2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the O.E.C.D. test guideline 471 with GLP compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,6-Tetrahydrophthalic anhydride, oligomeric reaction products with 2,2-dimethylpropane-1,3-diol

EC Number:

500-091-1

EC Name:

1,2,3,6-Tetrahydrophthalic anhydride, oligomeric reaction products with 2,2-dimethylpropane-1,3-diol

Cas Number:

36621-20-2

Molecular formula:

(C8H8O3.C5H12O2)x

IUPAC Name:

1,2,3,6-Tetrahydrophthalic anhydride, oligomeric reaction products with 2,2-dimethylpropane-1,3-diol

Constituent 2

Reference substance name:

1,3-Isobenzofurandione, 3a,4,7,7a-tetrahydro-, polymer with 2,2-dimethyl-1,3-propanediol

IUPAC Name:

1,3-Isobenzofurandione, 3a,4,7,7a-tetrahydro-, polymer with 2,2-dimethyl-1,3-propanediol

Details on test material:

As per IUCLID Sections 1.1. 1.2. and 4.1.

Method

Target gene:

Histidine and trytophan synthesis genes.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver derived S9 fraction.

Test concentrations with justification for top dose:

15, 50, 150, 500, 1500 and 5000 µg per plate in all tester strains.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2 aminoanthracene, CAS No. 613-13-8 with S9 activation.

Remarks:

The other positive controls without S9 activation are tester strain specific and are: 2-nitrofluorene, sodium azide, 9-aminoacridine and methyl methanesulfonate.

Details on test system and experimental conditions:

Salmonella tester strains were from Dr. Bruce Ames’ Master cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm at 37±2°C approximately 12 to 14 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter.

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize at least two promutagens to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose 6 phosphate, 4 mM ß nicotinamide adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4.

The test system was exposed to the test article via the preincubation methodology described by Yahagi et al. (1977). Minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L histidine, D biotin and L tryptophan solution to a final concentration of 50 µM each. Bottom agar was Vogel Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

One half (0.5) milliliter of S9 or sham mix, 100 µL of tester strain and 50 µL of vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 60±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar plates. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2 C.

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 are judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0 times the mean vehicle control value. Data sets for tester strains TA98, TA100 and E. coli WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

Statistics:

None

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

When tested up to the high concentration of 5000 ug/plate, there was no increase in reverse mutation frequency with and without a rat liver derive S9 metabolic activation preparation in any of the bacterial tester strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without S9 metabolic activation.

The test substance is not mutagenic when tested up to 5000 ug/plate in the Bacterial mutation test.

Executive summary:

The test substance, 1,2,3,6 -Tetrahydrophthalic anhydride, oligomeric reaction products with 2,2 -dimethylpropane-1,3 -diol, when tested in an O.E.C.D. test guideline 471 bacterial mutation assay by the preincubation method was not mutagenic under the conditions of the study.