Reaction product of tetrahydro-2,5-dimethoxy furan, ethanol and water

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Reaction product of tetrahydro-2,5-dimethoxy furan, ethanol and water

EC Number:

947-436-6

Molecular formula:

not applicable for UVCB

IUPAC Name:

Reaction product of tetrahydro-2,5-dimethoxy furan, ethanol and water

Test material form:

liquid

Method

Target gene:

histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 and was purchased from Trinova Biochem GmbH, 35394 Gießen, Germany. The S9 fraction is stored at -80°C. The S9 mix is freshly prepared on the day of the test.

Test concentrations with justification for top dose:

Six concentrations ranging from 8.8 to 1389 µg Reaction product of DMO-THF, water and ethanol/plate (corresponding to 0.316 to 50 µg dimethoxytetra¬hydrofuran/plate) were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
Based on cytotoxicity in pre-test.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-amino-anthracene,

Details on test system and experimental conditions:

1st independent experiment - Plate Incorporation Method
Sterile top agar containing 0.6% agar and 0.5% NaCl was molten on the day of the test. 10 mL of a sterile solution of 0.5 mM L-histidine HCl/0.5 mM biotin were added to 100 mL of molten agar. 2 mL of this top agar were distributed into culture tubes held at 45°C in a heating block. 0.1 mL of Salmonella cell suspension (containing approximately 108 viable cells in the late exponential or early stationary phase) 0.2 mL of test item (or 0.2 mL solvent or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer mentioned above.
The test components were mixed by vortexing the soft agar for 3 sec at low speed and then poured onto a coded 27.5 mL minimal glucose agar plate (Minimal Glucose Agar medium E). To achieve a uniform distribution of the top agar on the surface of the plate, the uncovered plate was quickly tilted and rotated and then placed on a level surface with the cover on and finally allowed to harden.
Immediately, the plates were inverted and placed in a dark 37°C incubator for 48 to 72 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter , and the presence of the background lawn on all plates was confirmed. A lawn that was thin compared with the lawn on the negative control plate confirmed bacterial toxicity.
Routine examination of the background lawn of bacterial growth resulting from the trace of histidine added to the top agar can be an aid in determining the presence of toxic effects. If massive cell death has occurred, the background lawn on the test plates will be sparse compared with control plates.
In this case more histidine is available to the individual surviving bacteria and they undergo more cell divisions, consequently appearing as small colonies which can be mistaken for revertants if the absence of a normal background lawn is not noted.

2nd independent experiment - Preincubation Method
The test item was preincubated with the test strain (containing approximately 108 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.2 mL of test item (or 0.2 mL solvent or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.

Evaluation criteria:

A test item is considered to show a positive response if
-the number of revertants is significantly increased (p  0.05, U-test according to MANN and WHITNEY, see Section 6, Reference 3) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
-in addition, a significant (p  0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient, see Section 6, Reference 3) is observed;
-positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Statistics:

The Mann and Whitney test (p ≤ 0.05, see section 6, reference 3.) may be used to determine statistical significance. The Spearman's rank correlation coefficient (section 6, reference 3.) may also be applied.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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

Additional information on results:

No increase in revertant colony numbers as compared with control counts was observed for Reaction product of DMO-THF, water and ethanol, tested up to a cytotoxic concentration of 1389 µg/plate (corresponding to 50 µg dimethoxy-tetrahydrofuran/plate) in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, Reaction product of DMO-THF, water and ethanol tested up to a cytotoxic concentration of 1389 µg/plate (corresponding to 50 µg dimethoxytetrahydrofuran/plate), caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

No increase in revertant colony numbers as compared with control counts was observed for Reaction product of DMO-THF, water and ethanol, tested up to acytotoxic concentrationof 1389 µg/plate (corresponding to 50 µg dimethoxy­tetrahydrofuran/plate) in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and thesensitivity of the test system.