2-furoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay (471)

Ames assay was performed to determine the mutagenic nature of 2-Furoic acid. The study was perfomed using Salmonella typhimurium strain TA100 and TA98. The test chemical was dissolved in spectral grade DMSO and used at dose levels of 0, 25, 50, 75 or 100µg/plate. The plates were incubated for 40 hrs and then observed for number of revertants/plate.2- Furoic acid did not induce reversion inSalmonella typhumurium TA100 and TA98and hence it is not likely to classify as a gene mutant in vitro.

Chromosomal Abberation (473)

CHL cells were treated with 0.125, 0.25 and 0.5 mg/ml test chemical for 24 and 48 hours. The incidences for polyploidy and structural aberrations of treated cells were not different from the negative controls, and hence the test chemical is considered to be non-mutagenic in nature.

Mammalian cell mutation (476)

The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the absence of S9 metabolic activation system and hence dose not classify for gene mutation in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Ames assay was performed to determine the mutagenic nature of test chemical

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

0, 25, 50, 75 or 100 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Spectral grade DMSO
- Justification for choice of solvent/vehicle: The test chemical dissolved in Spectral grade DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

spectral grade DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 40 hrs in dark
- Expression time (cells in growth medium): 40 hrs in dark
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other:

OTHER: Compounds showing any ambiguity in the assays were tested twice at concentrations of up to 100 /µg/plate.

The experiments on the effect of pH on the mutagenic activity of the 2-furoate derivatives were performed using a series of base agar plates buffered in the range of pH 6.2-7.2. Agar plates of varying pH were prepared using phosphate buffer.

Rationale for test conditions:

No data

Evaluation criteria:

Chemicals that caused dose-dependent induction of more than twice the number of spontaneous revertants were defined as mutagenic, and mutagenic activity is expressed as the number of revertants per nmol, which was calculated from the linear portion of the dose -response curve.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98 and TA100

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No detailed data available
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Test chemical did not induce reversion in Salmonella typhumurium TA100 and TA98 and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Ames assay was performed to determine the mutagenic nature of test chemical The study was perfomed using Salmonella typhimurium strain TA100 and TA98. The test chemical was dissolved in spectral grade DMSO and used at dose levels of 0, 25, 50, 75 or 100µg/plate. The plates were incubated for 40 hrs and then observed for number of revertants/plate.The test chemical did not induce reversion inSalmonella typhumurium TA100 and TA98and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

To determine the mutagenic potential of test chemical from WoE report number 8 and 9.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

8.
9. Default

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

8

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

9

Metabolic activation:

with and without

Metabolic activation system:

8. No data available
9.rat liver homogenate S9

Test concentrations with justification for top dose:

8.0.125, 0.25 and 0.5 mg/ml
9.10 mg/plate, resp 1.0 mg/mL (CHL cell test)

Vehicle / solvent:

8.Not specified
9.water: saline (CHL test)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

8.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

9.

Details on test system and experimental conditions:

8.Not specified
9.Not specified

Rationale for test conditions:

8.Not specified
9.Not specified

Evaluation criteria:

8.Not specified
9.Not specified

Statistics:

8.Not specified
9.Not specified

Species / strain:

Chinese hamster lung (CHL/IU)

Remarks:

8.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

9.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

8.Not specified
9.Not specified

Remarks on result:

other: No mutagenic potential observed

Conclusions:

Under the experimental conditions reported, lactic acid did not cause gene mutations. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration assay.

Executive summary:

Data available for the target chemical was reviewed to determine the mutagenic nature of 2 -Furoic acid. The studies are as mentioned below:

CHL cells were treated with 0.125, 0.25 and 0.5 mg/ml test chemical for 24 and 48 hours. The incidences for polyploidy and structural aberrations of treated cells were not different from the negative controls, and hence the test chemical is considered to be non-mutagenic in nature.

In a mammalian cell cytogenetics assay, chromosomal aberration tests were carried out on 242 food additives, using Chinese hamster fibroblast cell line (CHL). Chinese hamster lung fibroblasts cell cultures were exposed to lactic acid at concentrations of 10 mg/plate (equivalent to 1.0 mg/mL) with and/or without metabolic activation. There was no evidence of chromosome aberration and cytotoxicity. Therefore, the test item is considered to be non-clastogenic.

Based on the data available for the target chemical, 2 -Furoic acid does not exhibit gene mutation in chromosomal abberation study In-vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

To determine the mutagenic potential of test chemical from WoE report number 8 and 9.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

11.Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot. This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process. The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme. HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG. Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
12.Thymidine kinase

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

11

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Remarks:

12

Cytokinesis block (if used):

11.No data
12.No data

Metabolic activation:

with and without

Metabolic activation system:

11.S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
12.Liver S9 fraction was prepared from Aroclor 1254-induced male Sprague- Dawley rats.

Test concentrations with justification for top dose:

11.0, 0.5, 1.0, 2.5 or 5.0 mM
12.5-52 µg/mL

Vehicle / solvent:

11.Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl phenylacetate was easily dissolved in ethanol.
12.No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: N-ethyl-N-nitrosourea (ENU) - without S9

Remarks:

11

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

other: 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.

Remarks:

12

Details on test system and experimental conditions:

11.METHOD OF APPLICATION: In medium with pre-incubation

Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
Exposure duration: 3 hours
Expression time: 7 days
Selection time: 14 days
Fixation time: 7 days (harvest of cells)

SELECTION AGENT
(mutation assays):6-thioguanine (TG)
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

12.METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 6000000 cells

DURATION
- Preincubation period: No data available
- Exposure duration: 4 h
- Expression time (cells in growth medium):48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated cultures

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

11.The plates were scored for total number of colonies
12.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Statistics:

No data available

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

11

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

not valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

12

Additional information on results:

11.TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES:
Preliminary dose-finding/toxicity test
Completed without S9 metabolic activation. A range of test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assay MTT and the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since cytotoxicity was evident at the tested concentration in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 0.5 mM. Since the test chemical was dissolved in ethanol, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of ethanol. The test chemical could only be dissolved in 99.5% ethanol.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
12.No data

Remarks on result:

other: 11. No mutagenic potential 12.No mutagenic potential observed. The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Conclusions:

The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the absence of S9 metabolic activation system and hence dose not classify for gene mutation in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for mammalian cell mutation to a greater or lesser extent. The studies are summarized as below :

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different test concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at 2.5 mM in the absence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. No cytotoxic effects were observed when CHO cells were exposed to test chemical for 3 hrs. Based on the results of the study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and does not induce cytotoxic effects at concentrations of ≤ 5.0mM.

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 5 -52 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase .The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro. The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

Therefore based on above summarized studies it can be concluded that the test chemical in not mutagenic to mammalian cell line and thus the test chemical can be classified as 'Not Mutagenic' as per CLP regulation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Ames assay was performed to determine the mutagenic nature of test chemical

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

No data

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical dissolved in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other:

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for colonies revertant to histidine prototroph. Results were expressed as numbers of revertant colonies per test plate after substraction of about 150 colonies per control plate

Statistics:

No data

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not specified

Genotoxicity:

negative

Remarks:

upto 1 mg/Plate

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Test chemical did not induce reversion in Salmonella typhumurium TA100 and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Ames assay was performed to determine the mutagenic nature of test chemical. The study was perfomed using Salmonella typhimurium strain TA100 by the preincubation assay. The test chemical was dissolved in DMSO and was preincubated for 20 mins with 0.1M sodium phosphate buffer and overnight culture of TA100. Soft molten agar was added and mixed, poured immediately over 1.2% Vogel Bonner minimal medium E agar. The plates were incubated for 2 days at 37˚C and the number of histidine revertant colonies was counted. Test chemicaldid not induce reversion inSalmonella typhumurium TA100and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro: (471)

Data available for the target chemical was reviewed to determine the mutagenic nature of 2 -Furoic acid. The studies are as mentioned below:

1) Ames assay was performed by Ichikawa et al (Carcinogenesis, 1986) to determine the mutagenic nature of 2-Furoic acid. The study was perfomed using Salmonella typhimurium strain TA100 and TA98. The test chemical was dissolved in spectral grade DMSO and used at dose levels of 0, 25, 50, 75 or 100 µg/plate. The plates were incubated for 40 hrs and then observed for number of revertants/plate.2- Furoic acid did not induce reversion inSalmonella typhumurium TA100 and TA98and hence it is not likely to classify as a gene mutant in vitro.

2) Soska et al (Mutation Research, 1981) performed Ames assay to determine the mutagenic nature of 2-Furoic acid. The study was performed using the quantitative method using Salmonella typhimurium strain TA100. Spot test was performed as the preliminary study. The reversions were counted, and the counts were plotted against concentrations. From the initial linear part of the dose-response curve, the regression and correlation coefficients of the compound were calculated, and the mutagenic activity was expressed as the number of reversions per nanomole. 2- Furoic acid did not induce reversion inSalmonella typhumurium TA100and hence it is not likely to classify as a gene mutant in vitro.

3) In the same study performed by Soska et al (Mutation research, 1981), Bacterial repair test was performed to determine the mutagenic nature of 2-furoic acid. The bacteria E. coli B/r strains WP2, WP2 uvrA, WP67, WP100, CM561, CM571 and CM611, at 10⁸per plate, were suspended in M9 medium supplemented with tryptophan. In the middle of the plate, a 5-mm disk of 3 mm Whatman filter paper was placed. 5µl of solution to be tested containing 1000µg was soaked into the paper disk. The concentrations used exhibited a low, or just no, inhibition on the repair-proficient strain WP2. The plates were evaluated after 16 h incubation. The diameters of growth inhibition zones were measured and expressed in mm.2- Furoic acid did not induce mutation inE. coli WP2, WP2 uvrA, WP67, WP100, CM 561, CM 571 and CM 611and hence it is not likely to classify as a gene mutant in vitro.

4) Gene mutation study was performed on  strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 102 and TA 100 in the presence and absence of S9 metabollic activation system. Test chemical was non-mutagenic in the bacterial reverse mutation assay (Ames test) using strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 102 and TA 100.

5)The test chemical is an artificial flavouring substance in food products was studied for mutagenic properties by the use of the Salmonella/mammalian microsome test (Ames test). The test was performed by plate incorporation method at 5 different dosesupto 3600 µg/plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 with and without S9 metabolic activation system and the plates were incubated for 48hrs. Concurrent positive control chemicals were incorporated in the study. A reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency. Agents producing reproducible, dose-related and significant (P≤0.01) but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions.The test chemical did not cause a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency and hence the chemical is not mutagenic in the Salmonella/microsome AMES test performed using Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 in the presence and absence of S9 metabolic activation system.

6)In a study by Kitamura et al (Journal of Pharmacobio-Dyanamics, 1978), Ames assay was performed to determine the mutagenic nature of 2-Furoic acid. The study was perfomed using Salmonella typhimurium strain TA100 by the preincubation assay. The test chemical was dissolved in DMSO and was preincubated for 20 mins with 0.1M sodium phosphate buffer and overnight culture of TA100. Soft molten agar was added and mixed, poured immediately over 1.2% Vogel Bonner minimal medium E agar. The plates were incubated for 2 days at 37˚C and the number of histidine revertant colonies was counted. 2- Furoic acid did not induce reversion inSalmonella typhumurium TA100and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the target chemical, 2 -Furoic acid does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Chromosomal Abberation (473)

Data available for the target chemical was reviewed to determine the mutagenic nature of 2 -Furoic acid. The studies are as mentioned below:

8) CHL cells were treated with 0.125, 0.25 and 0.5 mg/ml test chemical for 24 and 48 hours. The incidences for polyploidy and structural aberrations of treated cells were not different from the negative controls, and hence the test chemical is considered to be non-mutagenic in nature.

9)In a mammalian cell cytogenetics assay, chromosomal aberration tests were carried out on 242 food additives, using Chinese hamster fibroblast cell line (CHL). Chinese hamster lung fibroblasts cell cultures were exposed to lactic acid at concentrations of 10 mg/plate (equivalent to 1.0 mg/mL) with and/or without metabolic activation. There was no evidence of chromosome aberration and cytotoxicity. Therefore, the test item is considered to be non-clastogenic.

Based on the data available for the target chemical, 2 -Furoic acid does not exhibit gene mutation in chromosomal abberation study In-vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Mammalian cell mutation (476)

In different studies, the given test chemical has been investigated for mammalian cell mutation to a greater or lesser extent. The studies are summarized as below :

11)An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different test concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at 2.5 mM in the absence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. No cytotoxic effects were observed when CHO cells were exposed to test chemical for 3 hrs. Based on the results of the study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and does not induce cytotoxic effects at concentrations of ≤ 5.0mM.

12)The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 5 -52 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase .The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro. The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

Therefore based on above summarized studies it can be concluded that the test chemical in not mutagenic to mammalian cell line and thus the test chemical can be classified as 'Not Mutagenic' as per CLP regulation.

13) In vitro unscheduled DNA synthesis was performed by Aaron et al (Mutation Research, 1989) to determine the mutagenic nature of 2-furoic acid. The study was performed using hepatocytes isolated from male Sprague Dawley rats the test chemical was exposed to the rat hepatocytes at dose level of 1, 3, 10, 30, 100, 300 or 1000µg/mL. Rats were housed in polypropylene cages with hardwood-chip bedding and maintained on a 12-h light: 12-h dark cycle. Rats received Purina Rodent Chow No. 5001 (Ralston Purina Co., St. Louis, MO) and water ad libitum. The livers were perfused by inserting a needle in the vena cava and allowing the perfusate to escape from the portal vein. The liver was first perfused with an EGTA wash solution at a flow rate of 40-45 ml/min for about 2-3 min. After perfusion with collagenase the liver was minced, stirred with the collagenase solution and decanted through a sterile gauze pad to remove tissue chunks and debris. The cell suspension was diluted to 50 ml with complete L15 (10% FBS, 50/µg/ml gentamicin, 20 mM Hepes, 10 mM insulin per ml and 0.75 /µg dexamethasone/ml) and allowed to settle on ice 10-12 min. The supernatant was discarded and the cells were resuspended in complete L15. The cell suspension was centrifuged at 50 x g and the cells were resuspended in L15 medium. Viability (trypan blue) and yield (cells/g of liver) were monitored and cell preparation with less than 70% viability was discarded. Typical yield under the conditions described here was 3-5 x 10⁷cells per g liver. Viable cells were plated onto tissue culture coverslips in 6-well plates at 3-5 X 10⁵cells per well and incubated at 73°C for 1-3 h to allow attachment of the cells to the coverslips. Immediately before the assay the test chemical was dissolved in solvent. After cell attachment the hepatocytes were treated with 2-furoic acid. The exposure time was 16-20 h. The final concentration of solvent was maintained at 1% to preclude the possibility of a cytotoxic effect in response to the solvent. Concurrently with the exposure to the test material, the cultures were exposed to 3H-TdR (10µCi/ml). After treatment the cells were washed, swollen, fixed, air-dried and the coverslips were attached to slides. Autoradiography was performed using NTB-2 emulsion. The dipped slides were exposed for 7 days, developed, coded and scored with a semiautomated procedure involving an Artek colony counter connected to an IBM-PC computer. 50 cells were scored per slide. Each nucleus scored was accompanied by scoring of two adjacent cytoplasmic areas. The highest cytoplasmic grain count was subtracted from the nuclear grain count to yield net grains per nucleus (N.G.).2- Furoic acid did not induce unscheduled DNA synthesis (UDS) in the rat hepatocytes and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available for the target chemical, 2 -Furoic acid (CAS no 88 -14 -2) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.