: Observation of the number of revertant colonies and bacterial lawns
Evaluation criteria:
The test item is considered non-mutagenic (negative) in the test system if the below criteria are not met.

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. A fold increase greater than two times the concurrent solvent control (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
Statistics:
Dunnett’s Regression Analysis (* = p < 0.05) to confirm statistical significance in increases in frequency of revertant colonies compared to the concurrent solvent control

Results and discussion

Test results
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: Sponsor stated that substance was completely soluble
- Precipitation and time of the determination: not reported
- Other confounding effects: not reported

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

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

Applicant's summary and conclusion

Conclusions:
Negative: under the conditions of this test Di-Trimethylolpropane was considered to be non-mutagenic.
Executive summary:

A bacterial reverse mutation test was performed by Covance Laboratories Limited, U.K., on behalf of Perstorp Holding AB, Sweden, to determine the potential of the test substance Di-Trimethylolpropane (Di-TMP) to induce gene mutation. The test was performed in accordance with OECD, EC, US EPA, Japanese, and other international test guidelines, and the test was carried out to GLP. Only one strain of Escherichia coli (WP2uvrA pKM101) was tested in this study to compliment previous testing performed on this (Jensen, 1992). No evidence of cytotoxicity or mutagenic activity was observed during two tests, either in the presence or absence of S9 metabolic activation at concentrations up to and including the limit concentration of 5000 ug/plate.