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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro studies performed according to OECD and GLP are available to address all potential mode-of-actions for genotoxicity: Ames, chromosome aberration and mouse lymphoma. The substance tested 'negative' with respect to genetic toxicity in all three tests performed.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May - 5 June, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene in S. typhimurium
Tryptophan gene in E. coli
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:
phenobarbital and ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
First and second mutation test: 100, 333, 1000, 3330 and 5000 µg/plate
The S9-mix contained 5% (v/v) S9 fraction in the dose range finding test and the first mutation test when tested in the presence of S9-mix.
The S9-mix contained 10% (v/v) S9 fraction in the second mutation test when tested in the presence of S9-mix.
Vehicle / solvent:
dimethyl sulfoxide
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance is soluble in DMSO and stable for at least 96 hours

Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA1535 without S9, 5 µg/plate in saline
Positive control substance:
sodium azide
Positive controls:
yes
Remarks:
for TA1537 without S9, 60 µg/plate in Milli-Q water
Positive control substance:
9-aminoacridine
Positive controls:
yes
Remarks:
for TA98 without S9, 10 µg/plate in DMSO
Positive control substance:
2-nitrofluorene
Positive controls:
yes
Remarks:
for TA100 without S9, 650 µg/plate in DMSO
Positive control substance:
methylmethanesulfonate
Positive controls:
yes
Remarks:
for WP2uvrA without S9, 10 µg/plate in DMSO
Positive control substance:
4-nitroquinoline-N-oxide
Positive controls:
yes
Remarks:
for all strains with S9 (5% and 10%)
Positive control substance:
other: 2-aminoanthracene
Remarks:
Concentrations range from 1-10 µg/plate in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- Other: precipitation of the test substance
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Key result
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:
no cytotoxicity
Untreated negative controls validity:
valid
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:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No reduction of the bacterial background lawn and no decrease or increase in the number of revertants were observed up to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Conclusions:
An AMES test was performed according to OECD/EC guidelines and GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.Based on these findings it is concluded that Ditetrahydrofurylpropane is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-Sep-2010 to 04-Nov-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 1843 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000 and 1843 µg/mL

First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 333, 1000 and 1843 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 400 and 600 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 150, 400 and 500 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 333, 1600 and 1843 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Positive control substance:
cyclophosphamide
Remarks:
with S9 in Hank's Balanced Salt Solution: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was only observed at the prolonged treatment period in the absence of S9-mix. In the other treatment periods no toxicity was observed up to the top dose of 1843 µg/mL (= 0.01 M), which is limit dose.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
Solvent control: 7.31
1843 µg/mL: 7.34
- Effects of osmolality: No
Solvent control: 400 mOsm/kg
1843 µg/mL: 402 mOsm/kg
- Precipitation: No precipitation was observed up to and including the top dose of 1843 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 1000 µg/mL and above in the absence of S9 for the continuous treatment of 24 hr and at dose levels of 333 µg/mL and above in the absence of S9 for the continuous treatment of 48 hr.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- For the continuous treatment of 24 and 48 hr, appropriate toxicity was reached at the dose levels selected for scoring.
- For the 3 hours treatment in the absence and presence of S9-mix, no toxicity was observed up to the top dose of 1843 µg/mL.

In the first cytogenetic assay, in the absence of S9-mix, Ditetrahydrofurylpropane increased the number of polyploid cells in a dose dependent manner (1 up to 4) and above the historical control data range (3 hr exposure). In the second cytogenetic assay, in the absence of S9 -mix, at the 24 hr continuous exposure time, Ditetrahydrofurylpropane increased the number of cells with endoreduplicated chromosomes (2) just above the historical control data range. This may indicate that Ditetrahydrofurylpropane has the potential to disturb mitotic processes and cell cycle progression.

Conclusions:
A chromosome aberration study was conducted according to OECD/EC guidelines and GLP principes. It is concluded that this test is valid and that Ditetrahydrofurylpropane is not clastogenic in human lymphocytes under the experimental conditions described in this report. Ditetrahydrofurylpropane may have the potential to disturb mitotic processes and cell cycle progression.
Executive summary:

A chromosome aberration study was conducted according to OECD/EC guidelines and GLP principes. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Ditetrahydrofurylpropane did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-Sep-2010 to 09-Nov-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000 and 1843 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 1000 and 1843 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 300, 500, 650, 800, 900, 1000, 1050 and 1100 µg/mL
With S9-mix, 3 hours treatment: 33, 300, 500, 800, 1000, 1100, 1150 and 1200 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 33, 100, 300, 500, 650, 800, 850 and 900 µg/mL
With S9-mix, 3 hours treatment: 100, 300, 500, 600, 700, 800, 900 and 1000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
(strain L5178Y/TK+/-3.7.2C)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
Solvent control: 7.31
1843 µg/ml: 7.34
- Effects of osmolality: No
Solvent control: 0.400 mOsm/kg
1843 µg/ml: 0.402 mOsm/kg
- Precipitation: No precipitation was observed up to and including the top dose of 1843 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 1843 µg/mL in the absence and presence of S9, 3 hours treatment and at dose levels of 1000 µg/mL and above in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.
Conclusions:
A mouse lymphoma assay was performed according to OECD/EC guidelines and GLP principles. It is concluded that Ditetrahydrofurylpropane is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.
Executive summary:

mouse lymphoma assay was performed according to OECD/EC guidelines and GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, Ditetrahydrofurylpropane did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Ditetrahydrofurylpropane did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

In a study performed according to OECD 471 test guidelines, the species S. typhimurium TA1535, 1537, 98 and 100 and the species E. coli WP2 uvr A were tested up to the limit concentration of 5000 microg/plate with and without metabolic activation. All bacterial strains showed negative responses over the entire dose range with and without metabolit activation, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that Ditetrahydrofurylpropane is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  

Chromosome aberration test:

In a study performed according to OECD 473 test guidelines, human lymphocytes were exposed to the test substance up to 1843 microg/ml (0.01M, limit dose) with and without metabolic activation. Ditetrahydrofurylpropane did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. Therefore it was concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions chosen. In the first cytogenetic assay, in the absence of S9-mix, Ditetrahydrofurylpropane increased the number of polyploid cells in a dose dependent manner and above the historical control data range (3 hr exposure). In the second cytogenetic assay, in the absence of S9 -mix, at the 24 h continuous exposure time, Ditetrahydrofurylpropane increased the number of cells with endoreduplicated chromosomes just above the historical control data range. This may indicate that Ditetrahydrofurylpropane has the potential to disturb mitotic processes and cell cycle progression. However, as the effects observed were only just above the historical control range and only observed without metabolic activation, no further investigation is considered necessary.  

Mouse lymphoma test:

In a study performed according to OECD 476 test guidelines, L5178Y mouse lymphoma cells were expsoed to the test substance up to 1843 microg/ml (=0.01M, limit dose) with and without metabolic activation. In the absence of S9-mix, Ditetrahydrofurylpropane did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Ditetrahydrofurylpropane did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. Based on the results of the test it was concluded that Ditetrahydrofurylpropane is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions chosen.

Justification for classification or non-classification

Based on the results of the genotoxicity studies, the substance does not need to be classified according to the CLP Regulation (EC) 1272/2008.