Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-30 - 2018-05-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Molecular Formula:
HOCH2C(Br)CH2OH
Molecular Weight:
245.91
Characteristics (Physical Appearance):
White crystalline powder
CAS No.:
3234-02-4
Batch Number:
1088
Purity:
100%

Method

Target gene:
selected histidine loci
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
triplicate cultures at the concentrations of 125, 40, 12.5, 4 and 1.25 μg/plate and 5000, 1500, 500, 150 and 50 μg/plate, in the absence and presence and of metabolic activation system (S9)
Test concentrations for this study were selected from the results obtained with solubility/ precipitation test and preliminary cytotoxicity test of the test item employing only TA100 with and without metabolic activation system.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
methylmethanesulfonate
other: ICR191, 2-Aminoanthracene, 2-Aminofluorene, Danthron
Details on test system and experimental conditions:
Solubility / Precipitation Test
trans-2,3-Dibromo-2-Butene-1,4-Diol was found to be completely soluble in DMSO at a concentration of 50 mg/ml. Precipitation was not observed in the final reaction mixture at the concentrations from 2.38 mg/ml to 0.0595 mg/ml corresponding to final test concentrations from to 5000 to 125 µg/plate respectively. Hence 5000 μg/plate showing no precipitation was the highest concentration selected for the preliminary cytotoxicity study.
Preliminary Cytotoxicity Test
Before commencing the study, the test item was assessed for cytotoxicity to the tester bacteria using tester strain TA100. Eight concentrations viz. 5000, 4000, 3000, 2000, 1000, 500, 250 and 125 μg/plate of the test item, formulated in DMSO, were tested for toxicity to bacterial cells.
No cytotoxicity was observed to revertant colonies and bacterial background lawn at the concentration 125 μg/plate and 5000 μg/plate in the absence and presence and of metabolic activation system (S9) respectively. Hence, selected as the maximum test concentration for main study.
EXPERIMENTAL PROCEDURE – PRE-INCUBATION ASSAY
Maximum concentration of 5000 μg/plate, which was found non cytotoxic to TA100 in the preliminary cytotoxicity, was selected as the highest concentration for main study, with four lower concentrations viz. 1500, 500, 150 and 50 μg/plate. Triplicate plating was performed at each dose level for an adequate evaluation of the variation.
PRE-INCUBATION ASSAY WITHOUT METABOLIC ACTIVATION
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. Sterile phosphate buffer (0.5 ml) and freshly prepared test item / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ºC for 20 minutes in orbital shaker incubator.
Finally 2 ml of top agar with histidine-biotin was added. The contents were mixed and poured over the surface of the Petri plates containing minimal glucose VB agar.
PRE-INCUBATION ASSAY WITH METABOLIC ACTIVATION
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. The S9 mixture (0.5 ml) and freshly prepared test item / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ºC for 20 minutes in orbital shaker incubator.
Finally 2 ml of top agar with histidine-biotin was added. The contents were mixed and poured over the surface of the Petri plates containing minimal glucose VB agar.
INCUBATION
Contents of all the plates were allowed to solidify after which the plates were inverted and incubated at 37 ºC for about 68 hours.
OBSERVATIONS
After incubation period, the plates were checked for sterility. The plates were observed for a uniform lawn of auxotrophs (his-) and the colonies for histidine revertants as the prototrophs (his+). Histidine revertant colonies per plate were counted and the mean number of colonies at each test point was calculated.
Evaluation criteria:
Criteria for Evaluation
1. Criteria for a Positive Response
A test item is considered to be positive (mutagenic), if it induces a concentration dependent increase and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate, in at least one strain with or without metabolic activation system, which is at least 2-fold (3-fold for TA1535) of that observed in the corresponding concurrent vehicle control.
2. Criteria for a Negative Response
A test item for which the results do not meet the above criteria is considered non-mutagenic in this test. In order a substance considered to be negative if the revertant colonies cannot be greater than 2 (or 3 for strain TA 1535), or less than 0.5 in at least 5 doses for all strains tested.
However, reproducibility of negative results was confirmed by repeat experimentation.
Criteria for an Equivocal Response
Occasionally, a test item cannot be judged to be positive or negative (e.g., concentration dependent increases that fail to reach 2-fold (3-fold for TA1535) control values, or ≥ 2-fold (3-fold for TA1535) increases that do not appear to be concentration dependent). In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified study plan such as appropriate spacing of dose levels.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Salmonella typhimurium, Reverse Mutation Assay of trans-2,3-Dibromo-2-Butene-1,4-Diol was carried out in compliance with the Organization for Economic co-operation and Development (OECD) Guidelines for Testing of Chemicals (Guideline No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted by the council on 21 July 1997.
Under the conditions described for this study, it is concluded that, trans-2,3-Dibromo-2-Butene-1,4-Diol is non-mutagenic in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535.
Executive summary:

‘Salmonella typhimurium, Reverse Mutation Assay of trans-2,3-Dibromo-2-Butene-1,4-Diol’ was carried out in compliance with the OECD Guidelines for Testing of Chemicals (Guideline No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted 21 July 1997 and as per mutually agreed study plan.

trans-2,3-Dibromo-2-Butene-1,4-Diol was evaluated in Ames test to determine its ability to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation system (S9). Based upon the preliminary tests conducted to assess the solubility / precipitation and cytotoxicity of trans-2,3-Dibromo-2-Butene-1,4-Diol, the tester strains were exposed to the test item in triplicate cultures at the concentrations of 125, 40, 12.5, 4 and 1.25 μg/plate and 5000, 1500, 500, 150 and 50 μg/plate, in the absence and presence and of metabolic activation system (S9) respectively. Liver S9, induced in Wistar rats with phenobarbitone and β - naphthoflavone, was used for this purpose.

DMSO was used as a vehicle. The exposed bacteria were plated onto minimal glucose agar medium supplemented with L-histidine. The plates were incubated at 37 ºC for about 48 hours for experiment1 and experiment 2 for 68 hours for after which the histidine revertant colonies were counted and their frequency was compared with that in the vehicle control group. Concurrent positive control groups were also included in the experiment, as specified by the test guideline.

Results of this test indicated that the frequencies of histidine revertant colonies at all concentrations of trans-2,3-Dibromo-2-Butene-1,4-Diol in strains TA1535, TA97a, TA98, TA100 and TA102, in the presence and absence of a metabolic activation system, were comparable to those observed in the vehicle control group, as per the criteria employed for evaluation of mutagenic potential, and this observation was confirmed by repetition of the experiments.

Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX PVT. LTD. They also compared well with the range reported in the literature. Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation

Analysis of the samples of the doses formulated for treatment revealed that the average measured lower concentrations of trans-2,3-Dibromo-2-Butene-1,4-Diol were 1.22 and 0.0120 mg/ml and higher concentration 48.49 and 0.48 mg/ml against the nominal concentrations of lower concentration 1.25.and 0.0125. mg/ml, higher concentration 50, 0.5 respectively. For above mentioned doses confirmed that the test system received an adequate dose of the Test Item.

It is concluded that, under the conditions of this study, trans-2,3-Dibromo-2-Butene-1,4-Diol is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.