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Toxicological information

Genetic toxicity: in vitro

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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
Study period:
7 Sept - 12 Sept 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed study equivalent to current OECD Guidelines with quality inspection; E. coli not tested

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli not tested
GLP compliance:
no
Remarks:
study performed before GLP
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Colorless liquid

Method

Species / strain
Species / strain / cell type:
other: Salmonella thyphimurium TA98, TA100, TA1535, TA1537, TA1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.005, 0.01, 0.1, 1.0, 5.0, 10.0, 25.0 and 50.0 µL/plate
Vehicle / solvent:
Distilled water
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
Positive controls:
yes
Positive control substance:
other: without S9 mix: TA98/1538 - 2-Nitrofluorene; TA100/1535 - Sodium azide; TA1537 - 9-Aminocaridine; with S9 mix: 2-Anthramine
Details on test system and experimental conditions:
- Dosage Selection
All tests are run at a minimum of 4 concnetration. In the standard plate test, at least 6 dose levels of the test material, dissolved in a suitable solvent, are added to the test system. The standard test doses are 0.005, 0.01, 0.1, 1.0, 5.0 and 10.0 µL/plate.

- Mutagenicity Testing
the procedure is based on the Ames publication (1975) and performed as follows:

(1) Nonactivation Assay
To a sterile test tube placed in a 43°C water bath the following is added in order:
(a) 2.00 mL of 0.6% agar containing 0.05 mM Histidine and 0.05 mM Biotin
(b) 0.05 mL of a solution of Diethylene glycol dimethyl ether to give approximate dose
(c) 0.1 mL - 0.2 mL of indicator organisms
(d) 0.5 mL of 0.01M Phosphate buffer, pH 7.4
This mixture is swirled gently and then poured into minimal agar plates . After the top agar has set, the plates are incubed at 37°C for approx. 2 days. The number of his+ revertant colonies growing on the plates is counted.

(2) Activation assay
The activation assay is run concurrently with the nonactivation assay. The only difference is the addition of 0.5 mL of S9 mix (S9 supernatant from Aroclor 1254 induced adult male Sprague-Dawley rats) to the tubes in place of 0.5 mL of Phosphate buffer which is added in nonactivation assays. All other details are similar to the procedure for nonactivation assays.

- Control compounds
A negative control consisting of the solvent (distilled water) used for the test material is performed in all cases. For negative controls, step (b) of Nonactivation assays is replaced by 0.05 mL of the solvent. The negative controls are employed for each indicator strain and are performed in the absence and presence of S9 mix.
Specific positive control compounds known to revert each strain are also used in the assays:
(a) Nonactivation
- Sodium azide (1 µg/plate): TA100, TA1535
- 2-Nitrofluorene (10 µg/plate): TA98, TA1538
- 9-Aminocridine (50 µg/plate): TA1537

(b) Activation
- 2-Anthramine (2.5 µg/plate): all strains



Evaluation criteria:
(1) Strains TA1535, TA1537 and TA1538
If the solvent control value is within the normal range, a test material that produces a positive dose response over three times concentrations with the highest increase equal to three times the solvent control values will be considered to be mutagenic.

(2) strains TA98 and TA 100
If the solvent control vallue is within the normal range, a test material that produces a positive dose response over three concnetrations with the highest increase equal to twice the solvent control value for TA98 and TA 100 will be considered to be mutagenic.

(3) Reproducibility
If a test material produces a response in a single test that cannot be reproduced in additional runs, the initial positive test data lose significance.

The preceding criteria are not absolute, and other extenuating factors may enter into a final evaluation decision. However, these criteria will be applied to the majority of situations.
Statistics:
None

Results and discussion

Test results
Species / strain:
other: Salmonella th. TA98, TA100, TA1535, TA1537 and 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of mean number of revertants in Salmonella typhimurium with and without metabolic activation (negative and positive controls)

Concentration

TA 98

TA 100

TA1535

TA 1537

[µL/plate]

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

0

25

42

121

121

21

13

13

21

0.05

24

36

126

104

10

8

19

16

0.01

22

37

128

98

15

10

13

11

0.1

34

36

109

109

15

10

15

16

1.0

25

27

127

130

16

6

7

14

5.0

10.0

25.0

50.0

27

30

35

36

35

32

40

45

130

137

173

173

110

146

109

146

15

18

-

-

14

5

-

-

9

10

-

-

9

14

-

-

 

 

 

 

 

 

 

 

 

Positive:

 

 

 

 

 

 

 

 

9-Aminoacridine

 

 

 

 

 

 

518

 

Sodium azide

 

 

1433

 

1119

 

 

 

2-Nitrofluorene

1170

 

 

 

 

 

 

 

2-Anthramine

 

2109

 

2631

 

592

 

499

 

Concentration

TA 1538

 

 

 

[µL/plate]

- S9 mix

+ S9 mix

 

 

 

 

 

 

0

21

23

 

 

 

 

 

 

0.05

14

29

 

 

 

 

 

 

0.01

11

19

 

 

 

 

 

 

0.1

19

22

 

 

 

 

 

 

1.0

14

33

 

 

 

 

 

 

5.0

10.0

25.0

50.0

19

20

-

-

36

22

-

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive:

 

 

 

 

 

 

 

 

9-Aminoacridine

 

 

 

 

 

 

 

 

Sodium azide

 

 

 

 

 

 

 

 

2-Nitrofluorene

1367

 

 

 

 

 

 

 

2-Anthramine

 

 

 

 

 

 

 

 

 

 

1876

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Diethylene glycol dimethyl ether was not genotoxic in any of the bacterial reverse mutation assays (Ames test) performed with Salmonella th. TA98, TA100, TA1535, TA1537 and TA1538.
Executive summary:

Diethylene glycol dimethyl ether was examined for mutagenic activity in Salmonella th. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats.The dose range employed for the evaluation of this compound was from 0.005 to 50 µL/plate.

Diethylene glycol dimethyl ether exhibited slight toxicity in the activation assay with the strain TA1535 at 10 µL/plate.

The results of the tests conducted on Diethylene glycol dimethyl ether in the presence and absence of metabolic activation were all negative.