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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

HPMA has been evaluated for genotoxic potential in bacterial cells in culture and was found not to be mutagenic in this assay system. Further, HPMA was found not to be mutagenic in mammalian cells in culture [CHO HGPRT assay]. HPMA was reported to cause chromosomal aberrations in mammalian cells in culture.

It is concluded that HPMA is not mutagenic in bacterial and mammalian cells in culture. Further, while HPMA may cause chromosomal aberrations in mammalian cells in culture, the material is not clastogenic in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, japanese study, only abstract english
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0, 313, 625, 1250, 2500 and 5000 ug/plate
Details on test system and experimental conditions:
IUCLID4 Type: Escherichia coli reverse mutation assay
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other: other: Escherichia coli WP2 uvrA
Remarks:
Migrated from field 'Test system'.
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 nutrient mix (GIBCO, Grand Island, New York) supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 25 µg/ml; GIBCO) and an additional 2 mM L-glutamine (GIBCO).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mixS9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
45.1, 90.1, 180.3, 360.5, 721, and 1442 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate and 20-methylcholanthrene
Details on test system and experimental conditions:
Each dose level was set up in duplicate from the time of treatment until the completion of the assay. The cultures were trypsinized at the end of the treatment and replated at a density of 1 x 10e6 cells/100 mm dish (at least two dishes/replicate) for phenotypic expression (O'Neill et al., 1977a; O'Neill and Hsie, 1979). In addition, 200 cells/60 mm dish (three dishes/replicate) were also plated to determine the toxicity and incubated for approximately 6-8 days to permit colony formation. During the phenotypic expression period (7-9 days), cells in the larger petri dishes were subcultured every 2-3 days and plated (at least two dishes/replicate) at a density of about 1 x 10e6 cells/100 mm petri dish.

At each subculture, cells from various dishes within each replicate were pooled prior to replating. At the end of the expression period, the cultures were trypsinized and plated at a density of 2 x 105 cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F-12 without hypoxanthine and with 6-thioguanine) for the determination of HGPRT- mutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficiency. The dishes were incubated for about 7-9 days and the colonies were fixed/stained with methanol/crystal violet. The mutation frequency (expressed as mutants per 106 clonable cells) for each replicate were calculated.
Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the solvent controls. An additional criteria, was that the mutant frequency in the solvent controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the solvent controls.
Statistics:
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutation frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was used as an estimate of variance (Kirkland, 1989). If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

There was no appreciable change in either the pH or osmolality at this concentration as compared to the culture medium with solvent alone.

RANGE-FINDING/SCREENING STUDIES: Concentration levels of 0 (solvent control), 45.1, 90.1, 180.3, 360.5, 721, and 1442 µg/ml of the test material were selected in the absence and presence of S9. In the absence of S9, little to no toxicity was observed with RCS values ranging from 74.7 to 113.8%. In the presence S9, cultures displayed moderate to no toxicity with RCS values ranging from 52.9 to 88.4%. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 were not significantly different from the concurrent solvent control values.

All mutant frequencies were within a reasonable range of historical background values.
A second confirmatory assay with treatment of cultures in the absence or the presence of S9 was not considered necessary since the results of the initial test yielded clearly negative results.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The results of the in vitro CHO/HGPRT forward gene mutation assay with hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

Hydroxypropyl methacrylate (2-methyl-2-propenoic acid monoester with 1,2- propanediol) was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthineguanine- phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 45.1 to 1442 µg/ml in the absence and presence of S9. The highest concentration was based on the assay system limit of 10 mM. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assay in the absence of S9 and 20- methylcholanthrene for assay in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. distilled water). The results of the in vitro CHO/HGPRT forward gene mutation assay with hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP
Principles of method if other than guideline:
Method: Guidelines for Screening Mutagenicity Testing of Chemicals (Japan)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimum essential medium (MEM)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver, induced with phenobarbital and 5,6- benzoflavone
Test concentrations with justification for top dose:
+ S9-mix/ -S9-mix: 0, 0.35, 0.70 and 1.4 mg/ml (short-term treatment); -S9-mix: 0, 0.18, 0.35, 0.70 mg/mL (continuous treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: S9 mix, Mitomycin C ; +S9 mix, Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 h
- Exposure duration: the proliferating cells were treated with the chemicals for 6 h in serum-free MEM with S9 mix (S9(+)), or without S9 mix (S9(-)), then cultured a further 18 h in the fresh MEM with serum. Moreover, the cells were also treated with the chemicals for 24 and 48 h continuously in the absence of S9 mix.
- Fixation time (start of exposure up to fixation or harvest of cells): 8 minutes

STAIN (for cytogenetic assays): 3% Giesma solution

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 2 x 10e4 plated

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: defined as a metaphase with more than 37 chromosomes, i.e. theoretical "hypertriploid cell': containing approximately three times the number of chromosomes than half the modal chromosome number (25) in CHL/IU cells.
Species / strain:
other: Chinese hamster lung (CHL/IU) cells)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
weakly
Additional information on results:
Lowest concentration producing cytogenetic effects in vitro:
Without metabolic activation (continuous treatment):                     
0.35 mg/ml (clastogenicity)                   
0.18 mg/ml (polyploidy) 
Without metabolic activation (short-term treatment):                   
1.4 mg/ml (clastogenicity) 
With metabolic activation (short-term treatment):                    
0.35 mg/ml (clastogenicity and polyploidy)
Remarks on result:
other: other: Chinese hamster lung (CHL/IU) cells)
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive

2-Hydroxypropyl methacrylate induced structural chromosomal aberrations in CHL/IU ce l Is with and without an exogeneous metabolic activation
system. Polyploidy was induced without an exogeneous metabolic activation system.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, japanese study, only abstract english
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver, induced with phenobarbital and 5,6- benzoflavone
Test concentrations with justification for top dose:
0, 313, 625, 1250, 2500 and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix, 2-(2-Fury!)-3-(5-nitro-2-fury1)acryIamide (TA100, WP2, TA98), Sod i um az i de (TA1535) and 9-Ami noacr id i ne (TAI 537), +S9 mix, 2-Ami noanthracene (five strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 2

OTHER: 3 plates/test
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:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA100, TA1535, TA98 and TA1537
Conclusions:
Interpretation of results (migrated information):
negative

2-Hydroxypropyl methacrylate was not mutagenic to Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with or without an exogeneous metabolic activation system.

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Documentation sufficient for assessment
Principles of method if other than guideline:
Somatic Mutation and Recombinogenic Test (SMART) to detect mitotic recombination and a diverse set of mutational events.
GLP compliance:
not specified
Type of assay:
somatic mutation and recombination test in Drosophila
Species:
Drosophila melanogaster
Sex:
male/female
Details on test animals or test system and environmental conditions:
The monomer genetic toxicities were accessed using the Standard Cross version of the wing SMART test: flr3 ⁄TM3, BdS females crossed with mwh ⁄mwh males. Eggs from this cross were collected for 8 hr on culture bottle containing a solid agar base (3% w⁄ v) enriched with a layer of live fermenting
baker’s yeast supplemented with sucrose. Three days later the larvae were transferred to vials containing 1.5 g of Drosophila Instant Medium (Carolina Biological Supply, Burlington, NC, USA) rehydrated with 5 ml of the test solutions.
Vehicle:
- Vehicle(s)/solvent(s) used: water
Details on exposure:
The monomer genetic toxicities were accessed using the Standard Cross version of the wing SMART test: flr3 ⁄TM3, BdS females crossed with mwh ⁄mwh males. Eggs from this cross were collected for 8 hr on culture bottle containing a solid agar base (3% w⁄ v) enriched with a layer of live fermenting
baker’s yeast supplemented with sucrose. Three days later the larvae were transferred to vials containing 1.5 g of Drosophila Instant Medium (Carolina Biological Supply, Burlington, NC, USA) rehydrated with 5 ml of the test solutions. Negative solvent controls were always included. The treated individuals remained in the vials until the emergence of the surviving adult flies.
Duration of treatment / exposure:
Flies were collected after eclosion
Frequency of treatment:
Eggs from the cross were collected for 8 hr. Three days later the larvae were transferred to vials containing 1.5 g of Drosophila Instant Medium rehydrated with 5 ml of the test solutions. Flies were collected after eclosion.
Remarks:
Doses / Concentrations:
HEMA diluted in distilled water – 0.675%, 1.25%, 1.875% and 2.5%
Basis:

No. of animals per sex per dose:
30 total
Control animals:
yes
Positive control(s):
ethyl methanesulfonate
Tissues and cell types examined:
presence of cell clones showing malformed wing hairs
number of spots as well as their type and size
Details of tissue and slide preparation:
After eclosion, the flies were collected from the treatment vials and stored in 70% ethanol. Subsequently, the wings were mounted on slides and scored under 400 X· magnification for the presence of cell clones showing malformed wing hairs. The number of spots as well as their type and size were recorded. In test larvae, two genotype configurations are possible: trans-heterozygous for the recessive wing cell markers [mwh and flr3 (mwh +⁄+ flr3)], and balancer- heterozygous (mwh ⁄TM3). Induced loss of heterozygosity on marker-heterozygous flies leads to two types of mutant clones: (i) single spots, either mwh and flr3, which can be produced by somatic point mutation, chromosome aberration as well as mitotic recombination and (ii) twin spots, consisting of both mwh and flr3 sub clones, which are originated exclusively from mitotic recombination. On balancer-heterozygous flies, mwh spots should reflect somatic point mutation and chromosome aberration, as mitotic recombination – involving the TM3 chromosome and its structurally normal homologue – is a lethal event.
Evaluation criteria:
We considered the treatment as positive if the frequency of mutant clones in the treated series was at least m (multiplication factor) times higher than that in the control series.
Statistics:
The conditional binomial test of Kastenbaun and Bowman [1970] was applied to assess differences between the frequencies of each spot type in treated and concurrent NC flies. The multiple decision procedure described by Frei and Wrgler [1988, 1995] was used to judge the overall response of an agent as positive, weakly positive, negative or inconclusive. We considered the treatment as positive if the frequency of mutant clones in the treated series was at least m (multiplication factor) times higher than that in the control series. As small single spots and total spots have a comparatively high spontaneous frequency, m was fixed as 2 (testing for a doubling of the spontaneous frequency). For large single spots and twin spots, which have a low spontaneous frequency, m = 5 was used. The recombinagenic action of the drugs was calculated comparing the standard frequency of clones per 105 cells obtained from mwh ⁄ flr3 and mwh ⁄TM3 genotypes. For an unbiased comparison of this frequency, only mwh clones in mwh single spots and in twin spots were used.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
HEMA did not have a significant effect on total spot frequencies in marker-heterozygous (mwh ⁄ flr3) flies analysed, suggesting that HEMA does not act as a genotoxin in the SMART assay.
Conclusions:
Interpretation of results (migrated information): negative
HEMA did not have a significant effect on total spot frequencies in marker-heterozygous (mwh ⁄ flr3) flies analysed, suggesting that HEMA does not act as a genotoxin in the SMART assay.
Executive summary:

The present in vivo study investigated the genotoxicity of hydroxyethylmethacrylate (HEMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly’s wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. HEMA had no statistically significant effect on total spot frequencies – suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Fullinsdorf CH-4414 Fullinsdorf/Basel, Switzerland
- Age at study initiation: 10 weeks
- Weight at study initiation: 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: singly in wire mesh top Makrolon cages
- Diet (e.g. ad libitum): pelleted standard diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 5 dyas

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 deg C
- Humidity (%): not monitored
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 1989-07-09 To: 1989-08-14
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: polyethylene glycol
- Justification for choice of solvent/vehicle: nontoxicity to animals
- Concentration of test material in vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was suspended in PEG on the day of the experiment
Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
72 hoursafter treatment
Remarks:
Doses / Concentrations:
2000 mg/kg (maximum tolerated dose) in suspension with polyethyleneglycol
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): good stability at room tepmperature
- Route of administration: gavage
- Doses / concentrations: 40 mg/kg in phys. saline in a volume of 10 mL/kg bw
Tissues and cell types examined:
The femora were removed, the epiphyses were cut off and the marrow was flushed out.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on the results of the preliminary study; maximum tolerated dose level

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Preparation of bone marrow: 24, 48 and 72 hours after administration

DETAILS OF SLIDE PREPARATION: The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 ml fetal calf serum, using a 5 ml syringe, into 1 ml fetal calf serum. The cell suspension was centrifuged at 1,000 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: NIKON microscopes with 100x oil immersion objectives. Five animals per sex and group were evaluated as described.

Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of - micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
Statistics:
The non-parametric Mann-Whitney test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a first pre-experiment 4 animals ( 2 males, 2 females) received orally a single dose of 5000 mg/kg b.w. Hydroxypropylmethacrylate suspended in PEG. The volume administered was 10 ml/kg b.w..
Two animals, one male and one female died after administration.

In a second pre-experiment 4 animals (2 males, 2 females) per dose level received orally a single dose of 2000, 3000, or 4000 mg/kg b.w., respectively, Hydroxypropylmethacrylate suspended in PEG. The volume administered was 10 ml/kg b.w.

2000 mg/kg:
All animals expressed toxic reactions: reduction of spontaneous , activity, abdominal position (two males only), eyelid closure (one male only), apathy (two males only).

3000 mg/kg:
All animals expressed toxic reactions: reduction of spontaneous activity, abdominal position, apathy, eyelid closure (two males only). One female died within 24 h after application.

4000 mg/kg:
All animals expressed toxic reactions: reduction of spontaneous activity, abdominal position, apathy. One male and one female died within 6 h after application.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
24 h: 0.06%
48 h: 0.06%
72 h: 0.15%
- Ratio of PCE/NCE (for Micronucleus assay):
24 h: 1000/691
48 h: 1000/605
72 h: 1000/771
- Appropriateness of dose levels and route: dose level is maximum tolerated dose
- Statistical evaluation: no statistical significance
Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

This study was performed to investigate the potential of Hydroxypropylmethacrylate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in PEG. This suspending agent was used as negative control. The volume administered orally was 10 ml/kg b.w.. At 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic - erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 2000 mg/kg b.w.. In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control, which showed a distinct increase of induced micronucleus frequency.

Additional information

In vitro studies:

 

HPMA has been evaluated for the ability to induce mutation in bacterial cells in culture. The study (Shibuya et al, 2001), conducted by the Japan Ministry of Health and Welfare and was assigned a K-1, reliable without restriction reliability rating. HPMA was not toxic and not mutagenic to Salmonella typhimurium tester strains (TA 1535, 1537, 1538, 98 and 100) or to E. Coli WP2 uvrA at levels up to and including 5000 microgram/plate in the presence or absence of activating enzymes. Rat liver S-9 induced with Phenobarbital and with Benzoflavone was the activation system. Appropriate positive controls for each test strain in the presence or absence of S-9 were included in the study protocol.

 

HPMA has also been evaluated for the ability to induce chromosomal aberrations in mammalian cells in culture. Kusakabe et al (2002) evaluated the clastogenic potential of HPMA along with a large number of other substances in Chinese hamster lung cells in culture. HPMA was reported to induce structural chromosome aberrations following 6 hr exposure of cells in the presence of S-9 only. Continuous exposure of cells for 24 hours hours or for 48 hours without S-9 also caused an elevated incidence of chromosome aberrations for HPMA. Polyploidy was reported after both short-term treatment and 48 hour continuous treatment exposures for HPMA. These effects were found at exposure levels which caused <50% cell death. The study was assigned a Klimisch rating of 2, reliable with restriction.

HPMA was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay (Dow, 2010). This study was assigned a K-1, reliable without restrictions reliability. The genotoxic potential of the test material was assessed in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 45.1 to 1442 µg/ml in the absence and presence of S9. The highest concentration was based on the assay system limit of 10 mM. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assay in the absence of S9 and 20-methylcholanthrene for assay in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e.distilled water). The results of the in vitro CHO/HGPRT forward gene mutation assay with hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

 

In vivo studies:

 

HPMA was reported to cause chromosomal aberrations in mammalian cells in culture, but an in vivo micronucleus study with HPMA indicated that this material was not clastogenic in vivo.

   

 

 


Short description of key information:
HPMA has been evaluated for genotoxic potential in bacterial cells in culture and was found not to be mutagenic in this assay system. Further, HPMA was found not to be mutagenic in mammalian cells in culture [CHO HGPRT assay]. HPMA was reported to cause chromosomal aberrations in mammalian cells in culture.

It is concluded that HPMA is not mutagenic in bacterial and mammalian cells in culture. Further, while HPMA may cause chromosomal aberrations in mammalian cells in culture, the material is not clastogenic in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

It is concluded that both HEMA and HPMA are not mutagenic in bacterial and mammalian cells in culture. Further, while HEMA and HPMA may cause chromosomal aberrations in mammalian cells in culture, these materials are not clastogenic in vivo.