Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-22 - 2017-12-21 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
his-
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
10 / 3 / 1.0 / 0.3 / 0.1 μL/plate (1st experiment)
10 / 5 / 2.5 / 1.25 / 0.63 / 0.31 μL/plate (2nd experiment)
The test item is an UVCB available as aqueous solution, so 10 µl correspond to 5000 µg solid test item, which is the registered substance. 5000 µg/plate is the top dose required by the guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: demin. water
- Justification for choice of solvent/vehicle: Demin. water was chosen as vehicle, because the test item was sufficiently soluble in it, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Remarks:
solvent controls
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO), batch: 246245640, for the positive controls nitrophenylendiamine, benzo-a-pyrene and 2-amino-anthracene. Demineralised water, batch: 20170309 and 20170805 for the positive control sodium azide and for the test item.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine / 2-Amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, 1st experiment); preincubation (2nd experiment)

DURATION
- Preincubation period: 20 min (2nd experiment)
- Exposure duration: 48h

SELECTION AGENT (mutation assays): his-minimal agar

NUMBER OF REPLICATIONS: Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used. Two independent experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn / number of revertant colonies
Rationale for test conditions:
as indicated by the guideline
Evaluation criteria:
Evaluation
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
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
- Effects of pH: none stated
- Effects of osmolality: none stated
- Water solubility: not exceeded
- Precipitation: no
- Other confounding effects: none stated

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not reduced.

Applicant's summary and conclusion

Conclusions:
The study was conducted under GLP according to OECD guideline 471 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. The substance was tested in the form in which it is distributed, i.e. the marketed aqueous solution. However, the applied amounts were corrected for the actual content of the registered substance, HBOPS-Na, in the aqueous solution, so that the limit dose of 5 mg/plate of HBOPS-Na was reached and the study can be considered valid. Hence, the results can be considered as reliable to assess the potential of HBOPS-Na to induce reverse mutations in bacteria.
The test item showed no increase in the number of revertants in all bacteria strains in both experiments.
All negative and nearly all 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.
Based on the results of this study it is concluded that HBOPS-Na is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

The determination of the mutagenic potential of 2-Butyne-1,4-diol, reaction products with 1,2-oxathiolane 2,2-dioxide and sodium hydroxide (HBOPS-Na) was assessed with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 under GLP

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item HBOPS-Na was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

In the first experiment, the test item (the solid dissolved in demin. water) was tested up to concentrations of 10 µL/plate (corresponding to 5 µg/plate of the solid) in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Based on the first experiment, the test item was tested up to concentrations of 10 µL/plate in the absence and presence of S9-mix in all bacteria strain using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that HBOPS-Na is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.