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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23-sept-2015 to 18-apr-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-chloroethyl) ether
EC Number:
203-870-1
EC Name:
Bis(2-chloroethyl) ether
Cas Number:
111-44-4
Molecular formula:
C4H8Cl2O
IUPAC Name:
1-chloro-2-(2-chloroethoxy)ethane
Test material form:
liquid
Specific details on test material used for the study:
TEST MATERIAL
- Name (as cited): 2,2`- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Batch No.: 20150706
- Expiration date of the lot/batch: 01 June 2016
- Purity test date: 28 August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%), protected from light

Method

Target gene:
hisD3052; hisG46; hisC3076; trpE
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of male Sprague Dawley rats, which were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. The S9 mix comprised 10% S9 fraction.
Test concentrations with justification for top dose:
78.1; 156.2; 312.5; 625; 1250; 2500; 5000 µg/plate. No inhibitory, cytotoxic effect of the test item was observed in the preliminary experiment.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble at 100 mg/mL concentration in Distilled water (stock solution); but the formulation at the same concentration using DMSO as vehicle (solvent) was a clear solution.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Without activation: 4-nitro-1,2-phenylene-diamine (TA98 only) / Na-azide (TA100, TA 1535) / 9-aminoacridine (TA1537) / Methyl-methanesulfonate (WP2 uvrA) With activation: 2-aminoanthracene (all Solmonella strains and E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 1.89-4.63E+09 CFU/mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
No statistics perfomed; evaluation based on criteria mentioned above

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Remarks:
but relatively low number of revertants
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Dose-dependent increase in the observed mutation factor values at 5000 and 2500 μg/plate (mutation factor values of 2.78 and 2.12, respectively; threshold value: 2)

Any other information on results incl. tables

In the Initial Mutation Test (using the plate incorporation method), slight positive effect was observed in E. coli WP2 uvrA strain with metabolic activation. The observed mutation factor value (MF=2.12) at the highest examined concentration of 5000 μg/plate was above the threshold limit of 2 of this strain, and dose dependence was also observed. In the Confirmatory Mutation Test, a clear positive effect was seen: the observed mutation factor values at the two highest examined concentrations of 5000 and 2500 μg/plate were above the threshold limit of 2 of this strain (mutation factor values of 2.78 and 2.12, respectively) and dose dependence was also observed. Additionally, dose-dependent increase in the number of revertant colonies was also observed in this strain with metabolic activation using the pre-incubation method, although the mutation factor values remained slightly below the relevant threshold limit (MF=2) in that case (highest mutation factor value was 1.83).

In the Confirmatory Mutation Test using the pre-incubation method, biologically relevant increases were also observed in S. typhimurium TA1535 strain with metabolic activation. The observed mutation factor values were 4.91 and 3.18 at 2500 and 1250 μg/plate concentration, respectively (i.e. above the threshold limit of 3 of this strain). The observed values in the examined concentration range were compatible with a dose response curve (slightly lower value was detected at 5000 μg/plate due to the observed cytotoxicity on the plates). These values also indicate a mutagenic effect, although the numbers of revertant colonies seen in the DMSO control plates of this strain were relatively low compared to the untreated control (as indicated by the MF: 1.50 value of the untreated control). However, even compared with the untreated control, the mutation factor at 2500 μg/plate had a positive result with a mutation factor of 3.27.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main test in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test. Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main test at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

No inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test using the plate incorporation method in any of the examined strains. However, inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development, pinpoint colonies were also detected in some cases) was observed in the Confirmatory Mutation Test using the pre-incubation method in all examined strains at 5000 μg/plate concentration with and without metabolic activation and in Salmonella typhimurium TA1537 strain at 2500 μg/plate without metabolic activation.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this test, the test item 2,2’-Dichlorodiethyl ether exhibit a weak mutagenic activity in S. typhimurium TA 1535 and E. coli WP2 uvrA with metabolic activation.
Executive summary:

The test substance was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and Escherischia coli WP2 uvrA, performed according to OECD TG 471. In the Initial Mutation Test a weak positive effect of the test item was obtained in Escherichia coli WP2 uvrA strain with metabolic activation using the plate incorporation method (the observed revertant colony numbers were above the respective biological threshold value, slight dose response was also indicated). Although the effect was weak, it was reproducible in the Confirmatory Mutation Test using the same test conditions. Furthermore, a weak positive effect of the test item was also observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation using the pre-incubation method.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains Escherichia coli WP2 uvrA and Salmonella typhimurium TA1535 strains in the presence of metabolic activation. In conclusion, the test item 2,2’-Dichlorodiethyl ether exhibit a weak mutagenic activity under the test conditions of this study.