Diisopentyl ether

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diisopentyl ether

EC Number:

208-857-4

EC Name:

Diisopentyl ether

Cas Number:

544-01-4

Molecular formula:

C10H22O

IUPAC Name:

3-methyl-1-(3-methylbutoxy)butane

Details on test material:

- Name of test material (as cited in study report): Diisopentyl ether
- Analytical purity: 99.2%
- Lot/batch No.: TAP 625
- Storage condition of test material: 2-8 °C

Method

Target gene:

his

Species / strain

Species / strain / cell type:

other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate

Test concentrations with justification for top dose:

- Experiment I (plate incorporation): 0, 50, 150, 501, 1504 and 5012 μg/plate in absence and presence of S9.
- Experiment II (pre-incubation method): 0, 313, 626, 1252, 2504 and 5008 μg/plate in absence and presence of S9.
- Experiment III (pre-incubation method): 0, 33, 100, 330, 1000, 3301 and 5003 μg/plate in absence and presence of S9.

Vehicle / solvent:

Ethanol

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Remarks:

TA97a, TA98 and TA102 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA1535 without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene

Remarks:

TA97a, TA100, TA102 and TA1535 with metabolic activation (S9)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 with metabolic activation (S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method (first experiment), preincubation method (second and third experiment)

DURATION
- Incubation period: 48 hours

NUMBER OF REPLICATIONS: four plates per strain and dose (with and without S9 mix)

DETERMINATION OF CYTOTOXICITY
- Method: background lawn

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

Both numerical significance and biological relevance were considered together in the evaluation. No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97

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

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

All concentrations (second and third experiment)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

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

Test results 4

Species / strain:

S. typhimurium TA 102

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

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

All concentrations (second and third experiment)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First experiment
The first experiment (plate incorporation method) showed no significant increase in the number of revertant colonies in any strain, both in the presence and in absence of metabolic activation. The first experiment is therefore considered negative for mutagenicity.

Second experiment
In the second experiment, performed to verify the results of the first experiment, a significant increase (>2 times the control value) in revertant colonies was observed in bacterial strains TA98 and TA1535 both in the presence and absence of metabolic activation (see table below). With strain TA98, an increase of about 7 times was observed at all concentrations tested, however without a dose response, both in the presence and in absence of metabolic activation. With strain TA1535, an increase of about 15 times was observed at all concentrations tested, however without a dose response, both in the presence and in absence of metabolic activation.

Third experiment
In the third experiment, performed to verify the results of the second experiment, a significant increase (>2 times the control value) in revertant colonies was observed in bacterial strains TA98 and TA1535 both in the presence and absence of metabolic activation (see table below). With strain TA98, an increase of about 7 times was observed at all concentrations tested, however without a dose response, both in the presence and in absence of metabolic activation. With strain TA1535, an increase of about 11 times was observed at all concentrations tested, however without a dose response, both in the presence and in absence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table: Number of revertants counted in strain TA98 and TA1535 in the bacterial reverse mutation test with diisopentyl ether (experiment 2)

	TA98 –S9	TA98 +S9	TA1535 –S9	TA1535 +S9
H2O	15 ± 2.2	14 ± 2.1	17 ± 2.4	13 ± 1.8
DMSO	15 ± 1.6	15 ± 3.1	17 ± 4.9	14 ± 3.0
ethanol	15 ± 1.9	16 ± 3.1	14 ± 3.0	14 ± 3.0
Positive control	361 ± 66	258 ± 36	234 ± 20	236 ± 18
5008 µg/plate	116 ± 2	117 ± 2	234 ± 3	247 ± 13
2504 µg/plate	113 ± 1	112 ± 3	250 ± 12	239 ± 14
1252 µg/plate	115 ± 2	113 ± 2	225 ± 10	223 ± 11
626 µg/plate	116 ± 2	113 ± 2	226 ± 12	203 ± 4
313 µg/plate	115 ± 3	112 ± 1	203 ± 8	197 ± 6

Table: Number of revertants counted in strain TA98 and TA1535 in the bacterial reverse mutation test with diisopentyl ether (experiment 3)

	TA98 –S9	TA98 +S9	TA1535 –S9	TA1535 +S9
H2O	16 ± 1	12 ± 3	16 ± 2	15 ± 5
DMSO	16 ± 1	12 ± 3	17 ± 3	17 ± 3
ethanol	12 ± 1.3	16 ± 2.4	15 ± 2.2	15 ± 2.1
Positive control	298 ± 39	267 ± 72	315 ± 18	323 ± 38
5003 µg/plate	113 ± 8	112 ± 13	167 ± 24	204 ± 22
3301 µg/plate	110 ± 4	105 ± 10	164 ± 20	208 ± 19
1000 µg/plate	111 ± 8	108 ± 12	142 ± 33	184 ± 13
330 µg/plate	106 ± 10	95 ± 7	167 ± 12	162 ± 19
100 µg/plate	100 ± 8	94 ± 4	168 ± 22	165 ± 11
33 µg/plate	94 ± 6	107 ± 11	156 ± 7	169 ± 11

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Under the conditions of the test, diisopentyl ether showed mutagenic effects towards Salmonella typhimurium, strains TA98 and TA1535.

Executive summary:

In a GLP compliant Ames test, performed according to OECD guideline 471, five Salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535) were used to test the mutagenic potential of diiosopentyl ether with and without metabolic activation. Diisopentyl ether was tested at concentrations up to and including 5000 μg/plate. Results from positive and negative controls were well within historical control ranges, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The first experiment (plate incorporation assay) showed no significant increase in the number of revertant colonies, both in the presence and in absence of metabolic activation. The first experiment is therefore considered negative for mutagenicity. These results were, however, not confirmed in the second independent repeat (pre-incubation method), in which increased numbers of revertant colonies were observed in two bacterial strains (TA98 and TA1535), however without a dose response relationship. The results of the second experiment were confirmed in a third experiment (pre-incubation method) and therefore it was concluded that diisopentyl ether showed mutagenic effects towards Salmonella typhimurium, strains TA98 and TA1535 under the conditions of the test.