2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, reaction products with phosphorus oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available results fromin vitrogenotoxicity assays with the test substance, HEMA-phosphate, as well as the read across substance, test substance is not considered to have mutagenic or clastogenic potential.

Link to relevant study records

Reference

Reference 1

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 March 10, 2004 to March 26, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name: PARAD Substance 139.
- Batch: 31104251.
- Molecular formula: C6H10O3.xH3O4P.
- Molecular weight: ~250.
- CAS number: 52628-03-2.
- Description: Slightly yellowish viscous liquid.
- Purity: Not available (mixture).
- Storage: At room temperature in the dark.
- Stability under storage conditions: Stable.
- Expiry date: December 31, 2004.

Target gene:

TA1537: hisC3076
TA98: hisD3052
TA1535: hisG46
TA100: hisG46

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The Salmonella typhimurium strains were 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 (ref.1). The strain was 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).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate S9 fraction

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1,000, 3,330 and 5,000 μg/plate
Mutation test:
Experiment I: 3, 10, 33, 100, 333, 1,000, 3,330 and 5,000 μg/plate
Experiment II: 100, 333, 1,000, 3,330 and 5,000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Stability in vehicle: Stable

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

TA98, TA100, TA1535, TA1537, WP2uvrA

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation (+S9 mix)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

TA1535

Positive control substance:

sodium azide

Remarks:

Without metabolic activation (-S9 mix)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation (-S9 mix)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

TA98

Positive control substance:

other: daunomycin

Remarks:

Without metabolic activation (-S9 mix)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

TA100

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (-S9 mix)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

WP2uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation (-S9 mix)

Details on test system and experimental conditions:

Cell culture:
Preparation of bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth 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 (10^9 cells/mL). Freshly grown cultures of each strain were used for a test.

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

Top agar:
Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride 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 the dark at 37±1°C. The temperature was monitored during the experiment.

Dose range finding test:
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1,000, 3,330 and 5,000 μg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of test substance used in the subsequent mutation assay was 5 mg/plate.

Mutation assay:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments. Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in dimethyl sulfoxide and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37±1°C for 48 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony counting:
The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50,000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

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 background 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:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
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) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) 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.

Key result

Species / strain:

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

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:

Dose range finding test:
Precipitate:
The test substance did not precipitate in the top agar. Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period in both tester strains.

Toxicity:
To determine the toxicity of test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. A reduction in the number of revertants equal to the minimal value of the historical control data range is not considered biologically relevant and, therefore, will not be considered cytotoxic. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.


Mutation assay:
Based on the results of the dose range finding test, test substance was tested up to concentrations of 5,000 μg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Test substance did not precipitate in the top agar. Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period.

Toxicity:
In both mutation assays, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Number of revertants:
All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

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

Conclusions:

Under the study conditions, the test substance was not found to be mutagenic either in the presence or absence of metabolic activation.

Executive summary:

A study was conducted to test the mutagenic potential of the test substance in a bacterial reverse mutation assay performed according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study included a preliminary concentration range finding test, an initial mutation test and a confirmatory mutation test. Based on the results of the dose range finding study, the substance was tested in the first mutation assay at a concentration range of 100 to 5,000 μg/plate in the absence and presence of 5% (v/v) S9-mix in Salmonella typhimurium strains TA 98, TA1535 and TA1537. In the second mutation assay, the substance was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in Salmonella typhimurium tester strains TA 98, TA100, TA1535, TA1537 and E. coli strain WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the five tester strains in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. The negative and strain-specific control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the findings of the study, it can be concluded that the substance was not mutagenic either in the presence or absence of metabolic activation (Verspeek-Rip CM, 2004).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2007

Justification for type of information:

Refer to section 13 for details on the read-across justification. The HPRT study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus of Chinese hamster ovary (CHO) cells

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

RANGE-FINDING/SCREENING STUDIES: CHO cells were exposed to solvent alone and nine concentrations of test article ranging from 0.5 to 5000 µg/mL in the absence and presence of S9 reaction mixture. There was no visible precipitate in the treatment medium at any concentration. The osmolality of the solvent control was 428 mmol/kg and the osmolality of the highest soluble concentration, 5000 µg/mL, was 412 mmol/kg. Cloning efficiency relative to the solvent controls (RCE) at 5000 µg/mL was 0% without activation and 56% with S9 activation. Based on the results of the toxicity test, the concentrations chosen for the mutagenesis assay ranged from 500 to 3000 µg/mL for the non-activated cultures and 1000 to 5000 µg/mL for the S9-activated cultures.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control articles have been characterized as per the Certificates of Analysis on file with the testing facility. The stability of the negative and positive control articles and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.

Remarks on result:

other: strain/cell type: CHO-K1 cells

Conclusions:

Based on the study results of the read across substance (QM-1326AP), the SoI is concluded to be not mutagenic in HGPRT mutation assay.

Executive summary:

A study was conducted to test the mutagenic potential of the read across substance, QM-1326AP in a mammalian cell gene mutation assay performed according to OECD Guideline 476 and EPA OPPTS 870.5300., and in compliance with GLP. Based on the study results of the read across substance, the SoI is concluded to be not mutagenic in in the in vitro HGPRT mutation assay. As explained in the read across justification, the differences in the structure of the components between the SoI and the read across substance are unlikely to lead to differences in toxicities in an in vitro HGPRT assay that are higher than typical experimental error of the test method.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

2007

Justification for type of information:

Refer to section 13 for details on the read-across justification. The in vitro chromosomal aberration study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes:

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

Remarks on result:

other: other: human peripheral blood lymphocytes

Conclusions:

Based on the findings of the in vitro mammalian chromosome aberration test with the read across substance, QM-1326AP, the SoI is considered to be negative for the induction of structural and numerical chromosome aberrations in both the non-activated and the S9-activated test systems.

Executive summary:

A study was conducted to determine the clastogenecity potential of the read across substance according to OECD Guideline 473 as well as in compliance with GLP. Based on the findings of the in vitro mammalian chromosome aberration test with the read across substance, QM-1326AP, the SoI is considered to be negative for the induction of structural and numerical chromosome aberrations in both the non-activated and the S9-activated test systems. As explained in the read across justification, the differences in the structure of the components between the SoI and the read across substance are unlikely to lead to differences in toxicities in an in vitro chromosomal aberration assay that are higher than typical experimental error of the test method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not required.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to test the mutagenic potential of HEMA-phosphate in a bacterial reverse mutation assay performed according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study included a preliminary concentration range finding test, an initial mutation test and a confirmatory mutation test. Based on the results of the dose range finding study, the substance was tested in the first mutation assay at a concentration range of 100 to 5,000μg/plate in the absence and presence of 5% (v/v) S9-mix inSalmonella typhimuriumstrains TA 98, TA1535 and TA1537. In the second mutation assay, the substance was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix inSalmonella typhimuriumtester strains TA 98, TA100, TA1535, TA1537 andE. Colistrain WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the five tester strains in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. The negative and strain-specific control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the findings of the study, it can be concluded that the substance was not mutagenic either in the presence or absence of metabolic activation (Verspeek-Rip CM, 2004).

 

A study was conducted to test the mutagenic potential of the read across substance in a mammalian cell gene mutation assay performed according to OECD Guideline 476 and EPA OPPTS 870.5300., and in compliance with GLP. In this study, the test substance,QM[SM1] -1326AP[CF2] , was tested in the CHO HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9 metabolic activation. The preliminary toxicity assay was used to establish the concentration range for the initial mutagenesis assay. The initial and independent repeat mutagenesis assays were used to evaluate the mutagenic potential of the test substance. Dimethyl sulfoxide (DMSO) was determined to be the solvent of choice based on solubility of the test article and compatibility with the target cells. The test substance was soluble in DMSO at approximately 500 mg/mL, the maximum concentration tested.

In the preliminary toxicity assay, the maximum concentration of QM-1326AP tested was 5000μg/mL. There was no visible precipitate in the treatment medium at any concentration. Selection of concentrations for the mutagenesis assay was based on the cloning efficiency relative to the solvent control. Substantial toxicity, i.e., cloning efficiency ≤ 50% of the solvent control, was observed at 5000μg/mL without S9 activation only. Based on these findings, the concentrations chosen for the initial mutagenesis assay ranged from 500 to 3000μg/mL for the non-activated cultures and 1000 to 5000μg/mL for the S9-activated cultures.

In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per l06 clonable cells, were observed. No visible precipitate was observed in treatment medium at any concentrations. No toxicity, i.e., cloning efficiency ≤50% of the solvent control, was observed at any concentration with or without S9 activation.

The concentrations chosen for the independent repeat assay ranged from 1000 to 5000μg/mL for both the non-activated and S9-activated cultures. In the independent repeat assay, no positive responses were observed. No visible precipitate was observed in treatment medium at any concentration. Toxicity was observed at concentrations ≥3000 pg/mL with and without S9 activation.

Under the conditions of this study, the read across test substance was concluded to be negative in the CHO/HGPRT Mutation Assay (Dow, 2007a).

 

A study was conducted to determine the clastogenicity potential of the read across substance according to OECD Guideline 473 as well as in compliance with GLP. In this study, the test substance,QM-1326AP[CF3] , was tested in thein vitromammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article. Dimethyl sulfoxide (DMSO) was the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the maximum dose tested was 2100 μg/mL (10 mM). Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor induced S9 activation system for 4 h and continuously for 20 h in the absence of S9 activation. The test substance was soluble in DMSO and in the treatment medium at all concentrations tested at the beginning and conclusion of the treatment period. Selection of dose levels for the chromosome aberration assay was based on a reduction in the mitotic index relative to the solvent control. Substantial toxicity (at least 50% reduction in mitotic index relative to the solvent control) was observed at doses ≥63 μg/mL in all three exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 5 to 100μg/mL for all three exposure groups.

In the chromosome aberration assay (main study), the cells were treated for 4 and 20 h in the non-activated test system and for 4 h in the S9-activated test system. All cells were harvested 20 h after treatment initiation. The test article was soluble in DMSO and in the treatment medium at all concentrations tested at the beginning and conclusion of the treatment period. Selection of doses for microscopic analysis was based on mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index, relative to the solvent control and two lower doses) in all harvests. The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not increased above that of the solvent control at any dose level (p>0.05, Fisher’s Exact test).

Based on the findings of the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes, the read across substance, QM-1326AP was concluded to be negative for the induction of structural and numerical chromosome aberrations in both the non-activated and the S9-activated test systems (Dow, 2007b).

Justification for classification or non-classification

Based on the available results fromin vitrogenotoxicity assays with the test substance, HEMA-phosphate, as well as the read across substance, the test substance does not require classification for this endpoint according to CLP criteria (EC 1272/2008)