Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 April, 2012 to 4 May, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Esterification products of acrylic acid and 2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol
EC Number:
800-838-4
Cas Number:
1384855-91-7
Molecular formula:
Molecular formula not available for this UVCB.
IUPAC Name:
Esterification products of acrylic acid and 2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): DPHA
- Substance type: Colourless to slightly yellow viscous liquid
- Physical state: Liquid
- Storage condition of test material: At room temperature protected from light
- Purity: UVCB, 100%
- Lot/batch No.: JBHA0020T
- Expiration date of the lot/batch: 06 March 2014

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate
Experiment 2:
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 : 650 µg/plate in DMSO for TA100
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA1537, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 1000 µg/plate and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Any other information on results incl. tables

Dose range finding test:

Precipitate: Precipitation of DPHA on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 μg/plate and upwards.

Toxicity: To determine the toxicity of DPHA, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with DPHA under all conditions tested.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, it was concluded that the test substance at concentrations up to 1000 μg/plate is mutagenic in tester strain TA1535 with metabolic activation only. No mutagenicity was observed with or without metabolic activation using other Salmonella typhimurium tester strains (TA1537, TA100 or TA98) and Escherichia coli strain.
Executive summary:

A study was conducted to determine the mutagenic potential of test substance DPHA in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat) according to the OECD Guideline 471 and EU Method B.13/14 in compliance with GLP. DPHA was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix. In the dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance precipitated on the plates at dose levels of 1000 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 10 to 1000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance precipitated on the plates at the top dose of 1000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the presence of S9-mix, the test substance induced 3.6 and 5.8-fold increases in tester strain TA1535 in the first and second experiment, respectively. The increases observed were above the laboratory historical control data range and the increases were greater than three times the concurrent control, were dose-related and observed in two independently repeated experiments. Therefore these increases are considered to be biologically relevant and DPHA is considered to be mutagenic in this strain with metabolic activation. All other bacterial strains showed negative responses over the entire dose range (no significant dose-related increase in the number of revertants in two independently repeated experiments). In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it was concluded that the test substance at concentrations up to 1000 μg/plate is mutagenic in tester strain TA1535 with metabolic activation only. No mutagenicity was observed with or without metabolic activation using other Salmonella typhimurium tester strains (TA1537, TA100 or TA98) and Escherichia coli strain (Verspeek-Rip, 2013).