Dimethyl ether

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:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

S. typhimurium tester strain TA 97a using ICR 191 as a positive control was used as a substitute for strain(s) TA 97 and/or TA 1537; E.coli WP uvrA (pKM 101) was the only E.coli tester used. (validity of the study not affected-as reported)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS Guidelines (Subpart H, 40CFR Part 799.9510, 1989)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Directive 92/69/EEC, EEC Method B. 12

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl ether

EC Number:

204-065-8

EC Name:

Dimethyl ether

Cas Number:

115-10-6

Molecular formula:

C2H6O

IUPAC Name:

dimethyl ether

Details on test material:

- Purity: 99.8%

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 97a, TA 98, TA 100 and TA 1535 and Escherichia coli strain WP2uvrA(pKM101)

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver S9

Test concentrations with justification for top dose:

First trial: 20%, 30%, 40%, 50%, 75%
[actual test substance conc: 16%, 22.5%, 31.4%, 35.4% , 40.1% in the absence of S9 and 18.9%, 25.7%, 32.6%, 40.2% , 60.7% in the presence of S9]
Second trial: 20%, 30%, 40%, 50%, 75% [actual test substance conc: 14.7%, 22.8%, 30.1%,, 42.9%, 61.3% in the absence of S9 and 15.5%, 22.9%, 32.8%, 48.4%, 68.3% in the presence of S9)

Vehicle / solvent:

Air

Controls

Untreated negative controls:

yes

Remarks:

filtered house-line air

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene;N-Ethhyl-N-nitro-N-nitrosoguanidine;sodium azide; ICR 191 Acridine mutagen; 9,10-dimethyl-1,2-benzanthracene; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: top agar plate incorporation method for gases (2 mL, 0.6% agar w/v, 0.6% NaCl w/v)

DURATION - Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 10E8 (0.1 mL culture)

The study consisted of 2 independent trials with and without a metabolic activation system. A third trial, utilizing S. typhimurium TA98 with S9 was used to confirm the results. Three replicates were plated for each tester strain, test concentration and condition. Treatments with activation were conducted by adding 0.1 mL of positive control (positive control only), 0.5 mL of S9 mix and 0.1 mL (containing 10E8 bacteria) of an overnight culture to 2 mL of top agar supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin for S. typhimurium or 0.05 mM L-tryptophan for the E. coli strain. These components were briefly mixed and poured onto a minimal glucose agar plate. Treatments in the absence of the metabolic activation system were identical to those in the presence of an exogenous metabolic activation system with the exception that 0.5 mL of sterile buffer was used as a replacement for the 0.5 mL volume of the exogenous metabolic activation system.

Plates were exposed to dilutions of the test gas after being placed on stainless steel racks specially designed to fit in 6-L glass chambers. Chambers were equipped with Teflon stopcocks and Viton o-ring gaskets. As gases, the test substance and filtered air flows were regulated using individual rotameters, and mixed prior to entry into chambers. A flow rate of approximately 6 L/minute for 5 minutes was used to create 5 volume changes to occur within the chambers to ensure homogeneous concentrations. Chambers were closed and 2-4 samplings of each chamber were taken and analyzed by GC to determine initial concentration of the test substance. Chambers were placed into an incubator at approximately 37°C for approximately 48 hours. Chambers were again sampled and analyzed to determine the ending test substance concentrations. The chambers were flushed with at least 5 chamber volumes of filtered air. The plates were then removed and refrigerated until evaluation.

DETERMINATION OF CYTOTOXICITY
Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation.

Evaluation criteria:

Any individual assay must have included a negative and positive control and at least three concentration levels per test substance for each tester strain and condition. A data point was excluded from analysis when acceptability criteria were not met. Such criteria are clearly defined as: tester strain integrity; tester strain titer; positive control values; revertant toxicity; rejection of plates, concentration levels, or assay.

Positive and negative classification are clearly stated in the classification criteria and are based on number of revertants in strains compared to the concurrent negative control. The test substance was classified as positive if the mean number of revertants in any strain (except TA1535) at any test substance concentration was at least two times greater than the mean number of revertants of the concurrent negative control, and there was a concentration-related increase in the mean number of revertants per plate in that same strain. For strain TA1535, there must have been a mean number of revertants that was at least three times greater than the mean of its concurrent negative control and a concentration-related increase in the mean number of revertants per plate. A test substance was classified as negative if all positive classification criteria for all strain were not met. Results not meeting criteria for either positive or negative classification were evaluated using scientific judgment and experience and may have been reported as equivocal.

Statistics:

Data for each tester strain were evaluated independently: the mean number of revertants and the standard deviation at each concentration in the presence of and absence of metabolic activation were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance related precipitate was observed at any concentration with any strain in any trial.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data are reported for tester strains used in the report study. Data reported are based on studies conducted in the period 1996-1998. All control solvents or diluents, metabolic activation based on Aroclor-induced rat liver S9 and all forms of study modification are included. The mean number of revertants observed in the negative control for each strain was within the prescribed acceptable historical control range.

Results:
In the first trial, there was an apparent leakage in one chamber at the high dose without S9. The other chamber concentrations decreased approximately 50% from the mean at 0-hr and 48-hr. Test substance-related toxicity, as evident by a concentration dependent reduction in mean revertant colonies per plate, was observed in all tester strains except S. typhimurium strains TA100 and TA1535 without S9. No evidence of mutagenicity was observed.

In a second trial, test substance related toxicity, as evidenced by a concentration-related reduction in the mean number of revertants per plate, was observed with all tester strains in the presence and absence of the metabolic activation system. The chamber concentrations were again decreased approximately 36% after 48 hours. Due to an equivocal response in S. typhimurium TA98 in the presence of S9 in trial 2, tester strain TA98 with a metabolic activation system was repeated. At one concentration level of strain TA98, a doubling of the mean revertant plate count was observed compared to the mean of the concurrent negative control. There was no concentration-related increase in the tester strain, and therefore the data was judged inconclusive.

A third trial was conducted with only TA98 with S9 at target concentrations of 45%, 55%, and 65%, again with a mean decrease in chamber concentration of 22% with no apparent chamber leakage. As this repeat of trial 2 was negative, again with evidence of toxicity, the conclusion that trial 2 also exhibited no evidence of mutagenicity was affirmed.

Any other information on results incl. tables

 

Trial #1 (Without Activation)	(Histidine) + Revertants Per Plate
	Strain TA97a	Strain TA98	Strain TA100	Strain TA1535	Strain E. coli pKM101
DME (target concentration)
0 %	137	18	123	12	187
20 %	122	16	127	16	136
30%	108	14	121	15	146
40%	88	10	101	13	68
50%	93	4	90	16	79
75%	71	8	108	14	98
ICR 191 (2 ug/plate)	2208
2NF(25 ug/plate)		1215
NAAZ(2 ug/plate)			658	588
ENNG(2 ug/plate)					1493

 

Trial #1 (With Activation)	(Histidine) + Revertants Per Plate
	Strain TA97a	Strain TA98	Strain TA100	Strain TA1535	Strain E. coli pKM101
DME (target concentration)
0 %	172	20	167	18	167
20 %	178	18	152	17	179
30%	149	15	139	13	146
40%	173	13	138	15	98
50%	140	12	129	9	83
75%	63	4	115	9	19
DMBA(20 ug/plate)	1076
2AA(2 ug/plate)		1206	1839	287	1416

 

 

Trial #2 (Without Activation)	(Histidine) + Revertants Per Plate
	Strain TA97a	Strain TA98	Strain TA100	Strain TA1535	Strain E. coli pKM101
DME (target concentration)
0 %	145	30	133	23	202
20 %	130	26	136	24	171
30%	140	19	133	22	164
40%	92	38	112	24	109
50%	33	31	100	26	33
75%	2	0	31	12	4
ICR 191(2 ug/plate)	2695
2NF(25 ug/plate)		1441
NAAZ(2 ug/plate)			1008	872
ENNG(2 ug/plate)					1714

 

Trial #2 (With Activation)	(Histidine) + Revertants Per Plate
	Strain TA97a	Strain TA98	Strain TA100	Strain TA1535	Strain E. coli pKM101
DME (target concentration)
0 %	178	23	152	21	210
20 %	194	23	151	17	207
30%	148	25	144	33	159
40%	161	25	133	16	102
50%	32	48	110	37	26
75%	0	13	45	12	0
DMBA(20 ug/plate)	1503
2AA(2 ug/plate)		1192	1778	265	1798

 

 

Trial #3 (With Activation)
	Strain TA98
DME
0 %	25
45%	15
55%	8
65%	4
2AA
25 ug/plate	1043

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, no evidence of mutagenic activity was detected in either trial with the substance. Based on the findings, the test substance was concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli, with and without metabolic activation. The study and the conclusions fulfil the quality criteria (validity, reliability, repeatability).

Executive summary:

The test substance was evaluated in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2uvrA (pKM101) in the presence and absence of an exogenous metabolic activation system at target concentrations of 0, 20, 30, 40, 50, and 75%.

Under the conditions of the study, no evidence of mutagenic activity was detected in either trial with the substance. Based on the findings, the test substance was concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli, with and without metabolic activation.