Reaction mass of 2-(((1-(methacryloyloxy)propan-2-yl)oxy)carbonyl)benzoic acid and 2-((2-(methacryloyloxy)propoxy)carbonyl)benzoic acid

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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-methyl-2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen phthalate

EC Number:

265-951-8

EC Name:

1-methyl-2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen phthalate

Cas Number:

65859-45-2

Molecular formula:

C15H16O6

IUPAC Name:

2-{[2-(methacryloyloxy)-1-methylethoxy]carbonyl}benzoic acid

Test material form:

liquid

Specific details on test material used for the study:

Test Substance:
Identification: Methacryloxyisopropyl Acid Phthalate
Synonym: Reaction mass of 2-[({1-[(2-methylprop-2-enoyl)oxy]propan-2-yl}oxy)carbonyl]benzoic acid and 2-({2-[(2-methylprop-2-enoyl)oxy]propoxy}carbonyl)benzoic acid
Purity: 62.8%
Molecular Weight: 292.3 g/mol
Lot Number: YY00GCV000
Description: Pale whitish yellow clear liquid
Storage Conditions: Room temperature, protected from light
Receipt Date: 22 February 2017

Method

Target gene:

Histidine and tryptophan locus

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:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Confirmatory Mutagenicity Assay:
Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:

DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance. The formulation prepared in the solubility test also was adjusted using a correction factor of 1.59.

Vehicle: DMSO
CAS Number: 67-68-5
Supplier: Sigma-Aldrich
Lot Number: SHBB9319V
Purity: 99.95%
Expiration Date: Nov 2019

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2 aminoanthracene

Details on test system and experimental conditions:

Dose Formulations:
Dose formulations were adjusted to compensate for the purity (62.8%) of the test substance, using a correction factor of 1.59. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Positive Controls:
Positive controls plated concurrently with each assay are listed in Table 1. All positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water. All subdivided solutions of positive controls were stored at -10 to 30°C.

Dose Formulation Collection and Analysis:
Dose formulation samples were collected from the confirmatory mutagenicity assay. The solution sampling, one set of samples was shipped (dry ice) to The Dow Chemical Company (Midland, MI) for analysis and the backups were stored at (-10 to -30 °C) at BioReliance. Backup samples were discarded upon acceptance of the analytical results by the Study Director.

Materials and Methods:
Test System:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976). Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Solubility Determination:
DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance. The formulation prepared in the solubility test also was adjusted using a correction factor of 1.59.

Preparation of Tester Strain:
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 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 0.3x10^9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Identification of Test System:
Each plate was identified by the BioReliance study number and a code system to designate the treatment condition, dose level and test phase, as described in detail in BioReliance's Standard Operating Procedures.

Metabolic Activation System:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3738, Exp. Date: 05 Jan 2019; Lot No. 3737, Exp. Date: 04 Jan 2019) was purchased commercially 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 benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared on the day of use.
The Sham mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.

Frequency and Route of Administration:
The test system was exposed to the test substance via the preincubation methodology described by Yahagi et al. (1977).

Initial Toxicity-Mutation Assay to Select Dose Levels:
The initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test substance, in duplicate, in the presence and absence of Aroclor induced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.

Confirmatory Mutagenicity Assay:
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and six dose levels of the test substance, in triplicate, in the presence and absence of Aroclor induced rat liver S9.

Treatment of Test System:
Media used in the treatment of the test system were as indicated in Table 2.
To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in each assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.
One half (0.5) milliliter of S9 or sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle or test substance 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 20±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. When plating the positive controls, the test substance aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Scoring:
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in Table 3. As appropriate, colonies were enumerated either by hand or by machine.

Tester Strain Verification:
On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.

Evaluation criteria:

Criteria for a Valid Test:
The following criteria must be met for each assay to be considered valid:
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls. The mean revertants per plate must be within the ranges (inclusive) indicated in Table 4. Untreated controls, when part of the design, must also be within the ranges cited in Table 4.
To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10^9 cells/mL.
The mean of each positive control must exhibit at least a 3.0 fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Statistics:

Evaluation of Test Results:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
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 as specified below:

Strains TA98, TA1535, TA1537 and WP2 uvrA:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA100:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative if it is neither positive nor equivocal.

Results and discussion

Test results

Key result

Species / strain:

other: The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose levels tested.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Sterility Results:
No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Initial Toxicity-Mutation Assay:
The maximum dose of 5000 µg per plate was achieved using a concentration of 100 mg/mL and a 50.0 µL plating aliquot.
No precipitate was observed. Toxicity was observed at 5000 µg per plate with TA100 and TA1535 in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Confirmatory Mutagenicity Assay:
Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity was observed.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Dose Formulation Analysis:
The results obtained from the concentration and stability analysis of Methacryloxyisopropyl Acid Phthalate in the dose solutions provided measured concentrations that ranged from 96.9 to 109% of target concentrations. Precision of the analyzed dose solutions ranged from 0.5 to 11.2% relative percent difference (RPD).The vehicle control was reported as less than the lowest level of quantitation ( In order to evaluate stability of Methacryloxyisopropyl Acid Phthalate in DMSO during dosing, samples of the 0.300 and 100 mg/mL dose solutions were collected and frozen immediately prior to the start of the dosing (Dose Start). These stability samples were compared with the 0.300 and 100 mg/mL concentration samples that were used for dosing and then frozen (Dose End). Approximate two hour “Dose End” stability was confirmed at 99.1 and 95.1% of the “Dose Start” for the lowest and highest dose solutions, respectively.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Methacryloxyisopropyl Acid Phthalate did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of an exogenous metabolic activation system.

Executive summary:

The test substance,Methacryloxyisopropyl Acid Phthalate, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed at 5000 µg per plate with TA100 and TA1535 in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate Methacryloxyisopropyl Acid Phthalate was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.