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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro mutagenicity tests are performed. All tests show no evidence of genetic toxicity in vitro. No classification according to CLP regulation (EC) No 1272/2008 is needed.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

histidine operon

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS; Cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258. Spontaneous mutation rate was continuously monitored.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was obtained from Trinova Biochem . S9 was collected from male rats.

Test concentrations with justification for top dose:

In the main study concentrations of 8.7, 17.4, 34.8, 69.5 and 139 µg Reaction product of DMO-THF, water and ethanol/mL medium were selected for the experiments without and with metabolic activation.
Based on cytotoxicity in the pre-test.

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

Mutagenicity
Experiments without metabolic activation
The mutation frequency of the solvent control highly purified water was 7.05 and 18.75 mutant colonies per 10E6 cells. Hence, the solvent controls were well within the expected range (see Text table 4.8-1).
The mutation frequency of the cultures treated with concentrations of 8.7, 17.4, 34.8 and 69.5 µg Reaction product of DMO-THF, water and ethanol/mL culture medium ranged from 6.90 to 19.12 mutant colonies per 10E6 cells. These results are within the normal range of the solvent controls.

Experiments with metabolic activation
The mutation frequency of the solvent control highly purified water was 32.94 and 33.67 mutant colonies per 10E6 cells. Hence, the solvent controls were well within the expected range (see Text table 4.8-1).
The mutation frequency of the cultures treated with concentrations of 8.7, 17.4, 34.8 and 69.5 µg Reaction product of DMO-THF, water and ethanol/mL culture medium ranged from 26.40 to 37.88 mutant colonies per 10E6 cells. These results are within the normal range of the solvent controls.
The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 532.59 to 921.33 mutant colonies per 10E6 cells in the case of EMS and ranging from 548.00 to 1037.58 mutant colonies per 10E6 cells in the case of DMBA, indicating the validity of this test system.

No relevant changes in pH or osmolality compared to the solvent control were noted up to a concentration of 8.78 mg/mL medium. A slight decrease in pH and an increase in osmolality was noted at the pronounced cytotoxic concentrations of 27.8 and 55.6 mg/mL medium.

Conclusions:

Under the present test conditions, Reaction product of DMO-THF, water and ethanol tested up to cytotoxic concentrations was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

Reaction product of DMO-THF, water and ethanol was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.The test item wascompletely dissolved inhighly purified water.

The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test.

In the main study concentrations of 8.7,17.4, 34.8, 69.5 and 139 µg Reaction product of DMO-THF, water and ethanol/mL medium were selected for the experiments without and with metabolic activation.

The mutation frequency of the cultures treated with concentrations of 8.7, 17.4, 34.8 and 69.5 µg Reaction product of DMO-THF, water and ethanol/mL culture medium was within the normal range of the solvent controls in both experiments without and with metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venipuncture from young (approximately 18 – 35 years of age), healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

CytoB (Cytochalasin B)

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was purchased from Trinova Biochem . S9 was collected from male rats.

Test concentrations with justification for top dose:

In the preliminary experiment without and with metabolic activation concentrations of 0.09, 0.28, 0.88, 2.78, 8.78, 27.8 and 55.6 mg Reaction product of DMO-THF, water and ethanol/mL medium were employed. Test item precipitation was noted at concentrations of 27.8 and 55.6 mg/mL in both experiments. In the experiments without and with S9 mix (24- or 4-hour exposure, respectively) cytotoxicity was noted at all tested concentrations. No relevant changes in pH or osmolality of the test item formulations at concentrations of 0.09 to 55.6 mg/mL medium were noted. Hence, 139 µg test item/mL (corresponding to 5 µg dimethoxytetrahydrofuran/mL medium) were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour or 24-hour exposure.

Vehicle / solvent:

purified water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

At least 500 cells per replicate cell culture (two cultures per concentration in the main study, one culture per concentration in the preliminary test) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
Thus, an RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis.
All slides, including those of the solvent controls, were independently coded before the microscopic analysis.
The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate. Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

Statistics:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined. Individual culture data were provided.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
•at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
•the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
•any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Species / strain:

lymphocytes: human peripheral lymphocytes

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

Conclusions:

In conclusion, under the present test conditions,Reaction product of DMO-THF, water and ethanol tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. The results for the vehicle controls were within historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.
Therefore, the test is considered valid.

Executive summary:

Tests without metabolic activation (4- and 24-hour exposure):

The micronucleus frequencies of cultures treated with the concentrations of 17.4, 34.8, 69.5 and 139 µg Reaction product of DMO-THF, water and ethanol/mL medium in the absence of metabolic activation (4- and 24-hour exposure) ranged from 4.5 to 8.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 139 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test, the following frequencies were observed: vehicle control: 5.5 or 6.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure, respectively. The vehicle result was within the historical control ranges.

Test with metabolic activation (4-hour exposure):

The micronucleus frequencies of cultures treated with the concentrations of 8.7, 17.4, 34.8 and 69.5 µg Reaction product of DMO-THF, water and ethanol/mL medium (4-h exposure) in the presence of metabolic activation ranged from 2.0 to 3.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 69.5 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test, a mean frequency of 3.0 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification necessary based on negative outcome of experimental in vitro studies.