Dioctyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

genetic toxicity in vitro (Ames test): negative
genetic toxicity in vitro (Mouse lymphoma test): negative
genetic toxicity in vitro (chromosome aberration): 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

Study period:

2014

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Cetiol OE
- Physical state: liquid
- Lot/batch No.: DENNECKE00090 Charge 0011189321

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

33µg-5000 µg/plate (standard plate test), 33µg-5000 µg/plate (preincubation test)

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other:

Remarks:

With S9 mix: 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%) (60 μg/plate, dissolved in DMSO); Without S9 mix: 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%) (5 μg/plate, dissolved in DMSO)

Details on test system and experimental conditions:

For testing, a deep-frozen (-70°C to -80°C) bacterial culture (E. coli WP2 uvrA) is thawed at room temperature, and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. Fresh cultures of bacteria should be grown up to late exponential or early stationary phase of growth (approximately 109 dells per mL). This culture grown overnight is kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strain mentioned is in accordance with the current scientific recommendations for the conduct of this assay. The Escherichia coli strain was obtained from Merck KGaA, Darmstadt, Germany (09 Sep 1991).

Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

E. coli WP2 uvrA is checked for UV sensitivity. Tryptophan auxotrophy is checked in each experiment via the spontaneous rate.

The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALACapproved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and -naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 30 - 70%. The day/night rhythm was 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours). Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9 000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (so-called S9 fraction) were stored at -70°C to -80°C.

The S9 mix was prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 volume of S9 fraction is mixed with 9 volumes of S9 supplement (cofactors). This preparation, the so-called S9 mix, was kept on ice until used.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with
benzo(a)pyrene.

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for the tester strain
• The sterility controls revealed no indication of bacterial contamination
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling of the spontaneous mutation rate in the tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for the tester strain was within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the Standard plate test and in the 2nd-PIT, test substance precipitation was found from about 2500 μg/plate onward with and without S9 mix.

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in several experiments carried out independently of each other (standard plate test and preincubation assay). However, the slight increase in the number of trp+ revertants observed in the preincubation test without S9 mix was not reproduced in a repeat experiment, and so these findings have to be regarded as biological irrelevant. Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative control was within or marginally below the range of the historical negative control data for the tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control

data.

Conclusions:

Dioctyl ether is not considered to be mutagenic in this test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only 4 strains tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Di-n-Octylether
- Physical state: liquid
- Analytical purity oder Active matter: 99.9%
- Lot/batch No.: 7/91

Target gene:

Salmonelle typhimurium LT 2

Species / strain / cell type:

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

Remarks:

and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

tween 80/bidest. water

Species / strain:

other: TA 1535, TA 1537, TA 1538, TA 98 and 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

Conclusions:

Dioctyl ether is not considered to be mutagenic in this test.

Executive summary:

The test substance was tested with the bacterial tester strains S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98 in the presence and absence of enzymes obtained from the livers of Aroclor 1254 pre-treated rats (S9 mix). Suspensions of the test compounds were prepared in Tween 80/bidest.water and diluted with bidest water just before use. The following concentrations were tested: 8, 40, 200, 1000 and 5000µg/plate. The test subsatnce did not induce reverse mutations in the tested strains of S. typhimurium in this bacterial mutagenicity test, neither with nor without metabolic activation by S9 mix. This result was confirmed in a repetitioustest.Thus, no indications exist for the test substance to have any potential to induce gene mutations.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): SAT 001193
- Physical state: liquid
- Analytical purity/active matter: 99%
- Lot/batch No.: CB00940009

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

2.5, 5 and 10 µg/mL

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

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

Conclusions:

The substance is not clastogenic in vitro.

Executive summary:

The test substance dissolved in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in-vitro in two independent experiments according to OECD guideline 473. The following study design was performed:

 

without S9-mix				with S9-mix
	exp. I	exp. II		exp. I	exp. II
Exposure period	4 h	18 h	28 h	4 h	4 h
Recovery	14 h	-	-	14 h	24 h
Preparation interval	18 h	18 h	28 h	18 h	28 h

 

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase cells were scored for structural chromosome aberrations, except for the positive control in experiment II (without S9, interval 28 h) where only 50 metaphase cells were scored. The highest applied concentration in the pre‑test on toxicity (2500 µg/ml; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Due to strong toxicity in the absence of S9 mix a second range finding pre-test was performed. Test item concentrations between 0.2 and 20 µg/ml were applied in the absence of S9 mix only. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment. No increase in the frequencies of polyploid metaphases was found after treatment as compared to the frequencies of the controls. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in-vitro.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): C-SAT 080029
- Physical state: liquid
- Analytical purity/active matter: 99.1%
- Lot/batch No.: CE72530027

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1.56 to 25 µg/mL

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 3-Methylcholanthrene (with S9 mix)

Species / strain:

mouse lymphoma L5178Y cells

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:

According to the evaluation criteria for this assay, these findings indicate that dioctyl ether tested up to a cytotoxic concentration of 25 µg/mL in the absence and presence of metabolie activation did neither induce mutations nor had any chromosomal aberration potential.

Executive summary:

The test substance was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK+/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out twice. Acetone was used as solvent for the test item and as the negative reference item. In the preliminary experiment without and with metabolic activation pronounced cytotoxicity (decreased survival) was noted from a concentration of 25 μg/mL onwards. Hence, in the experiments without or with metabolic activation the concentration range of 1.56 to 25 μg/mL was used. In the main study, cytotoxicity (decreased survival) was noted at the top concentration of 25 μg/mL immediately after treatment (plating efficiency step 1) in the experiments without and with metabolic activation. Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene in the presence of exogenous metabolic activation. The mean values of mutation frequencies of the solvent controls ranged from 21.43 to 21.98 per 106 clonable cells in the experiments without metabolic activation, and from 21.19 to 21.50 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with the test substance ranged from 19.21 to 36.96 per 106 clonable cells (3 hours exposure) and 19.55 to 33.08 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and 21.14 to 37.97 per 106 clonable cells (3 hours exposure, first assay) and 19.94 to 36.31 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the solvent controls and, hence, no mutagenicity was observed according to the criteria for assay evaluation. In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.50 to 1.75 for the test substances treated cells and from 0.88 to 1.59 for the solvent controls. Under the present test conditions,the test substance, tested up to a cytotoxic concentration of 25 µg/mL in the absence and presence of metabolie activation in two independent experiments, was negative with respect to the mutant frequency in theLK5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, the test substance also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that the test substance tested up to a cytotoxic concentration of 25 µg/mL in the absence and presence of metabolie activation did neither induce mutations nor had any chromosomal aberration potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames assay

The test substance was tested with the bacterial tester strains S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98 in the presence and absence of enzymes obtained from the livers of Aroclor 1254 pre-treated rats (S9 mix). Suspensions of the test compounds were prepared in Tween 80/bidest.water and diluted with bidest water just before use. The following concentrations were tested: 8, 40, 200, 1000 and 5000µg/plate. The test subsatnce did not induce reverse mutations in the tested strains of S. typhimurium in this bacterial mutagenicity test, neither with nor without metabolic activation by S9 mix. This result was confirmed in a repetitioustest.Thus, no indications exist for the test substance to have any potential to induce gene mutations.

Based on the reliability and relevance, this study has been used as key study.

Furthermore, the test substance was tested for its mutagenic potential in the Escherichia coli strain WP2 uvrA . Under the experimental conditions of this study, the test substance is also not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Mouse lymphoma assay:

The test substancewas assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK+/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out twice. Acetone was used as solvent for the test item and as the negative reference item.In the preliminary experiment without and with metabolic activation pronounced cytotoxicity (decreased survival) was noted from a concentration of 25 μg/mL onwards. Hence, in the experiments without or with metabolic activation the concentration rangeof 1.56 to 25 μg/mL was used. In the main study, cytotoxicity (decreased survival) was noted at the top concentration of 25 μg/mL immediately after treatment (plating efficiency step 1) in the experiments without and with metabolic activation. Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene in the presence of exogenous metabolic activation.The mean values of mutation frequencies of the solvent controls ranged from 21.43 to 21.98 per 106 clonable cells in the experiments without metabolic activation, and from 21.19 to 21.50 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with the test substanceranged from 19.21 to 36.96 per 106 clonable cells (3 hours exposure) and 19.55 to 33.08 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and 21.14 to 37.97 per 106 clonable cells (3 hours exposure, first assay) and 19.94 to 36.31 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the solvent controls and, hence, no mutagenicity was observed according to the criteria for assay evaluation.In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.50 to 1.75 for the test substancestreated cells and from 0.88 to 1.59 for the solvent controls.Under the present test conditions,the test substance, tested up to a cytotoxic concentration of 25 µg/mL in the absence and presence of metabolie activation in two independent experiments, was negative with respect to the mutant frequency in theLK5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, the test substancealso did not exhibit clastogenic potential at the concentration-range investigated.According to the evaluation criteria for this assay, these findings indicate that the test substancetested up to a cytotoxic concentration of 25 µg/mL in the absence and presence of metabolie activation did neither induce mutations nor had any chromosomal aberration potential.

Based on the reliability and relevance, this study has been used as key study.

Chromosome aberration:

The test substancedissolved in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in-vitro in two independent experiments according to OECD guideline 473. The following study design was performed:

 

without S9-mix				with S9-mix
	exp. I	exp. II		exp. I	exp. II
Exposure period	4 h	18 h	28 h	4 h	4 h
Recovery	14 h	-	-	14 h	24 h
Preparation interval	18 h	18 h	28 h	18 h	28 h

 

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase cells were scored for structural chromosome aberrations, except for the positive control in experiment II (without S9, interval 28 h) where only 50 metaphase cells were scored. The highest applied concentration in the pre‑test on toxicity (2500 µg/ml; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Due to strong toxicity in the absence of S9 mix a second range finding pre-test was performed. Test item concentrations between 0.2 and 20 µg/ml were applied in the absence of S9 mix only. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment. No increase in the frequencies of polyploid metaphases was found after treatment as compared to the frequencies of the controls.In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in-vitro.

Based on the reliability and relevance, this study has been used as key study.

Justification for classification or non-classification

Based on results of the key studies the substance does not need to be classified according to GHS (Regulation (EU) 1272/2008).