Reaction products of 2-hydroxyethyl methacrylate and diphosphorous pentoxide and water

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Weight of evidence: data inconclusive, further information required, 2021

(1) negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2018

(2) positive, in vitro chromosome aberration test with human lymphocytes (with and without S-9 activation), OECD TG 473, 2021

(3) negative, in vitro mammalian gene mutation test (with and without S-9 activation), OECD TG 490, 2021

(4) More information required, : intent to read-across to source substance OECD TG 489, COMET Assay (in vivo mammalian cell study: DNA damage and/or repair) currently under test proposal assessment. Source substance: Reaction products of 2-hydroxyethyl methacrylate and diphosphorus pentaoxide ; EC/List Number: 810-703-1 / CAS Number: 1187441-10-6

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-01-2018 to 21-03-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Range Finding Experiment 1 (direct plate assay): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Definitive Experiment 1 (direct plate assay): 52, 164, 512, 1600, 5000 μg/plate
Definitive Experiment 2 (pre-incubation assay): 52, 164, 512, 1600, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item was soluble in dimethyl sulfoxide. Test item concentrations were used within 3 hours of preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 ; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies were counted.
Experiment 2. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies were counted.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants

Rationale for test conditions:

Selection of an adequate range of doses was based on the first experiment (seven concentrations in triplicate) with all five tester strains, both with and without S9-mix considering cytotoxicity and precipitation. See tables for more information.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done. See 'Any other information on materials and methods' and 'evaluation criteria' for details on the acceptability and evaluation criteria of the assay.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

Test item was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain at up to the top dose of 5000 µg/plate.
Experiment 2: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain at up to the top dose of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
Experiment 1 (direct plate assay) served as a range finding test in all species/strains for subsequent pre-incubation assays.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control values were within the laboratory historical control data ranges. The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1535 in the absence of S9-mix in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 170 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study. The test item indicated no toxicologically significant dose-related increases in the number of revertants in two independently repeated experiments. Any increases were within the historical control range.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:
negative
Under the conditions of this study, the test itme was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD TG 471 and EU Method B.13/14 in accordance with GLP, to evaluate the mutagenic activity of the test item in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (incorporating the plate incorporation and pre-incubation methods) in both the presence and absence of S-9 metabolic activation system. The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose level of 5000 μg/plate. In all three experiments the test item did not precipitate on the plates at up to 5000 μg/plate dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a toxicologically significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in two independently repeated experiments. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-05-2020 to 25-01-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

: Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 20 November 2006

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable (chromosome aberration test)

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines OECD, EC, METI). Whole blood samples obtained from healthy male subjects were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals. Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined. Further details on the donors is available in the full study report. Therefore the exposure time for the experiments for all volunteer donors was between ca. 1.5 to 1.7 x AGT at 24 hours. Further details on the donors is available in the full study report.

CELLS USED
- Type and source of cells: Human lymphocytes from volunteers
- Suitability of cells: Cells used in accordance with OECD TG 473. Details on volunteers provided below.
- Normal cell cycle time (negative control): See below ; (13.2 to 14.8 hours = 1.0 normal cell cycle time ; ca. 24 hours = ca. 1.7 times normal cell cycle time).

For lymphocytes:
- Sex, age and number of blood donors: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (volunteers are typically: aged 18-35), sex not specified ; who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes for the donors used in this study: was considered to be approximately 14.4 hours (see above for range of AGT). Therefore using this average, it can be considered that the in-house exposure time for the experiments for 1.5 x AGT is ca. 21.6 hours and 1.7 x AGT is ca. 24.48 hours . Further details on the donors is available in the full study report.
- Whether whole blood or separated lymphocytes were used: Whole blood ; cells were cultured as follows : whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added to stimulate the lymphoctyes to divide. Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin. All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 42 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 - 38.8°C) during the study.
- Whether blood from different donors were pooled or not: None-pooled.
- Mitogen used for lymphocytes: (9 mg/mL) phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cells were cultured as follows : cells were grown in RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin. All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 42 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 - 38.8°C) during the study.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: from Recognised supplier (origin and dates within full study report)
- method of preparation of S9 mix: Documented in the full study report. S9-mix was prepared immediately before use and kept refrigerated. Mixing S9, 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. Filtered (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. Final S9 in exposure medium was 1.8% (v/v).
- concentration or volume of S9 mix and S9 in the final culture medium: 1.8%(v/v) S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to use, each batch of S9 was tested for its capability to activate known mutagens documented outside the full study report. Additionally, concurrent positive control substances all produced a positive response (within the historic control range) and demonstrated the validity of the experiment and the integrity of the S9-mix.

Test concentrations with justification for top dose:

See 'any other information on materials and methods incl. tables' for further information.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed in a preceding gene mutation assay (citedn the full study report) in which the test item proved to be insoluble in culture medium. The test item formed a clear colourless solution in dimethyl sulfoxide (DMSO). Within the present study, the stock solution was pre-treated with ultrasonic pre-treatment until the test item had completely dissolved, except in experiment 1B and the second experiment where no ultrasonic pre-treatment was considered necessary. To protect the test item from light, amber-colored glassware was used for test item preparations. The final concentration of the solvent in the culture medium was 1.0% (v/v). The pH and the osmolarity of the culture medium containing several tested concentrations were recorded. Test item concentrations were used within 2.5 hours after preparation.
- Other: The pH and osmolarity of several test item concentrations and the solvent control was measured in the solubility test and in the first and second cytogenetic assay. See tables within 'any other information on materials and methods incl. tables' for further information.

Untreated negative controls:

other:

Remarks:

Vehicle control served as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation, 0.5 and 0.75 μg/ml for a 3 h, 0.2 and 0.3 μg/ml for a 24 h exposure and 0.1 and 0.15 μg/ml for the 48 h exposure periods, respectively

Untreated negative controls:

other:

Remarks:

Vehicle control served as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation 10 μg/ml for a 3 h exposure period

Details on test system and experimental conditions:

METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components: Lymphocytes (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin). Cultured for 48 hours and thereafter exposed to respective doses of test item for the defined exposure durations with fixation consisting of centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for the defined fixation period.

DURATION
- Preincubation period: Not reported.
- Exposure duration: See ‘any other information on materials and methods incl. tables’.

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/mL)

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

NUMBER OF CELLS EVALUATED: A total of 1000 lymphocyte cell nuclei per culture were counted and the number of cells in metaphase per 1000 cells recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported.
- Other: Scoring: Where possible, the first 100 consecutive well-spread metaphases from each concentration (200 per duplicate) were assessed for observations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

Positive response criteria
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Negative response criteria
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistical analysis is also performed (see: ‘Statistics’). The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Statistics:

Statistical analysis was performed. The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

See 'any other information on results incl. tables' for further information.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH and osmolarity of several test item concentrations and the solvent control was measured in the solubility test and in the first and second cytogenetic assay. See tables within 'any other information on materials and methods incl. tables' for further information. In the absence of S9-mix at the 3 h and 24 h exposure time, the test item induced a statistically significant increase in the number of cells with chromosome aberrations, both when gaps were included and excluded in the first cytogenetic assay and where gaps were included in the second cytogenetic assay. A dose related trend was observed (Cochran Armitage trend test p= 0.008 and p <0.001) and moreover the number of cells with chromosome aberrations was above the 95% control limits of the distribution of the historical negative control database. The increase is therefore considered biologically relevant. However, care should be taken with the interpretation of the results especially in the first cytogenetic assay since the pH of the test item was low in comparison with the solvent control (approximately pH 6 in comparison to pH 7.5 for the solvent control). The low pH may lead to artefactual positive results, i.e. chromosome damage not caused by direct interaction between the test item and chromosomes.
- Effects of osmolality: See above.
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: A precipitate of the test item was observed at 5000 μg/mL in the culture medium. No precipitates were reported in the first or second cytogenecity assays.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 5000 μg/mL. The maximum dose was limited based on solubility and cytotoxicity in culture media. The selection of the maximum dose level was based on toxicity for the main test.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. For data that was considered acceptable and used for scoring and subsequent analysis. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: For each exposure group (that may fall) outside the laboratory historical control data range was compared to that of the solvent control using statistical analyses.

Main Test:

Experiment 1/1B

Within EXP 1B, although a concentration of 2700 μg/mL showed not enough cytotoxicity (43%) and a concentration of 2900 μg/mL was too toxic for scoring (73%) it was considered appropriate to score these concentrations for chromosome aberrations.

Table 2. Chromosome Aberrations in Human Lymphocyte Cultures Treated with test item in the Absence of S9-Mix in Cytogenetic Assay 1B (3 H Exposure Time, 24 H Fixation Time) (EXP 1B)

Conc	DMSO (1.0% v/v)				100 µg/mL				2500 µg/mL				2700 µg/mL				2900 µg/mL				MMC-C 0.5 µg/mL
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				98				95				57				27				72
No. of Cells scored	150  150 300				150  150 300				150  150 300				150  150 300				150  150 300				150  150 300
No. of Cells with aberrations (+ gaps) a)	0	1		1	0	1		1	1	0		1	10	21		***^) 31	13	34		***^) 47	37	40		***^) 77
No. of Cells with aberrations (- gaps)	0	1		1	0	1		1	1	0		1	10	21		***^) 31	13	34		***^) 47	37	40		***^) 77
g’
g”
b’						1							6	25			16	34			23	20
b”		1				1			1				2	8			4	15			15	10
m’													1					6
m”																		2
exch.													1	7			5	18			8	11
dic
d’
misc.									endo poly		2poly		5poly,p 3endo		5endo		7poly 6endo		4poly 4endo		poly		p
total aberr (+ gaps)	0	1			0	2			1	0			11	40			25	75			46	42
total aberr (- gaps)	0	1			0	2			1	0			11	40			25	75			46	42

a) Abbreviations used for various types of aberrations are listed in the full study report
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration.

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

 

Table 3. Chromosome Aberrations in Human Lymphocyte Cultures Treated with test item in the Presence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time) (EXP 1)

Conc	DMSO (1.0% v/v)				100 µg/mL				2500 µg/mL				3000 µg/mL				CP 10 µg/mL
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				97				72				49				50
No. of Cells scored	150  150 300				150  150 300				150  150 300				150  150 300				150  150 300
No. of Cells with aberrations (+ gaps) a)	0	0		0	0	0		0	0	0		0	0	0		0	29	26		***^) 55
No. of Cells with aberrations (- gaps)	0	0		0	0	0		0	0	0		0	0	0		0	29	26		***^) 55
g’
g”
b’																	18	15
b”																	5	4
m’
m”																	2	1
exch.																	6	6
dic
d’
misc.									2 poly		endo		2 poly endo		poly 2 endo				poly intra
total aberr (+ gaps)	0	0			0	0			0	0			0	0			31	27
total aberr (- gaps)	0	0			0	0			0	0			0	0			31	27

a) Abbreviations used for various types of aberrations are listed in the full study report
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration.

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

 

Table 4. Chromosome Aberrations in Human Lymphocyte Cultures Treated with test item in the Absence of S9-Mix in the Second Cytogenetic Assay (24 H Exposure Time, 24 H Fixation Time) (EXP 2A)

Conc	DMSO (1.0% v/v)				100 µg/mL				500 µg/mL				1250 µg/mL				MMC-C 0.2 µg/mL
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				64				71				49				49
No. of Cells scored	150  150 300				150  150 300				150  150 300				150  150 300				150  150 300
No. of Cells with aberrations (+ gaps) a)	0	1		1	0	0		0	0	1		1	5	6		**^) 11	38	24		***^) 62
No. of Cells with aberrations (- gaps)	0	1		1	0	0		0	0	1		1	3	3		6	36	24		***^) 60
g’													2	3			4
g”
b’		1											4	3			15	11
b”										1							6	2
m’
m”
exch.																	19	14
dic
d’
misc.
total aberr (+ gaps)	0	1			0	0			0	1			6	6			44	27
total aberr (- gaps)	0	1			0	0			0	1			4	3			40	27

a) Abbreviations used for various types of aberrations are listed in the full study report
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

 

Table 5. Chromosome Aberrations in Human Lymphocyte Cultures Treated with test item in the Absence of S9-Mix in the Second Cytogenetic Assay (48 H Exposure Time, 48 H Fixation Time) (EXP 2A)

Conc	DMSO (1.0% v/v)				10 µg/mL				100 µg/mL				200 µg/mL				300 µg/mL				MMC-C 0.1 µg/mL
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				96				63				53				37				43
No. of Cells scored	150  150 300				150  150 300				150  150 300				150  150 300				150  150 300				150  150 300
No. of Cells with aberrations (+ gaps) a)	0	0		0	0	0		0	0	0		0	0	0		0	0	0		0	33	37		***^) 70
No. of Cells with aberrations (- gaps)	0	0		0	0	0		0	0	0		0	0	0		0	0	0		0	33	37		***^) 70
g’																						1
g”																					1
b’																					20	10
b”																					5	11
m’
m”
exch.																					16	22
dic
d’
misc.
total aberr (+ gaps)	0	0			0	0			0	0			0	0			0	0			42	44
total aberr (- gaps)	0	0			0	0			0	0			0	0			0	0			41	43

a) Abbreviations used for various types of aberrations are listed in the full study report
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

Conclusions:

Interpretation of results:
Positive (precautionary conclusion, due to potentially artefactual positive results due to low pH of the test item replicates in comparison to the solvent control replicates)
Under the conditions of this study, the test item was considered to be clastogenic to human lymphocytes in vitro.

Executive summary:

The study was performed to the requirements of OECD TG 473 under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of peripheral human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, multiple exposure conditions were investigated; a 3-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1.8% final concentration with cell harvest after a 24-hour fixation period, 3-hour exposure in the absence of metabolic activation (S9) with a 24-hour fixation period. A 24-hour exposure in the absence of metabolic activation with a 24-hour fixation period and a 48-h exposure 48-hour fixation period in the absence of metabolic activation (S9). In the first cytogenetic assay, the test item was tested up to 2900 and 3000 µg/mL for a 3 h exposure with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix, respectively. Toxicity was reached at these dose levels. In the second cytogenetic assay, the test item was tested up to 1250 µg/mL for a 24 h continuous exposure with a 24 h fixation time and up to 300 µg/mL for a 48 h continuous exposure with a 48 h fixation time in the absence of S9-mix. Toxicity was reached at these dose levels. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. All acceptability criteria were met. It was therefore concluded that the test conditions were adequate, and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, the test item induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded in the first cytogenetic assay and where gaps were included in the second cytogenetic assay. These increases were observed at the 3 h and 24 h exposure time. The number of cells with chromosome aberrations was above the historical control data range both when gaps were included and excluded. The increases were therefore considered biologically relevant. However, care should be taken with the interpretation of the results especially in the first cytogenetic assay since the pH of the test item was low in comparison with the solvent control (approximately pH 6 in comparison to pH 7.5 for the solvent control). The low pH may lead to artefactual positive results, i.e. chromosome damage not caused by direct interaction between the test item and chromosomes. The test item did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9 mix at the 48 h exposure time and in the presence of S9-mix at the 3 h exposure time. In the first cytogenetic assay the test item increased the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix in a dose dependent manner. This may indicate that the test item has the potential to disturb mitotic processes and cell cycle progression. It can be concluded that the test item has the potential to disturb mitotic processes and cell cycle progression and induce numerical chromosome aberrations under the experimental conditions described. Under the conditions of this study, the test item was considered to be clastogenic to human lymphocytes in vitro. Care should be taken with the interpretation of the results especially in the first cytogenetic assay since the pH of the test item was low in comparison with the solvent control (approximately pH 6 in comparison to pH 7.5 for the solvent control). The low pH may lead to artefactual positive results, i.e. chromosome damage not caused by direct interaction between the test item and chromosomes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-06-2020 to 11-01-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C) protected from light
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The final concentration of the solvent in the exposure medium was 1%(v/v). The test item did not precipitate in the exposure medium up to and including the concentration of 5000 μg/mL. Therefore, this concentration was used as the highest test item concentration for the dose-range finding test.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item formulated in DMSO. Serial dilutions prepared from stock.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Liquid test item dissolved in stock.
- Final preparation of a solid: Not applicable.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle, of the test item.

OTHER SPECIFICS: Not applicable

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Recognised supplier (documented in full study report).
- Suitability of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line has been used successfully in in vitro experiments for many years and is guideline specified (OECD TG 490). The test system has been extensively validated.
- Cell cycle length, doubling time or proliferation index: Doubling time ca. 12 hours. Determined under normal growth conditions.
- Sex, age and number of blood donors if applicable: Not applicable.
- Whether whole blood or separated lymphocytes were used if applicable: Not applicable.
- Number of passages if applicable: Not applicable.
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: The karyotype for the cell line has been published and the modal chromosome number is 40 (ref: OECD TG 490).
- Normal (negative control) cell cycle time: Doubling time ca. 12 hours.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stocks of cells are stored in the freezer at approximately -150 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (50 units/mL), Streptomycin (50 U/mL), Sodium pyruvate (1 mM) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly.
- Properly maintained: Yes.
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically checked for karyotype stability: Yes.
- Periodically 'cleansed' against high spontaneous background: Yes. Before the stocks of cells were cleansed of homozygous (TK -/-) mutants by culturing in R10 medium for 24 hours. This medium contained Thymidine (1.6 x10^-5 M), Hypoxanthine (1.0 x10^-4 M) and Aminopterine (2.0 x10^-7 M). For the following 48 hours the cells were cultured in R10 medium containing Hypoxanthine (1.0 x10^-4 M) and Thymidine (1.6 x10^-5 M) only before being returned to R10 medium for a further 24 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: from Recognised supplier (origin and dates within full study report)
- method of preparation of S9 mix: Documented in the full study report. S9-mix was prepared immediately before use and kept refrigerated. Mixing S9, 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. Filtered (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. Final S9 in exposure medium was 4% (v/v).
- concentration or volume of S9 mix and S9 in the final culture medium: 4%(v/v) S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to use, each batch of S9 was tested for its capability to activate known mutagens documented outside the full study report. Additionally, concurrent positive control substances all produced a positive response (within the historic control range) and demonstrated the validity of the experiment and the integrity of the S9-mix.

Test concentrations with justification for top dose:

The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The test item did not precipitate in the exposure medium up to and including the concentration of 5000 μg/mL. Therefore, this concentration was used as the highest test item concentration for the dose-range finding test. The pH and osmolarity of the test item concentrations in culture medium were measured and are indicated in table 1.

I. Preliminary toxicity test: 0 (control), 313, 625, 1250, 2500, 5000 μg/mL
Within three exposure groups:
i) 3-hours exposure to the test item without S9-mix
ii) 3-hours exposure to the test item with S9-mix (4%)
iii) 24-hour exposure to the test item without S9-mix
No precipitate of the test item was observed.

II. Main Test:
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test (Experiment 1) in the absence and presence of S9-mix (4% v/v) for 3 hours exposure: 75, 150, 300, 600, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 200, 2200 and 2400 μg/mL exposure medium.
However, since no dose level with a cell survival of approximately 10-20% was observed the above were rejected and an additional experiment was performed (Experiment 1A) as detailed below.
3-hour without S9: 0 (control), 125, 250, 500, 1000, 1500, 2000, 2150, 2300, 2450, 2600, 2750 and 2900 μg/mL and MMS 15 μg/mL (Experiment 1A)
3-hour with S9: 0 (control), 125, 250, 500, 1000, 1500, 2000, 2150, 2300, 2450, 2600, 2750 and 2900 μg/mL and CP 5 μg/mL (Experiment 1A)
The above were the “accepted” test assays that passed the relevant validity criteria.
24-hour without S9: 0 (control), 50, 100, 400, 500, 600, 700, 1000, 1100, 1200, 1250, 1300 and 1400 μg/mL and MMS 5 μg/mL (Experiment 2)
where:
MMS = Methylmethanesulfonate
CP = Cyclophosphamide
The cultures were then daily sub-cultured subject to acceptable limits of mean cell count. Giving total of 48 hours for expression period.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The final concentration of the solvent in the exposure medium was 1%(v/v). The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The test item did not precipitate in the exposure medium up to and including the concentration of 5000 μg/mL. Therefore, this concentration was used as the highest test item concentration for the dose-range finding test. The pH and osmolarity of the test item concentrations in culture medium were measured and are indicated in table 1.
Applicant assessment indicates: DMSO is a guideline accepted vehicle with an available laboratory historic control data set.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

Full details of the positive control substances, is available in the full study report

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation with test item.
- Cell density at seeding (if applicable): Preliminary Test: 3-hour exposure: 1 x10^6 cells/mL ; 24-hour exposure: 1.25 x10^5 cells/mL ; Definitive Test: 3-hour exposure: 1 x10^6 cells/mL; 24-hour exposure: 1.25 x10^5 cells/mL

DURATION
- Preincubation period: None.
- Exposure duration: Treatment was for 3 hours or 24 hours, respectively at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): 5-Trifluorothymidine at 5 μg/mL

SPINDLE INHIBITOR (cytogenetic assays): Not applicable.

STAIN (for cytogenetic assays): Not applicable.

NUMBER OF REPLICATIONS: The study conducted in single cultures but with two replicates (A and B) at in solvent and positive control groups.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

NUMBER OF CELLS EVALUATED: 2000 cells/well were seeded with selection agent. Cells were also diluted and plated for viability (%V) analysis. Scoring of plates daily for %RSG and %V to obtain RTG. Mutation scoring of plates was ultimately performed for the presence of mutant colonies; large and small colonies analyses was additionally conducted.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Not applicable.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) and cloning efficiency via viability (%)
- Any supplementary information relevant to cytotoxicity: percentage relative suspension growth (%RSG) (post exposure toxicity during the expression period); viability (%) from non-selective medium cultures.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable.

Rationale for test conditions:

See tables. The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (single cultures and/or duplicate controls). In the absence of precipitation: optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Additionally, The pH and osmolarity of the test item concentrations in culture medium were measured.

See information provided in “any other information on materials and methods incl. tables”

Evaluation criteria:

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH effects were observed in pre-solubility testing (see table 1) and pH effects with concentration were recorded. However, no significant effect of the pH on the test were observed at up to optimal (cyto)toxicity or above (i.e. excessive cytotoxicity) in the definitive test.
- Effects of osmolality: Not applicable.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable.
- Precipitation: The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The final concentration of the solvent in the exposure medium was 1%(v/v). The test item did not precipitate in the exposure medium up to and including the concentration of 5000 μg/mL. Therefore, this concentration was used as the highest test item concentration for the dose-range finding test.
- Definition of acceptable cells for analysis: The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (single cultures and/or duplicate controls). In the absence of precipitation: optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES (if applicable): See tables.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See tables.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible:
1. Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 x 10^-6 .
2. A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. Any observed increase should be biologically relevant and will be compared with the historical control data range.
3. A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- Statistical analysis: conducted where applicable (see above: biological relevance)
- Any other criteria: Acceptability:
A mutation assay is considered acceptable if it meets the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: see tables.
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency : RSG% and RTG. ; see tables.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Information provided in the full study report. Between n=113 (3 hour treatment) to n=101 (24 hour treatment) and n=113 (3 hour treatment; + S9-mix) are presented.
- Negative (solvent/vehicle) historical control data: Information provided in the full study report. Between n=114 (3 hour treatment) to n=100 (24 hour treatment) and n=113 (3 hour treatment; + S9-mix) are presented.
- Other: The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Table 1: Results of range-finding test

Dose (μg/ml)		Cell count after 24 hours sub-culture (cells/mL x 10^5)		Cell count after 48 hours sub-culture (cells/mL x 10^5)	SG (1)	RSG (%) (2)
3-hours without metabolic activation
SC		3.8		9.1	17	100
313		3.6		9.8	18	102
625		2.4		9.5	11	66
1250		3.4		5.5	9	54
2500		0.8	(4)	1.5	1	3
5000		0.8	(4)	0.6	0	1
3-hours with metabolic activation
SC		4.4		8.6	21	100
313		5.9		8.0	24	114
625		5.4		7.6	21	99
1250		4.9		7.3	18	87
2500		1.0	(4)	0.9	0	2
5000		0.6	(4)	0.9	0	1
24-hours without metabolic activation
SC		9.6		3.9	24	100
313		7.4		3.5	17	69
625		5.7		2.8	10	43
1250		3.0		1.5	3	12
2500		0.8	(4)	0.7	0	1
5000		0.8	(4)	0.8	0	2

SC = solvent control = DMSO

(1) = suspension growth

(2) = relative suspension growth

(3) = the test item precipitated in the exposure medium

(4) = since less than 1.25 x 10^5 c/ml were present, no subculture was performed

 

Table 2: Definitive Test - summary of results: 3-hour exposure - with and without S9

Dose (μg/ml)	RSG (%)	CE day2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6 survivors
					total	small	Large
3-hours without metabolic activation (Experiment 1A)
SC1	100	89	100	100	79	9	69
SC2		78			99	24	72
250	97	85	102	99	81	9	72
500	106	83	99	105	74	10	62
1000	97	101	121	117	76	13	61
1500	83	76	91	76	62	12	48
2000	62	77	92	57	93	25	66
2150	50	86	103	51	113	28	80
2300	26	94	113	29	96	19	74
2450	7	89	106	7	124	40	78
MMS	61	44	52	32	860	406	375
3-hours with metabolic activation (Experiment 1A)
SC1	100	105	100	100	74	18	53
SC2		93			88	18	67
125	127	85	86	109	86	15	69
250	111	86	87	97	82	13	66
500	131	97	98	128	75	12	62
1000	131	102	103	136	76	16	58
1500	108	85	86	93	74	17	55
2000	82	84	85	70	94	21	70
2150	48	81	82	39	83	25	56
2300	10	52	53	5	185	70	106
CP	71	55	55	39	475	255	190

 

Table 3: Definitive Test - summary of results: 24-hour exposure - without S9

Dose (μg/ml)	RSG (%)	CE day2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6 survivors
					total	small	Large
24-hours without metabolic activation
SC1	100	95	100	100	147	30	109
SC2		95			106	26	76
50	109	116	122	132	93	28	60
100	103	77	81	83	194	61	120
400	63	95	100	63	138	44	85
500	53	89	93	49	199	62	121
600	50	91	96	48	154	49	96
700	43	93	97	41	143	47	88
1000	16	78	82	13	238	68	151
1300	11	65	68	8	461	115	287
MMS	109	64	67	74	1152	328	595

 

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.

Executive summary:

The study was performed according to the requirements of OECD TG 490 under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at 313, 625, 1250, 2500, 5000 μg/mL concentration in DMSO vehicle (selection was based on absence of precipitation in exposure medium at up to 5000 μg/mL), a definitive test was performed whereby L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item. Exposures were conducted using a 3-hour exposure with and without metabolic activation (4% S9), and a 24-hour exposure without metabolic activation. The following concentrations were utilised and/or considered acceptable for assessment under the test acceptability criteria: 3-hour without and with S9: 0 (control), 125, 250, 500, 1000, 1500, 2000, 2150, 2300, 2450, 2600, 2750 and 2900 μg/m and 24-hour without S9: 0 (control), ), 50, 100, 400, 500, 600, 700, 1000, 1100, 1200, 1250, 1300 and 1400 μg/mL.In the first experiment, the test item was tested up to concentrations of 2450 and 2300 µg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. In the absence of S9-mix, relative total growth (RTG) was reduced to 7% and 29% at concentrations of 2450 and 2300 µg/mL respectively. In the presence of S9-mix the RTG was reduced to 5 % and 39% at concentrations of 2300 and 2150 µg/mL, respectively. In the second experiment, the test item was tested up to concentrations of 1300 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 8% and 13% at concentrations of 1300 and 1000 µg/mL respectively. Excessive toxicity was observed at 1300 µg/mL dose level in the absence S9-mix. Acceptable toxicity was observed at 1000 µg/mL dose level. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. This result was confirmed in an independent experiment with modification in the duration of treatment. After the 24 hour treatment, the test item induced an increase in the mutation frequency. The observed increase was above the positive threshold of MF(control) + GEF (i.e. above 253 x 10-6) at the highest dose level tested. However, the increase in the mutation frequency at the TK locus after a 24 hour treatment period was only observed at excessively toxic dose levels (RTG 8%). According to the OECD TG 490 guideline a result would not be considered positive if the increase in MF occurred only at less than or equal to 10% RTG. Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In accordance with REACH Regulation (EC) No. 1907/2006 Annex VII, column 2 section 8.4 : appropriate in vivo mutagenicity studies shall be considered in case of positive result in any of the genotoxicity studies in Annex VII or VIII. With further reference to REACH Regulation (EC) No. 1907/2006 : Article 25(1) and Article 40 and in lieu of formal testing proposal on the present substance, the registrant intends to firstly adapt the information requirement according to REACH Regulation (EC) No. 1907/2006 Annex XI : section 1.5 : Grouping of substances and read-across approach, by utilising data generated associated with a test proposal already under agency (ECHA) assessment under REACH Regulation (EC) No. 1907/2006 : Article 40 and Article 41.

 

The registrant has contacted the agency (ECHA) and/or is contacting the test proposer in order to acquire access to the proposed and/or yet to be conducted study/studies.

 

NON-CONFIDENTIAL NAME OF (SOURCE) SUBSTANCE AND DETAILS OF TEST PROPOSAL

(associated with the test proposal not made by the current registrant, but which is intended to be used within the current registration):

- Name/identifiers: Reaction products of 2-hydroxyethyl methacrylate and diphosphorus pentaoxide (POEMA) ; EC/List Number: 810-703-1 / CAS Number: 1187441-10-6

- Details on the test proposal: According to the test proposer: “it is proposed to address the potential genotoxic properties of POEMA in an in vivo Comet assay. This assay addresses all potential mode-of-actions (the COMET assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death), performance of this in vivo study is considered to be sufficient to conclude on this endpoint. The oral route is considered to be the most appropriate route. As no sex-specific toxicity is expected, the test is performed in a single species. As no tissue-specific toxicity is expected, blood and liver samples will be tested (sic).”

- Test Guideline: OECD TG 489 /

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

- GLP compliance: YES

- Status: Under Agency (ECHA) assessment (2020/2021)

 

For the target substance: the aforementioned study will address the positive chromosome aberrations (precautionary conclusion, due to potentially artefactual positive results due to low pH of the test item replicates in comparison to the solvent control replicates) detected in an available OECD TG 473 in vitro chromosome aberration assay. This would provide further information in accordance with the requirements of REACH Regulation (EC) No. 1907/2006 Annex VIII, column 2 section 8.4 and/or REACH Regulation (EC) No. 1907/2006 Annex XI : section 1.5 : Grouping of substances and read-across approach. And is a required step prior to the proposal to test on the present (target) substance. The demonstration of ‘chemical similarity’ is provided between source and target is given in Section 13 of the current registration dossier.

 

References:

1. REACH Regulation (EC) No. 1907/2006 Annex VIII, column 2 section 8.4

2. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.7, July 2017)

3. REACH Regulation (EC) No. 1907/2006 Annex XI : section 1.5 : Grouping of substances and read-across approach

4. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.6: QSARs and grouping of chemicals, May 2008)

5. ECHA Read-Across Assessment Framework (RAAF), 2017 (as amended)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Registrant indicates - intention to read-across to test currently under (test proposal) assessment by the agency (ECHA). For further information see: ‘Justification for type of information’

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex VII, column 2 section 8.4 : appropriate in vivo mutagenicity studies shall be considered in case of positive result in any of the genotoxicity studies in Annex VII or VIII. With further reference to REACH Regulation (EC) No. 1907/2006 : Article 25(1) and Article 40 and in lieu of formal testing proposal on the present substance, the registrant intends to firstly adapt the information requirement according to REACH Regulation (EC) No. 1907/2006 Annex XI : section 1.5 : Grouping of substances and read-across approach, by utilising data generated associated with a test proposal already under agency (ECHA) assessment under REACH Regulation (EC) No. 1907/2006 : Article 40 and Article 41.

The registrant has contacted the agency (ECHA) and/or is contacting the test proposer in order to acquire access to the proposed and/or yet to be conducted study/studies.

NON-CONFIDENTIAL NAME OF (SOURCE) SUBSTANCE AND DETAILS OF TEST PROPOSAL
(associated with the test proposal not made by the current registrant, but which is intended to be used within the current registration):
- Name/identifiers: Reaction products of 2-hydroxyethyl methacrylate and diphosphorus pentaoxide (POEMA) ; EC/List Number: 810-703-1 / CAS Number: 1187441-10-6
- Details on the test proposal: According to the test proposer: “it is proposed to address the potential genotoxic properties of POEMA in an in vivo Comet assay. This assay addresses all potential mode-of-actions (the COMET assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death), performance of this in vivo study is considered to be sufficient to conclude on this endpoint. The oral route is considered to be the most appropriate route. As no sex-specific toxicity is expected, the test is performed in a single species. As no tissue-specific toxicity is expected, blood and liver samples will be tested (sic).”
- Test Guideline: OECD TG 489 /
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- GLP compliance: YES
- Status: Under Agency (ECHA) assessment (2020/2021)

For the target substance: the aforementioned study will address the positive chromosome aberrations (precautionary conclusion, due to potentially artefactual positive results due to low pH of the test item replicates in comparison to the solvent control replicates) detected in an available OECD TG 473 in vitro chromosome aberration assay. This would provide further information in accordance with the requirements of REACH Regulation (EC) No. 1907/2006 Annex VIII, column 2 section 8.4 and/or REACH Regulation (EC) No. 1907/2006 Annex XI : section 1.5 : Grouping of substances and read-across approach. And is a required step prior to the proposal to test on the present (target) substance. The demonstration of ‘chemical similarity’ is provided between source and target is given in Section 13 of the current registration dossier.

References:
1. REACH Regulation (EC) No. 1907/2006 Annex VIII, column 2 section 8.4
2. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.7, July 2017)
3. REACH Regulation (EC) No. 1907/2006 Annex XI : section 1.5 : Grouping of substances and read-across approach
4. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.6: QSARs and grouping of chemicals, May 2008)
5. ECHA Read-Across Assessment Framework (RAAF), 2017 (as amended)

Reason / purpose for cross-reference:

read-across: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable. Mode of action is inconclusive based on current dataset.

Additional information

Key study : OECD TG 471, 2018 : The study was performed to the requirements of OECD TG 471 and EU Method B.13/14 in accordance with GLP, to evaluate the mutagenic activity of the test item in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (incorporating the plate incorporation and pre-incubation methods) in both the presence and absence of S-9 metabolic activation system. The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose level of 5000 μg/plate. In all three experiments the test item did not precipitate on the plates at up to 5000 μg/plate dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a toxicologically significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in two independently repeated experiments. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Key study :OECD TG 473, 2021 : The study was performed to the requirements of OECD TG 473 under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of peripheral human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, multiple exposure conditions were investigated; a 3-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1.8% final concentration with cell harvest after a 24-hour fixation period, 3-hour exposure in the absence of metabolic activation (S9) with a 24-hour fixation period. A 24-hour exposure in the absence of metabolic activation with a 24-hour fixation period and a 48-h exposure 48-hour fixation period in the absence of metabolic activation (S9). In the first cytogenetic assay, the test item was tested up to 2900 and 3000 µg/mL for a 3 h exposure with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix, respectively. Toxicity was reached at these dose levels. In the second cytogenetic assay, the test item was tested up to 1250 µg/mL for a 24 h continuous exposure with a 24 h fixation time and up to 300 µg/mL for a 48 h continuous exposure with a 48 h fixation time in the absence of S9-mix. Toxicity was reached at these dose levels. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. All acceptability criteria were met. It was therefore concluded that the test conditions were adequate, and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, the test item induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded in the first cytogenetic assay and where gaps were included in the second cytogenetic assay. These increases were observed at the 3 h and 24 h exposure time. The number of cells with chromosome aberrations was above the historical control data range both when gaps were included and excluded. The increases were therefore considered biologically relevant. However, care should be taken with the interpretation of the results especially in the first cytogenetic assay since the pH of the test item was low in comparison with the solvent control (approximately pH 6 in comparison to pH 7.5 for the solvent control). The low pH may lead to artefactual positive results, i.e. chromosome damage not caused by direct interaction between the test item and chromosomes. The test item did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9 mix at the 48 h exposure time and in the presence of S9-mix at the 3 h exposure time. In the first cytogenetic assay the test item increased the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix in a dose dependent manner. This may indicate that the test item has the potential to disturb mitotic processes and cell cycle progression. It can be concluded that the test item has the potential to disturb mitotic processes and cell cycle progression and induce numerical chromosome aberrations under the experimental conditions described. Under the conditions of this study, the test item was considered to be clastogenic to human lymphocytes in vitro. Care should be taken with the interpretation of the results especially in the first cytogenetic assay since the pH of the test item was low in comparison with the solvent control (approximately pH 6 in comparison to pH 7.5 for the solvent control). The low pH may lead to artefactual positive results, i.e. chromosome damage not caused by direct interaction between the test item and chromosomes.

Key study : OECD TG 490, 2021 : The study was performed according to the requirements of OECD TG 490 under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at 313, 625, 1250, 2500, 5000 μg/mL concentration in DMSO vehicle (selection was based on absence of precipitation in exposure medium at up to 5000 μg/mL), a definitive test was performed whereby L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item. Exposures were conducted using a 3-hour exposure with and without metabolic activation (4% S9), and a 24-hour exposure without metabolic activation. The following concentrations were utilised and/or considered acceptable for assessment under the test acceptability criteria: 3-hour without and with S9: 0 (control), 125, 250, 500, 1000, 1500, 2000, 2150, 2300, 2450, 2600, 2750 and 2900 μg/m and 24-hour without S9: 0 (control), ), 50, 100, 400, 500, 600, 700, 1000, 1100, 1200, 1250, 1300 and 1400 μg/mL.In the first experiment, the test item was tested up to concentrations of 2450 and 2300 µg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. In the absence of S9-mix, relative total growth (RTG) was reduced to 7% and 29% at concentrations of 2450 and 2300 µg/mL respectively. In the presence of S9-mix the RTG was reduced to 5 % and 39% at concentrations of 2300 and 2150 µg/mL, respectively. In the second experiment, the test item was tested up to concentrations of 1300 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 8% and 13% at concentrations of 1300 and 1000 µg/mL respectively. Excessive toxicity was observed at 1300 µg/mL dose level in the absence S9-mix. Acceptable toxicity was observed at 1000 µg/mL dose level. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. This result was confirmed in an independent experiment with modification in the duration of treatment. After the 24 hour treatment, the test item induced an increase in the mutation frequency. The observed increase was above the positive threshold of MF(control) + GEF (i.e. above 253 x 10-6) at the highest dose level tested. However, the increase in the mutation frequency at the TK locus after a 24 hour treatment period was only observed at excessively toxic dose levels (RTG 8%). According to the OECD TG 490 guideline a result would not be considered positive if the increase in MF occurred only at less than or equal to 10% RTG. Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.

Justification for classification or non-classification

The substance has an inconclusive conclusion under the classification criteria under Regulation (EC) No 1272/2008 for mutagenicity

 

More information required, see above information: intent to read-across to source substance OECD TG 489, COMET Assay (in vivo mammalian cell study: DNA damage and/or repair) currently under test proposal assessment. Source substance: Reaction products of 2-hydroxyethyl methacrylate and diphosphorus pentaoxide ; EC/List Number: 810-703-1 / CAS Number: 1187441-10-6