Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 3 May 2007 and 20 June 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

EC Number:

217-062-1

EC Name:

Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]

Cas Number:

1732-96-3

Molecular formula:

C20H10O10

IUPAC Name:

2-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyloxy)ethyl 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxylate

impurity 1

Reference substance name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

EC Number:

209-008-0

EC Name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

Cas Number:

552-30-7

Molecular formula:

C9H4O5

IUPAC Name:

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

impurity 2

Reference substance name:

not assignable

IUPAC Name:

not assignable

Specific details on test material used for the study:

Substance referred to as RIKACID TMEG-100
Chemical name is given as 1,2,4-Benzenetricarboxylic acid, ester with 1,2-ethanediol, but substance is actually Ethylene bis[1,3-dihydro-1,3-dioxoisobenzofuran-5-carboxylate]
Lot No. 0020
CAS No. 71342-70-6
Appearance: Powder of white color
Purity: 99. 2%

Method

Target gene:

The histidine operon in the Salmonella typhimurium strains
The tryptophan operon in the Escherichia coli strains

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

The preliminary dose range-finding test was performed at 5 dose levels of the test substance, 312.5, 625, 1250, 2500 and 5000 μg/plate including negative and strain-specific positive control groups.
As a result of preliminary dose range-finding test, cytotoxicity and deposition were not observed in all strains in the absence and presence of metabolic activation.
According to the preliminary dose range-finding test, the highest dose in bacterial reverse mutation test was selected as 5000 μg/plate in the absence and presence of metabolic activation and was sequentially diluted by a geometric ratio of 2 to produce 4 additional lower dose levels for respective strains accompanying a negative and positive control.

Vehicle / solvent:

Through the preliminary solubility test to determine the solubility and dispersion properties of the test substance with reference to the information from the Sponsor, Dimethyl sulfoxide (DMSO, Lot No. : K33960231 504, Merck, USA) was chosen as a vehicle for this study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

5.0 µg/plate for strain TA98

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1.5 µg/plate for strains TA100 and TA 1535

Positive control substance:

sodium azide

Remarks:

Without S9-mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

80.0 µg/plate for strain TA 1537

Positive control substance:

9-aminoacridine

Remarks:

Without S9-mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1.0 µg/plate for strain WP2uvrA

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1.0 µg/plate for strains TA98 and TA100, 2.0 µg/plate for strains TA1535, TA 1537 and WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): > 1 x 10^9

Each 100 μL of test substance solutions, negative control and positive control was placed in glass tubes sterilized by dry oven. 500 μL of 0.1 mol/L sodium phosphate buffer (pH 7.4) for the treatment in the absence of metabolic activation or 500 μL of
S9 mix for the treatment in the presence of .metabolic activation was added and followed by 100 μL of pre-incubated bacterial suspension (1 x 10^9 cells/mL). This mixture was incubated in a shaking water bath at at 37 °C for 20 minutes. Then, 2 mL of warmed top agar was added and mixed thoroughly with a vortex mixer. Finally, the mixture solution was poured into the minimal glucose agar plate and the overlaid agars were allowed to solidify. After solidification of the top agar, the minimal glucose agar plate was cultured in the incubator (DK-LI020-P, Daiki scientific Co., LTD., Korea) at 37 °C for 48 hours whilst turned over. Duplicate plate of each dose levels of the test substance in the absence and presence of metabolic activation including negative and strain-specific positive controls was used.

Confirmation test
The microbial contaminations were examined as follows.
The highest dose of the test substance or S9 mix was placed in sterilized glass tubes and incubated in a shaking water bath at 37 °C for 20 minutes. 2 mL of warmed top agar was added and mixed thoroughly with a vortex mixer. And, the mixture solution was poured into the nutrient broth agar plate and the overlaid agars were allowed to solidify. The plate was cultured at 37 °C for 48 hours whilst turned over.


Observation and measurement
(1) Colony counting
After the cultivation, the number of revertant colonies was automatically counted by the colony counter (ProtoCOL, SINBIOSIS, UK). If automatic counting is considered not to be performed accurately due to the deposition of the test substance and the growth inhibition of the test strain, the number of revertant colonies was counted macroscopically.
Individual plate count and the mean number of revertant colonies for preliminary dose range-finding test and the bacterial reverse mutation test were recoi:ded at all dose levels of the test substance.
(2) Observation of background lawn
Background lawn was examined by the microscope.

Evaluation criteria:

The test substance was considered positive if the following conditions are met:
- The number of revertant colonies in each dose level was increased dose dependently at least twice as compared with that of the negative control groups in the absence and presence of metabolic activation.

Statistics:

Other than the calculations of mean value and standard deviation for the number of colonies, no statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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:

not applicable

Positive controls validity:

valid

Additional information on results:

The preliminary dose range-finding test was performed at 5 dose levels of the test substance, 312.5, 625, 1250, 2500 and 5000 μg/plate including negative and positive control groups.
As a result of preliminary dose range-finding test, cytotoxicity and deposition were not observed in all strains in the absence and presence of metabolic activation. According to the preliminary dose range-finding test, the highest dose in bacterial reverse mutation test was determined to 5 000 μg/plate in the absence and presence of metabolic activation and was sequentially diluted by a geometric ratio of 2 to produce 4 additional lower dose levels including a negative and positive control.
As a result, the number of revertant colonies in the test strains was not increased more than twice as compared with that of the negative control group in the absence and presence of metabolic activation. In addition, cytotoxicity and deposition were not observed in all strains in the absence and presence of metabolic activation. In the positive control, the number of the revertant colonies was significantly increased as compared with that of the negative control group.

Applicant's summary and conclusion

Conclusions:

In conclusion, the test substance, RIKACID TMEG-100 did not showed the mutagenic potential under the conditions of this study.

Executive summary:

This study was designed to examme the mutagenic potential of RIKACID TMEG-100 using Salmonella typhimurium (TA98, TAlOO, TA1535 and TA1537) and Escherichia coli (WP2uvrA(pKM101))strains.

The preliminary dose range-finding test was performed at 5 dose levels of the test substance, 312.5, 625, 1250, 2500 and 5000 μg/plate including negative and strain-specific positive control groups.

As a result of preliminary dose range-finding test, cytotoxicity and deposition were not observed in all strains in the absence and presence of metabolic activation.

According to the preliminary dose range-finding test, the highest dose in bacterial reverse mutation test was selected as 5000 μg/plate in the absence and presence of metabolic activation and was sequentially diluted by a geometric ratio of 2 to produce 4 additional lower dose levels for respective strains accompanying a negative and positive control.

As a result, the number of revertant colonies in all strains was not increased more than twice as compared with that of the negative control group in the absence and presence of metabolic activation.

The cytotoxicity and deposition were not observed in all strains in the absence and presence of metabolic activation.

In the positive control, the number of revertant colonies was increased as compared with that of the negative control group.

In conclusion, the test substance, RIKACID TMEG-100, did not show the mutagenic potential under the conditions of this study.