2-methylglutaric acid

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

Study period:

from 2014-06-09 to 2014-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study, OECD 471 compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

2013-05-06

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylglutaric acid

EC Number:

210-521-7

EC Name:

2-methylglutaric acid

Cas Number:

617-62-9

Molecular formula:

C6H10O4

IUPAC Name:

2-methylglutaric acid

Test material form:

solid: crystalline

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate with and without S9-mix.
Experiment 1 and 2: 52, 164, 512, 1600 and 5000 µg/plate with and without S9-mix.

Vehicle / solvent:

Milli-Q water (Millipore Corp., Bedford, MA., USA)

Controls

Untreated negative controls:

yes

Remarks:

Milli-Q water (Millipore Corp., Bedford, MA., USA)

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water (Millipore Corp., Bedford, MA., USA)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria

Source: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Merck) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Acros Organics).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Merck) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 32.4 – 38.9°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Any variation were evaluated and maintained in the raw data.

All the test were evaluated in triplicate.

Evaluation criteria:

Acceptability of the assay

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Data evaluation and statistical procedures

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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 2

Key result

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:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: the substance was soluble at all concentration tested
- Precipitation: no precipitation observed

RANGE-FINDING: yes
In the dose range finding test, 2-Methylglutaric acid was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. 2-Methylglutaric acid did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.


COMPARISON WITH HISTORICAL CONTROL DATA: yes
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table1: Dose range finding test: Mutagenic response of 2-Methylglutaric acid in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

 

Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain.
	TA100					WP2uvrA
	Without S9-mix
Positive control	985	±	8		(7.8)	1548	±	14		(45.5)
Solvent control	127	±	9			34	±	8
1.7	119	±	26		(0.9)	29	±	8		(0.9)
5.4	117	±	16		(0.9)	32	±	6		(0.9)
17	117	±	11		(0.9)	29	±	7		(0.9)
52	123	±	8		(1.0)	38	±	7		(1.1)
164	123	±	17		(1.0)	30	±	4		(0.9)
512	122	±	16		(1.0)	30	±	7		(0.9)
1600	126	±	10		(1.0)	33	±	3		(1.0)
5000	136	±	8	^n NP	(1.1)	38	±	9	^n NP	(1.1)
	With S9-mix^1
Positive control	998	±	263		(8.8)	113	±	14		(3.6)
Solvent control	113	±	8			31	±	5
1.7	124	±	29		(1.1)	42	±	11		(1.4)
5.4	118	±	21		(1.0)	33	±	6		(1.1)
17	121	±	19		(1.1)	41	±	3		(1.3)
52	100	±	13		(0.9)	29	±	5		(0.9)
164	115	±	12		(1.0)	40	±	6		(1.3)
512	125	±	10		(1.1)	41	±	11		(1.3)
1600	117	±	17		(1.0)	41	±	8		(1.3)
5000	112	±	16	^n NP	(1.0)	34	±	5	^n NP	(1.1)

 

^1	Plate incorporation assay (5% S9)
NP	No precipitate
n	Normal bacterial background lawn   The fold induction of the number of revertants compared to the solvent control group is presented between brackets

 

Table2: Experiment 1: Mutagenic response of 2-Methylglutaric acid in the Salmonella typhimurium reverse mutation assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimurium.
	TA1535					TA1537					TA98
	Without S9-mix
Positive control	730	±	21		(25.2)	495	±	22		(55.0)	969	±	67		(42.1)
Solvent control	29	±	9			9	±	5			23	±	6
52	28	±	2		(1.0)	7	±	3		(0.8)	23	±	4		(1.0)
164	33	±	5		(1.1)	12	±	7		(1.3)	25	±	5		(1.1)
512	26	±	6		(0.9)	12	±	5		(1.3)	22	±	6		(1.0)
1600	32	±	10		(1.1)	10	±	1		(1.1)	23	±	7		(1.0)
5000	33	±	9	^n NP	(1.1)	13	±	4	^n NP	(1.4)	21	±	9	^n NP	(0.9)
	With S9-mix^1
Positive control	321	±	16		(21.4)	410	±	40		(45.6)	1029	±	132		(29.4)
Solvent control	15	±	1			9	±	2			35	±	6
52	13	±	3		(0.9)	7	±	3		(0.8)	25	±	7		(0.7)
164	15	±	2		(1.0)	14	±	2		(1.6)	29	±	8		(0.8)
512	17	±	6		(1.1)	16	±	13		(1.8)	27	±	3		(0.8)
1600	18	±	2		(1.2)	11	±	1		(1.2)	31	±	3		(0.9)
5000	14	±	6	^n NP	(0.9)	7	±	4	^n NP	(0.8)	28	±	4	^n NP	(0.8)

 

^1	Plate incorporation assay (5% S9)
NP	No precipitate
n	Normal bacterial background lawn The fold induction of the number of revertants compared to the solvent control group is presented between brackets

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Based on the results of this study, it is concluded that Methylglutaric acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

Evaluation of the mutagenic activity of 2-Methylglutaric acid in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

2-Methylglutaric acid was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test substance was dissolved in Milli-Q water. In the dose range finding test, 2-Methylglutaric acid was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. 2-Methylglutaric acid did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the mutation assay.

Based on the results of the dose range finding test, 2-Methylglutaric acid was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, 2-Methylglutaric acid was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

2-Methylglutaric acid did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 2-Methylglutaric acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.