Furan-2,5-dicarboxylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A chromosome aberration study, an AMES test (Salmonella typhimurium reverse mutation assay and an Escherichia coli reverse mutation assay) and a mouse lymphoma assay were conducted with and without metabolic activation according to current OECD/EC guidelines and GLP principles. Based on the results of these three different in vitro assays, it is concluded that FDCA is not mutagenic with or without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 14, 2011 - April 04, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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 S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2: TA1535, TA1537, TA98, TA100 and WP2uvrA:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was 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: 650 µg/plate in DMSO for TA100

Negative solvent / vehicle controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Negative solvent / vehicle controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9: 10 µg/plate in DMSO for WP2uvrA

Negative solvent / vehicle controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9: 5 µg/plate in saline for TA1535

Negative solvent / vehicle controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

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:
- The presence of precipitation of the test compound on the plates was determined.

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 repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

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:

cytotoxicity

Remarks:

In the first mutation experiment, a reduction of the bacterial background lawn, was only observed in tester strain TA100 in the presence of S9. In the second experiment, toxicity was observed in all strains at the concentration of 3330 and 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the first mutation experiment, there was no reduction of the bacterial background lawn. In the second experiment, toxicity was observed in the presence of S9 mix at the concentration of 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 3330 and 5000 μg/plate in the presence of S9-mix. In tester strain WP2uvrA and TA100 without S9-mix, no toxicity was observed up to and including the top dose of 5000 µg/plate.
- No mutagenicity was observed up to and including the top dose of 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix (first and second experiment; positive control). Evaluation: The values (874 and 859) were above the limit of the range (756), however these values were more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate counts of the positive control had no effect on the results of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Second experiment:
TA1535, TA1537, TA98 and TA100: without S9 and with S9: 3330 and 5000 µg/plate
WP2uvrA: with S9: 5000 µg/plate
- No mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

A Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and an Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) with FDCA was performed according to OECD 471 guideline and GLP principles in two independent experiments. It is concluded that FDCA is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and an Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) with FDCA was performed according to OECD 471 guideline and GLP principles in two independent experiments.

In the first mutation experiment, a reduction of the bacterial background lawn, was only observed in tester strain TA100 at dose levels of 3330 and 5000 μg/plate in the presence of S9-mix. In the second experiment, toxicity was observed in all strains at the concentration of 3330 and 5000 μg/plate, except in tester strain WP2uvrA in the absence of S9-mix.

FDCA did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

It is concluded that FDCA is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 23, 2012 - August 22, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

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 S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 1561 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000 and 1561 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 1561 µg/mL
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 100, 1000 and 1561 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 100, 1000 and 1561 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 400 and 700 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 100, 300 and 600 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 100, 1000 and 1561 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was 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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

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.

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

3 h exposure time

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

highest concentration was the recommended 0.01 M

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

24 h and 48 h exposure time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest concentration based on toxicity, inhibition of the mitotic index of about 50%

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Yes
Solvent control: 7.64
1561 µg/ml: 5.26
1000 µg/ml: 6.02
333 µg/ml: 6.82
100 µg/ml: 7.19
- Effects of osmolality: No
Solvent control: 421 mOsm/kg
1561 µg/ml: 419 mOsm/kg
1000 µg/ml: 426 mOsm/kg
- Precipitation: No precipitation was observed up to and including the top dose of 1561 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, 3 hr treatment/24 hr fixation; at dose levels of 1000 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr and at the dose level of 1561 µg/ml in the presence of S9, 3 hours treatment, 24 hours fixation

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. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring. The highest concentration analysed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater (24 and 48 h exposure time), or the recommended 0.01 M.

OTHER:
Although both in the absence and presence of S9-mix one duplicate culture treated with 1561 μg/ml showed an increase in the number of chromosome aberrations above the historical control data range, this increase was not observed in the duplicate culture. The number of chromosome aberrations per 200 cells was well within the historical control data range. In addition no dose related increase was observed. Therefore these increases were considered not biologically relevant.

Conclusions:

A chromosome aberration study with FDCA was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that FDCA is not clastogenic in human lymphocytes.

Executive summary:

A chromosome aberration study with FDCA was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

Both in the absence and presence of S9-mix FDCA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No biologically relevant effects of FDCA on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that FDCA does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. It is concluded that FDCA is not clastogenic in human lymphocytes when tested up to 1561 µg/mL (= 0.01 M) with and without S9 -mix.

Reference 3

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:

22 July, 2012 - 23 October, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

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

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 1561 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 1000 and 1561 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 10, 50, 100, 500, 1000, 1100 and 1200 µg/mL
With S9-mix, 3 hours treatment: 10, 50, 100, 350, 500, 700 and 800 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 333, 560, 750, 1000, 1050, 1150, 1200 and 1250 µg/mL
With S9-mix, 3 hours treatment: 3, 10, 33, 100, 333, 560, 750 and 800 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate at a concentration of 15 and 5 μg/ml for a 3 and 24 hours treatment period, respectively.

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide at a final concentration of 10 μg/mL.

Remarks:

with S9

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:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10E6 survivors, and for CP not below 700 per 10E6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

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.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP 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.

Species / strain:

mouse lymphoma L5178Y cells

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: Yes
Solvent control: 7.64
1561 µg/ml: 5.26
- Effects of osmolality: No
Solvent control: 421 mOsm/kg
1561 µg/ml: 419 mOsm/kg
- Precipitation: At the end of 3 hour treatment period in the presence of S9-mix, FDCA precipitated in the exposure medium at dose levels of 1000 μg/ml and above in the dose range finding test, at 1150 μg/ml and above in mutation experiment 1 without S9-mix. No precipitation was observed in mutation experiment 1 with S9-mix (highest dose level 1100 μg/ml) and mutation experiment 2 (highest dose level 1000 μg/ml).

RANGE-FINDING/SCREENING STUDIES:
- In the absence of S9-mix after 3 hours treatment, the relative suspension growth was 74% at the test substance concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 1561 μg/ml.
In the presence of S9-mix after 3 hours tretament, the relative suspension growth was 26% at the test substance concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 1561 μg/ml.
In the absence of S9-mix after 24 hours treatment, the relative suspension growth was 40% at the test substance concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 1561 μg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency of one solvent control (first experiment; in the presence of S9-mix) was recorded to be outside the range of ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors. Evaluation: The value of 46 per 10E6 survivors was just below the lower limit of the range and clear negative results were obtained, therefore this deviation in the mutation frequency had no effect on the results of the study.
In the other experiments, 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:
In the first experiment (incubation time 3 hours), FDCA was tested up to cytotoxic levels of 65 and 50% in the absence and presence of S9-mix, respectively. In the second experiment, FDCA was tested up to cytotoxic levels of 91 and 95% in the absence and presence of S9-mix, respectively.

In the absence of S9-mix, FDCA did not induce a significant increase in the mutation frequency in the first experiment after a 3 hour treatment period. FDCA induced a 3.1-fold increase in the mutation frequency at the TK locus after a prolonged treatment period. The mutation frequency of 209 x 10-6 at the TK locus at the highest tested dose level in the second experiment was above the GEF + MF(controls) (= 193 x 10-6). However the increase was only observed at the highest tested concentration of 1250 μg/ml with severe toxicity (relative total growth (RTG) of 9%). The next lower dose level of 1200 μg/ml showed a toxicity of 85% (RTG 15%) and no biologically relevant increase in the mutation frequency. Furthermore it must be noted that an increase observed at dose levels with a RTG below 10% give rise to false positive conclusions. Therefore, this increase was considered not relevant and FDCA is considered to be not mutagenic in the absence of S9-mix.
In the presence of S9-mix, FDCA did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

FDCA was tested in the mouse lymphoma assay according to OECD 476 guideline and GLP principles. It is concluded that FDCA is not mutagenic.

Executive summary:

FDCA was tested in the mouse lymphoma assay according to OECD 476 guideline and GLP principles. Two independent experiments were performed up to cytotoxic concentrations both in the absence and presence of S9 -mix.

In the absence of S9-mix, FDCA did not induce a significant increase in the mutation frequency in the first experiment after a 3 hour treatment period. FDCA induced a 3.1-fold increase in the mutation frequency at the TK locus after a prolonged treatment period. The mutation frequency of 209 x 10-6 at the TK locus at the highest tested dose level in the second experiment was above the GEF + MF(controls) (= 193 x 10-6). However the increase was only observed at the highest tested concentration of 1250 μg/ml with severe toxicity (relative total growth (RTG) of 9%). The next lower dose level of 1200 μg/ml showed a toxicity of 85% (RTG 15%) and no biologically relevant increase in the mutation frequency. Furthermore it must be noted that an increase observed at dose levels with a RTG below 10% give rise to false positive conclusions. Therefore, this increase was considered not relevant and FDCA is considered to be not mutagenic in the absence of S9-mix.

In the presence of S9-mix, FDCA did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

It is concluded that FDCA is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

A Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and an Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) with FDCA was performed according to OECD 471 guideline and GLP principles in two independent experiments.

In the first mutation experiment, a reduction of the bacterial background lawn, was only observed in tester strain TA100 at dose levels of 3330 and 5000 μg/plate in the presence of S9-mix. In the second experiment, toxicity was observed in all strains at the concentration of 3330 and 5000 μg/plate, except in tester strain WP2uvrA in the absence of S9-mix.

FDCA did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

It is concluded that FDCA is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration study:

A chromosome aberration study with FDCA was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. No cytotox, no precipitation, but tested up to max concentration 0.01M.

Both in the absence and presence of S9-mix FDCA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No biologically relevant effects of FDCA on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that FDCA does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. It is concluded that FDCA is not clastogenic in human lymphocytes when tested up to 1561 µg/mL (= 0.01 M) with and without S9 -mix.

Mouse lymphoma assay:

FDCA was tested in the mouse lymphoma assay according to OECD 476 guideline and GLP principles. Two independent experiments were performed up to cytotoxic concentrations, both in the absence and presence of S9 -mix.

In the absence of S9-mix, FDCA did not induce a significant increase in the mutation frequency in the first experiment after a 3 hour treatment period. FDCA induced a 3.1-fold increase in the mutation frequency at the TK locus after a prolonged treatment period. The mutation frequency of 209 x 10-6 at the TK locus at the highest tested dose level in the second experiment was above the GEF + MF(controls) (= 193 x 10-6). However the increase was only observed at the highest tested concentration of 1250 μg/ml with severe toxicity (relative total growth (RTG) of 9%). The next lower dose level of 1200 μg/ml showed a toxicity of 85% (RTG 15%) and no biologically relevant increase in the mutation frequency. Furthermore it must be noted that an increase observed at dose levels with a RTG below 10% give rise to false positive conclusions. Therefore, this increase was considered not relevant and FDCA is considered to be not mutagenic in the absence of S9-mix.

In the presence of S9-mix, FDCA did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

It is concluded that FDCA is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the available data, FDCA does not have to be classified for mutagenicity according to Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.