Dibenzyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A valid Ames test according OECD TG 471, a valid MNT according OECD TG 487 and a valid HPRT test according OECD TG 476 are available.
Several not assignable mutagenicity tests were published.

Dibenzyl oxide is not considered as a potential mutagenic in regards to negative results obtained in various mutation assay and chromosome aberration assay. No mutagenic effects have been observed in vitro or in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

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:

other: GLP guideline study according OECD TG 471

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of dibenzyl ether, CAS 103-50-4 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The histidine dependent strains are derived from Salmonella typhimurium strain LT2 through mutations in the histi¬dine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-mi¬nus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine, methyl methane sulfonate, 2-aminoanthracene

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

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:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test item dibenzyl ether, CAS 103-50-4 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate.

The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment II in all strains without S9 mix at 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with dibenzyl ether, CAS 103-50-4 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Conclusions:

Interpretation of results: negative

Executive summary:

This study was performed to investigate the potential of dibenzyl ether, CAS 103-50-4 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment II in all strains without S9 mix at 5000 µg/plate.

The plates incubated with the test item showed reduced background growth in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with dibenzyl ether, CAS 103-50-4 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, dibenzyl ether, CAS 103-50-4 is negative in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

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:

other: OECD Guideline for the Testing of Chemicals No. 487 “In vitro Mammalian Cell Micronucleus Test”, adopted 26 September 2014

Principles of method if other than guideline:

The test item dibenzyl ether, CAS 103-50-4, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1984.0 µg/ml in experiment I and II

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without metabolic activation: mitomycin, demecolcin; with metabolic activation: cyclophosphamide

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: In Exp. I in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In Exp. I in the presence of S9 mix and in Exp. II in the absence and presence of S9 mix concentrations showing clear cytogenetic damage

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test item dibenzyl ether, CAS 103-50-4, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 1984.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

No precipitation of the test item in the culture medium was observed.

No relevant influence on osmolarity or pH value was observed.

Phase separation was observed in Experiment I with and without S9 mix and in Experiment II without S9 mix at 69.1 µg/mL and above. In Experiment II phase separation was observed at 100.0 mg/mL and above at the end of treatment.

In Experiment I in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (54.5 % cytostasis). In Experiment I in the presence of S9 mix and in Experiment II in the absence and presence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item in Experiment I with S9 mix and Experiment II with and without S9 mix (0.00 – 1.60 % micronucleated cells) were close to the range of the solvent control values (0.05 – 1.55 % micronucleated cells) and within the range of the laboratory historical control data.

In Experiment I in the absence of S9 mix one single increase in the number of micronucleated cells (1.50 %), slightly above the range of the laboratory historical solvent control data (0.15 – 1.40 %) was observed after treatment with 22.6 µg/mL. Since this value is not statistically significant and no dose-dependency was observed, this finding has to be regarded as biologically irrelevant.

In both experiments, either Demecolcin (125.0 ng/mL), MMC (2.0 µg/mL) or CPA (12.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Conclusions:

Interpretation of results: negative

Executive summary:

The test item dibenzyl ether, CAS 103-50-4, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1984.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In Experiment I in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In Experiment I in the presence of S9 mix and in Experiment II in the absence and presence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.

However, in Experiment I in the absence of S9 mix one single increase in the number of micronucleated cells (1.50 %), slightly above the range of the laboratory historical solvent control data (0.15 – 1.40 %) was observed after treatment with 22.6 µg/mL. Since this value is not statistically significant and no dose-dependency was observed, this finding has to be regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, dibenzyl ether, CAS 103-50-4 is negative in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.

Reference 3

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)

Principles of method if other than guideline:

The study was performed to investigate the potential of dibenzylether to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. Two independent experiments were performed. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The mutation frequency was examined in both main experiments.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus in V79 cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: The cells have a stable karyotype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

exposure S9
period mix
concentrations in µg/mL
Experiment I
4 hours - 1.9 3.9 7.8 15.5 31.0 62.0
4 hours + 1.9 3.9 7.8 15.5 31.0 62.0
Experiment II
24 hours - 1.9 3.9 7.8 15.5 31.0 62.0
4 hours + 3.9 7.8 15.5 31.0 62.0 124.0

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Relevant cytotoxic effects occurred in Ep. I at 62 µg/ml and in Exp.II at 124 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test item dibenzyl ether, CAS 103-50-4 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical general experimental procedures.

In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The main experiments were evaluated at the following concentrations:

exposure period

Experiment I

4 hours -S9 mix: 3.9, 7.8, 15.5, 31.0, 62.0 [concentrations in µg/ml]

4 hours + S9 mix: 3.9, 7.8 15.5, 31.0 62.0 [concentrations in µg/ml]

Experiment II

24 hours -S9 mix: 3.9, 7.8, 15.5, 31.0, 62.0 [concentrations in µg/ml]

4 hours +S9 mix: 7.8, 15.5, 31.0, 62.0, 124.0 [concentrations in µg/ml]

Phase separation of the test item was noted in the first experiment at 62.0 µg/mL with and without metabolic activation. In the second experiment phase separation occurred at 62.0 and 124.0 µg/mL with and at 31.0 and 62.0 µg/mL without metabolic activation. So, the concentration range of both main experiments was limited by the solubility of the test item in culture medium.

Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 62.0 µg/mL in the absence of metabolic activation (4 hours treatment). In the second experiment cytotoxic effects as described above were noted at 124.0 µg/mL in the presence of metabolic activation (4 hours treatment).

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls.

The threshold of three times the mutation frequency of the corresponding solvent control was exceeded in the first culture of the first experiment at intermediate concentrations of 7.8, 15.5, and 62.0 µg/mL without metabolic activation. These effects however, were judged as biologically irrelevant as they were based upon a rather low solvent control of just 4.2 mutant colonies/10E6 cells. There was no dose dependent increase of the mutation frequency as indicated by a lacking statistical significance and the mutation frequency remained within the solvent control range of this study.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.2 up to 26.1 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.7 up to 25.2 mutants per 10E6 cells.

EMS (150 µg/mL) and DMBA (2.2 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

Interpretation of results: negative

Executive summary:

The study was performed to investigate the potential of dibenzylether to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the pre-experiment (1984 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, dibenzylether is negative in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Several not assignable in-vivo mutagenicity tests were published.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an Ames test dibenzyl ether, CAS 103-50-4 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Dibenzyl ether, CAS 103-50-4 was negative in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Three published not assignable Ames tests were also negative.

Additionally dibenzyl ether, CAS 103-50-4, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. Under the experimental conditions, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, dibenzyl ether, CAS 103-50-4 is negative in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.

In a further test the potential of dibenzylether to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was evaluated. Two independent experiments were performed. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The mutation frequency was examined in both experiments. Dibenzylether is negative in this HPRT assay.

Justification for classification or non-classification

All available genotoxicity tests, including a valid Ames test, a valid MNT in human lymphocytes and a valid HPRT were negative.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.