Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), 2-propenoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 07, 2016 to June 17, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch no.: JBIJ0009V
Purity: 100% (UVCB)
Appearance: yellowish liquid

Target gene:

histidine

Species / strain / cell type:

other: S. Typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced by Aroclor 1254)

Test concentrations with justification for top dose:

Based on a pre-test, the following nominal concentrations were prepared for experiments 1a and 1b: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, Based on a pre-test, the following nominal concentrations were prepared for experiments 1a and 1b: 5000, 1500, 500, 150 and 50 μg/plate.
The following nominal concentrations were prepared for experiment 2: 5000, 2500, 1250, 625, 313, 156 and 78 μg/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-Amino-anthracene, 4-nitro-1,2-phenylene diamine,

Details on test system and experimental conditions:

Mutagenicity test:
Top agar was prepared for the Salmonella strains by mixing 100 mL agar (0.6% agar, 0.5% NaCl) with 10 mL of a 0.5 mM histidine-biotin solution. The following ingredients were added (in order) to x mL of molten top agar at 45°C:
- 0.1 mL of an overnight nutrient broth culture of the bacterial tester strain
- 0.1 mL test compound solution
- 0.5 mL S-9 Mix (if repuired) or buffer
After mixing, the liquid was poured into a petridish with minimal agar (1.2% agar, Vogel-Bonner E medium with 2% glucose). After incubation for 48 to 72 hours at 37°C in the dark, colonies (his+ revertants) were counted.

Rationale for test conditions:

Toxicity experiments and dose range finding

Evaluation criteria:

Number of his+ revertants

Statistics:

The number of colonies per plate with each strain as well as mean values of 3 plates, corrected to the next whole number

Key result

Species / strain:

other: Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535

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:

cfr "Any other information on results incl. tables"

Remarks on result:

other: not mutagenic

Experiment 1a:

The test substance (dissolved in DMSO) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA100, TA102 and TA1535 using the plate incorporation method. The test substance showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test substance showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations caused a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 1b:

The test substance (dissolved in DMSO) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strain TA98 using the plate incorporation method. The bacteria strain TA98 was tested separately from the other strains, because of a pipetting error in the experiment 1a. The test substance showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test substance showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed again that none of the tested concentrations caused a significant increase in the number of revertants in the bacteria strain, in the presence and absence of metabolic activation.

Experiment 2:

On the base of the experiment 1a, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strain using the pre-incubation method. The test substance showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test substance showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test substance caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test substance did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it was concluded that the test substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Conclusions:

Under the study conditions, the test substance was not mutagenic in a bacterial reverse mutation test with and without metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B13/14 (bacterial reverse mutation assay), in compliance with GLP. Five Salmonella typhimurium strains (i.e. TA 97a, TA 98, TA 100, TA 102, TA 1535) were exposed to the test substance for 48 to 72 h, at concentrations of 50 to 5000 µg/plate (with 3 plates per condition). At the end of the incubation period, the number of His+ revertants was counted. The test substance did not cause a significant increase in the number of revertant colonies with any of the tester strains, either in the absence or presence of metabolic activation. Positive and negative (vehicle) controls gave expected results; the experiment was therefore considered valid. Under the study conditions, the test substance was not mutagenic in a bacterial reverse mutation study with and without metabolic activation (Andres, 2016).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

supporting study

Study period:

From March 14, 1984 to April 24, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

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:

bacterial forward mutation assay

Target gene:

Thymidine kinase (TK)

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:

1st experiment: 0.25; 0.5; 1.0; 2.0 nL/mL (without metabolic activation)
1st experiment: 2.0; 6.0; 12.0; 16.0; 20.0; 24.0; 32.0 nL/mL (with metabolic activation)
2nd experiment: 1.9; 5.0; 10.0; 12.5; 15.0; 20,0; 25.0 nL/mL (with metabolic activation)
Doses were chosen based on a preliminary cytotoxicity experiment.

Vehicle / solvent:

The test substance was immiscible in 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:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulfonate (EMS) and 3-Methylcholanthrene (MCA)

Details on test system and experimental conditions:

- Preliminary cytotoxicity experiment to establish the doses: 4h exposure at 37°C. After the exposure, the cells are resuspended in growth medium and incubated for 24h; the cell count is then determined (cell growth).
- Experiment without metabolic activation: 3.0E06 cells, 4h exposure, 37°C. A expression period of 2 days is used to allow recovery, growth and expression of the TK -/- phenotype. Cells density are determined after 24 and 48h. The appearance of treated cultures are recorded as well. The cloning efficiency is measured with 1.0E06 cells after 10 to 14 days.
- Experiment with metabolic activation: identical to the 1st exp. with the addition of S9-mix (Arolor 1254 induced rat liver homogenate).

Rationale for test conditions:

- Preliminary testing
- Guidelines

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is at least 150% of the concurrent background frequency. 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 to evaluate the mutagenicity of a test substance. 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, 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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In the preliminary cytotoxicity assay, the test material was lethal at 15.6 nL/mL without activation and highly toxic at 31.3 nL/mL in the presence of metabolic activation.
- The test substance 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 substance 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 tested 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 substance 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 substance was tested 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.

Conclusions:

Based on the results of the read across study, the test substance was considered to be mutagenic in mammalian cells with metabolic activation (lymphoma forward mutation assay).

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance, TMP(EO)TA, according to a mouse lymphoma forward mutation assay (Clive, 1975 and 1979), in compliance with GLP. The test was performed to evaluate a test substance’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 was tested in the presence of 5-trifluorothymidine and with a test substance concentration of 0.25 to 2 nL/mL without metabolic activation and of 1.9 to 32 nL/mL with metabolic activation (S9-mix - Arolor 1254 induced rat liver homogenate). Two experiments were initiated but the 2nd experiment was only performed in the presence of metabolic activation. Under non-activation conditions, three treatments were assayed and moderate toxicities were induced (percent relative growths, 59 to 37.8%). Higher toxicities were not assayed because a small increase in concentration from 1.0 nL/mL to 2.0 nL/mL was excessively toxic. In preliminary cytotoxicity assay, the test material appeared less toxic, but only because the assay was based on 24-h growth. The test substance induced delayed toxicity which was not apparent until 48 h. In order for a treatment to be considered mutagenic in this assay, a mutant frequency exceeding 30.5E-6 was required. None of the assayed treatments induced this level of mutant action. The test substance was therefore considered non-mutagenic in this assay without activation at concentrations that approached excessive toxicity. In the presence of metabolic activation, moderate to very high toxicities were induced (percent relative growths, 41.1 to 2.7%). Treatments with relative growths below 10% were not used in the analysis. Of the remaining three treatments, one (12 nL/mL, 18.9% relative growth) induced a mutant frequency that exceeded the minimum criterion of 44.8E-6. The increase was 2.7-fold above background mutant frequency (average solvent controls). However, this increase was not sufficient evidence to consider a test substance mutagenic in one assay. Another activation assay was therefore performed. In this activation assay, treatments from 1.88 nL/mL to 20 nL/mL were assayed for mutant induction and non-detectable to moderate toxicities were induced (percent relative growths, 113 to 27%). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 44.1E-6. The two highest concentrations (15 nL/mL and 20 nL/mL) induced mutant frequencies that exceeded the minimum criterion. The increases confirmed the activity observed in the previous experiment under metabolic activation. The test substance was therefore considered mutagenic with activation in this assay. In the assays used in this evaluation, the average cloning efficiencies for the solvent control varied from 73.2% without activation to 65.6 and 82.4% with activation which demonstrated acceptable cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background. The positive control mutant frequency in the activation portion of the experiment 1 was lower than usual, but there was a large increase in mutant colonies. Based on the results of the read across study, the test substance induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- cells only in the presence of metabolic activation (Cifone, 1984).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

From December 07, 2005 to February 17, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: United Kingdom Environmental Mutagen Society

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

according to UK GLP

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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
(Doses were selected based on the results of the preliminary toxicity test for cytotoxicity).

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Method of application: in suspension
Durations:
- Preliminary test (with 5.0E05 cells/mL or 1.5E05 cells/mL): exposures of 4 h (with and without S9) and 24 h (without S9)
- Preliminary test (with 2.0E05 cells/mL): incubation of 24 h to evaluate the suspension growth values
(Fixation time (start of exposure up to fixation or harvest of cells): 24- 48 h)
- Experiment 1 (1.0E06 cells/mL): 4 h incubation
- Experiment 2 (1.0E06 cells/mL): 4 h incubation
- Expression time (cells in growth medium): 48 h
Selection agent (mutation assays): 5-trifluorothymidine (TFT)
Staining (for cytogenetic assays): MTT solution
Number of replicates: Two

Rationale for test conditions:

- Preliminary cytotoxicity test
- guidelines

Evaluation criteria:

- Vehicle control values greater than 250 x 10E-06 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 10E-06 will be considered positive.

Statistics:

No data

Key result

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

Additional information on results:

- No precipitation was observed throughout the study.
- 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 substance produced cytotoxicity, criteria (iii) was used for dose selection in the main study.
- Experiments 1 and 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.
- 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 controls induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Conclusions:

Based on the results of the read across study, the test substance was considered to be mutagenic in mammalian cells with and without metabolic activation (lymphoma forward mutation assay).

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 476 and EU Method B.17 (In Vitro Mammalian Cell Gene Mutation Test, in compliance with GLP. 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 - DMSO) and positive controls (EMS and CPA). The entire experiment was repeated to confirm the result of the first experiment. Four hour exposures were used both with and without metabolic 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 controls 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. Based on the results of the read across study, the test substance was considered to be mutagenic in mammalian cells with and without metabolic activation (Flanders, 2006).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

supporting study

Study period:

From November 02, 1998 to January 16, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL 2 due to RA

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

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: 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 substance 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), of an intermediate and of a low dose of the test substance. The route of administration was chosen to maximize the chance of the test substance to reach the target tissue. The dosing volume was 10 mL/kg bw.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

- 24h and 48h: treatment groups
- 48h: positive conrol

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

nominal concentration

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

No. of animals per sex per dose:

5 (two groups per dosage)

Control animals:

yes, concurrent vehicle

Positive control(s):

- positive control: cyclophosphamide (CPA)
- Route of administration: intraperitoneal
- Doses / concentrations: 50mg/kg bw

Tissues and cell types examined:

- Bone marrow
- 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:

- A 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

Key result

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

Remarks on result:

other: toxicity signs (at 125 and 250 mg/kg bw): lethargic behaviour and rough coat

Remarks:

no abnormalities at 62.5 mg/kg bw

Additional information on results:

- No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of the animals treated with the test substance. The animals of the groups that received 250 mg/kg bw showed slight decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time compared to the ratio observed in the animals treated with 62.5 mg/kg bw. This result reflected a toxic effect of the test substance on the erythropoiesis, implying that it reached the bone marrow cells of the mouse.
- Several toxicity signs such as lethargic behaviour and rough coat were observed in mice following the administration of 125.0 and 250.0 mg/kg bw. No abnormalities was recorded in the groups that had received 62.5 mg/kg bw test substance.

Conclusions:

Based on the results of the read across study, the test substance was considered to be not clastogenic in mouse (in vivo Mammalian Erythrocyte Micronucleus Test).

Executive summary:

A study was conducted to determine the in vivo toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 474 (in vivo Mammalian Erythrocyte Micronucleus Test), in compliance with GLP. Six groups each comprising 5 male and 5 female NMRI mice, received a single intraperitoneal injection of 10 mL/kg bw test substance. Two groups were dosed with 250 mg/kg bw, two groups were dosed with 125 mg/kg bw and two groups were dosed with 62.5 mg/kg bw. One group treated with the vehicle alone (corn oil) or with cyclophosphamide (CPA) at 50 mg/kg bw served as negative and positive controls, respectively. The tissue examined was the bone marrow. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. Mortality and toxicity signs were recorded daily. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of the animals treated with the test substance. The animals of the groups that received 250 mg/kg bw showed slight decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time compared to the ratio observed in the animals treated with 62.5 mg/kg bw. This result reflected a toxic effect of the test substance on the erythropoiesis, implying that it reached the bone marrow cells of the mouse. Several toxicity signs such as lethargic behaviour and rough coat were observed in mice following the administration of 125.0 and 250.0 mg/kg bw. No abnormalities were recorded in the groups that had received 62.5 mg/kg bw test substance. Based on the results of the read across study, the test substance was considered to be not clastogenic in mouse (Bertens, 2001).

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

supporting study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Once

Post exposure period:

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

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)
- 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 substance, i.e. 100 mg/kg bw (leading to clinical signs such as hunched posture and ptosis) was selected for use in the main test, with 50 and 25 mg/kg bw 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 at either 24 or 48 h 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.

Key result

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 the range-finding test
- 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 substance 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 substance 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 the main 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 substance groups when compared to their concurrent vehicle control group. 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 group.
- 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 substance 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
	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:

Based on the results of the read across study, the test substance was considered to be not clastogenic in mouse (in vivo Mammalian Erythrocyte Micronucleus Test).

Executive summary:

A study was conducted to determine the in vivo toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 474 and EU Method B.12 (in vivo Mammalian Erythrocyte Micronucleus Test), in compliance with GLP. This experiment was performed in groups of Crl:CD-l(ICR)BR mice. A range-finding test was performed to find suitable dose levels of the test substance, route of administration and to investigate difference in toxic response between the sexes. As there was no marked difference in toxicity 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 femoral bone marrow was extracted, and smear preparations were prepared and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice (each of 7 animals) were given a single intraperitoneal dose of arachis oil and represented vehicle control groups. Additionally, 5 mice orally dosed with cyclophosphamide served as positive control. Vehicle control animals were killed 24 or 48 h later, and positive control animals 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. Based on the results of the read across study, the test substance was considered to be not mutagenic in mouse (Durward and Flanders, 2006).

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

From July 01, 2002 to August 08, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL 2 due to RA

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Batch no.: B 02011
Appearance: Light yellow liquid

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: 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 substance 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:

Preparations 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

Post exposure period:

24 or 48 hours

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

nominal concentrations

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

nominal concentrations

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 substance 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:

Femora bone marrow and erythrocytes

Details of tissue and slide preparation:

- Criteria for dose selection:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg bw 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 and the dose of 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as lower 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 for slides preparation:
The two femora were prepared by dissection and removing of 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) 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 and may 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.

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.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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:

Based on the results of the read across study, the test substance was considered to be not clastogenic in mouse (in vivo Mammalian Erythrocyte Micronucleus Test).

Executive summary:

A study was conducted to determine the in vivo toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 474 and EU Method B.12 (in vivo Mammalian Erythrocyte Micronucleus Test), in compliance with GLP. The test substance was tested for chromosomal damage (clastogenicíty) and for its ability to induce spindle poison effects (aneugenic activity) in male NMRI mice. For this purpose, the test substance, dissolved in DMSO, was administered once orally to male animals at dose levels of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg bw in a volume of 4 ml/kg bw. As negative control group, mice were administered the vehicle by the same route. 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 2000 mg/kg bow and in the vehicle control groups. In the test groups 1000 mg/kg bw and 500 mg/kg bw and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 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. The results obtained for the negative control group 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 did not show any clinical signs of toxicity. The administration of the test substance was tolerated by all animals without any signs or symptoms. Based on the results of the read across study, the test substance was considered to be not clastogenic in mouse (Engelhardt, 2002).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro study with the test substance

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B13/14 (bacterial reverse mutation assay), in compliance with GLP. Five Salmonella typhimurium strains (i.e. TA 97a, TA 98, TA 100, TA 102, TA 1535) were exposed to the test substance for 48 to 72 h, at concentrations of 50 to 5000 µg/plate (with 3 plates per condition). At the end of the incubation period, the number of His+ revertants was counted. The test substance did not cause a significant increase in the number of revertant colonies with any of the tester strains, either in the absence or presence of metabolic activation. Positive and negative (vehicle) controls gave expected results; the experiment was therefore considered valid. Under the study conditions, the test substance was not mutagenic in a bacterial reverse mutation study with and without metabolic activation (Andres, 2016).

In vitro studies with a read across substance

A study was conducted to determine the in vitro genetic toxicity of the read across substance, TMP(EO)TA, according to a mouse lymphoma forward mutation assay (Clive, 1975 and 1979), in compliance with GLP. The test was performed to evaluate a test substance’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 was tested in the presence of 5-trifluorothymidine and with a test substance concentration of 0.25 to 2 nL/mL without metabolic activation and of 1.9 to 32 nL/mL with metabolic activation (S9-mix - Arolor 1254 induced rat liver homogenate). Two experiments were initiated but the 2nd experiment was only performed in the presence of metabolic activation. Under non-activation conditions, three treatments were assayed and moderate toxicities were induced (percent relative growths, 59 to 37.8%). Higher toxicities were not assayed because a small increase in concentration from 1.0 nL/mL to 2.0 nL/mL was excessively toxic. In preliminary cytotoxicity assay, the test material appeared less toxic, but only because the assay was based on 24-h growth. The test substance induced delayed toxicity which was not apparent until 48 h. In order for a treatment to be considered mutagenic in this assay, a mutant frequency exceeding 30.5E-6 was required. None of the assayed treatments induced this level of mutant action. The test substance was therefore considered non-mutagenic in this assay without activation at concentrations that approached excessive toxicity. In the presence of metabolic activation, moderate to very high toxicities were induced (percent relative growths, 41.1 to 2.7%). Treatments with relative growths below 10% were not used in the analysis. Of the remaining three treatments, one (12 nL/mL, 18.9% relative growth) induced a mutant frequency that exceeded the minimum criterion of 44.8E-6. The increase was 2.7-fold above background mutant frequency (average solvent controls). However, this increase was not sufficient evidence to consider a test substance mutagenic in one assay. Another activation assay was therefore performed. In this activation assay, treatments from 1.88 nL/mL to 20 nL/mL were assayed for mutant induction and non-detectable to moderate toxicities were induced (percent relative growths, 113 to 27%). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 44.1E-6. The two highest concentrations (15 nL/mL and 20 nL/mL) induced mutant frequencies that exceeded the minimum criterion. The increases confirmed the activity observed in the previous experiment under metabolic activation. The test substance was therefore considered mutagenic with activation in this assay. In the assays used in this evaluation, the average cloning efficiencies for the solvent control varied from 73.2% without activation to 65.6 and 82.4% with activation which demonstrated acceptable cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background. The positive control mutant frequency in the activation portion of the experiment 1 was lower than usual, but there was a large increase in mutant colonies. Based on the results of the read across study, the test substance induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- cells only in the presence of metabolic activation (Cifone, 1984).

A study was conducted to determine the in vitro genetic toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 476 and EU Method B.17 (In vitroMammalian Cell Gene Mutation Test, in compliance with GLP. 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 - DMSO) and positive controls (EMS and CPA). The entire experiment was repeated to confirm the result of the first experiment. Four hour exposures were used both with and without metabolic 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 controls 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. Based on the results of the read across study, the test substance was considered to be mutagenic in mammalian cells with and without metabolic activation (Flanders, 2006).

In vivo studies with a read across substance

A study was conducted to determine the in vivo toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 474 (in vivo Mammalian Erythrocyte Micronucleus Test), in compliance with GLP. Six groups each comprising 5 male and 5 female NMRI mice, received a single intraperitoneal injection of 10 mL/kg bw test substance. Two groups were dosed with 250 mg/kg bw, two groups were dosed with 125 mg/kg bw and two groups were dosed with 62.5 mg/kg bw. One group treated with the vehicle alone (corn oil) or with cyclophosphamide (CPA) at 50 mg/kg bw served as negative and positive controls, respectively. The tissue examined was the bone marrow. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. Mortality and toxicity signs were recorded daily. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of the animals treated with the test substance. The animals of the groups that received 250 mg/kg bw showed slight decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time compared to the ratio observed in the animals treated with 62.5 mg/kg bw. This result reflected a toxic effect of the test substance on the erythropoiesis, implying that it reached the bone marrow cells of the mouse. Several toxicity signs such as lethargic behaviour and rough coat were observed in mice following the administration of 125.0 and 250.0 mg/kg bw. No abnormalities were recorded in the groups that had received 62.5 mg/kg bw test substance. Based on the results of the read across study, the test substance was considered to be not clastogenic in mouse (Bertens, 2001).

A study was conducted to determine the in vivo toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 474 and EU Method B.12 (in vivo Mammalian Erythrocyte Micronucleus Test), in compliance with GLP. The test substance was tested for chromosomal damage (clastogenicíty) and for its ability to induce spindle poison effects (aneugenic activity) in male NMRI mice. For this purpose, the test substance, dissolved in DMSO, was administered once orally to male animals at dose levels of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg bw in a volume of 4 ml/kg bw. As negative control group, mice were administered the vehicle by the same route. 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 2000 mg/kg bow and in the vehicle control groups. In the test groups 1000 mg/kg bw and 500 mg/kg bw and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 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. The results obtained for the negative control group 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 did not show any clinical signs of toxicity. The administration of the test substance was tolerated by all animals without any signs or symptoms. Based on the results of the read across study, the test substance was considered to be not clastogenic in mouse (Engelhardt, 2002).

A study was conducted to determine the in vivo toxicity of the read across substance, TMP(EO)TA, according to OECD Guideline 474 and EU Method B.12 (in vivo Mammalian Erythrocyte Micronucleus Test), in compliance with GLP. This experiment was performed in groups of Crl:CD-l(ICR)BR mice. A range-finding test was performed to find suitable dose levels of the test substance, route of administration and to investigate difference in toxic response between the sexes. As there was no marked difference in toxicity 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 femoral bone marrow was extracted, and smear preparations were prepared and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice (each of 7 animals) were given a single intraperitoneal dose of arachis oil and represented vehicle control groups. Additionally, 5 mice orally dosed with cyclophosphamide served as positive control. Vehicle control animals were killed 24 or 48 h later, and positive control animals 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. Based on the results of the read across study, the test substance was considered to be not mutagenic in mouse (Durward and Flanders, 2006).

Evidence from the literature

A review by Johannsenet al.(2008) shows that there was no evidence of point mutations when acrylic acid or over 60 acrylates and methacrylates were investigated inSalmonellabacterial tests or in HPRT mutation tests with mammalian cells. Also, no evidence of a mutagenic effects was seen in whole animal clastogenicity and/or aneuploidy (chromosomal aberration/micronucleus) studies. Consistent with thein vivotesting results, acrylic acid exhibited no evidence of carcinogenicity in chronic rodent cancer bioassays. Nevertheless, positive results were often observed in the mouse lymphoma assay and/or otherin vitromammalian cell assays designed to detect clastogenicity. However, the biological relevance of thisin vitroresponse is questioned based on the non-concordance ofin vitroresults with those ofin vivostudies addressing the same mutagenic endpoint (clastogenicity). Thein vitromouse lymphoma results are therefore likely to represent false positives.

Overall assessment

Considering the fact that the acrylates are known to produce false positives in the mouse lymphoma assay and/or other in vitro mammalian cell assays designed to detect clastogenicity, both tests results from mouse lymphoma assay with TMP(EO)TA are considered to be less reliable. Further, the clastogenic effects (observed in the form of small colonies in the Flanders, 2006 assay) can be overruled by the result of the three in vivo micronucleus assays conducted in mice. Therefore, based on the available weight evidence, the test substance is overall not considered to be genotoxic and no further testing is necessary.

Justification for classification or non-classification

Based on the overall weight of evidence, the test substance does not require classification for mutagenicity according to CLP (EC 1272/2008) criteria.