Iron(II) fumarate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial cell gene mutation assay

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to bacterial tester strains under both conditions of metabolic activation.

In vitro chromosomal aberration study

Based on the above studies and applying the weight of evidence approach, the test chemical can be considered to be negative for mutachromosomal effects in mammalian cells. Hence, it can be considered to be non-mutagenic in nature.

In vitro mammalian cell gene mutation assay

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to mouse lymphoma cell line when tested in vitro under both conditions of metabolic activation.

Link to relevant study records

Reference

Reference 1

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

Remarks:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

other: Chinese hamster lung-derived fibroblasts (CHL)

Remarks:

Study 5,6

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970),

For cell lines:
- Absence of Mycoplasma contamination: No data
- Number of passages if applicable: 4-day passages
- Methods for maintenance in cell culture: The cell line was maintained in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Cell cycle length, doubling time or proliferation index : The doubling time was approximately 15 hr.
- Modal number of chromosomes: The modal chromosome number is 25

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

5. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting.

Metabolic activation:

without

Metabolic activation system:

5, 6. no metabolic activation systems were applied

Test concentrations with justification for top dose:

5. 0, 0.5 µg/ml. Top dose was expected to produce a 50% inhibition of cell growth based on data from a pre-experiment.
6. 0.001 mg/ml, Top dose was expected to produce a 50% inhibition of cell growth based on data from a pre-experiment.

Vehicle / solvent:

5,6. - Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Physiological saline

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

Study 5,6

Details on test system and experimental conditions:

5,6. DURATION
- Exposure duration: 24 & 48 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: 50% cell growth inhibition
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

5,6. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%

Statistics:

No data

Species / strain:

other: Chinese hamster lung-derived fibroblasts (CHL)

Remarks:

Study 4

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

5,6 . Chromosome aberration test (CA) in mammalian cells:

o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : 0% polypliody was observed.

Remarks on result:

other: No mutagenic potential

Conclusions:

Based on the above studies and applying the weight of evidence approach, the test chemical can be considered to be negative for mutachromosomal effects in mammalian cells. Hence, it can be considered to be non-mutagenic in nature.

Executive summary:

Various studies have been reviewed to evaluate the mutagenic potential of the test chemical. The results are mentioned below:

In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.5 µg/ml for 24 and 48 hr. Physiological saline was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivocal if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

This result is further strengthened by a similar in vitro mammalian chromosome aberration study conducted for the test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.001 mg/ml for 24 and 48 hr. Physiological saline was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivocal if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

Based on the above studies and applying the weight of evidence approach, the test chemical can be considered to be negative for mutachromosomal effects in mammalian cells. Hence, it can be considered to be non-mutagenic in nature.

Reference 2

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:

data from handbook or collection of data

Remarks:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on various test chemicals

GLP compliance:

not specified

Type of assay:

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

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

L5178Y TK± 3.7.C

Details on mammalian cell type (if applicable):

Type and identity of media: Fischer's medium, supplemented with 2mM L-glutamaine, sodium pyruvate, 110 µg/ml, 0.05% pluronic F68, antibiotics and 10% heat-inactivated donor horse-serum (v/v)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Remarks:

Study 8

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

Study 9

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

8, 9. Aroclor induced rat liver S9

Test concentrations with justification for top dose:

8. At least five concentrations up to 6000 µg/mL; 2 cultures per concentration
9. Test 1: 0, 156, 624, 1249, 2498, 3997, 4996 µg/mL
Test 2: 0, 2573, 3431, 4288, 5146, 6861 µg/mL
Test 3: 0, 4977, 5806, 6636, 7465, 8295 µg/mL

Vehicle / solvent:

8. - Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : DMSO

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:
9. - Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : water

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Remarks:

Study 8

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

Study 9

Details on test system and experimental conditions:

8. METHOD OF APPLICATION: in medium;
Exposed culture consist of 6x10E5 cells

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 11-14d
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16d

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: two cultures per test compound concentration, experiment repeated.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in cloning efficiency ; other: reduction in cell population growth in expression period

9. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) ; One or 2 replicates were performed for tests 1 and 2, and 3 replicates were performed for test 3.
- Number of independent experiments : 3

Rationale for test conditions:

No data

Evaluation criteria:

8. Compliance with pre-determined quality control criteria was required. The response categories used were:
Positive response: dose related trend and response at one of the three highest acceptable doses;
Negative response: no dose related trend and no statistically significant response at any dose, and acceptable positive control. Negative responses were classified as toxic or non-toxic.
9. Mutant frequencies were measured and compared with control

Statistics:

8. colonies were counted with an Artek 880 Automated colony counter. Colony size distribution was not assessed. Statistical analyses were based on the Lee & Caspary method, consisting of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

L5178Y TK1/- 3.7.C

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

8. TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: A significant response was observed in one experiment without S9 at a concentration of 6000 µg/ml, well above the saturation concentration.
- Precipitation:Precipitation was observed at 150 µg/ml.
9. Gene mutation study in mammalian cells: The test chemical did not produce repeatable increases in mutant frequency at the TK locus in L5178Y mouse lymphoma cells under the conditions of S-9 microsomal activation and adjustment of the assay mixture to a neutral pH range (pH 7.0 to pH 7.4). Concentrations from 156-8295 µg/mL (with pH adjustment) induced, at best, moderate toxicity. Sporadic increases in mutant frequency were observed, but could not be confirmed in replicate treatments and/or at higher concentrations of the test substance. Hence, the test chemical was considered to be negative with neutral pH to mouse lymphoma cell line.

Remarks on result:

other: negative

Conclusions:

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to mouse lymphoma cell line when tested in vitro under both conditions of metabolic activation.

Executive summary:

Various studies have been summarized to assess the mutagenic potential of the test chemical. The results are mentioned below:

Mouse lymphoma assay was performed to evaluate the mutagenic potential of the test chemical. Mouse lymphoma cells L5178Y TK± were used for the study. Prior to the mutagenicity test a toxicity test was conducted with the highest concentration of 5000µg/ml. The mutagenicity assay was performed in duplicates. 3 -methylcholanthrene and ethylmethanesulfonate were used as positive controls. Compliance with pre-determined quality control criteria was required. The response categories used were: Positive response: dose related trend and response at one of the three highest acceptable doses; Negative response: no dose related trend and no statistically significant response at any dose, and acceptable positive control. Negative responses were classified as toxic or non-toxic. Colonies were counted with an Artek 880 Automated colony counter. Colony size distribution was not assessed. Statistical analyses were based on the Lee & Caspary method, consisting of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. A significant response was observed in one experiment without S9 at a concentration of 6000 µg/ml, well above the saturation concentration. Hence, the test chemical was considered to be not mutagenic to mouse lymphoma cells under both conditions of metabolic activation.

This is supported by another mouse lymphoma assay was performed according to the method of Clive and Spector, 1975, to evaluate the mutagenic potential of the test chemical. Mouse lymphoma L5178Y/ TK cells were used for the study. Aroclor-induced rat liver S-9 was used as exogenous metabolic activation system. The chemical was dissolved in water. Three experiments were performed and concentrations used for the 3 experiments were experiment 1- 0, 156, 624, 1249, 2498, 3997, 4996 µg/mL; Experiment 2: 0, 2573, 3431, 4288, 5146, 6861 µg/mL and experiment 3: 0, 4977, 5806, 6636, 7465, 8295 µg/mL . One or 2 replicates were performed for tests 1 and 2, and 3 replicates were performed for test 3.  Mutant frequencies were measured and compared with control. Concurrent negative and positive controls were tested. The negative control was water and the positive control was dimethylnitrosamine . The test chemical did not produce repeatable increases in mutant frequency at the TK locus in L5178Y mouse lymphoma cells under the conditions of S-9 microsomal activation and adjustment of the assay mixture to a neutral pH range (pH 7.0 to pH 7.4). Concentrations from 156-8295 µg/mL (with pH adjustment) induced, at best, moderate toxicity. Sporadic increases in mutant frequency were observed, but could not be confirmed in replicate treatments and/or at higher concentrations of the test substance. The above mentioned sporadic increases in the mutant frequency in cells (positive effects) was due to the other factors like change in pH and osmolality of the test medium and not due to the test chemical exposure.Hence, the test chemical was considered to be negative with neutral pH to mouse lymphoma cell line under both conditions of metabolic activation.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to mouse lymphoma cell line when tested in vitro under both conditions of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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

Remarks:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA 98, TA100,TA 102, TA1535, TA1537, and TA1538

Metabolic activation:

with and without

Metabolic activation system:

2. Type and composition of metabolic activation system:
Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight.
- source of S9
- method of preparation of S9 mix
:The post-mitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. (26). The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix.
- concentration or volume of S9 mix and S9 in the final culture medium
: For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
3. Type and composition of metabolic activation system:
The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co .) pretreated 5 days before with
polychlorinated biphenyls (500mg/kg body weight of Kanechlor KC-400 in olive oil, ip) .
- source of S9
- method of preparation of S9 mix
:The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4 mM-NADPH, 4 mM-NADH, 33 mNt-KCl, 8 mM-MgCl2, 100 mM-phosphate buffer (pH 7 .4) and 3 .75 ml S-9 (129 mg protein) in a total volume of 12 .5 ml.

Test concentrations with justification for top dose:

2.up to and including 10,000 µg Fe/plate
3. Maximum dose tested 10 mg/plate

Vehicle / solvent:

2.- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Dimethyl Sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

- Justification for choice of solvent/vehicle: No data available

3. - Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Dimethyl Sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

- Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Study 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

Study 3

Details on test system and experimental conditions:

2. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
: triplicate
- Number of independent experiments
: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
20 minutes
- Exposure duration/duration of treatment:
48 hours
- Harvest time after the end of treatment (sampling/recovery times):



METHODS FOR MEASUREMENTS OF GENOTOXICITY

: All plates were counted with a MiniCount automated colony counter (Imaging Products, International, Inc., Chantilly, VA), which was calibrated
prior to use
- OTHER: Initially the test chemical was tested in the Salmonella typhimurium plate incorporation assay with tester strains TA100 and TA98 at dose levels up to and including 10,000 mg Fe/plate without metabolic activation and with liver S9 preparations from Sprague- Dawley rats and Syrian golden hamsters.In the absence of a mutagenic response in either strain, the test chemical was further tested with strains TA1535, TA1537, and TA1538 using the same dose levels and activation systems. The test chemical was also tested using strain TA102 using both the plate incorporation and preincubation
procedures.

3. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
: duplicate
- Number of independent experiments
: single

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
20 minutes
- Exposure duration/duration of treatment:
48 hours
- Harvest time after the end of treatment (sampling/recovery times):



- OTHER: Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating . Duplicate plates were used for each of six different concentrations of the sample

Evaluation criteria:

2.The criteria used to evaluate a test stipulated that a test article must induce at least a doubling (TA97a, TA98, TA100, TA102, and TA1535) in the mean number of revertants per plate of at least
one tester strain for it to be considered positive. This increase in the mean revertants per plate must be accompanied by a dose response to increasing concentrations of the test chemical. If the study shows a dose–response, but with a less than 3-fold increase on TA1537 or TA1538, the response must be confirmed in a repeat experiment
3. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated) . If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays (Yoshikawa, Nakadate, Watabe er al. 1980) were performed .

Statistics:

2. Mean ± Standard Deviation

Species / strain:

S. typhimurium, other: TA 98, TA100,TA 102, TA1535, TA1537, and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

2. RANGE-FINDING/SCREENING STUDIES (if applicable):
The test chemical was initially tested in the Salmonella typhimurium plate incorporation assay with tester strains TA100 and TA98 at dose levels up to and including 10,000 microgram/plate without metabolic activation and with liver S9 preparations from Sprague- Dawley rats and Syrian golden hamsters

Ames test: The test chemical failed to elicit a positive response with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, TA1537, TA 1538 strains in the presence and absence of rat and hamster liver S9 mix.

3.
Ames test:
The test chemical failed to induce mutagenic activity in Salmonella Typhimurium tester strains TA92, TA1535, TA100, TA1537, TA94 and TA98 under both conditions of metabolic activation system.

Remarks on result:

other: Not mutagenic

Conclusions:

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to bacterial tester strains under both conditions of metabolic activation.

Executive summary:

Various studies have been reviewed to evaluate the mutagenic potential of the test chemical. These inculude in vitro studies performed on various bacterial tester strains for the test chemical. The results are mentioned below:

A Gene mutation toxicity study was performed by the modified pre-incubation method to evaluate the mutagenic potency of the test chemical.Salmonella typhimurium histidine auxotrophs TA98, TA100, TA102, TA 1535, TA 1537, TA1538 were obtained from Dr. Bruce Ames, University of California, Berkeley, CA. Strain TA102 was included because of its ability to detect oxidative mutagens [Levin et al., 1982]. Cultures were grown overnight in Oxoid nutrient broth No. 2 and were removed from incubation when they reached a density of 1–2 x10⁹cells/ml. On the day of use, all tester strain cultures were checked for genetic integrity as recommended [Ames et al., 1975]. Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The post-mitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. The components of the S9 mix were 8 mM MgCl₂, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix. For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added. The test chemical was initially tested in the Salmonella typhimurium plate incorporation assay with tester strains TA100 and TA98 at dose levels up to and including 10,000 microgram/plate without metabolic activation and with liver S9 preparations from Sprague- Dawley rats and Syrian golden hamsters. In the absence of a mutagenic response in either strain, the test chemical was further tested with strains TA1535, TA1537, and TA1538 using the same dose levels and activation systems. The test chemical was also tested using  strain TA102 using both the plate incorporation and pre-incubation procedures.The initial preincubation assay was performed according to the procedures described earlier. In the test with metabolic activation, 500 ml of S9 mix was added to 13 * 100 mm glass culture tubes preheated to 37 6 2ºC. To these tubes were added 100 ml of the appropriate tester strain and 50 ml of vehicle or the appropriate concentration of test chemical. When the chemical was tested without metabolic activation, 500 ml of the cofactor mix without S9 was substituted for the complete S9 mix. After vortexing, the mixture was incubated for 20 min at 37± 2ºC. Top agar (2 ml) was then added to each tube and the mixture was overlaid onto 25 ml of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for approximately 48 hr at 37± 2ºC. All plates were counted with a MiniCount automated colony counter (Imaging Products, International, Inc., Chantilly, VA), which was calibrated prior to use.The test chemical failed to elicit a positive response with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, TA1537, TA 1538 strains in the presence and absence of rat and hamster liver S9 mix.

This result is supported by another Gene mutation toxicity study was performed by the pre-incubation method to evaluate the mutagenic potency of the test chemical. Reverse mutation assays using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 were carried out according to the method of Ames, McCann & Yamasaki (1975). The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co .) pretreated 5 days before with polychlorinated biphenyls (500mg/kg body weight of Kanechlor KC-400 in olive oil, ip) . The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4 mM-NADPH, 4 mM-NADH, 33 mNt-KCl, 8mM-MgCl2, 100 mM-phosphate buffer (pH 7 .4) and 3 .75 ml S-9 (129 mg protein) in a total volume of 12 .5 ml. The test chemical was dissolved in DMSO and tested upto the maximum dose of 10 mg/plate. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating . Duplicate plates were used for each of six different concentrations of the sample. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated) . If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays (Yoshikawa, Nakadate, Watabe er al. 1980) were performed . The test chemical failed to induce mutagenic activity in Salmonella Typhimurium tester strains TA92, TA1535, TA100, TA1537, TA94 and TA98 under both conditions of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to bacterial tester strains under both conditions of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro bacterial cell gene mutation assay

Various studies have been reviewed to evaluate the mutagenic potential of the test chemical. These inculude in vitro studies performed on various bacterial tester strains for the test chemical. The results are mentioned below:

A Gene mutation toxicity study was performed by the modified pre-incubation method to evaluate the mutagenic potency of the test chemical.Salmonella typhimurium histidine auxotrophs TA98, TA100, TA102, TA 1535, TA 1537, TA1538 were obtained from Dr. Bruce Ames, University of California, Berkeley, CA. Strain TA102 was included because of its ability to detect oxidative mutagens [Levin et al., 1982]. Cultures were grown overnight in Oxoid nutrient broth No. 2 and were removed from incubation when they reached a density of 1–2 x10⁹cells/ml. On the day of use, all tester strain cultures were checked for genetic integrity as recommended [Ames et al., 1975]. Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The post-mitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. The components of the S9 mix were 8 mM MgCl₂, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix. For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added. The test chemical was initially tested in the Salmonella typhimurium plate incorporation assay with tester strains TA100 and TA98 at dose levels up to and including 10,000 microgram/plate without metabolic activation and with liver S9 preparations from Sprague- Dawley rats and Syrian golden hamsters. In the absence of a mutagenic response in either strain, the test chemical was further tested with strains TA1535, TA1537, and TA1538 using the same dose levels and activation systems. The test chemical was also tested using  strain TA102 using both the plate incorporation and pre-incubation procedures.The initial preincubation assay was performed according to the procedures described earlier. In the test with metabolic activation, 500 ml of S9 mix was added to 13 * 100 mm glass culture tubes preheated to 37 6 2ºC. To these tubes were added 100 ml of the appropriate tester strain and 50 ml of vehicle or the appropriate concentration of test chemical. When the chemical was tested without metabolic activation, 500 ml of the cofactor mix without S9 was substituted for the complete S9 mix. After vortexing, the mixture was incubated for 20 min at 37± 2ºC. Top agar (2 ml) was then added to each tube and the mixture was overlaid onto 25 ml of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for approximately 48 hr at 37± 2ºC. All plates were counted with a MiniCount automated colony counter (Imaging Products, International, Inc., Chantilly, VA), which was calibrated prior to use.The test chemical failed to elicit a positive response with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, TA1537, TA 1538 strains in the presence and absence of rat and hamster liver S9 mix.

This result is supported by another Gene mutation toxicity study was performed by the pre-incubation method to evaluate the mutagenic potency of the test chemical. Reverse mutation assays using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 were carried out according to the method of Ames, McCann & Yamasaki (1975). The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co .) pretreated 5 days before with polychlorinated biphenyls (500mg/kg body weight of Kanechlor KC-400 in olive oil, ip) . The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4 mM-NADPH, 4 mM-NADH, 33 mNt-KCl, 8mM-MgCl2, 100 mM-phosphate buffer (pH 7 .4) and 3 .75 ml S-9 (129 mg protein) in a total volume of 12 .5 ml. The test chemical was dissolved in DMSO and tested upto the maximum dose of 10 mg/plate. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating . Duplicate plates were used for each of six different concentrations of the sample. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated) . If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays (Yoshikawa, Nakadate, Watabe er al. 1980) were performed . The test chemical failed to induce mutagenic activity in Salmonella Typhimurium tester strains TA92, TA1535, TA100, TA1537, TA94 and TA98 under both conditions of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to bacterial tester strains under both conditions of metabolic activation.

In vitro chromosomal aberration study

Various studies have been reviewed to evaluate the mutagenic potential of the test chemical. The results are mentioned below:

In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.5 µg/ml for 24 and 48 hr. Physiological saline was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivocal if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

This result is further strengthened by a similar in vitro mammalian chromosome aberration study conducted for the test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.001 mg/ml for 24 and 48 hr. Physiological saline was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivocal if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

Based on the above studies and applying the weight of evidence approach, the test chemical can be considered to be negative for mutachromosomal effects in mammalian cells. Hence, it can be considered to be non-mutagenic in nature.

In vitro mammalian cell gene mutation study

Various studies have been summarized to assess the mutagenic potential of the test chemical. The results are mentioned below:

Mouse lymphoma assay was performed to evaluate the mutagenic potential of the test chemical. Mouse lymphoma cells L5178Y TK± were used for the study. Prior to the mutagenicity test a toxicity test was conducted with the highest concentration of 5000µg/ml. The mutagenicity assay was performed in duplicates. 3 -methylcholanthrene and ethylmethanesulfonate were used as positive controls. Compliance with pre-determined quality control criteria was required. The response categories used were: Positive response: dose related trend and response at one of the three highest acceptable doses; Negative response: no dose related trend and no statistically significant response at any dose, and acceptable positive control. Negative responses were classified as toxic or non-toxic. Colonies were counted with an Artek 880 Automated colony counter. Colony size distribution was not assessed. Statistical analyses were based on the Lee & Caspary method, consisting of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. A significant response was observed in one experiment without S9 at a concentration of 6000 µg/ml, well above the saturation concentration. Hence, the test chemical was considered to be not mutagenic to mouse lymphoma cells under both conditions of metabolic activation.

This is supported by another mouse lymphoma assay was performed according to the method of Clive and Spector, 1975, to evaluate the mutagenic potential of the test chemical. Mouse lymphoma L5178Y/ TK cells were used for the study. Aroclor-induced rat liver S-9 was used as exogenous metabolic activation system. The chemical was dissolved in water. Three experiments were performed and concentrations used for the 3 experiments were experiment 1- 0, 156, 624, 1249, 2498, 3997, 4996 µg/mL; Experiment 2: 0, 2573, 3431, 4288, 5146, 6861 µg/mL and experiment 3: 0, 4977, 5806, 6636, 7465, 8295 µg/mL . One or 2 replicates were performed for tests 1 and 2, and 3 replicates were performed for test 3.  Mutant frequencies were measured and compared with control. Concurrent negative and positive controls were tested. The negative control was water and the positive control was dimethylnitrosamine . The test chemical did not produce repeatable increases in mutant frequency at the TK locus in L5178Y mouse lymphoma cells under the conditions of S-9 microsomal activation and adjustment of the assay mixture to a neutral pH range (pH 7.0 to pH 7.4). Concentrations from 156-8295 µg/mL (with pH adjustment) induced, at best, moderate toxicity. Sporadic increases in mutant frequency were observed, but could not be confirmed in replicate treatments and/or at higher concentrations of the test substance. The above mentioned sporadic increases in the mutant frequency in cells (positive effects) was due to the other factors like change in pH and osmolality of the test medium and not due to the test chemical exposure.Hence, the test chemical was considered to be negative with neutral pH to mouse lymphoma cell line under both conditions of metabolic activation.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic to mouse lymphoma cell line when tested in vitro under both conditions of metabolic activation.

Justification for classification or non-classification

Based on the above studies the test chemical can be regarded to be non-genotoxic and classified under the category "Not Classified" as per CLP Regulation.