Tetrahydrofuran

Genetic toxicity: in vitro

Administrative data

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:

20 August 2009 to 12 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted following internationally recognized guidelines and with full GLP compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrofuran

EC Number:

203-726-8

EC Name:

Tetrahydrofuran

Cas Number:

109-99-9

Molecular formula:

C4H8O

IUPAC Name:

tetrahydrofuran

Details on test material:

- Name of test material (as cited in study report): Tetrahydrofuran- Physical state/appearance: liquid, colorless, clear- Analytical purity: >/= 99.8% (Certificate of Analysis, Merck KGaA, Germany dated 13 August 2008)- Impurities (identity and concentrations): specific organic impurities not identified; peroxide content

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Substrain K3Stocks of the CHO cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Experiment 1, without S9 mix, 4-hour exposure:93.8, 187.5, 375.0 and 750.0 microg/mLExperiment 1, with S9 mix, 4-hour exposure:93.8, 187.5, 375.0 and 750.0 microg/mLExperiment 2, without S9 mix, 24-hour exposure:93.8, 187.5, 375.0 and 750.0 microg/mLExperiment 2, with S9 mix, 4-hour exposure:250.0, 375.0, 500.0 and 750.0 microg/mLDuplicate cultures (A and B) were used for all experimental groups

Vehicle / solvent:

Ham's F12 culture medium

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 300 microg/mL

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: 10 and 20 microg/mL

Details on test system and experimental conditions:

Culture media:All media were supplemented with:- 1% (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL)- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)Treatment medium (4-hour exposure period):Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No. FG 0815).Culture medium and Treatment medium (24-hour exposure):Ham's F12 medium containing L-glutamine source and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS).Pretreatment medium ("HAT" medium):Ham's F12 medium supplemented with:- hypoxanthine (13.6 x 10^-3 mg/mL)- aminopterin (0.18 x 10^-3 mg/mL)- thymidine (3.88 x 10^-3 mg/mL)- 10% (v/v) fetal calf serum (FCS)Selection medium ("TG" medium):Hypoxanthine-free Ham's F12 medium supplemented with:- 6-thioguanine (10 μg/mL)- 1% (v/v) L-glutamine (200 mM)- 10% (v/v) fetal calf serum (FCS)Cell culture:For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL Ham's F12 medium including 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% humidity up to the approximate confluence and subcultured twice weekly (routine passage in 75 cm2 plastic flasks).Routine passage (preparation of a single cell suspension from a 75 cm² flask):- Cell medium was removed and washed with 5 mL PBS or HBSS (both Ca-Mg-free).- Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks.- This reaction was stopped by adding 6 mL culture medium.- Cells were pipetted to separate the cells and to prepare a homogeneous single cell suspension.- Cells were counted in a counting chamber or using a cell counter.- Cell suspension was diluted with complete culture medium to the desired cell count.Preparation of test cultures:The stocks of cells (1.0-mL portions) were thawed at 37°C in a water bath. 0.5 mL was pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). The medium was replaced after 24 hours to remove any dead cells. After at least2 passages, cells were taken for the experiment, and for these there was another passage to prepare test cultures.Pretreatment of cells with "HAT" medium:During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 3 – 5x10^5 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. After that, a passage in Ham's F12 medium incl. 10% (v/v) FCS was followed with a subsequent incubation for a further 3 - 4 days.Attachment period:For each test group, about 1x10^6 logarithmically growing cells per flask (175 cm²; after the 2nd passage) were seeded into about 20 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours. Two flasks (one flask referred to as A and one flask referred to as B) were used for each test group.Exposure period:After the attachment period, the medium was removed from the flasks and the treatment medium was added (see table 1 below). The cultures were incubated for the exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% humidity.Expression period:The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).Selection period:For selection of the mutants, six 75 cm2 flasks with 3x10^5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.Cytotoxicity determination:Cloning efficiency (CE) (pre-experiment) The procedure for the determination of the cloning efficiency in the pre-experiment wassimilar to that described for the determination of the cloning efficency 1 (CE1) in the main experiments, except every dose group contained only two cultures.Cloning efficiency 1 (CE1; survival):For the determination of the influence of the test item directly after the exposure period, per dose group about 200 cells were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were treated with the vehicle, test item or positive control for 4 hours or 24 hours. The exposure periods were completed by rinsing several times with HBSS. Then the flasks were topped up with 5 mL Ham's F12 medium incl. 10% (v/v) FCS.Cloning efficiency 2 (CE2; viability):The mutation rate after the expression period was determined in parallel to the selection of mutants. For each dose group about 200 cells were taken in duplicate, seeded in 25 cm2 flasks using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. In all cases, after seeding of the cells the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group as follows:Cytotoxicity (CE, CE1, CE2):The cloning efficiency (CE, %) was calculated for each test group as follows:CE(absolute) = total number of colonies in the test group / total number of seeded cells in the test group x 100CErelative = CE(absolute) of the test group / CE(absolute) of the vehichle control x 100The number of colonies in every flask was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CE(absolute), CE1 absolute and/or CE2 absolute) were calculated. Based on these values the relative cloning efficiencies (CE(relative), CE1 relative and/or CE2 relative) of the test groups were calculated and given in percentage compared with the respective CE(absolute) value of the corresponding vehicle/negative control (vehicle/negative control = 100%).Mutant frequency:The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to 10^6 cells seeded. The uncorrected mutant frequency (MFuncorr.) per 106 cells was calculated for each test group as follows:MF(uncorr.) = total number of mutant colonies / number of seeded cells x 106The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):MF(corr.) = MF(uncorr.) / CE2 absolute x 100Check or determination of further parameters:pHChanges in the pH were recorded by a change in the indicator color in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the negative controls with and without S9 mix.OsmolarityOsmolarity was measured, at least for the top dose and for the negative controls with and without S9 mix.SolubilityTest item precipitation was checked immediately after treatment of the test cultures and at the end of treatment.Cell morphologyThe test cultures of all test groups were examined microscopically at the end of exposureperiod with regard to cell morphology, which allows conclusions to be drawn about the attachment of the cells.

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0- 15 mutants per 106 clonable cells (see Appendix 6).• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data; see Appendix 7).• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.A test item is considered as positive if the following criteria are met:• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 6).• Evidence of reproducibility of any increase in mutant frequencies.• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.The test item is considered non-mutagenic if the following criteria are met:• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

The number of mutant colonies in each flask of the dose groups and the positive controls were compared with that of the solvent control groups using the Fisher-Pitman Test for the hypothesis of equal means. It was assumed for this test that the 6 flasks were independent within each group. If the results were significant, labels (* for p ≤ 0.05 and ** for p ≤ 0.01) were printed with the group values (uncorrected mutant frequency) in the tables. The test was performed one-sided. However, both, biological and statistical significance should be considered together.

Results and discussion

Test results

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutant Frequency:

No biologically relevant increases in the number of mutant colonies were observed either with or without S9 mix. In both experiments after 4 hours treatment, the values for the corrected mutation frequencies were close to the respective control values (see the following table).

Summary of Results

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity**MF(corr)[per 10^6 cells]	Cytototoxicity***
						CE1 [%]	CE2 [%]
1	4 hrs	Negative	-	-	5.28	100.0	100.0
		93.8 microg/mL	-	-	1.31	105.2	100.6
		187.5 microg/mL	-	-	7.33	100.6	89.2
		375.0 microg/mL	-	-	0.36	101.9	95.3
		750.0 microg/mL	-	-	1.14	96.1	85.4
		Positive control^1	-	-	119.09s	99.6	94.3
2	24 hrs	Negative	-	-	1.85	100.0	100.0
		93.8 microg/mL	-	-	0.91	99.5	98.6
		187.5 microg/mL	-	-	1.81	101.6	87.9
		375.0 microg/mL	-	-	0.00	97.1	91.5
		750.0 microg/mL	-	-	0.31	97.8	97.3
		Positive control^1	-	-	360.72s	67.7	60.0
1	4 hrs	Negative	+	-	3.04	100.0	100.0
		93.8 microg/mL	+	-	7.32	97.8	87.9
		187.5 microg/mL	+	-	3.86	93.5	96.0
		375.0 microg/mL	+	-	7.22	94.1	84.0
		750.0 microg/mL	+	-	15.03s	98.3	94.3
		Positive control^2	+	-	51.82s	99.5	91.0
		Positive control^3	+	-	99.59s	92.5	86.7
2	4 hrs	Negative	+	-	1.15	100.0	100.0
		250.0 microg/mL	+	-	0.95	94.6	95.1
		375.0 microg/mL	+	-	1.22	100.0	94.0
		500.0 microg/mL	+	-	2.75	99.9	93.5
		750.0 microg/mL	+	-	0.34	95.1	85.7
		Positive contorl^2	+	-	60.28s	103.5	79.4
		Positive control^3	+	-	93.54s	94.6	60.9

* Precipitation

** Mutant frequency

*** Cloining efficiency

Pos Cntl: 1) EMS, 2) MCA, 10 microg/mL, 3) MCA, 20 microg/mL

s Mutant frequency statistically signifiacantly higher than the corresponding control value

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeThus, under the experimental conditions of this study, tetrahydrofuran is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.