Tetrahydrofurfuryl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-09-04 - 2013-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU method B.49 (In vitro Mammalian Cell Micronucleus Test), dated July 06, 2012.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrofurfuryl methacrylate

EC Number:

219-529-5

EC Name:

Tetrahydrofurfuryl methacrylate

Cas Number:

2455-24-5

Molecular formula:

C9H14O3

IUPAC Name:

tetrahydrofuran-2-ylmethyl methacrylate

Details on test material:

- Name of test material (as cited in study report): Tetrahydrofurfuryl methacrylate

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human, healthy donors not receiving medication

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX, supplemented with penicillin/streptomycin (100 U/mL/100 Lg/mL), the mitogen PHA (3 Lg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not relevant
- Periodically checked for karyotype stability: not relevant
- Periodically "cleansed" against high spontaneous background: not relevant

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I (4 h exposure, 16 h recovery, fixation time 40 h, without S9): 11.0, 19.3, 33.8, 59.2, 103.6, 181.3, 317.2, 555.1, 971.4, 1700.0 µg/mL
Experiment I (4 h exposure, 16 h recovery, fixation time 40 h, with S9): 11.0, 19.3, 33.8, 59.2, 103.6, 181.3, 317.2, 555.1, 971.4, 1700.0 µg/mL

Experiment II (20 h exposure, fixation time 40 h, without S9): 11.0, 19.3, 33.8, 59.2, 103.6, 181.3, 317.2, 555.1, 971.4, 1700.0 µg/mL
Experiment II (4 h exposure, 16 h recovery, fixation time 40 h, with S9): 103.6, 181.3, 317.2, 555.1, 971.4, 1700.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, final concentration in medium 0.5%
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other: (without metabolic activation, pulse treatment)

Controls 2

Positive controls:

yes

Positive control substance:

other: Demecolcin (without metabolic activation, continuous treatment)

Controls 3

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I:
4 hours pulse treatment (with and without metabolic activation), expression phase 16 hours, cytokinesis block 20 hours.
Experiment II:
4 hours pulse treatment (with metabolic activation), expression phase 16 hours, cytokinesis block 20 hours
20 hours continuous treatment (without metabolic activation), cytokinesis block 20 hours
- Expression time (cells in growth medium): 20 h (+ cytochalasin B)
- Fixation time (start of exposure up to fixation or harvest of cells): 40

A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.

SPINDLE INHIBITOR (cytogenetic assays): 4 µg/mL cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per experiment
NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage

DETERMINATION OF CYTOTOXICITY
- Method: CBPI (Cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis

Evaluation criteria:

The micronucleus assay is considered acceptable if it meets the following criteria:
- The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data
- The positive control substances should produce significant increases in the number of cells with micronuclei.

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
-no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

Chi square test (alpha < 0.05)

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Exp. I: no cytotoxicity with and without S9; Exp II: cytotoxic without S9, no cytotoxicity with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.6 / 7.8 for solvent control, 7.7 / 7.8 for test item in experiment I / II -> no relevant influence
- Effects of osmolality: 376 / 385 for solvent control, 375 / 381 for test item in experiment I / II -> no relevant influence
- Precipitation: No precipitation of the test item in the culture medium was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: all results were within the range of the laboratory historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix the highest applied concentration showing clear cytotoxic effects was not evaluable.

Any other information on results incl. tables

 

	Proliferation index CBPI, mean of 2 cultures	Cytostasis [%]	Micronucleated cells [%]
Experiment I; Exposure period 4 hrs without S9 mix
Solvent control, 0.5% DMSO	2.01	---	0.85
Positive control, 2.0 µg/mL MMC	1.70	30.8	15.50
Test item 555.1 µg/mL	1.95	5.4	0.60
Test item 971.4 µg/mL	1.77	24.0	0.80
Test item 1700.0 µg/mL	1.89	11.8	0.75
Experiment I; Exposure period 4 hrs with S9 mix
Solvent control, 0.5% DMSO	1.86	---	0.95
Positive control, 17.5 µg/mL CPA	1.87	n.c.	3.10
Test item 555.1 µg/mL	1.91	n.c.	0.35
Test item 971.4 µg/mL	1.92	n.c.	0.50
Test item 1700.0 µg/mL	1.83	3.5	0.45
Experiment II; Exposure period 20 hrs without S9 mix
Solvent control, 0.5% DMSO	1.82	---	0.50
Positive control, 125 ng/mLDemecolcin	1.47	43.2	5.55
Test item 317.2 µg/mL	1.88	n.c.	0.25
Test item 555.1 µg/mL	1.75	8.6	0.40
Test item 971.4 µg/mL	1.71	14.0	0.40
Experiment II; Exposure period 4 hrs with S9 mix
Solvent control, 0.5% DMSO	1.92	---	0.55
Positive control, 15 µg/mL CPA	1.70	23.7	3.10
Test item 555.1 µg/mL	1.94	n.c.	0.65
Test item 971.4 µg/mL	1.95	n.c.	0.70
Test item 1700.0 µg/mL	1.96	n.c.	0.60

 

n.c.= Not calculated as the CBPI is equal or higher than the solvent control value

 

 

Applicant's summary and conclusion

Conclusions:

In this test under the experimental conditions reported, the test item Tetrahydrofurfuryl methacrylate did not induce micronuclei in human lymphocytes in vitro when tested up to cytotoxic or the highest evaluable concentration.

Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 478 (adopted July 22, 2010) and EU method B.49, dated July 06, 2012., primary human lymphocyte cultures were exposed toTetrahydrofurfuryl methacrylate(99.0%) in DMSO with and without metabolic activation (S9 mix).

The following concentrations were evaluated (calculations were not adjusted to purity):

 

Experiment I:

4 h exposure, 16 h recovery, fixation time 40 h, without S9: 555.1, 971.4, 1700.0 µg/mL

4 h exposure, 16 h recovery, fixation time 40 h, with S9: 555.1, 971.4, 1700.0 µg/mL

Experiment II:

20 h exposure, fixation time 40 h, without S9: 317.2, 555.1, 971.4 µg/mL

4 h exposure, 16 h recovery, fixation time 40 h, with S9: 555.1, 971.4, 1700.0 µg/mL

 

The test item was tested up to cytotoxic or the guideline limit concentration of 10 mM (corresponding to 1700.0 µg/mL). No cytotoxicity was observed in Experiment I with and without metabolic activation as well as in Experiment II with metabolic activation. In Experiment II in the absence of S9 mix the highest applied concentration showing clear cytotoxic effects was not evaluable.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. 

Positive controls induced the appropriate response. 

There was no evidence of micronucleated cells induced over background. Therefore,Tetrahydrofurfuryl methacrylate is considered to be non-clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or the guideline limit concentration.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.