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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Induced DNA Damage by Dental Resin Monomers in Somatic Cells
Author:
Arossi, G.A., Lehmann, M., Dihl, R.R., Reguly, M.L.and de Andrade, H.H.R.
Year:
2009
Bibliographic source:
Basic and Clinical Pharmacology and Toxicology 106, 124-129

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Reference substance 001
Details on test material:
- Name of test material (as cited in study report): HEMA

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.