Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

Propylidynetrimethanol, ethoxylated, esters with acrylic acid

EC number: 500-066-5 | CAS number: 28961-43-5 1 - 6.5 moles ethoxylated

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

GENETIC TOXICITY IN VITRO

Gene mutation in bacteria:

S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, with and without metabolic activation (Aroclor-induced), OECD 471/472: weakly positive for TA1535 with metabolic activation negative for all other (BASF, 1992)

S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, with and without metabolic activation (Aroclor-induced) OECD 471/472: negative (BASF, 2002 (a))

S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, with and without metabolic activation (Aroclor-induced) OECD 471/472: negative (BASF, 2002m (b))

S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, with and without metabolic activation (phenobarbitone/ß-naphthoflavone -induced): negative (Cytec, Safepharm, 2006)

Gene mutation in mammalian cells:

Mouse Lymphoma, L5178Y, with and without metabolic activation (phenobarbital/p-naphthoflavone), OECD 476: mutagenic with and without metabolic activation (Cytec, Safepharm Laboratories, 2006).

Mouse Lymphoma, L5178Y, with and without metabolic activation (phenobarbital/p-naphthoflavone), OECD 476: mutagenic with and without metabolic activation (Cytec, Litton, 1984).

In vitro mammalian cell gene mutation test using the Hprt and xprt genes, V79 cells of the Chinese hamster, OECD 476: non-mutagenic with and without metabolic activation (ICCR-Roßdorf GmbH, 2019).

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: [DMSO]
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had
been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix for strains TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

without S-9 mix for strains TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

without S-9 mix for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S-9 mix for strain E.coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: the standard plate test and preincubation test were used

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours of incubation

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer)

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one
tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

no data

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

Remarks:

>= 500-2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 500-2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A slight decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation test bacteriotoxicity was
observed depending on the strain and test conditions from about 500 µg - 2500 µg/plate onward .

Conclusions:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains , i .e . Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA, in a reverse mutation assay according to OECD guideline 471. The test was performed ass standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix) with a concentration range of 20 µg - 5,000 µg/plate.

The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay

Executive summary:

Toxicity: A decrease in the number of revertants was occasionally observed in the standard plate test . In the preincubation test bacteriotoxicity was observed depending on the strain and test conditions from about 500 Ng -2,500 pg/plate onward

Mutagenicity: An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix for strains TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

without S-9 mix for strains TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S-9 mix for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S-9 mix for strain E.coli WP2 uvrA

Details on test system and experimental conditions:

Evaluation criteria:

Statistics:

no data

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

Remarks:

>= 2500 µg/plate (preincubation assay only)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2500 µg/plate (preincubation assay only)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A slight, dose-dependent decrease in the number of revertants was occasionally observed in the standard plate test.
In the preincubation assay bacteriotoxicity (slight reduction in the titer and/or slight decrease in the number revertants) was observed depending on
the strain and test conditions from about 2,500 pg/plate onward.

Conclusions:

Interpretation of results (migrated information):
negative

not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay

Executive summary:

The substance "confidential substance name" was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains , i .e . Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli

WP2 uvrA, in a reverse mutation assay according to OECD guideline 471. The test was performed ass standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix) with a concentration range of 20 µg - 5,000 µg/plate.

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 µg/plate for all strains and 0, 1000, 2000, 3000, 4000 and 5000 µg/plate in an additional experiment for TA 1535

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: The test substance is insoluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix; for strains: TA 100, TA 98, TA 1537, TA 1535 and E.coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitroso-N-nitroso-guanidine (MNNG)

Remarks:

without S-9 mix; for strains TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (NPD)

Remarks:

without S-9 mix, for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix, for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix, for strain E.coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: the standart plate test and the preincubation test were used

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours at 37°C (incubation)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Statistics:

no data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

other: Strain TA 1535 : With metabolic activation weakly positive reaction; other strains were negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Occasionally a slight decrease in the number of revertant colonies was observed at doses >= 2500 ug/plate.

Strain TA 1535 : With metabolic activation weakly positive reaction in the standard plate test and in the preincubation test from about 500 µg - 2000 µg/plate (factor 1.8 - 2.3) onward; increase in the number of mutant colonies by a factor of 2.7 - 4.6 at 2500 µg - 5000 µg/plate.

Strains TA 100, TA 1537, TA 98 as well as E.coli WP2 uvrA: No increase in the number of his+ or trp+ revertants.

Conclusions:

The test substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test and in the Escherichia coli - reverse mutation assay according to OECD 471 and 472.
The test substances was positive with metabolic activation only in Strain TA 1535 but negative without metabolic activation.

According to the results of the present study, the test substance is weakly mutagenic in the Ames test but not in the Escherichia coli - reverse mutation assay under the experimental conditions chosen.

According to the applicant the positive effect with metabolic activation was seen at cytotoxic concentrations without dose dependency. As this result is also not confirmed by other bacteria mugenicity test with the same strain it is rated as less of relevance.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test and in the Escherichia coli - reverse mutation assay according to OECD 471 and 472. Strains TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvr A were used with a dose range for the test substance of 10µg - 5000µg/plate solubilised in DMSO. The test was performed as standard plate test and preincubation test both with and without metabolic activation (Aroclor induced rat liver S-9 mix).

A cytotoxic effect was observed occasionally (slight decrease in the number of revertant colonies) at doses > 2500 µg/plate. No increase in the number of his+ or trp+ revertants were observed for TA 100, TA 1537, TA 98 and E . coli WP2 uvrA with and without metabolic activation. TA 1535 shows with metabolic activation weakly positive reaction in the standard plate test and in the preincubation test appears from about 500 µg - 2000 µg/plate (factor 1 .8 - 2 .3) and also increases in the number of mutant colonies by a factor of 2 .7 - 4 .6 at 2500 µg - 5000 µg/plate, but no increase was reported without metabolic activation.

According to the results of the present study, the test substance is weakly mutagenic in the Ames test but not in the Escherichia coli - reverse mutation assay under the experimental conditions chosen.

According to the applicant the positive effect with metabolic activation was seen at cytotoxic concentrations without dose dependency. As this result is also not confirmed by other bacteria mugenicity test with the same strain it is rated as less of relevance.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP conforme guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was prepared from phenobarbitone/ß-naphthoflavone treated Sprague-Dawley rat liver

Test concentrations with justification for top dose:

0; 0.15; 0.5; 1.5; 5; 15; 50; 150; 500; 1500; 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle :The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in
dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle of choice

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine; 9-Aminoacridine; 4-Nitroquinoline-1 -oxide; 2-Aminoanthracene; Benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension; The test was performed by mixing bacterial culture, molten, trace histidine or tryptophan supplemented, top agar, test material formulation (or positive control or vehicle) and S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar.

DURATION
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 48h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

other:
- additiional sterility test was performed

Evaluation criteria:

according to guideline e.G.
- dose-related increase in the dose range tested
- a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation

Statistics:

significant differences were determined if necessary

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:

valid

Positive controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix

Conclusions:

A mutagenicity test according to OECD Guideline 471 was performed to access the mutagenic potential of the test substance. Therefore the test substance diluted in DMSO was applied at concentration of 50 to 5000 µg/plate to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- in triplicate, both with and without the addition of a rat liver homogenate metabolising system (received from phenobarbitone/ß-naphthoflavone treated Sprague-Dawley rat liver).

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Toxicity: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ng/plate.

Mutagenicity: No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 5

Endpoint:: in vitro cytogenicity / micronucleus study
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented study conducted from a know test laboratory under GLP

Qualifier:

no guideline followed

Principles of method if other than guideline:

Clive , D. and Spector, J.F.S.: "Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma
cells , " Mutation Res., 31:17-29, 1975. and Clive, D., Johnson, K.O., Spector, J.F.S., Batson A.G. and Brown, M.M.M.:
Validation and characterization of the L5178Y TK +/- mouse lymphoma mutagen assay system. Mutation Research, 59:61-108, 1979.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Arolor 1254 induced rat liver homogenate)

Test concentrations with justification for top dose:

0.25; 0.5; 1.0; 2.0 ng/ml (without metabolic activation)
2.0; 6.0; 12.0; 16.0; 20.0; 24.0; 32.0 ng/ml (with metabolic activation)
1.9; 5.0; 10.0; 12.5; 15.0; 20,0; 25.0 ng/ml (with metabolic activation; 2nd experiment)

Vehicle / solvent:

The test material, C-661, was immiscible with water at 100 µL/ml but formed a clear colorless liquid in dimethyl sulfoxide (DMSO) at the same
concentration. The solvent of choice was therefore DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulfonate; 3-Methylcholanthrene

Details on test system and experimental conditions:

see study report

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment will be a mutant frequency that is at least 150% of the concurrent background frequency plus 10x10e-6.
The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test material as a mutagen. The following test results must be obtained to reach this conclusion.
- A dose-related or toxicity-related increase in mutant frequency should be observed.
- If an increase of about two times the minimum criterion or greater is observed for a single dose near the highest testable toxicity, as defined
in the-Assay Acceptance Criteria, the test material will be considered mutagenic.
- For some test materials, the correlation between toxicity and applied concentration
is poor......Therefore, either parameter,
applied concentration or toxicity (percent relative growth), can be used to establish whether the mutagenic activity is related to an increase in
effective treatment.
- Treatments that induce less than 10% relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates
to risk assessment.
For a detailed description see original study report.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

A mouse lymphoma forward mutation assay was performed to evaluates a test article's mutagenic potential in a specific locus (thymidine kinase (TK)) mutation assay using mammalian cells. Therefore the colony growth of L5178Y TK+/- mouse lymphoma cells in the presence of 5-trifluorothymidine and with test substance concentration of 0.25 to 2 ng/ml without metabolic activation and 1.9 to 32ng/ml with metabolic activation (via aroclor induced S9 mix) was examined. Two trials of the mutation assay were initiated but trial 2 was only performed in the presence of metabolic activation.The test material induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- cells only in the presence of metabolic activation. In conclusion, the test material is considered active in the Mouse Lymphoma Forward Mutation Assay only in the presence of metabolic activation

Executive summary:

The test material induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- cells only in the presence of metabolic activation. Under nonactivation conditions, the test material was assayed from 0.25 nl/ml to 1.0 nl/ml and low to moderate toxicities were induced without inducing significant increases in the mutant frequency. Highly toxic treatments were not assayed because a small increase in concentration from 1.0 nl/ml to 2.0 nl/ml was excessively toxic. In the presence of metabolic activation, the test material was converted to a less toxic form or forms that induced increases in the mutant frequency that ranged from 2.1-fold to 3.1-fold above the background The test material was assayed from 2.0 nl/ml to 24.0 nl/ml. The two highest concentrations (15.0 nl/ml and 20.0 nl/ml) induced mutant frequencies that exceeded the minimum criterion.

The test material is therefore considered active in the Mouse Lymphoma Forward Mutation Assay only in the presence of metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 07, 2005 to February 17, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is according to OECD Guideline 476 and EU Method B.17 in compliance with Good Laboratory Practices of UK

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC

Deviations:

Qualifier:

according to guideline

Guideline:

other: United Kingdom Environmental Mutagen Society

Deviations:

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

according to UK GLP

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine Kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically "cleansed" against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Pβ/PNF S9 (2% )

Test concentrations with justification for top dose:

Preliminary toxicity test:
- 4 h without S9: 0, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL
- 4 h with S9: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
- 24 h without S9: 0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL
Mutagenicity test:
Experiment 1:
- Without S9: 0, 0.13, 0.25, 0.5, 1, 2, 3, 4 and 5 µg/mL;
- With S9: 0, 1.25, 2.5, 5, 7.5, 10, 20, 30, 40, 50 and 60 µg/mL
Experiment 2:
- Without S9: 0, 0.13, 0.25, 0.5, 1, 2, 3, 4 and 5 µg/mL;
- With S9: 0, 10, 20, 30, 35, 40, 45, 50 and 55 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

absence of metabolic activation Migrated to IUCLID6: 400 µg/mL

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

presence of metabolic activation Migrated to IUCLID6: 1.25 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In suspension

DURATION
-Exposure duration: 24 h
Preliminary study:
- Expression duration- Preliminary test: 4 h (with and without S9); 24 h (without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24- 48 h
Experiment 1: 4 h incubation
Experiment 2: 48 h incubation
- Expression time (cells in growth medium): 48 h

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): MTT solution

NUMBER OF REPLICATIONS: Two

Evaluation criteria:

- Vehicle control values greater than 250 x 10(-6) mutant frequency per survivor are not normally acceptable and will be repeated.
- Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
- Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126 x 10(-6) will be considered positive.

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: The dose range in the preliminary toxicity test was 0.16 to 40 µg/mL in the 4 h without S9 cultures, 19.53 to 5000 µg/mL in the 4 h with S9 cultures and 0.08 to 20 µg/mL in the 24 h without S9 cultures.
Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 µg/mL or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).

As the test material produced cytotoxicity, therefore, criteria (iii) was used for dose selection in the main study.

RESULT DETAILS: IRR 560 induced weak but reproducible toxicologically significant dose-related increases in the mutant frequency both with and without metabolic activation, in the first and second experiment. The mutagenic response was only observed in dose levels approaching the limit of acceptable toxicity. The increase in mutant frequency was predominantly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage.

None

Conclusions:

A study was conducted to assess the mutagenic potential of the test substance on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted according to OECD Guideline 476 and EU Method B.17 in compliance with principles of GLP UK. L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to ten dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. 4 h exposures were used both with and without activation in Experiment 1 and Experiment 2.

The test substance induced weak but reproducible toxicologically significant dose-related increases in the mutant frequency both with and without metabolic activation, in the first and second experiment. The mutagenic response was only observed in dose levels approaching the limit of acceptable toxicity. The increase in mutant frequency was predominantly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage.

In conclusion, the test substance was considered to be mutagenic to L5178Y cells with and without metabolic activation.

Executive summary:

The dose range of the test substance, plated for expression of mutant colonies, was selected following the results of a preliminary toxicity test and was 0.13 to 4 µg/mL without activation and 7.5 to 50 µg/mL with activation for the first experiment. For the second experiment the dose range was 0.13 to 5 µg/mL without activation and 10 to 55 µg/mL with activation.

The maximum dose level used was limited by the test substance induced toxicity. No precipitate of the test substance was observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test substance induced weak but reproducible toxicologically significant dose-related increases in the mutant frequency both with and without metabolic activation, in the first and second experiment. The mutagenic response was only observed in dose levels approaching the limit of acceptable toxicity. The increase in mutant frequency was predominantly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage.

In conclusion, the test substance was considered to be mutagenic to L5178Y cells with and without metabolic activation.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

Principles of method if other than guideline:

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Identification: Propylidynetrimethanol, ethoxylated, esters with acrylic acid
Appearance: Clear Liquid
Batch: 180330317
Purity/Composition: 99.96%
Test item storage: At room temperature
Stable under storage conditions until: 29 March 2019 (expiry date)

Test Facility Test Item Number: 209397/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 220°C
Chemical name (IUPAC, synonym or trade name): Propylidynetrimethanol, ethoxylated, esters with acrylic acid; Trade name: MIRAMER M3130
CAS No: 28961-43-5
EC No: 500-066-5

Target gene:

Gene mutations at the HPRT locus in V79 cells. HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for approx. 6 days. The expression period is terminated by adding 6-TG to the culture medium.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium (4). Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×106 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.

All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

The PBS was composed as follows (per litre):
NaCl 8000 mg; KCl 200 mg; KH2PO4 200 mg; Na2HPO4 150 mg

The "saline G" solution had the following constituents (per litre):
NaCl 8000 mg; KCl 400 mg; Glucose•H2O; 1100 mg; Na2HPO4•2H2O192 mg; KH2PO4 150 mg

The pH was adjusted to 7.2.

Cytokinesis block (if used):

No information

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. The S9 was prepared and stored according to the currently valid version of the ICCR-Roßdorf SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).

The protein concentration of the S9 preparation was 30.4 mg/mL (Lot. No.: 210618) in the pre-experiment and the main experiment.

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) will be performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-Test was not performed since all mean mutant frequencies were well within the 95% confidence interval of our laboratory’s historical negative control data.

However, both, biological and statistical significance is considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

The main experiment was analysed for gene mutation at the following concentrations:

- without S9 mix: 0.16; 0.31; 0.63; 1.3; 2.5, 5.0; and 7.5 μg/mL

- with S9 mix: 3.9; 7.8; 15.6; 31.3; 62.5, and 125.0 μg/mL

No relevant increase in the mean mutant colony numbers/106 cells was observed in the main experiment up to the maximum concentration.

The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical laboratory solvent control data.

The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.

The mean mutant frequency obtained for the solvent controls in the main experiment was 5.1 mutants per 106 cells in the absence of metabolic activation, and 10.2 mutants per 106 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of our laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476.

The range of the mutant frequencies (mean values) of the groups treated with the test item was from 3.5 up to 14.1 mutants per 106 cells.

EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

The study was performed to investigate the potential of TMPeoTA to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in accordance to OECD 476. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment was 2000 μg/mL according to OECD 476. The concentration range of the main experiment was limited by cytotoxicity and phase separation of the test item observed in the pre-experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion, it can be stated that under the experimental conditions reported TMPeoTA did not induce gene mutations at the HPRT locus in V79 cells (OECD 476). Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

TMPeoTA was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster in accordance to OECD 476. The treatment period was 4 hours with and without metabolic activation.

The main experiment was analysed for gene mutation at the following concentrations:

- without S9 mix: 0.16; 0.31; 0.63; 1.3; 2.5, 5.0; and 7.5 μg/mL

- with S9 mix: 3.9; 7.8; 15.6; 31.3; 62.5, and 125.0 μg/mL

No relevant increase in the mean mutant colony numbers/10⁶ cells was observed in the main experiment up to the maximum concentration.

The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical laboratory solvent control data.

The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.

The mean mutant frequency obtained for the solvent controls in the main experiment was 5.1 mutants per 10⁶ cells in the absence of metabolic activation, and 10.2 mutants per 10⁶ cells in the presence of metabolic activation. The values were well within the 95% confidence interval of the laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476.

The range of the mutant frequencies (mean values) of the groups treated with the test item was from 3.5 up to 14.1 mutants per 10⁶ cells. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion, it can be stated that under the experimental conditions reported TMPeoTA did not induce gene mutations at the HPRT locus in V79 cells (OECD 476). Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

GENETIC TOXICITY IN VIVO

Micronucleus test in vivo, NMRI mice, oral, 2,000, 1,000 and 500 mg/kg body weight, OECD 474: negative (BASF, 2002)

Micronucleus test in vivo, NMRI mice, i.p., 250, 125 and 62.5 mg/kg body weight, OECD 474: negative (Cray Valley, NoTox, 2001).

Micronucleus test in vivo, Crl:CD-l(ICR)BR mice, i.p. 100, 50 and 25 mg/kg bw, OECD 474: negative (Cytec, Safepharm Laboratories, 2006)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: about 29 g
- Assigned to test groups randomly: yes, under following basis: randomized plan prepared with an appropriate computer program
- Housing: Makrolon cages, type MI, housed individually from start of the treatment until the end of the test
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the hydrolytical sensitivity of the test substance in water, DMSO was selected as the vehicle, which
had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 12.5 g/100 ml, 25.0 g/100 ml and 50.0 g/100 ml.
- Amount of vehicle: 4 ml/kg bw.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration.
The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once, single dose

Post exposure period:

24 or 48 hours

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals per dose for the 24 hour sacrifices and 5 animals per dose for the 48 hour sacrifices

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine sulphate (VCR)
- Justification for choice of positive controls: The stability of CPP and VCR is well-defined under the selected conditions, since both positive control
articles are well-defined clastogens and aneugens respectively.
- Route of administration: The positive controls, both, dissolved in purified water were administered to male animals once orally or intraperitoneally
each in a volume of 10 ml/kg body weight.
- Doses / concentrations: 20 mg CPP//kg body weight for clastogenic effects and 0.15 mg VCR/kg body weight for aneugenic effects.

Tissues and cell types examined:

In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored .

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD
Guideline were survived by all animals (male and female) without any clinical signs. Thus, only male animals were used for the cytogenetic
investigations. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the híghest dose group of
2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control groups,the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the
diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed
thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified
water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an
index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration andmay serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression
may be identified and excluded from the evaluation.
- Number of small micronuclei (dD/4) (d = diameter of micronucleus, D= cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative
values can occur.

Evaluation criteria:

The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes.
- The proportion of cells with micronuclei in negative control animals was within the normal range of the historical control data.
- The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei within the range of
the historical control data or above.

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
- The frequencies of cells containing micronuclei were within the historical control range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN.
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians . Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <=0.05, ** for p<=0.01) were printed with the group means in the tables. This test was performed one-sided.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Single oral administration of the test substance did not
result in any increase in the number of polychromatic
erythrocytes containing either small or large micronuclei.
The rate of micronuclei was always close to the range as
that of the concurrent negative control and within the range
of historical control data. No inhibition of erythropoesis
determined from the ratio of polychromatic to normochromatic
erythrocytes was detected.
According to the authors, the test substance did not have
any chromosome-damaging (clastogenic) effect, and there was no indication
of any impairment of chromosome distribution in the course
of mitosis (aneugenic activity) in bone marrow cells in vivo.

Conclusions:

The test substance was tested for chromosomal damage (clastogenicíty) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to OECD guideline 474. For this purpose, the test substance, dissolved in DMSO, was administered once orally to male animals at dose levels of 500 mg/kg, 1,000 mg/kg and 2,000 m g/kg body weight in a volume of 4 ml/kg body weight in each case. As a negative control, male mice were administered merely the vehicle, DMSO, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, ie. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Under the experimental conditions, the test substance has no chromosome-damaging (clastogenic) effect nor does it leadto any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance was tolerated by all animals without any signs or symptoms.The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the híghest dose group of 2,000 mg/kg body weight and in the vehicle controls . In the test groups of 1,000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only . After staining of the preparations, 2,000 polychromátic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micrónuclei occurring per 2,000 polychromatic erythrocytes were also recorded. According to the results of the present study, the single oral administration of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei . The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impaìrment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 5-7 weeks old
- Weight at study initiation: 27.2-34.8
- Housing: housed in labelled polycarbonate cages containing purified sawdust
- Diet (e.g. ad libitum): standard pelleted laboratory animal diet; ad libitum
- Water (e.g. ad libitum):tap-water; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30-70%.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 250mg/10ml; 125mg/10ml and 62.5mg/10ml

Details on exposure:

The mice received a single intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of PURIFIED TMPEOTA. The route of administration was chosen to maximize the chance of the test article reaching the target tissue. The dosing volume was 10 ml/kg body weight.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once, single dose

Post exposure period:

toxicity signs once a day,
24h, 48h treatment groups
48h positiv conrol

Remarks:

Doses / Concentrations:
250, 125, 62.5 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5
two groups per dosage

Control animals:

yes, concurrent vehicle

Positive control(s):

- positiv control: cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 50mg/kg bw

Tissues and cell types examined:

The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes.

Details of tissue and slide preparation:

The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

Evaluation criteria:

The test substance is considered positive in the micronucleus test if:
It Induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

Statistics:

yes, e.G, Wilcoxon Rank Sum Test; two-sided test at P < 0.05

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

lethargic and rough coat; no abnormalities at 62.5 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

TMPeoTA was tested in the Micronucleus Test in mice according to OECD guideline 474, to evaluate its genotoxic effect on erythrocytes in bone marrow. Based on the results, it was concluded that TMPeoTA is not mutagenic in the micronucleus test under the experimental conditions.

Executive summary:

TMPeoTA was tested in the Micronucleus Test in mice according to OECD guideline 474, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection. Two groups (A and B) were dosed with 250 mg/kg body weight, two groups (C and D) were dosed with 125 mg/kg body weight and two groups (E and F) were dosed with 62.5 mg/kg body weight. One group (G) treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with PURIFIED TMPEOTA. The animals of the groups treated with 250 mg TMPeoTA/kg body weight showed slight decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time compared to the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time of the animals treated with the lowest dose group of 62.5 mg/kg body weight. This phenomenon, reflects a toxic effect of PURIFIED TMPEOTA on the erythropoiesis, implying that TMPeoTA reached the bone marrow cells of the mouse.

It is concluded that TMPeoTA is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl:CD-l(ICR)BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5-8 wks
- Weight at study initiation: 23-30 g
- Housing: Groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet: Certified Rat and Mouse Diet Code 5LF2, BCM, IPS Ltd., London, UK, ad libitum
- Water: Ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity (%): 30-70 %
- Air changes: 15 per h
- Photoperiod: 12 h dark / h light

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis oil
- Lot/batch no.: SN365

Details on exposure:

Not applicable

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Single treatment

Post exposure period:

24 h (for 100, 50 and 25 mg/kg bw dose groups); 48 h (for 100 mg/kg bw dose group)

Remarks:

Doses / Concentrations:
100, 50 and 25 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Tissue: Femoral bone marrow
Cell types: Erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of a range-finding toxicity test, the maximum tolerated dose of the test material, 100 mg/kg bw, with the clinical signs of hunched posture and ptosis was selected for use in the main test, with 50 and 25 mg/kg as the lower dose levels.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24 or 48 h following dosing

DETAILS OF SLIDE PREPARATION: Both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared
following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCEblue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 h kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

The data was analysed following a √(x +1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No significant effects except hunched posture and ptosis

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Maximum tolerated dose: 100 mg/kg bw i.p. No toxicity was observed by oral route, therefore, systemic absorption could not be confirmed using this dose route.
- Clinical signs of toxicity in test animals: In animals dosed with the test material via the intraperitoneal route premature deaths (animals killed in extremis) occurred at and above 200 mg/kg bw, and clinical signs were observed at and above 100 mg/kg bw as follows: Hunched posture, ptosis, lethargy, ataxia, increased salivation, piloerection, decreased respiratory rate, laboured respiration, hypothermia, splayed gait, prostration and elevated tail.
- Other: The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48 h test material groups when compared to their concurrent vehicle control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the dose groups when compared to their concurrent vehicle control groups.
- Other: Mortality data and clinical observations: There were no premature deaths seen in any of the dose groups. Following clinical signs were observed in animals dosed with the test material at 100 mg/kg bw in both the 24 and 48 h groups: Hunched posture and ptosis.

Table 1: Micronucleus Test - Summary of Group Mean Data

Treatment Group	Number of PCE with Micronuclei per 2000 PCE		PCE/NCE Ratio
Treatment Group	Group Mean	SD	Group Mean	SD
Vehicle Control (Arachis oil) 10 mL/kg bw 48 h Sampling Time	1.1	1.2	0.83	0.38
Vehicle Control (Arachis oil) 10 mL/kg bw 24 h Sampling Time	1.1	1.2	0.76	0.23
Positive Control 50 mg/kg bw 24 h Sampling Time	86.4***	27.8	1.35	0.27
Test substance 100 mg/kg bw 48 h Sampling Time	1.3	0.8	0.9	0.33
Test substance 100 mg/kg bw 24 h Sampling Time	1.4	1.9	0.78	0.34
Test substance 50 mg/kg bw 24 h Sampling Time	0.1	0.4	0.89	0.26
Test substance 25 mg/kg bw 24 h Sampling Time	1.9	1.6	0.71	0.51

where,

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

*** = P < 0.001

Conclusions:

A study was performed to investigate the potential of test substance to induce micronuclei in the bone marrow of the Crl:CD-l(ICR)BR mouse according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and EU Method B.12 (Mutagenicity - In vivo Mammalian Erythrocyte Micronucleus Test). Based on the results. it was concluded that the test substance was considered to be non-genotoxic in the micronucleus test in the mouse.

Executive summary:

A range-finding test was performed to find suitable dose levels of the test substance, route of administration and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of "confidential substance name" between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice at the maximum tolerated dose of 100 mg/kg bw and with 50 and 25 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 h later, the bone marrow extracted, and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice (each of 7 mice) were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 h later, and positive control animals were killed after 24 h.

There were no premature deaths seen in any of the dose groups. Hunched posture and ptosis were observed in animals dosed with test substance at 100 mg/kg bw. No statistically significant decreases in the PCE/NCE ratio were observed in the test groups when compared to concurrent control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in test groups when compared to the concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

In conclusion, the test substance was considered to be non-genotoxic in the micronucleus test in the mouse.

Reference 4

Endpoint:

genetic toxicity in vivo

Type of information:

other: Supportive information from a literature reference: Regulatory Toxicology and Pharmacology 50 (2008) 322–335 used for weight of evidence

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented publication

Qualifier:

no guideline followed

Principles of method if other than guideline:

Supportive information from a literature reference: Regulatory Toxicology and Pharmacology 50 (2008) 322–335

GLP compliance:

Type of assay:

other: Supportive information from a literature reference: Regulatory Toxicology and Pharmacology 50 (2008) 322–335, describing the results from a range of in vitro and in vivo mutagenicity studies

Sex:

not specified

Remarks on result:

other: Supportive information from a literature reference: Regulatory Toxicology and Pharmacology 50 (2008) 322–335, describing the results from a range of in vitro and in vivo mutagenicity studies.

Additional information on results:

Supportive information from a literature reference: Regulatory Toxicology and Pharmacology 50 (2008) 322–335, describing the results from a range of in vitro and in vivo mutagenicity studies.

"Esters of acrylic acid and methacrylic acid, more commonly known as acrylates and methacrylates, respectively, are key raw materials in the coatings and printing industry, with several of its chemical class used in food packaging. The results of over 200 short-term in vitro and in vivo mutagenicity studies available in the open literature have been evaluated. Despite differences in acrylate or methacrylate functionality or in the number of functional groups, a consistent pattern of test response was seen in a typical regulatory battery of mutagenicity tests. No evidence of point mutations was observed when acrylic acid or over 60 acrylates and methacrylates were investigated in Salmonella bacterial tests or in hprt mutation tests mammalian cells, and no evidence of a mutagenic effect was seen when tested in whole animal clastogenicity and/or aneuploidy (chromosomal aberration/micronucleus) studies. Consistent with the in vivo testing results, acrylic acid exhibited no evidence of carcinogenicity in chronic rodent cancer bioassays. In contrast, acrylic acid and the entire acrylate and methacrylate chemical class produced a consistently positive response when tested in the mouse lymphoma assay and/or other in vitro mammalian cell assays designed to detect clastogenicity. The biological relevance of this in vitro response is questioned based on the non-concordance of in vitro results with those of in vivo studies addressing the same mutagenic endpoint (clastogenicity). Thus, in short-term mutagenicity tests, the acrylates and methacrylates behave as a single chemical category, and genotoxicity behavior of a similar chemical can be predicted with confidence by inclusion within this chemical class, thus avoiding unnecessary testing."

Please see original publication for further details (due to copyright reason detailed information is not presented here)

Abstact:

"Esters of acrylic acid and methacrylic acid, more commonly known as acrylates and methacrylates, respectively, are key raw materials in the coatings and printing industry, with several of its chemical class used in food packaging. The results of over 200 short-term in vitro and in vivo mutagenicity studies available in the open literature have been evaluated. Despite differences in acrylate or methacrylate functionality or in the number of functional groups, a consistent pattern of test response was seen in a typical regulatory battery of mutagenicity tests. No evidence of point mutations was observed when acrylic acid or over 60 acrylates and methacrylates were investigated in Salmonella bacterial tests or in hprt mutation tests mammalian cells, and no evidence of a mutagenic effect was seen when tested in whole animal clastogenicity and/or aneuploidy (chromosomal aberration/micronucleus) studies. Consistent with the in vivo testing results, acrylic acid exhibited no evidence of carcinogenicity in chronic rodent cancer bioassays. In contrast, acrylic acid and the entire acrylate and methacrylate chemical class produced a consistently positive response when tested in the mouse lymphoma assay and/or other in vitro mammalian cell assays designed to detect clastogenicity. The biological relevance of this in vitro response is questioned based on the non-concordance of in vitro results with those of in vivo studies addressing the same mutagenic endpoint (clastogenicity). Thus, in short-term mutagenicity tests, the acrylates and methacrylates behave as a single chemical category, and genotoxicity behavior of a similar chemical can be predicted with confidence by inclusion within this chemical class, thus avoiding unnecessary testing."

Conclusions:

Mouse lymphoma test with acrylates may led to false positive results.

Executive summary:

Abstract:

" Esters of acrylic acid and methacrylic acid, more commonly known as acrylates and methacrylates, respectively, are key raw materials in the coatings and printing industry, with several of its chemical class used in food packaging. The results of over 200 short-term in vitro and in vivo mutagenicity studies available in the open literature have been evaluated. Despite differences in acrylate or methacrylate functionality or in the number of functional groups, a consistent pattern of test response was seen in a typical regulatory battery of mutagenicity tests. No evidence of point mutations was observed when acrylic acid or over 60 acrylates and methacrylates were investigated in Salmonella bacterial tests or in hprt mutation tests mammalian cells, and no evidence of a mutagenic effect was seen when tested in whole animal clastogenicity and/or aneuploidy (chromosomal aberration/micronucleus) studies. Consistent with the in vivo testing results, acrylic acid exhibited no evidence of carcinogenicity in chronic rodent cancer bioassays. In contrast, acrylic acid and the entire acrylate and methacrylate chemical class produced a consistently positive response when tested in the mouse lymphoma assay and/or other in vitro mammalian cell assays designed to detect clastogenicity. The biological relevance of this in vitro response is questioned based on the non-concordance of in vitro results with those of in vivo studies addressing the same mutagenic endpoint (clastogenicity). Thus, in short-term mutagenicity tests, the acrylates and methacrylates behave as a single chemical category, and genotoxicity behavior of a similar chemical can be predicted with confidence by inclusion within this chemical class, thus avoiding unnecessary testing".

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

Valid experimental data are available to assess the genetic toxicity of ethoxylated trimethylolpropane triacrylate (TMPeoTA). These are described below:

GENETIC TOXICITY IN VITRO

Gene mutation in bacteria:

OECD conform studies:

The substance TMPeoTA was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test and in the Escherichia coli - reverse mutation assay according to OECD 471 and 472 (BASF, 1992). Strains TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvr A were used with a dose range for the test substance of 10µg - 5000µg/plate solubilized in DMSO. The test was performed as standard plate test and preincubation test both with and without metabolic activation (Aroclor induced rat liver S-9 mix). A cytotoxic effect was observed occasionally (slight decrease in the number of revertant colonies) at doses > 2500 µg/plate. No increase in the number of his+ or trp+ revertants were observed for TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with and without metabolic activation. TA 1535 shows with metabolic activation weakly positive reaction in the standard plate test and in the preincubation test appears from about 500 µg - 2000 µg/plate (factor 1 .8 - 2 .3) and also increases in the number of mutant colonies by a factor of 2 .7 - 4 .6 at 2500 µg - 5000 µg/plate, but no increase was reported without metabolic activation. According to the results of the present study, the test substance is weakly mutagenic in the Ames test but not in the Escherichia coli - reverse mutation assay under the experimental conditions chosen.

In a further study (BASF, 2002(a)) another batch of TMPeoTA was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e . Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA, in a reverse mutation assay according to OECD guideline 471. The test was performed as standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix) with a concentration range of 20 µg - 5,000 µg/plate. Toxicity: A decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation test toxicity was observed depending on the strain and test conditions from about 2,500 µg/plate onward. Mutagenicity: An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

In another study (BASF, 2002(b)) another batch of TMPeoTA was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains , i .e . Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA, in a reverse mutation assay according to OECD guideline 471. The test was performed ass standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix) with a concentration range of 20 µg - 5,000 µg/plate. Toxicity: A decrease in the number of revertants was occasionally observed in the standard plate test . In the preincubation test bacteria toxicity was observed depending on the strain and test conditions from about 500 ng - 2,500 pg/plate onward. Mutagenicity: An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

In a further ames test the mutagenic potential according to OECD Guideline 471 was determined for TMPeoTA (Cytec, Safepharm). Therefore the test substance diluted in DMSO was applied at concentration of 50 to 5000 µg/plate to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- in triplicate, both with and without the addition of a rat liver homogenate metabolising system (received from phenobarbitone/ß-naphthoflavone treated Sprague-Dawley rat liver). Toxicity: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ng/plate. Mutagenicity: No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Gene mutation in mammalian cells:

OECD conform studies:

A study was conducted to assess the mutagenic potential of TMPeoTA on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted according to OECD Guideline 476 and EU Method B.17 in compliance with principles of GLP (Cytec, Safepharm Laboratories, 2006, Flanders L.). L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to ten dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. 4 h exposures were used both with and without activation in Experiment 1 and Experiment 2. The dose range of the test substance, plated for expression of mutant colonies, was selected following the results of a preliminary toxicity test and was 0.13 to 4 µg/mL without activation and 7.5 to 50 µg/mL with activation for the first experiment. For the second experiment the dose range was 0.13 to 5 µg/mL without activation and 10 to 55 µg/mL with activation. The maximum dose level used was limited by the test substance induced toxicity. No precipitate of the test substance was observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance induced weak but reproducible toxicologically significant dose-related increases in the mutant frequency both with and without metabolic activation, in the first and second experiment. The mutagenic response was only observed in dose levels approaching the limit of acceptable toxicity. The increase in mutant frequency was predominantly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage. In conclusion, TMPeoTA was considered to be mutagenic to L5178Y cells with and without metabolic activation.

In another study, TMPeoTA was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster in accordance with OECD 476. The treatment period was 4 hours with and without metabolic activation. The main experiment was analysed for gene mutation at the following concentrations:

- without S9 mix: 0.16; 0.31; 0.63; 1.3; 2.5, 5.0; and 7.5 μg/mL

- with S9 mix: 3.9; 7.8; 15.6; 31.3; 62.5, and 125.0 μg/mL

Relevant cytotoxic effect indicated by the mean relative adjusted cloning efficiency I below 50% was observed at a concentration of 5.0 μg/mL and above in the absence of metabolic activation and at 62.5 μg/mL and above in the presence of metabolic activation. No relevant increase in the mean mutant colony numbers/10⁶cells was observed in the main experiment up to the maximum concentration.

The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical laboratory solvent control data.

The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups. The mean mutant frequency obtained for the solvent controls in the main experiment was 5.1 mutants per 10⁶cells in the absence of metabolic activation, and 10.2 mutants per 10⁶cells in the presence of metabolic activation. The values were well within the 95% confidence interval of the laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476.

The range of the mutant frequencies (mean values) of the groups treated with the test item was from 3.5 up to 14.1 mutants per 10⁶cells. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Tests according to other guidelines:

Another mouse lymphoma forward mutation assay was performed to evaluates a test article's mutagenic potential in a specific locus (thymidine kinase (TK)) mutation assay using mammalian cells (Cytec, Litton Bionetics, 1984). The test follows the guide published of Clive, nD. et al., 1979 and Clive , D. and Spector, J.F.S, 1975. Therefore the colony growth of L5178Y TK+/- mouse lymphoma cells in the presence of 5-trifluorothymidine and with test substance concentration of 0.25 to 2 ng/ml without metabolic activation and 1.9 to 32ng/ml with metabolic activation (via aroclor induced S9 mix) was examined. Two trials of the mutation assay were initiated but Trial 2 was only performed in the presence of metabolic activation. The test material, TMPeoTA, induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- cells only in the presence of metabolic activation. Under no activation conditions, the test material was assayed from 0.25 nl/ml to 1.0 nl/ml and low to moderate toxicities were induced without inducing significant increases in the mutant frequency. Highly toxic treatments were not assayed because a small increase in concentration from 1.0 nl/ml to 2.0 nl/ml was excessively toxic. In the presence of metabolic activation, the test material was converted to a less toxic form or forms that induced increases in the mutant frequency that ranged from 2.1-fold to 3.1-fold above the background the test material was assayed from 2.0 nl/ml to 24.0 nl/ml. The two highest concentrations (15.0 nl/ml and 20.0 nl/ml) induced mutant frequencies that exceeded the minimum criterion. The test material is therefore considered active in the Mouse Lymphoma Forward Mutation Assay only in the presence of metabolic activation.

GENETIC TOXICITY IN VIVO

In vivo cytogenicity:

OECD conform studies:

The substance TMPeoTA was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to OECD guideline 474 (BASF, 2002). For this purpose, the test substance, dissolved in DMSO, was administered once orally to male animals at dose levels of 500 mg/kg, 1,000 mg/kg and 2,000 mg/kg body weight in a volume of 4 ml/kg body weight in each case. As a negative control, male mice were administered merely the vehicle, DMSO, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i .e . cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance was tolerated by all animals without any signs or symptoms. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2,000 mg/kg body weight and in the vehicle controls. In the test groups of 1,000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2,000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2,000 polychromatic erythrocytes were also recorded. According to the results of the present study, the single oral administration of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. Thus, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Purified TMPeoTA was tested in a further micronucleus test in mice according to OECD guideline 474, to evaluate its genotoxic effect on erythrocytes in bone marrow (, NoTox, 2001). Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection. Two groups (A and B) were dosed with 250 mg/kg body weight, two groups (C and D) were dosed with 125 mg/kg body weight and two groups (E and F) were dosed with 62.5 mg/kg body weight. One group (G) treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test substance. The animals of the groups treated with 250 mg TMPeoTA/kg body weight showed slight decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time compared to the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time of the animals treated with the lowest dose group of 62.5 mg/kg body weight. This phenomenon reflects a toxic effect of the test substance on the erythropoiesis, implying that the substance reached the bone marrow cells of the mouse. It is concluded that Purified TMPeoTA is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Another study was performed to investigate the potential of TMPeoTA to induce micronuclei in the bone marrow of the Crl:CD-l(ICR)BR mouse according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and EU Method B.12 (Mutagenicity - In vivo Mammalian Erythrocyte Micronucleus Test) (Cytec, Safepharm Laboratories, 2006).

A range-finding test was performed to find suitable dose levels of the test substance, route of administration and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test substance between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice at the maximum tolerated dose of 100 mg/kg bw and with 50 and 25 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 h later, the bone marrow extracted, and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice (each of 7 mice) were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 h later, and positive control animals were killed after 24h.

In conclusion, the test substance was considered to be non-genotoxic in the micronucleus test in the mouse.

Evidence from Literature:

It is published that no evidence of point mutations was observed when acrylic acid or over 60 acrylates and methacrylates were investigated in Salmonella bacterial tests or in hprt mutation tests mammalian cells, also no evidence of a mutagenic effect was seen when tested in whole animal clastogenicity and/or aneuploidy (chromosomal aberration/micronucleus) studies. Consistent with the in vivo testing results, acrylic acid exhibited no evidence of carcinogenicity in chronic rodent cancer bioassays. Nevertheless positive results were often observed for when tested in the mouse lymphoma assay and/or other in vitro mammalian cell assays designed to detect clastogenicity. But the biological relevance of this in vitro response is questioned based on the non-concordance of in vitro results with those of in vivo studies addressing the same mutagenic endpoint (clastogenicity). This issue is address in the publication by F.R. Johannsen et al. / Regulatory Toxicology and Pharmacology 50 (2008) 322–335 underlining the less relevance of a positive mouse lymphoma for assessment of genetic toxicity.

Assessment of genetic toxicity:

According to the testing strategy for mutagenicity, first in vitro tests were conducted to assess the mutagenic potential.

There are four studies available assessing the gene mutation in vitro of TMPeoTA in bacteria. All studies are equal in reliability and relevance. Three studies report negative results whereas one study (BASF, 1992) reports about a weak positive result for TA 1535 with metabolic activation. According to the applicant the positive effect with metabolic activation was seen at cytotoxic concentrations without dose dependency. As this result is also not confirmed by other bacteria mutagenicity test with the same strain it is rated as less of relevance. In a weight of evidence approach all mutagenicity test are taken into account, leading to no increased mutagenic potential.

The next step to confirm the absence of an increased mutagenicity is to perform a in vitro cytogenicity or gene mutation test with mammalian cell. The mouse lymphoma test is normally the preferred test this test was conducted. A reliable and relevant in vitro mouse lymphoma test (Cytec, Safepharm Laboratories, 2006) leads to positive results. In another study, TMPeoTA was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster in accordance to OECD 476 (ICCR-Roßdorf GmbH, 2019). TMPeoTA was found to be non-mutagenic with and without metabolic activation. Based on these results, an in vivo test was conducted for confirmation.

This test, assessing the cytogenicity of TMPeoTA in vivo using the OECD 474 micronucleus mouse bone marrow test, reveals no positive results and was performed a few times in reliable and relevant studies. As it is known from the literature that mouse lymphoma assay and/or other in vitro mammalian cell assays designed to detect clastogenicity are yield false positive results with acrylates the positive test result seen in the mouse lymphoma were rated as less of relevance and overruled by the result of the in vivo micronucleus assay and the evidence from literature. Following the integrated mutagenicity testing strategy the next step in conformation of the absence of mutagenic potential could be a second in vivo test (like rodent bone marrow clastogenicity or comet assay) but this is not mandatory. As there is additional (negative) information from less reliable dermal (lifetime) carcinogenicity study in mice (Cytec, Kettering Laboratory, 1987) no further concerns of an increased genetic toxicity are expected. Therefore and according to animal welfare no further in vivo test is proposed.

In summary, due to the test results and information from literature the substance is considered as not genetic toxic in vivo.

Key study assignment:

There are four studies available assessing the gene mutation of TMPeoTA in bacteria. All studies are equal in reliability and relevance. Three studies report negative results whereas one study (BASF, 1992) reports about a weak positive result for TA 1535 with metabolic activation. As the result of this Ames test is questionable all studies assessing the mutagenicity in bacteria are integrated as weight of evidence and discussed in assessment section of the endpoint summary.

Assessing the gene mutation in vitro in mammalian cells there is 2 studies available conducted according to OECD guideline (Cytec, Safepharm Laboratories, 2006 and ICCR-Roßdorf GmbH, 2019). These studies are identified as key studies showing both mutagenic and non-mutagenic effects.

Assessing the in vivo cytogenicity there are three good documented and conducted OECD guideline studies available. As all studies show equal results, all studies can be used as key studies. Nevertheless the BASF study investigates the oral route which can be assumed as the most critical route, therefore the BASF study (BASF, 2002) is integrated as key study. But all other studies are also entered as supportive study and the results are taken into account for risk assessment.

Justification for classification or non-classification

As the positive result in one bacteria mutagenicity test (one strain) is questionable and not confirmed by other tests this result is rated as less reliable and in summary no positive result in bacteria mutagenicity test is concluded.

The results of an in vivo mutagenicity, the information from literature and the supporting information from a less reliable lifetime dermal carcinogenicity test did not reveal mutagenic or carcinogenic effects.

In summary, there are no indications for mutagenicity as obtained from experiments in mammals and TMPEOTA does not fulfil the requirement for germ cell mutagens cat. 2 according to GHS (Regulation (EU) 1272/2008)

Labelling mutagenic:

GHS: no labelling

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

Propylidynetrimethanol, ethoxylated, esters with acrylic acid

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

Referenceopen allclose all

Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

Referenceopen allclose all

Endpoint conclusion

Additional information

Justification for classification or non-classification

Referenceopen all close all

Referenceopen all close all