2-methoxyethyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA: negative with and without metabolic activation (according to OECD 471)
Chromosome aberration (in-vitro mammalian chromosome aberration test): positive with cultured peripheral human lymphocytes with metabolic activation; negative without metabolic activation (according to OECD 473).
Gene mutation (in-vitro mammalian cell gene mutation test): positive with L5178Y mouse lymphoma cells in the mutant frequency at the TK +/- locus with and without metabolic activation (according to OECD 476).

Link to relevant study records

Reference

Reference 1

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:

10 Sep - 01 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

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:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: cleansing medium (THG medium): medium containing thymidine (9 µg/mL), hypoxanthine (15 µg/mL) and glycine (22.5 µg/mL); THGM: TGM medium with methotrexate (0.3 µg/mL); growth medium (R10 medium): RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum; R20 medium: growth medium with 20% donor horse serum; R0 medium: growth medium without serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats daily treated with oral doses of a mixture of phenobarbitone (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for 3 consecutive days prior to sacrifice

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
4 h treatment (-S9 mix): 0.64, 1.27, 2.54, 5.08, 10.16, 20.31, 40.63, 81.25 and 162.5 µg/mL
4 h treatment (+S9 mix): 10.16, 20.31, 40.63, 81.25, 162.5, 325, 650, 975 and 1300 µg/mL
24 h treatment (-S9 mix): 0.16, 0.32, 0.64, 1.27, 2.54, 5.08, 10.16, 20.31 and 40.63 µg/mL

Main experiment:
4 h treatment (-S9 mix): 0.63, 1.25, 2.5, 5, 10, 20, 30, 40 µg/mL
4 h treatment (+S9 mix): 20.25, 40.5, 81, 162, 324, 432, 540 and 648 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

cyclophosphamide, 2 µg/mL in R0 medium, +S9; ethylmethanesulphonate, 400 µg/mL in R0 medium, -S9

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (± S9 mix)
- Expression time (cells in growth medium): 2 days after the end of treatment, cells were plated for determination of cell viability and the mutation frequency in 96-well microtitre plates containing TFT-selective medium.
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-16 days

SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures in one experiment in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: relative suspension growth and viability

OTHER EXAMINATIONS: Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal aberrations.

Evaluation criteria:

The test substance was considered mutagenic if:
- a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value was observed.
- any test item concentration has a mutation frequency value that is greater than the corresponding vehicle control by the GEF (global evaluation factor; Moore et al. (2006) Mouse Lymphoma Thymidine Kinase Locus Gene Mutation Assay: Follow-up international workshop on genotoxicity test procedures, New Orleans Louisiana. Environ. Mol. Mutagen. 40: 292-299) of 126E-06 and demonstrates a positive linear trend.

However, if a test item produced a modest increase in mutant frequency, which only marginally exceeded the global evaluation factor (GEF) value and was not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induced modest reproducible increases in the mutation frequencies that do not exceed the GEF value, then scientific judgement was applied. If the reproducible responses were significantly dose-related and included increases in the absolute numbers of mutant colonies, then they may be considered to be toxicologically significant. Small significant increases designated by the UKEMS statistical package were reviewed using the above criteria, and may be disregarded.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which followed the statistical guidelines recommended by the UKEMSA (Robinson et al., 1989). A test for linear trend was performed to identify a dose-response relationship.

Reference:
Robinson W D et al. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preliminary cytotoxicity test: ≥ 5.08 µg/mL (-S9) and ≥ 162.5 µg/mL (+S9); Main experiment: ≥ 10 µg/mL (-S9) and ≥ 324 µg/mL (+S9)

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: based on the toxicity observed in a previous chromosome aberration test, the concentration range used in the preliminary toxicity test was 0.64 to 162.5 µg/mL for the 4-h exposure in the absence of metabolic activation, 10.16 to 1300 μg/mL (corresponding to approx. 10 mM) for the 4-h exposure in the presence of metabolic activation, and 0.16 to 40.63 µg/mL for the 24-h exposure in the absence of metabolic activation. In all three exposure groups, there were marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. After 4-h treatment, cytotoxicity was observed at concentrations ≥ 5.08 µg/mL without S9 mix and ≥ 162.5 µg/mL with S9 mix. After 24-h exposure in the absence of S9 mix, cytotoxicity was noted at ≥ 2.54 µg/mL (RSG: 72%). Due to the steep nature of the toxicity curve at the higher concentrations (RSG: ≤ 10), it was difficult to achieve the optimum toxicity for all exposure conditions.

COMPARISON WITH HISTORICAL CONTROL DATA: the frequency of chromosomal aberrations in the negative and positive control was within the historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the main experiment, there was evidence of marked concentration-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the reduction in %RSG and RTG (relative total growth) values. Toxicity occurred at concentrations ≥ 10 µg/mL in the absence of S9 mix and at concentrations ≥ 324 µg/mL in the presence of S9 mix. Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The toxicity observed at 40 µg/mL in the absence of metabolic activation, and at 648 μg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%. Therefore, these concentrations were excluded from the statistical analysis.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration [mg/mL]	Relative suspension growth [%]	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Mutants per 1E+06 surviving cells		Proportion small colony mutants
					Small colonies	Large colonies
0 (Medium)	100	1	143.78	1	47.8	88.3	0.36
0.63	100	NA	NA	NA	NA	NA	NA
1.25	92	NA	NA	NA	NA	NA	NA
2.5	87	0.76	196.49	1.37	66.9	116.7	0.37
5	85	0.78	193.76	1.35	70.2	110.3	0.40
10	77	0.62	227.23*	1.58	100.7	109.9	0.48
20	49	0.47	240.24*	1.67	90.4	128.3	0.42
30	29	0.13	407.18*	2.83	239.7	132.2	0.63
40#	13	0.04	974.72	6.78	568.0	295.7	0.64
Linear trend			***
EMS, 400	59	0.34	1237.59	8.61	373.3	524.0	0.43

EMS = ethylmethanesulphonate; NA = not analysed; # = excluded from statistical analysis due to overt toxicity

*p < 0.05; ***p < 0.001

Table 2: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [mg/mL]	Relative suspension growth [%]	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Mutants per 1E+06 surviving cells		Proportion small colony mutants
					Small colonies	Large colonies
0 (Medium)	100	1	173.24	1	68.0	94.0	0.42
20.25	102	NA	NA	NA	NA	NA	NA
40.5	99	NA	NA	NA	NA	NA	NA
81	93	1.02	167.72	0.97	88.7	67.5	0.56
162	84	0.78	228.559	1.32	123.2	87.2	0.58
324	68	0.76	199.2	1.15	107.1	75.4	0.58
432	43	0.37	297.77**	1.72	141.2	127.2	0.52
540	20	0.11	444.21**	2.56	238.9	163.9	0.59
648#	9	0.03	768.32	4.44	568.0	147.7	0.78
Linear trend			***
CP, 2.0	63	0.35	1328.84	7.67	883.6	193.0	0.78

CP = cyclophosphamide; NA = not analysed; # = excluded from statistical analysis due to overt toxicity

*p < 0.05; ***p < 0.001

DIFFERENTIATION OF LARGE AND SMALL COLONIES

The increases in mutant frequency observed in the absence and presence of metabolic activation were mainly due to small colony formation indicating a clastogenic response (see Table 1 and 2).

Conclusions:

Interpretation of results (migrated information):
positive

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats daily treated with oral doses of a mixture of phenobarbitone (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for 3 consecutive days prior to sacrifice

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
4 and 24 h treatment (±S9 mix): 5.08, 10.16, 20.31, 40.63, 81.25, 162.5, 325, 650 and 1300 μg/mL
Main experiment:
4 h treatment (-S9 mix): 10*, 20*, 30, 40*, 60 and 80 µg/mL
4 h treatment (+S9 mix): 80, 160, 320*, 480*, 640* and 960 µg/mL
*These concentrations were used for chromosomal analysis (960 µg/mL was too toxic for metaphase analysis).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (for positive control cyclophosphamide)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

cyclophosphamide, 5 µg/mL in DMSO, +S9; mitomycin C, 0.4 µg/mL in Minimal Essential Medium, -S9

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (± S9 mix)
- Expression time (cells in growth medium): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.1 μg/mL
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency, a dose response relationship was required and appropriate statistical tests were applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps, gaps alone and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

other: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in the presence of S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Mitotic index of preliminary cytotoxicity test: ≥ 10.16 µg/mL (-S9) and ≥ 325 µg/mL (+S9); Mitotic index of main experiment: ≥ 30 µg/mL (-S9) and ≥ 480 µg/mL (+S9)

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: a preliminary cytotoxicity test with concentrations ranging from 5.08 to 1300 µg/mL (corresponding to approx. 10 mM) was performed in lymphocyte cultures for an exposure period of 4 h with and without S9 mix and for 24 h with S9 mix, respectively. After 4 h exposure, a significant decrease in mitotic index (MI < 50% compared to control) was observed at 81.25 µg/mL (MI = 13%) in the absence of S9 mix and at 650 µg/mL (MI = 29%) in the presence of S9 mix. The optimum toxicity (MI < 50% compare to control) after 20-h treatment was achieved at a concentration of 20.31 µg/mL. Based on these cytotoxicity data, the selection of the maximum concentration for the 4 h treatment in the main experiment was 80 and 960 µg/mL in the absence and presence of S9 mix, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: the frequency of chromosomal aberrations in the negative and positive control was within the historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the main experiment, a dose-related inhibition of mitotic index was observed after 4 h treatment with the test substance. A 49% mitotic inhibition was achieved at 40 µg/mL in the absence of S9 mix, whereas in the presence of S9 mix the toxicity curve was relatively steep with 35% mitotic inhibition at 640 µg/mL and 96% mitotic inhibition at 960 µg/mL. The maximum concentration selected for metaphase analysis was 40 µg/mL in the absence of S9, where optimum toxicity was achieved. In the presence of S9, the maximum concentration selected for metaphase analysis was 640 µg/mL as the concentration above (960 µg/mL) was considered too toxic for metaphase analysis.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

RESULTS OF PH AND OSMOLALITY MEASUREMENT

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (see Table 1).

Table 1. Results of pH and osmolality measurements

Parameter	Concentrations [µg/mL]
	0	5.08	10.16	20.31	40.63	81.25	162.5	325	650	1300
pH	7.38	7.38	7.35	7.34	7.42	7.43	7.38	7.39	7.39	7.43
Osmolality [mOsm]	281	280	-	-	279	280	-	-	-	295

RESULTS OF CHROMOSOMAL ANALYSIS

Table 2. Test results of experiment I

Test item	Concentration in µg/mL	Mitotic index in %	Aberrant cells in %		Polyploid cells in %
			with gaps	without gaps
Exposure period 4 h, fixation time 24 h, without S9 mix (2%)
NC	0	100	0	0	0
MMC	0.4	77	1.5	14**	0
Test item	10	82	0	0	0
	20	83	0	0	0.5
	40	51	0.5	1	0
Exposure period 4 h, fixation time 24 h, with S9 mix (2%)
NC	0	100	1	1	0
CP	5	50	16	35**	1
Test item	320	103	0	0.5	0
	480	71	4.5*	1	1.5
	640	65	4	7.5**	0.5

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls); NC: Negative control; * = P < 0.05; ** = P < 0.001

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

Reference 3

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:

30 May - 16 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium) and trp operon (E. coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg bw/day, orally, for 3 days prior to sacrifice

Test concentrations with justification for top dose:

Preliminary cytotoxicity test (TA 100 and WP2 uvrA): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9 mix
Main assay: Experiment I and II:
Salmonella strains: 5, 15, 50, 150, 500, 1500, 5000 µg/plate, with and without S9 mix
E.coli strain: 50, 150, 500, 1500, 5000 µg/plate, with and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: fully soluble in sterile distilled water at 50 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: ENNG (2 µg/plate: WP2uvrA; 3 µg/plate: TA 100; 5 µg/plate: TA 1535); 9AA (80 µg/plate: TA 1537); 4NQO (0.2 µg/plate: TA 98); +S9: 2AA (1 µg/plate: TA 100; 2 µg/plate: TA 1535 and TA 1537; 10 µg/plate: WP2uvrA); BP (5 µg/plate TA 98)

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine; 9AA: 9-aminoacridine; 4NQO: 4-nitroquinoline-N-oxide; 2AA: 2-aminoanthracene; BP: benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION I: experiment I and preliminary cytotoxicity test: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

METHOD OF APPLICATION II: experiment II: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: inspection of bacterial background lawn; determination of the number of revertant colonies using a colony counter

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al. 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Reference
Mahon G.A.T. et al. (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.), Cambridge University Press Report, 26-65.

Statistics:

Means and standard deviations of mean revertant colonies per plate were calculated. Statistical analysis of the data was performed according to UKEMS (Mahon et al. 1989).

Reference
Mahon G.A.T. et al. (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.), Cambridge University Press Report, 26-65.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1500 µg/plate (± S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1500 µg/plate (± S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1500 µg/plate (± S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1500 µg/plate (± S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity test, S. typhimurium strain TA 100 and E. coli WP2 uvrA were treated with concentrations ranging from 5 to 5000 µg/plate in the presence and absence of metabolic activation. The test item was initially toxic to TA 100 at 1500 µg/plate, but non-toxic to WP2uvrA as indicated by the number of mean revertants per plate and the inspection of bacterial background lawn. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: vehicle and positive control values were within the expected historical ranges of each tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the Salmonella tester strains, initially from 1500 µg/plate in the absence and presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology (plate incorporation or preincubation). No toxicity was noted in Escherichia coli strain WP2 uvrA at any test item concentration in either the absence or presence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Test results of experiment I

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± SD)
EXPERIMENT I (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
SC (water)	124 ± 9	25 ± 10	30 ± 3	21 ± 4	20 ± 5
Test item
5 µg	120 ± 8	29 ± 10	N/T	19 ± 2	13 ± 1
15 µg	131 ± 5	26 ± 4	N/T	23 ± 6	13 ± 9
50 µg	107 ± 10	23 ± 6	23 ± 2	17 ± 6	11 ± 3
150 µg	119 ± 19	23 ± 8	32 ± 5	17 ± 0	10 ± 1
500 µg	107 ± 6	16 ± 6	28 ± 10	17 ± 12	13 ± 2
1500 µg	86 ± 10	15 ± 8	36 ± 8	2 ± 2	7 ± 2
5000 µg	25 ± 11	2 ± 1	20 ± 2	0 ± 0#	4 ± 1#
PC
ENNG (3 µg)	506 ± 42	-	-	-	-
ENNG (5 µg)	-	288 ± 29	-	-	-
ENNG (2 µg)	-	-	530 ± 36	-	-
4NQO (0.2 µg)	-	-	-	141 ± 19	-
9AA (80 µg)	-	-	-	-	473 ± 103
S9-Mix	With
Concentration (per plate)	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
SC (water)	128 ± 6	14 ± 2	36 ± 7	25 ± 3	11 ± 6
Test item
5 µg	122 ± 7	16 ± 1	N/T	21 ± 2	9 ± 6
15 µg	109 ± 15	12 ± 7	N/T	23 ± 6	8 ± 1
50 µg	121 ± 5	12 ± 1	36 ± 10	25 ± 5	10 ± 2
150 µg	108 ± 8	12 ± 1	33 ± 3	21 ± 2	14 ± 2
500 µg	123 ± 3	9 ± 2	41 ± 2	18 ± 3	8 ± 2
1500 µg	95 ± 4	9 ± 2	31 ± 7	25 ± 4	9 ± 7
5000 µg	38 ± 16	0 ± 0	39 ± 1	6 ± 2#	2 ± 1#
PC
2-AA (1 µg)	1413 ± 75	-	-	-	-
2-AA (2 µg)	-	292 ± 30	-	-	313 ± 19
2-AA (10 µg)	-	-	429 ± 42	-	-
BP (5 µg)	-	-	-	222 ± 31	-
SC = Solvent control; PC = Positive control substances; SD = standard deviation; ENNG: N-ethyl-N-nitro-N-nitrosoguanidine; 9AA: 9-aminoacridine; 4NQO: 4-nitroquinoline-N-oxide; 2AA: 2-aminoanthracene; BP: benzo(a)pyrene # partial absence of bacterial background lawn N/T: not tested at this dose level

Table 2. Test results of experiment II

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± SD)
EXPERIMENT II (preincubation)
S9-Mix	Without
Concentration (per plate)	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
SC (water)	112 ± 17	24 ± 4	35 ± 2	26 ± 3	9 ± 2
Test item
5 µg	85 ± 8	19 ± 5	N/T	18 ± 3	9 ± 3
15 µg	95 ± 5	23 ± 1	N/T	24 ± 5	8 ± 1
50 µg	135 ± 1	22 ± 8	37 ± 5	25 ± 6	10 ± 2
150 µg	91 ± 18	20 ± 2	30 ± 2	30 ± 5	8 ± 3
500 µg	90 ± 6	14 ± 6	29 ± 13	19 ± 6	10 ± 1
1500 µg	58 ± 8	8 ± 1	43 ± 3	15 ± 3	5 ± 4#
5000 µg	14 ± 11	0 ± 0	34 ± 9	2 ± 2#	1 ± 1#
PC
ENNG (3 µg)	488 ± 56	-	-	-	-
ENNG (5 µg)	-	335 ± 39	-	-	-
ENNG (2 µg)	-	-	774 ± 80	-	-
4NQO (0.2 µg)	-	-	-	135 ± 8	-
9AA (80 µg)	-	-	-	-	572 ± 24
S9-Mix	With
Concentration (per plate)	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
SC (water)	148 ± 21	14 ± 2	34 ± 3	26 ± 6	12 ± 1
Test item
5 µg	112 ± 3	12 ± 4	N/T	26 ± 3	10 ± 2
15 µg	105 ± 6	11 ± 6	N/T	27 ± 6	11 ± 2
50 µg	124 ± 8	13 ± 6	30 ± 2	25 ± 2	11 ± 2
150 µg	101 ± 17	11 ± 6	35 ± 9	23 ± 2	11 ± 2
500 µg	109 ± 9	14 ± 2	41 ± 6	29 ± 4	13 ± 2
1500 µg	100 ± 20	1 ± 1	33 ± 2	12 ± 4	5 ± 3#
5000 µg	75 ± 12	2 ± 1	32 ± 13	5 ± 5#	2 ± 3#
PC
2-AA (1 µg)	1897 ± 62	-	-	-	-
2-AA (2 µg)	-	374 ± 34	-	-	509 ± 48
2-AA (10 µg)	-	-	672 ± 76	-	-
BP (5 µg)	-	-	-	288 ± 13	-
SC = Solvent control; PC = Positive control substances; SD = standard deviation; ENNG: N-ethyl-N-nitro-N-nitrosoguanidine; 9AA: 9-aminoacridine; 4NQO: 4-nitroquinoline-N-oxide; 2AA: 2-aminoanthracene; BP: benzo(a)pyrene # partial absence of bacterial background lawn N/T: not tested at this dose level

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mammalian alkaline comet assay: negative (according to OECD 489; 3 concentrations (2 x 120, 2 x 240 and 2 x 480 mg/kg bw) orally administered to male rats)

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Nov 2015 - 24 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TOAGOSEI CO., LTD
- Expiration date of the lot/batch: 31 Mar 2016
- Purity test date: 16 Sep 2015


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark, under Nitrogen
- Stability under test conditions: confirmed by chemical analytics during conduct of the study
- Solubility and stability of the test substance in the solvent/vehicle: confirmed by chemical analytics during conduct of the study

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited, UK
- Age at study initiation: 7 - 12 weeks
- Weight at study initiation: 150 - 200 g
- Assigned to test groups randomly: No
- Fasting period before study: No
- Housing: 5 animals of the same sex per cage in propylene cages with stainless stell mesh lids, bedding soft wood flakes
- Diet: Rodent 2014C Teklad Global Certified Diet (Envigo RMS Limited, UK), ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: 24 Nov 2015 - 24 Mar 2016

ADDITIONAL INFORMATION: One animal (120 mg/kg) marginally exceeded the upper body weight range. Since the difference was very small, (less than 1.5% overweight) it was considered to have no impact on the study outcome.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Phosphate buffered saline (PBS)
- Concentration of test material in vehicle: 12, 24, 48 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was freshly prepared before each administration as a solution at the appropriate concentration in phosphate buffered saline.

Duration of treatment / exposure:

2 single treatments within 24 hours

Frequency of treatment:

at 0 and 24 hours

Post exposure period:

Animals were sacrificed 4 hours after final treatment

Dose / conc.:

120 mg/kg bw/day (nominal)

Dose / conc.:

240 mg/kg bw/day (nominal)

Dose / conc.:

480 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 (negative control and test item groups)
5 (positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

N-methyl-N-nitrosourea
- Justification for choice of positive control(s): mentioned as positive control in OECD 489
- Route of administration: oral
- Doses / concentrations: 25 mg/kg bw

Tissues and cell types examined:

Liver, non-glandular and glandular stomach

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range finding study was performed to find maximum tolerated dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were treated two times at 0 and 24 hours. 28 hours after first treatment, animals were sacrificed and tissues were prepared. Sub-samples of the liver, glandular and non-glandular stomach were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathological examination.

DETAILS OF THE SLIDE PREPARATION: Tissue samples were processed to provide single cell suspensions. Cell suspensions were mixed using low melting point agarose and placed onto pre-coated slides. Four slides/animal for each tissues were prepared. After cell lysis the slides were transferred to electrophoresis to allow the DNA to unwind for 20 minutes. Electrophoresis was conducted at approximately 0.7 V/cm, 300 mA for 20 minutes. Slides were fixed in 100% methanol after electrophoresis and stained with propidium iodide (20 µg/mL).

METHOD OF ANALYSIS: Two slides for each tissue per animal were analyzed using a fluorescence microscope combined with a CCD camera attached to a PC-based image analysis program (Comet IV version 4.3.1.) and a maximum of 200 cells per tissue and animal were scored primary for relative tail intensity. Tail length and tail moment were also considered. Each slide was assessed for the incidence of hedgehog to give an indication of cell integrity.

OTHER: Due to statistical increase on percentage tail intensity in glandular and non-glandular stomach tissue, additional histopathological examinations of the non-glandular and glandular stomach were conducted. Therefore tissue samples were processed to paraffin wax, sectioned and stained with hematoxylin and eosin.

Evaluation criteria:

EVALUATION CRITERIA:
Acceptability Criteria:
- The concurrent negative control is comparable with the laboratory historical negative control range.
- The positive controls induce responses that are comparable with those in the laboratory positive control range.
- Adequate numbers of cells and doses have been analysed.
- The highest dose level selected meets the requirements of the guideline and the study plan.

Evaluation of results
Clearly negative if:
- None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
- There is no evidence of a dose- related response.
- The results are within the laboratory historical vehicle control range.
- There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved.

Clearly positive if:
- At least one of the test doses exhibits a statistical significant increase compared to the concurrent negative control.
- The response is considered to be dose- related.
- The results are substantially outside the laboratory historical vehicle control range.

Statistics:

Students t-test on transformed data using a transformation √(x+1)

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

Statistically significant increase in the % tail intensity in the mid and high dose groups

Toxicity:

yes

Remarks:

1/7 animals died in the high dose group. Hunched posture was observed in the remaining 6/7 animals of the high dose group 1 hour after dosing

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 24 hours and to select the most appropriate sex for use in the main test. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg bw.
- Dose range: 400, 480, 500, 600 mg/kg bw
- Clinical signs of toxicity in test animals: mortality was observed in males (2/3 animals, one animal was dosed only once) at 600 mg/kg and females at 500 (1/1 animal) and 600 mg/kg bw (1/1 animal), hunched posture was observed at 480-600 mg/kg bw
- Rationale for exposure: starting dose based on oral LD50 in rats (404 mg/kg bw)

RESULTS OF DEFINITIVE STUDY
- Percentage tail intensity: No significant increase in percentage tail intensity was observed in liver tissue of treatment groups compared with the negative control (Table 3).

A significant increase in the mean of median percentage tail intensity in the glandular stomach tissue was noted in all dose groups, and in the mean percentage tail intensity in the mid- and high dose group, compared with the control group respectively (1.4 - 3.6 fold compared with mean of controls) (Table 1). However, the increase fell within the historical negative control values (2.67 - 12.74% mean % tail intensity).
A significant increase in the percentage tail intensity was also observed in the non-glandular stomach tissue of the mid- and high-dose group, compared with the control group (1.7 - 2.3 fold compared to mean of controls, for mean percentage tail intensity and mean of median percentage tail intensity, Table 2). The individual variation in each dose group and in the control groups was quite high in stomach tissues.
Histopathological findings (Table 4):
- Glandular stomach: Erosion of the glandular epithelium observed in males treated with 240 mg/kg bw (4/7 animals) or 480 mg/kg bw (6/6 animals), inflammation of submucosa observed in males treated with 240 mg/kg bw (3/7 animals) and 480 mg/kg bw (6/6 animals), ulceration in 1 male treated with 480 mg/kg bw, myofiber degeneration observed in males treated with 480 mg/kg bw (4/6 animals)
- Non-glandular stomach: Ulceration observed in males (3/6 animals) treated with 480 mg/kg bw, erosion observed in males (2/6 animals) treated with 480 mg/kg bw, vacuolation of the non-glandular epithelium in males treated with 480 mg/kg bw (3/6 animals) and in 1 male treated with 240 mg/kg bw; Ulceration of the limiting ridge in males treated with 240 mg/kg bw(2/7 animals) and 480 mg/kg bw (4/6 animals), mucosal necrosis in males treated with 480 mg/kg bw (2/6 animals), vacuolation of the epithelium at the limiting ridge in males treated with 120 mg/kg bw (3/7 animals) or 240 mg/kg bw (3/7 animals) , epithelial hyperplasia in 1 male treated with 240 mg/kg bw, inflammation observed treated with 240 mg/kg bw (1/7 animals) and 480 mg/kg bw (6/6 animals), myofiber degeneration in males treated with 480 mg/kg bw (6/6 animals)
- Statistical evaluation: Significant increase in percentage tail intensity (mean percentage tail intensity and mean of median percentage tail intensity) was observed in the glandular- and non-glandular stomach tissues at 240 and 480 mg/kg bw, and at 120 mg/kg bw in the glandular stomach tissue (mean of median percentage tail intensity).

Table 1: Summary Table Comet Assay – Glandular Stomach

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	3.84 ± 1.44	2.05 ± 0.62	0.69 ± 0.42
480 mg/kg bw	2.65 ± 1.03	3.98 ± 1.71^a	2.52 ± 1.61^b
240 mg/kg bw	4.18 ± 1.33	2.92 ± 0.79^b	1.22 ± 0.63^c
120 mg/kg bw	4.00 ± 1.40	2.72 ± 0.99	1.18 ± 0.64^c
Positive (MNU)	6.04 ± 1.01	21.09 ± 1.81^a	19.28 ± 1.88^a

^a= P < 0.001

^b= P < 0.01

^c= P < 0.05

 

Table 2: Summary Table Comet Assay – Non-Glandular Stomach

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	6.12 ± 2.32	6.68 ± 1.88	4.35 ± 1.74
480 mg/kg bw	6.41 ± 1.07	11.42 ± 3.16^a	9.30 ± 3.87^a
240 mg/kg bw	3.93 ± 1.04	11.92 ± 3.58^a	10.29 ± 3.97^a
120 mg/kg bw	4.83 ± 1.28	7.92 ± 2.42	5.92 ± 2.42
Positive (MNU)	7.78 ± 1.71	41.68 ± 3.60^a	41.90 ± 4.21^a

^a= P < 0.001

Table 3: Summary Table Comet Assay – Liver

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	1.16 ± 0.71	0.34 ± 0.06	0.01 ± 0.01
480 mg/kg bw	0.25 ± 0.27	0.34 ± 0.08	0.01 ± 0.01
240 mg/kg bw	0.21 ± 0.26	0.32 ± 0.08	0.00 ± 0.00
120 mg/kg bw	0.69 ± 0.26	0.41 ± 0.23	0.01 ± 0.00
Positive (MNU)	1.04 ± 1.01	16.08 ± 3.85^a	15.56 ± 4.07^a

^a= P < 0.001

 

Table 4: Histopathological results of glandular and non glandular stomach (No affected animals/total No. examined)

Organ	Effect	control	480 mg/kg bw	240 mg/kg bw	120 m/kg bw
Glandular stomach	Erosion - minimal - slight	0/7 0/7	2/6 4/6	4/7 0/7	0/7 0/7
	Ulceration - slight	0/7	1/6	0/7	0/7
	Inflammation submucosa - minimal - slight	0/7 0/7	6/6 0/6	3/7 0/7	0/7 0/7
	Myofiber degeneration - minimal - slight	0/7 0/7	3/6 1/6	0/7 0/7	0/7 0/7
Non-glandular stomach	Ulceration - slight - marked	0/7 0/7	1/6 2/6	0/7 0/7	0/7 0/7
	Erosion - slight - moderate	0/7 0/7	1/6 1/6	0/7 0/7	0/7 0/7
	Vascuolation - minimal - slight	0/7 0/7	3/6 0/6	0/7  1/7	0/7 0/7
	Vacuolation limiting ridge - minimal - slight	0/7 0/7	0/6 0/6	1/7 2/7	3/7 0/7
	Epithelial Hyperplasia - slight	0/7	0/6	1/7	0/7
	Ulceration, Limiting Ridge - minimal - slight	0/7 0/7	2/6 2/6	1/7 1/7	0/7 0/7
	Mucosal necrosis, Limiting ridge - slight	0/7	2/6	0/7	0/7
	Inflammation submucosa - minimal - slight	0/7 0/7	0/6 6/6	0/7 1/7	0/7 0/7
	Myofiber Degeneration - minimal - slight - moderate	0/7 0/7 0/7	2/6 2/6 2/6	0/7 0/7 0/7	0/7 0/7 0/7

Conclusions:

The results in non-glandular stomach were considered to be an intrinsic genotoxic response.

Executive summary:

A Comet Assay was conducted according to the OECD TG 489 and in compliance with GLP. The primary target tissues of this assay were liver, glandular stomach and non-glandular stomach. A range-finding test was performed to find suitable dose levels of the test item and to investigate if there were any differences between the sexes. The initial dose selection for the range-finding test was based on the documented LD50 data from an oral rat study. The comet assay main test was performed using male rats only and groups, each of seven rats for the vehicle control group and the test item dose groups, and five rats for the positive control group were dosed twice at 0 and 24 hours with the test item via the oral route. The Comet assay main test was conducted with the test item at the maximum tolerated dose (MTD) 480 mg/kg with 240 mg/kg and 120 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration and samples of the liver, glandular stomach and non- glandular stomach tissues were processed to produce single cell suspensions. These were processed to produce the comet slides which were subsequently scored for the presence of Comets.

Further groups of rats were given a double oral dose of Phosphate Buffered Saline (seven rats) or N-Nitroso-N-methylurea (five rats), to serve as vehicle and positive controls, respectively.

The primary target tissues of this assay were liver, glandular stomach and non-glandular stomach.

Formulation analysis was performed on the test item formulations of the main test.

 

In the liver, no significant change in the % tail intensity was observed between the treatment groups and control group.

In the glandular stomach tissue, a dose-related significant increase in the mean of median % tail intensity was noted in all dose groups, and in the mean % tail intensity in the mid and high dose groups, compared with the control group. However, the increase fell within the range of the historical control data, which is composed by a limited dataset (only 11 animals) thus its adequacy is questionable.

In the non-glandular stomach tissue, a significant increase in the % tail intensity was also observed of the mid and high dose groups. At the site of contact: inflammation and degeneration of the glandular and non-glandular stomach tissues in the mid and high dose animals are considered to be the result of the corrosive properties of the substance and were more severe in non-glandular stomach than in glandular stomach. A statistically significant increase in the mean % tail intensity in the non-glandular stomach was observed already at the lowest dose, showing only minimal concomitant histopathological findings in the non-glandular stomach. Moreover, in the non-glandular stomach, the increase in cytotoxicity was clearly dose-related, but did not correlate with an increased genotoxic response. The genotoxicity effects were higher at the mid dose. If the genotoxicity effects were only the result of a cytotoxic response, the highest % tail intensity in the Comet assay would have been expected to be in the highest dose group, but this is not the case. Marked cytotoxicity including ulceration and necrosis were only observed at 480 mg/kg bw (highest dose) and not at 240 mg/kg bw (mid dose).Tthese results suggest that the genotoxic response cannot only be explained by cytotoxicity. Therefore, the results in non-glandular stomach were considered to be an intrinsic genotoxic response.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Genetic toxicity: in-vitro

A gene mutation assay in bacteria (Ames test) was conducted with 2-methoxyethyl acrylate in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA according to OECD 471 (Thompson, 2012). Based on a preliminary cytotoxicity test, concentrations ranging from 5 to 5000 µg/plate for S. typhimurium strains and from 50 to 5000 µg/plate for the E. coli strain were selected for treatment in the presence and absence of a metabolic activation system in two independent experiments. No increase in the mean revertant number of colonies was observed at any of the concentrations tested in both experiments. However, the test substance caused cytotoxicity, indicated as a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies, in all Salmonella tester strains, initially from 1500 µg/plate in the absence and presence of S9-mix. No toxicity was noted in Escherichia coli strain WP2 uvrA at any test concentration, neither in the absence nor in the presence of S9-mix. Based on these results, the test substance was not considered to be mutagenic in the Ames test with and without metabolic activation.

In an additional gene mutation assay in bacteria, however with only 4 tester strains, the in-vitro genetic toxicity of 2-MEA was also tested (NTP, 1991). Similar to OECD 471, the preincubation method or the plate test with vapour from the test substance was conducted with the S. typhimurium strains TA 100, TA 1535, TA 97 and TA98 in the presence and absence of a metabolic activation system. The test concentrations ranged from 10 to 3333 µg/plate for the preincubation method and from 0.5 to 50 mL/chamber for the plate test. 2-MEA did not induce reversions in any of the S. typhimurium strains with or without metabolic activation. Cytotoxic effects were observed for the plate test with TA 98 at the highest test concentrations with and without S9 mix (30% S9 mix).

The potential of 2-MEA to induce chromosomal aberrations was tested in cultured peripheral human lymphocytes according to OECD 473 (Morris, 2012). The lymphocytes were exposed to 2-MEA for 4 h with or without metabolic activation followed by 20 h culture in treatment-free media prior to cell harvest. The concentration range of the test substance was 10 to 80 µg/mL in the absence of S9 mix and 80 to 960 µg/mL in the presence of S9 mix.

A concentration-related inhibition of the mitotic index was observed with and without S9 mix: a 49% mitotic inhibition was achieved at 40 µg/mL in the absence of S9 mix, whereas in the presence of S9 mix the mitotic inhibition at 640 µg/mL was 35% and at 960 µg/mL 96%.The maximum concentration selected for metaphase analysis was 40 µg/mL without S9 mix and 640 µg/mL with S9 mix, where optimum toxicity was achieved.

The test substance did not induce any statistically significant increases in the frequency of cells with aberrations in the absence of S9 mix. However, in the presence of metabolic activation the test substance induced a statistically significant increase in the frequency of cells with aberrations, at a dose level of 640 μg/mL. The concentration below, 480 μg/mL demonstrated a statistically significant increase in gap-type aberrations which was considered to be indicative of the initial stages of the positive response. The test substance was therefore considered to be clastogenic to human lymphocytes under the conditions of the test.

2-MEA was also tested for its potential to cause gene mutations in the mouse lymphoma assay according to OECD 476 (Flanders, 2012). The potential mutagenicity of the test substance on the thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line was investigated after 4 h exposure. The concentration range of the test substance was 0.63 to 40 µg/mL in the absence of metabolic activation and 20.25 to 648 µg/mL in the presence of metabolic activation. Cytotoxicity occurred at concentrations ≥ 10 µg/mL without S9 mix and at concentrations ≥ 324 µg/mL with S9 mix. The toxicity observed at 40 µg/mL in the absence of metabolic activation, and at 648 μg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%. Therefore, these concentrations were excluded from the statistical analysis. The test substance induced toxicologically significant dose-related increases in the mutant frequency at the TK +/- locus in L5178Y cells both with and without metabolic activation. The increases in mutant frequency observed were mainly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage. 2-MEA is therefore considered to be mutagenic under the conditions of the test. The clastogenic potential observed in the chromosome aberration test of Morris (2012) was taken as confirmatory evidence for the mutagenicity of the test substance under in-vitro test conditions.

 

Genetic toxicity: in-vivo

An in-vivo mammalian alkaline comet assay was performed with 2-methoxyethyl acrylate on male rats, according to OECD guideline 489 and under GLP conditions (Morris, 2016).

Male rats (7/dose) were administered 120, 240 and 480 mg/kg bw of the test substance in phosphate buffered saline (PBS), or PBS alone, for 2 consecutive days (at 0 and 24 h). The highest dose level was the maximum tolerated dose (MTD). Five male rat administered 25 mg/kg bw N-methyl-N-nitrosourea according to the same protocol as the treatment groups served as the positive control. The animals were sacrificed 4 hours after the second dose administration and samples of the liver, glandular stomach and non-glandular stomach tissues were taken from each animal. Slides with single cell suspensions were prepared from each tissue sample. Following electrophoresis the slides were stained and at least 200 cells per tissue per animal were scored for percentage tail intensity and assessed for the incidence of ‘hedgehog’ cells. Because of the ambiguous result of the comet assays, preserved tissue samples of the non- glandular stomach and glandular stomach were processed and stained with haematoxylin and eosin prior to the samples being assessed by histological examination for cytotoxicity, i.e. inflammation effects and degeneration of the epithelium. 1/7 animals in the high-dose group died within 24 h; no reason for the mortality was given in the report. The remaining 6/7 rats had a hunched posture for approximately 1 h after each dosing. The positive control substance produced a marked increase in the % tail intensity value, showing the test system is reliable and sufficiently sensitive. No significant change in the percentage tail intensity in the liver tissue was observed between the treatment groups and control group. A significant increase in the mean of median percentage tail intensity in the glandular stomach tissue was noted in all dose groups, and in the mean percentage tail intensityin the mid- and high dose group,compared with the control group respectively. However, the increase fell within the range of the historical negative control data and the result is therefore not considered to be biologically relevant.

In the non-glandular stomach tissue, a significant increase in the % tail intensity was also observed of the mid and high dose groups. At the site of contact: inflammation and degeneration of the glandular and non-glandular stomach tissues in the mid and high dose animals are considered to be the result of the corrosive properties of the substance and were more severe in non-glandular stomach than in glandular stomach.  A statistically significant increase in the mean % tail intensity in the non-glandular stomach was observed already at the lowest dose, showing only minimal concomitant histopathological findings in the non-glandular stomach. Moreover, in the non-glandular stomach, the increase in cytotoxicity was clearly dose-related, but did not correlate with an increased genotoxic response. The genotoxicity effects were higher at the mid dose. If the genotoxicity effects were only the result of a cytotoxic response, the highest % tail intensity in the Comet assay would have been expected to be in the highest dose group, but this is not the case. Marked cytotoxicity including ulceration and necrosis were only observed at 480 mg/kg bw (highest dose) and not at 240 mg/kg bw (mid dose). These results suggest that the genotoxic response cannot only be explained by cytotoxicity. Therefore, the results in non-glandular stomach were considered to be an intrinsic genotoxic response.

 

Overall summary for genetic toxicity

The integrated testing strategy (ITS) for mutagenicity outlined in chapter R7.7.1 (including Figure R.7.7 Flow chart of the mutagenicity testing strategy) of ‘Guidance on information requirements and chemical safety assessment, Chapter R.7a: endpoint specific guidance. Version 4.1 October 2015’ was applied to assess the genetic toxicity of 2-methoxyethyl acrylate.

According to the ITS, the overall conclusion regarding the genetic toxicity of a substance for which the in vitro cytogenetic assay in mammalian cells and in vitro gene mutation assay in mammalian cells were positive, and for which the in vivo assay was negative, should be based on the relevance of the data and the result of the in vivo assay. All the in vitro and in vivo genetic toxicity studies performed with 2-methoxyethyl acrylate are of high quality and reliability. The in vivo assay (in vivo mammalian alkaline comet assay, OECD guideline 489) was conducted in line with the decision made by ECHA (Decision number TPE-D-2114300801-66-01).

Justification for classification or non-classification

Harmonised classification :

2-methoxyethyl acrylate has been inserted into ATP15. The following classification applies:

- Germ cell mutagenicity, category 2; H341 “Suspected of causing genetic defects“

 

The two in vitro mutagenicity tests (MLA +/- metabolic activation) and chromosome aberration assay (+ metabolic activation) show a positive mutagenic effect of 2-methoxyethyl acrylate.

RAC noted that the in vivo Comet assay with 2-methoxyethyl acrylate indicates negative results in liver, equivocal results in the glandular stomach and positive effects in the forestomach in rats. When comets are seen at the initial site of contact (forestomach), but not at a distant site (here the liver), the classification as mutagenic needs to be carefully considered. On the one hand, classification as a germ cell mutagen is important, because in the absence of carcinogenicity data it indirectly highlights the potential for carcinogenicity. On the other hand, it is not appropriate to classify substances that are not germ cell mutagens. Where the data is limited, it can be difficult to judge whether a classification for germ cell mutagenicity is appropriate. For germ cell mutagenicity hazard assessment and classification purposes the study designs exposing the bone marrow are still considered to be the most informative. When a substance is known to be distributed around the body, and especially one that is toxic to reproduction, such tests are still the most logical choice for further evaluation of in vitro mutagens.

Nevertheless, taking the arguments presented by the DS (see above) into account and the fact that humans have comparable squamous epithelial tissues in the oral cavity and the upper two-thirds of the oesophagus as in the rat forestomach, the mutagenicity effect observed in the Comet assay are considered to be relevant for humans and should be regarded as positive e (CLP guidance 2017, page 381). Therefore, the substance is considered to have genotoxic potential that may also be expected in humans at the route of entry.

No human data are available with 2-methoxyethyl acrylate, therefore classification as Muta. 1A is not appropriate.

The classification in category 1B was considered. There is neither in vivo heritable germ cell mutagenicity test nor tests in human germ cells available with 2-methoxyethyl acrylate. However, in the combined oral (gavage) repeated dose toxicity study with the reproduction/developmental toxicity screening test (see above), the following histopathological findings in male reproduction organs were observed:

- Enlarged cells
- Chronic active inflammation
- Most stages of spermatogenesis missing - Multiple acrosomes
- Individual cell necrosis
- Spermatidic giant cells

which shows the potential of the substance or its metabolite to reach the germ cells. However, the evidence from this test is not sufficient on its own to assess the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells. Therefore Muta 1B is not considered appropriate.

Overall, RAC considers that the classification criteria in CLP for Muta, category 1B, are not met, while the criteria for classification in category 2 (Table 3.5.1) are met based on “Positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from: (...) Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays”.

In conclusion, RAC is of the opinion that 2-methoxyethyl acrylate should be classified to for germ cell mutagenicity, category 2; H341 “Suspected of causing genetic defects“ based on the positive in vitro and in vivo data.