COASOL 290 PLUS

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

14-May-2001 to 11-Feb-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl glutarate

EC Number:

214-277-2

EC Name:

Dimethyl glutarate

Cas Number:

1119-40-0

Molecular formula:

C7H12O4

IUPAC Name:

dimethyl glutarate

Details on test material:

Clear liquid
Purity: 99.61%
Batch Number: H931363-A
Supplier: DuPont

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 78-5000 ug/L (halving concentrations)
-S9: 315-5000 ug/L (5 halving concentrations) in Test 1 and Test 2
+S9: 400, 500, 600, 700, 800, and 900 ug/L in Test 1
+S9: 600, 800, 900, 1000, 1100 and 1200 ug/L 9 in Test 2

Vehicle / solvent:

DMSO

Controls

Controls 1

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control in absence of S9

Controls 2

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Positive contol in presence of S9

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Details on test system and experimental conditions:

Dimethyl glutarate was evaluated for its mutagenic potential in Chinese hamster ovary cells. The test system evaluated the potential of DMG to induce a forward mutation at the functionally hemizygous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. A prelimiary toxicity test was performed on cell suspensions in nutrient medium in presence and absence of S9. Cells were incubated for 20 hours at 37C in a humid atmosphere fo 5% CO2 in the air prior to exposure to the test substance on Day 1. Two mL of S9 or nutirent medium were added to the cell suspension followed by 120 ul of the test material or control solvent. The treated cell suspension were returned to the incubator for an additional 4 hour. Cell suspension were covered during incubation to prevent loss of test material.

At the end of treatment, the cells were harvested, washed and seeded on 3 60 mm dishes (200 cells per dish) with nutrient medium. The plates were incubated for at least 7 days, then growing colonies were fixed stained and counted. Cell survival was expressed as the plating efficiency relative to solvent controls. Concentrations for the main test were chosen based on these results.

For the main test, cell suspensions were prepared as described above except that duplicate cultures were used for each test concentration and positive control and quadruplicate culture for solvent controls. At least 5 serial dilutions were used at concentrations expected to span LC80 to LC0. Following exposure to test substance for 4 hours and plating as described above, 10x6 cells were seeded in a flask and incubated for 7 days to allow expression of the mutant phenotype. The cultures were subcultured on days 4 or 5 and after a total of 7 days were harvested by trypsinization (Day 8). the cells were harvested, washed and seeded on 3 60 mm dishes (200 cells per dish) with nutrient medium. The plates were incubated for at least 7 days, then growing colonies were fixed stained and counted. Cell survival was expressed as the plating efficiency relative to solvent controls. Concentrations for the main test were chosen based on these results.

Evaluation criteria:

Cytotoxicity - [Total colonies on plates (treated)/total conlonies on plates (untreated)] x 100

Plating efficiency (PE)- [Total number of viable colonies for each treated group/number of plates scored for colony formation x 200] x 100

Mutation frequency (MF) - [Total number of mutant colonies x 5*/PE x number ofuncontaminated plates] x 100

*5 represents a correction factor for the number of uncontaminated plates which normally equals 5, but may have been less

Criteria for a positive response were:
1) Demonstration of a statistically significant increase in mutation frequency
2) Evidence of a dose-relationship over at least two dose-levels
3) Demonstration of reproduibility in any increases in mutant frequency
4) Mean mutation frequency should fall outside the upper limit of historical control range of 20 mutants per million survivors with acorresponding survival rate of 20% or greater

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Test 1: Dimethyl Glutarate in the absence of S9

Concentration	Mean Relative Survival Day 1	Mean Relative Survival Day8	Mean Mutation Frequency
0	100	100	7.47
313	119	100	9.34
625	120	109	2.21
1250	93	94	6.92
2500	81	96	4.58
5000	78	95	8.60
EMS ¿Positive control	99	80	392.67*

*p<0.001

Test 2: Dimethyl Glutarate in the absence of S9

Concentration	Mean Relative Survival Day 1	Mean Relative Survival Day8	Mean Mutation Frequency
0	100	100	11.35
313	105	98	12.69
625	94	102	9.81
1250	104	99	11.88
2500	97	95	7.9
5000	83	102	20.06
EMS ¿Positive control	76	94	449.50*

*p<0.001

Test 1: Dimethyl Glutarate in the presence of S9

Concentration	Mean Relative Survival Day 1	Mean Relative Survival Day8	Mean Mutation Frequency
0	100	100	10.51
400	100	96	2.89
500	88	92	8.79
600	82	92	2.96
700	82	98	2.84
800	29	94	4.09
900	82	97	9.59
3-MC- positive control	100	94	202.53*

*p<0.001

Applicant's summary and conclusion

Conclusions:

Dimethyl glutarate did not demonstrate mutagenic potential in the in vitro HPRT cell mutation assay in the presence and absence of S9 metabolic activation.

Executive summary:

Dimethyl glutarate was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells.

The test was repeated more than twice, but several tests, particularly in the presence of S9 mix, were rejected, because there were insufficient mutagenicity data to claim a valid test. The results of four independent tests, two in the absence of exogenous metabolic activation (S9 mix), and two in the presence of S9 mix are reported.

In the preliminary toxicity test, cultures were exposed to 7 halving concentrations ranging from 78- 5000 µg/ml. In the absence of S9 mix, cultures were exposed to 5 halving concentrations ranging from 313-5000 µg/ml in both mutagenicity tests. In the presence of S9 mix, cultures were exposed to 400, 500, 600, 700, 800 or 900 µg/ml in the first mutagenicity test, and to 600, 800, 900, 1000, 1100 and 1200 µg/ml in the second. In all tests exposure was for 4 hours.

The preliminary toxicity test showed Day 1 relative cell survival of 65% after exposure to 5000 µg/ml in the absence of S9 mix and no survival after exposure to 1250 µg/ml or higher concentrations in the presence of S9 mix. In the main mutagenicity tests, slight toxicity was observed after exposure to dimethyl glutarate in tests in the absence of S9 mix, so that Day 1 relative cell survival was reduced to 78% or 83% by 5000 µg/ml in the first and second main test respectively. In the presence of S9 mix, Day1 relative cell survival was reduced to 82% by 900 µg/ml in the first main test, and to 27% by 1200 µg/ml in the second.

No significant increases in mutant frequency were observed in any of the tests either in the absence or presence of S9 mix, nor was there evidence of a concentration-response relationship. In all tests the positive control substances increased mutant frequencies significantly (P<0.001)

In the presence of S9 mix, dimethyl glutarate failed to reduce the Day1 cell survival to 10-20% of solvent control values, but it is considered that a sufficient number of high concentrations in a toxic range were tested to assess mutagenic potential adequately.

It was concluded that dimethyl glutarate did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay in either the absence or presence of S9 metabolic activation, under the experimental conditions described.