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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:
from 05 December 2006 to 06 March 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to international test guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Species / strain
Species / strain / cell type:
bacteria, other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and the Escherichia coli strain WP2 uvrA.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-Naphthoflavone induced rat liver S9 is used as the metabolic activation system.
Test concentrations with justification for top dose:
Concentrations tested in Experiment I (plate incorporation test, with and without metabolic activation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.
Concentrations tested in Experiment II (pre-incubation test, with and without metabolic activation): 33, 100, 333, 1000, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Solvent used: DMSO (dimethylsulfoxide)
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details below
Details on test system and experimental conditions:
POSITIVE CONTROLS:
Without metabolic activation: Sodium azide for strains TA1535 and TA100 (10µg/plate), 4-nitro-o-phenylene-diamine for strains TA1537 (50µg/plate) and TA98 (10 µg/plate), methyl methane sulfonate for strain WP2 uvrA (3µg/plate).
With metabolic activation:2-aminoanthracene 2.5µg/plate for strains TA1535, TA100, TA1537 and TA98 and 10 µg/plate for strain WP2 uvrA .

TEST SYSTEM:
Characterisation of the Salmonella typhimurium Strains and E. coli Strain:
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine 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-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
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. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Storage:
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures:
From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective agar:
The plates with the selective agar were obtained from E. Merck, D-64293 Darmstadt.

Overla agar:
The overlay agar contains per litre:
for Salmonella strains:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine×HCl×H2O*
12.2 mg Biotin*

for Escherichia coli:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
2.5 mg Tryptophan*

* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121° C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX:
Preparation of S9:
Phenobarbital/-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar HanIbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and -Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquotes of the supernatant are frozen and stored in ampoules at -80° C until use.

The protein concentration in the S9 preparation was 38.1 mg/mL in both experiments.

S9 Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10% v/v in the cultures. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

PRE-EXPERIMENT FOR TOXICITY:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

DOSE SELECTION:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I since no toxic effects were observed and 5000 Lg/plate were chosen as maximal concentration.

The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

EXPERIMENTAL PERFORMANCE:
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar.

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

DATA RECORDING:
The colonies are counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control,
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data,
- the positive control substances should produce a significant increase in mutant colony frequencies.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. This evaluation is based on literature data and scientific experience.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
bacteria, other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and the Escherichia coli strain WP2 uvrA.
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:
see summary tables below.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Experiment I

 

Metabolic activation

Test group

Dose level (µg/plate)

Revertant colony counts (mean)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without activation

DMSO

16

12

31

123

62

Untreated

18

15

30

127

65

Dimethyl 2-methyl glutarate

3 µg

20

12

22

113

66

10 µg

28

16

27

120

63

33 µg

25

10

26

134

63

100 µg

20

14

25

134

59

333 µg

20R

14R

24R

132R

62R

1000 µg

24R

13R

22R

106R

60R

2500 µg

26R

19R

28R

125R

51R

5000 µg

26R

15R

29R

132R

61R

NaN3

10 µg

1916

 

 

2122

 

4-NOPD

10 µg

 

 

347

 

 

4-NOPD

50 µg

 

101

 

 

1432

MMS

3.0 µL

 

 

 

 

 

With activation

DMSO

18

18

30

160

80

Untreated

18

17

44

156

88

Dimethyl 2-methyl glutarate

3 µg

33

17

30

165

61

10 µg

26

13

26

165

66

33 µg

29

15

29

160

78

100 µg

23

10

31

142

77

333 µg

26

12

41

145

82

1000 µg

14

17

28

148

61

2500 µg

19

14

34

129

53

5000 µg

25

10

34

143

54

2-AA

2.5 µg

392

225

1389

2569

 

2-AA

10.0 µg

 

 

 

 

215

 

Summary of Results Experiment II

  

Metabolic activation

Test group

Dose level (µg/plate)

Revertant colony counts (mean)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without activation

DMSO

13

11

23

115

53

Untreated

19

15

24

128

50

Dimethyl 2-methyl glutarate

33 µg

17

16

26

122

48

100 µg

9

16

27

112

43

333 µg

19

12

24

110

38

1000 µg

12

11

26

125

41

2500 µg

19

13

26

108

38

5000 µg

17

13

23

101

30

NaN3

10 µg

1801

 

 

2077

 

4-NOPD

10 µg

 

 

406

 

 

4-NOPD

50 µg

 

109

 

 

 

MMS

3.0 µL

 

 

 

 

307

With activation

DMSO

21

18

33

123

60

Untreated

17

24

37

137

67

Dimethyl 2-methyl glutarate

33 µg

16

24

37

128

54

100 µg

18

17

37

123

61

333 µg

16

18

38

127

57

1000 µg

18

24

31

119

48

2500 µg

14

20

42

122

48

5000 µg

18

20

39

130

42

2-AA

2.5 µg

243

122

905

1312

 

2-AA

10.0 µg

 

 

 

 

262

 

Key to Positive Controls:

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

 

Key to Plate Postfix Codes:

R: Reduced background growth

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Dimethyl 2-methyl glutarate is considered to be non-mutagenic in Ames test, with and without metabolic activation.
Executive summary:

This study was performed to investigate the potential of Dimethyl 2-methyl glutarate to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and 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:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 Lg/plate

In experiment I, reduced background growth was observed from 333 up to 5000 Lg/plate without metabolic activation in all strains used. No reduction of the background growth was observed in the presence of metabolic activation, and with and without metabolic activation in experiment II.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with dimethyl 2 -methyl glutarate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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.

This study is classified as acceptable. It satisfies the OECD 471 guideline requirement for a bacterial reverse mutation test.