Methacrylic acid, monoester with propane-1,2-diol

Endpoint summary

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

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.03.2010 - 19.05.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Supplier: Rohm and Haas Company,
- Analytical purity: 98.96%
- Lot/batch No.: 0005398178

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.

Key result

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

Result table see under attachments

Conclusions:

Interpretation of results: 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:

In the in vitro Chinese hamster ovary cell/hypoxanthineguanine- phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay, Hydroxypropyl methacrylate (2-methyl-2-propenoic acid monoester with 1,2- propanediol) was evaluated in concentrations between 45.1 and 1442 µg/ml in the absence and presence of an externally supplied metabolic activation (S9) system. 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

Japanese version: Guidelines for Screening Mutagenicity Testing of Chemicals, 1996

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

Specific details on test material used for the study:

Supplier: Mitsubishi Rayon Co., LTD. (Tokyo, Japan),
Purity 98%,
Lot No. 0348402.
Imurities: <=2 % of dipropylene glycol monomethacrylate
The test solution prepared was confirmed to be stable for 4 hours.

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:

cyclophosphamide

mitomycin C

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.

Rationale for test conditions:

-Dosage :
Continuous treatment without S9 mix (treatment time 24 or 48 hr); 0, 180,
350, 700 micro-g/mL
Short-term treatment with and without S9 mix (treatment - recovery time, 6
- 18 hr); 0, 350, 700, 1400 (as 10 mmol/L) micro-g/mL
The doses were calculated with the results of a cell growth inhibition test.
The concentration that cause cell growth inhibition by approx. 60 % was
700, 1200 and 1400 micro-g/mL at the continuous treatment without S9
mix, the short-term treatment with and with S9 mix, respectively.

Evaluation criteria:

Number of examined cells, type and number of abnormal cells,
and number of polyploid cells were obtained for every dose group.

Statistics:

Judgement of clastogenicity by the test substance was based on the
results of Fisher's exact test (p < 0.05) and Cochran and Armitage's trend
test (p < 0.05).

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)

Table 1 Chromosomal analysis of Chinese hamster lung (CHL/IU)cells treated with methacrylic acid, monoester with propane-1,2-diol (Continuous treatment without S9 mix)

Group	Conc. µg/ml	Exposure time (h)	No. of cells analyzed	Rate of cells with aberrations (%)	Polyploid b)
Control			200	1.0	0.38
Acetone	0	24	200	0.5	0.25
HPMA	180	24	200	1.5	1.88*
HPMA	350	24	200	2.5	2.38*
HPMA	700	24	200	18.5*	3.50*
MMC	0.05	24	200	59.0	0.50
Judgement c)				+	+
Acetone	0	48	200	3.0	0.25
HPMA	180	48	200	3.0	1.82* d)
HPMA	350	48	200	10.5*	2.83* e)
HPMA	700	48	200	62.5*	4.22* f)
MMC	0.05	48	200	46.5	0.25
Judgement c)				+	+

MMC: mitomycin C (positive control)

1. a) Acetone was used as solvent.


2. b) Eight hundred cells were analyzed in each group.


3. c) Cochran-Armitage's test was done at p<0.05.


4. d) Seven hundred and seventy-one cells were analyzed.


5. e) Seven hundred and seventy-eight cells were analyzed.


6. f) Six hundred and eighty-eight cells were analyzed.

*; Significantly different from historical solvent control data with respect to

aberration at p<0.05 by Fisher's exact test using a Bonferroni correction for

multiple comparisons.

 

Table 2 Chromosomal analysis of Chinese hamster lung (CHL/IU) cells

treated with methacrylic acid, monoester with propane-1,2-diol

(Short-term treatment with and without S9 mix)

 

Group	Conc. µg/ml	Exposure time (h)	S9-mix	No. of cells analyzed	Rate of cells with aberrations (%)	Polyploid b)
Control				200	0.0	0.38
Acetone	0	6-18	-	200	2.5	0.63
HPMA	350	6-18	-	200	3.5	0.75
HPMA	700	6-18	-	200	2.5	1.13
HPMA	1400	6-18	-	200	1.5	3.88*
CPA	0.05	6-18	-	200	2.0	0.50
Judgement c)					NT	+
Acetone	0	6-18	+	200	0.5	0.50
HPMA	180	6-18	+	200	6.5*	4.38*
HPMA	350	6-18	+	200	21.0*	1.63*
HPMA	750	6-18	+	200	69.0*	0.88
CPA	0.05	6-18	+	200	47.0	0.00
Judgement c)					+	+

Exposure time (hr); 6 - 18 = treatment - recovery time

CPA: cyclophosphamide

1. a) Acetone was used as solvent.


2. b) Eight hundred cells were analyzed in each group.


3. c) Cochran-Armitage's test was done at p<0.05.

*; Significantly different from historical solvent control data with respect to

aberration at p<0.05 by Fisher's exact test using a Bonferroni correction for

multiple comparisons.

Conclusions:

Interpretation of results : 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.

Executive summary:

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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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

Specific details on test material used for the study:

Suppliere: Mitsubishi Rayon Co., LTD. (Tokyo, Japan),
Purity 98%, Lot No. 0348402.
Impurity: <=2 % of dipropylene glycol monomethacrylate
Storage: in a refrigerator before use.
The test solution prepared was confirmed to be stable for 4 hours.Purity: 98 %

Target gene:

his, trp

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:

9-aminoacridine

sodium azide

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 2

OTHER: 3 plates/test

Evaluation criteria:

Judgement of mutagenicity positive: It was judged as positive when the
number of average revertant colonies was more than twice of that of
control in at least one strain of + or - S9 mix, reproducibly or dose responsibly

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Key result

Species / strain:

S. typhimurium 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:

valid

Key result

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:

valid

Additional information on results:

No cytotoxicity was observed up to 5000 ug/plate in the five strains with
either the -S9 mix or the +S9 mix.

Conclusions:

Interpretation of results: 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.

Executive summary:

A reverse bacterial gene mutation assay (Ames-Test, plate incorporation assay) according to OECD Guideline 471 with 2-Hydroxypropyl methacrylate was negative up to the limit concentration of 5000 μg/plate with and without mammalian metabolic activation (rat liver S9-mix) in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA (pKM101). 

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

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.

   

 

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

4. DATA MATRIX

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Somatic Mutation and Recombinogenic Test (SMART) to detect mitotic recombination and a diverse set of mutational events.

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: negative

In an in vivo genetic toxicity test with Drosophila melanogaster with the structurally similar substance hydroxyethyl methacrylate (HEMA) the test substance 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. By read across this result also expected for HPMA.

Executive summary:

Genetic toxicity in vivo of 2-hydroxypropyl methacrylate was predicted by read across in an analogue approach with the structurally related substance substance 2-hydroxyethyl methacrylate.

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-07-7 to 1989-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1983-05-26

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Supplier: Röhm GmbH, Darmstadt
Batch No. 89-3 (BT-103)
Purity: 99.14 %
Stabilizer: 206 ppm Hydroquinone monomethylether
Storage: 4 °C, protected from light
In vehicle stable for approx. 3 hours, in water

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals: 84 (42 males/42 females)
- 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 hours after treatment

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

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.

Key result

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.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

4. DATA MATRIX

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Sex:

male

Evaluation criteria:

.

Statistics:

For the analysis of the percentage of PCEs, Student's t-test were applied. For the incidence of MNPCEs, tables of Kastenbaum and Bowman were applied.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no significant differences in the incidence of MNPCE's between any treatment group and the negative control group. There were significant increases in the incidences of PCE's between 1000 mg/kg group and the negative control group (p < 0.05).  Clinical signs were not observed with all of test substance groups.  The negative control incidences of MNPCE's among tests was within the range of our laboratory  background data and positive control ones showed remarkable increase.  These findings indicated that the test was conducted appropriately.  This chemical does not induce micronuclei under the test conditions employed.

Conclusions:

The results of an in vivo micronucleus test with the structurally similar substance hydroxyethyl methacrylate indicate that under the conditions of this study, hydroxypropyl methacrylate is not expected to induce micronuclei.

Executive summary:

Genetic toxicity in vivo of 2-hydroxyethyl methacrylate was predicted by read across in an analogue approach with the structurally related substance substance 2-hydroxypropyl methacrylate.

A bone marrow micronucleus study in seven week old, Sprague Dawley rats was conducted to assess the mutagenic potential of the test substance. The test substance was administered twice at 24 -hour interval by oral gavage at three doses of 500, 1000 and 2000 mg/kg. Twenty four hours after the final administration bone marrow samples were prepared, and were examined for incidence of micronucleated polychromatic erythrocytes. The test substance did not induce significant increases in the micronucleated polychromatic erythrocytes in any treated groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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.