Benzyl butyrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial gene mutation study

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 both in the presence and absence of exogenous metabollic activation system.

In vitro cytogenicity study in mammalian cells

Based on the available results and applying the weight of evidence approach, the test chemical can failed to induce chromosomal aberrations when tested in vitro in the presence and absence of metabolic activation study.

In vitro mammalian cell gene mutation study

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

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:

2. Histidine
3.Histidine locus in the genome of Salmonella typhimurium and tryptophan locus of Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

Study 2

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

Study 3

Details on mammalian cell type (if applicable):

In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of either the uvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium) results in a deficient DNA excision repair system, which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin for growth.

Salmonella typhimurium tester strains also contain the rfa wall mutation, which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall.

Tester strains TA98 and TAlOO also contain the pKMlOl plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism by which this plasmid increases sensitivity to mutagens is by modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TAlOO, TA1535, and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

2. Type and composition of metabolic activation system: Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the preparation of the liver fractions.
- method of preparation of S9 mix : Aroclor 1254 (200 mg/ml in corn oil) was administered ip at 500 mg/kg 5 days prior to decapitation (EGG, SRI) or cervical dislocation (CWR). The animals were deprived of food 12-24 hr immediately preceding death; otherwise food and water were provided ad libitum. The livers were removed aseptically, washed in ice-cold 0.15 M KCl, and minced and homogenized (3 ml of 0.15 M KC1 per gm of wet tissue) in a Potter-Elvehjem apparatus with a
Teflon pestle. CWR initially used a Waring blender, but switched to a Potter-Elvehjem apparatus. The S-9 fraction was obtained by centrifugation of the liver homogenate for 10 min at 9,000 g at 4°C. The S-9 fraction was dispensed into freezing ampules and stored in a -70°C freezer, or in liquid nitrogen.
- source of S9:
- concentration or volume of S9 mix and S9 in the final culture medium :The S-9 mix was prepared immediately prior to each assay and consisted of the following, per milliliter: S-9 fraction, 0.10 ml; 0.04 M MgC12,0.02 ml; 1.65 M KCl,0.02 ml; 0.04 M P-nicotinamide adenine dinucleotide phosphate (NADP), 0.10 ml;0.05 M glucose-6-phosphate, 0.10 ml; 1.0 M NaH2P04, (pH 7.4), 0.10 ml; and distilled water, 0.56 ml. Other levels of S-9 in the S-9 mix were used for some Aliquots.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
3. Type and composition of metabolic activation system: S9 Homogenate (Aroclor) in S9 Mix

Test concentrations with justification for top dose:

2. 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate
3. Salmonella tester strains (with S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Salmonella tester strains (without S9 mix): 3.33, 10.0, 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Escherichia coli tester strain (with and without S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate

Cytotoxicity was observed in the dose range finding study, and the highest dose level of test article used in the subsequent mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn.

Vehicle / solvent:

- 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: The chemical was soluble in DMSO
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)] : DMSO

- Justification for choice of solvent/vehicle: The test article was .observed to form a transparent, colorless solution at a concentration of 100 mg per mL in dimethylsulfoxide (DMSO). DMSO was selected as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution prepared for the mutagenicity assay, the test article was observed to form a transparent, non-viscous, colorless solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.

- 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:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine, 2-aminoanthracene

Remarks:

Study 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene - TA100, TA1535, TA1537, WP2uvrA with S9 Mix; ICR-191 - TA1537 without S9 Mix

Remarks:

Study 3

Details on test system and experimental conditions:

2.NUMBER OF REPLICATIONS:

- Number of cultures per concentration (single, duplicate, triplicate): All assays were repeated in duplicate one week after completion of the initial test. At least five dose levels of the chemicals were tested, with three plates per dose level.
- Number of independent experiments : 3

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:At least one toxic dose was incorporated into the first mutagenicity test, the repeat test(s) occasionally had the doses adjusted so that an apparent toxic dose was not reached.

3. NUMBER OF REPLICATIONS:

- Number of cultures per concentration (single, duplicate, triplicate) : All assays were repeated in triplicates

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: plate incorporation

- OTHER:At least one toxic dose was incorporated into the first mutagenicity test, the repeat test(s) occasionally had the doses adjusted so that an apparent toxic dose was not reached.Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls and positive controls were plated in triplicate.

Rationale for test conditions:

3. Experimental materials, methods and procedures are based on those described by Ames et al. (1975) and Green and Muriel (1976). The assay design is based on the OECD Guideline 471, updated and adopted 21 July 1997.

Evaluation criteria:

2.1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.
3. The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.
Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.

Revertant colonies were counted by automated colony counter or by hand.

Statistics:

2. Mutagenic responses of Salmonella tester strains TA100, TA1535, TA1537,TA97, and TA98 (mean SEM) to test chemicals

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

2. 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: The chemical was tested initially with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mgjplate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive controls used generated a positive response.

Ames test: The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system
- Signs of toxicity
- Individual plate counts
- Mean number of revertant colonies per plate and standard deviation

3. Dose Range Finding Assay

Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 ug per plate were tested.

Cytotoxicity was observed with tester strain TA100 at 333 ug per plate and above in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertants per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA in the presence or absence of S9 mix.

Mutagenicity Assay

In the initial mutagenicity assay, first trial (B1), all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

In the confirmatory mutagenicity assay, second trial (C1), contamination was observed on many of the assay plates and several of the plates were observed to have reduced or absent bacterial background lawns. Due to the multiple technical problems observed the data generated were not used in the evaluation of the test article (the results have not been included).

The confirmatory assay was repeated in third trial (D1). In the repeat confirmatory mutagenicity assay, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In this trial, a 2.7-fold increase was observed with tester strain WP2uvrA in the presence of S9 mix, however, this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix in fourth trial (D2). Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TAI100 at the same doses in the absence of S9 mix. In the fourth trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.

All criteria for a valid study were met.

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 both in the presence and absence of exogenous metabollic activation system.

Executive summary:

Various studies have been reviewed to determine the mutagenic potential of the test chemical. The results are mentioned as follows:

A bacterial cell gene mutation assay was performed using Salmonella strains TA1535, TA1537, TA98, and TA100 with and without Aroclor 1254-induced rat and hamster metabolic activation systems to assess the mutagenic potential of the test chemical. Since the test chemical was insoluble in water, DMSO was used as the solvent.Salmonella strains TA1535, TA1537, TA97, TA98, and TA100 were obtained by the individual laboratories from Dr. Bruce Ames and were stored according to the protocol described in AMES et.al, 1975. The test chemical was assayed for mutagenicity in the preincubation assay [Haworth et al, 1983]. To each of 13 X 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.5 ml or 2.0 ml of molten (45°C) top agar supplementedwith 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubeswere mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel andBonner, 19561 in 15 X 100-mm plastic petri dishes. When the top agar had solidified, theplates were inverted and incubated at 37°C for 48 hr. The doses of the test chemical tested were 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate. The following mutagens were used as concurrent positive controls: sodium

azide for TA1535 and TA 100,4-nitro-o-phenylenediaminef or TA98, and 9-aminoacridine for TA97 and TA1537; 2-aminoanthracene was used with all strains with hamster and rat liver metabolic activation systems. The criteria for a positive response was as described: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

This result is supported by another similar AMES assay performed according to OECD 471 Guidelines(1997) to determine the mutagenic potential of the test chemical.Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were used for the study.Doses tested in the mutagenicity assay were selected based on results of the dose range finding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 ug per plate were tested.Cytotoxicity was observed in the dose range finding study, and the highest dose level of test article used in the subsequent mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn which in this study was 5000 microgram/plate. S9 Homogenate (Aroclor) in S9 Mix was used as the metabolic activation system.Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls and positive controls were plated in triplicate.The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.Revertant colonies were counted by automated colony counter or by hand.The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of Aroclor™ induced rat liver (S9).

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 both in the presence and absence of exogenous metabollic activation system.

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

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

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Study 5

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5a medium with 10% fetal calf serum, L-glutamine, and
antibiotics
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

Study 6

Details on mammalian cell type (if applicable):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture: The cells were cultured with Eagle’s minimum essential medium supplemented with 10% heat inactivated bovine serum.

Cytokinesis block (if used):

No Data Available

Metabolic activation:

with and without

Metabolic activation system:

5. Type and composition of metabolic activation system: The S9 mix consisted of 15 microliter/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/
ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium
6.Type and composition of metabolic activation system: Liver 9,000×g supernatant (S9) fractions were prepared from homogenates of livers obtained from 7-week-old male Sprague–Dawley rats that had been pretreated with phenobarbital and 5,6-benzoflavone (Oriental Yeast Co., Ltd., Tokyo, Japan).
- method of preparation of S9 mix : The S9 mix was then prepared by mixing the S9 fraction with cofactor C (Oriental Yeast Co., Ltd.).

Test concentrations with justification for top dose:

5. without S9 160- 1600 µg/mL
with S9 500-5000 µg/mL
6. The preliminary test indicated that the concentration at which approximately 50% cell growth was inhibited was 92 μg/mL for the 6-h treatment, 178 μg/mL for the metabolic activation method, and 97 μg/mL for the continuous treatment. Consequently, the main test was carried out at doses of 0–170 μg/mL.

Vehicle / solvent:

5. - 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 or Ethanol or acetone solvent was not specified.
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)] : 50 μL of negative control (DMSO) or test chemical solution was used in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Study 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Study 6

Details on test system and experimental conditions:

5. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments
: without S9 - 3 trials and with S9 -2 trials

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:

- Exposure duration/duration of treatment:
C ells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9
- Harvest time after the end of treatment (sampling/recovery times):
For aberrations, standard harvest time was 14 hr

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive controls
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
6.NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, 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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
The direct (6-h treatment with 18-h recovery or 24-h continuous treatment)
- Harvest time after the end of treatment (sampling/recovery times):
The direct (6-h treatment with 18-h recovery

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.: Chromosome specimens were prepared using the conventional method (Ishidate and Odashima, 1977) and coded
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were then stained with Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 300 metaphases (150 metaphases/dish) were examined under a microscope with a magnification of ×600 for structural aberrations (excluding gaps) and polyploidy
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:

Rationale for test conditions:

No data available

Evaluation criteria:

5. Chromosomal aberrations were noted, where cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes)
6. 300 metaphases (150 metaphases/dish) were examined under a microscope with a magnification of ×600 for structural aberrations (excluding gaps) and polyploidy

Statistics:

5. For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et a1 [1983, pp 714-7151. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.
6. Significant differences in the frequencies of structural aberrations and polyploid cells were tested using Fisher’s exact test (Siegel and Castellan, 1988) and the Cochran-Armitage test (Agresti, 2002).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

5. RANGE-FINDING/SCREENING STUDIES: doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment. Doses were based on observations of cell confluence and mitotic cell availability in the SCE test.
STUDY RESULTS: In the aberration test, slight increases were seen, but these were not statistically significant
6.
STUDY RESULTS
- Concurrent vehicle negative and positive control data
: The frequencies of chromosomal aberration in the negative and positive control groups were within the 95% probability distribution of background data, consequently those in the positive control groups were significantly increased.

Chromosome aberration test (CA) in mammalian cells:
However, since no significant increases in chromosomal aberrations were seen at these doses, it was considered that the test chemical does not induce chromosomal aberrations in vitro.
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps
: All the treatment groups had ≤ 1.0% structural aberrations and ≤ 1.3% polyploidy, and exhibited no significant differences in the frequencies of chromosomal aberrations from the negative controls or dose-dependency. The RPDs of the 6-h and 24-h treatments were 21% and 23%, respectively.

Remarks on result:

other: No mutagenic potential of the test chemical was observed.

Conclusions:

Based on the available results and applying the weight of evidence approach, the test chemical can failed to induce chromosomal aberrations when tested in vitro in the presence and absence of metabolic activation study.

Executive summary:

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

 

The test chemical was tested for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells.Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc- Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The S9 mix consisted of 15 microliter/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium. Doses were chosen for the aberration assay based on a preliminary test of cell survival 24 hours after treatment or on observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of aberrations. The chromosome aberration assay was performed using both short and long-term incubation periods and test chemical was tested at doses up to the cytotoxic level. In the presence of S9 mix ,cells were exposed to the test chemical at doses ranging from 500-5000 µg/mlfor 2 hours at the beginning of the test period.In tests without S9 mix,the test chemical was added at concentration range 160-1600 µg/ml and was left in culture until colcemid addition. The cells were harvested 14 hours after the beginning of treatment.Cells were collected by mitotic shake-off and slides were stained with Giemsa. Hundred cells were scored from each of the three highest dose groups and from positive (mitomycin C without S9, or cyclophosphamide with S9) and solvent controls. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaksand terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. Gaps and endore duplications were recorded but were not included in the totals. In the aberration test, slight increases were seen, but these were not statistically significant. Hence, the test chemical can be considered to be non-mutagenic when tested in-vitro in CHO cells in the presence or absence of S9 metabolic activation system.

This result is supported by another similarin vitro chromosome aberration assay conducted according to the OECD test guideline (OECD TG 473) to assess the clastogenic potential of the test chemical either with or without metabolic activation (S9 mix) in Chinese hamster lung cells. The cytotoxic nature of the test chemical was assessed in a preliminary test, and the highest test dose was selected as the one which caused 50% cytotoxicity. A cytotoxic index was produced by calculating the relative population doubling (RPD) at each dose compared with the PDs in negative control groups. The preliminary test indicated that the concentration at which approximately 50% cell growth was inhibited was 92 μg/ml for the 6-hours treatment without metabolic activation, 178 μg/ml for the metabolic activation method, and 97 μg/ml for the continuous treatment. Consequently, the main test was carried out at doses of 0–170 μg/ml. The aberration assay was performed employing either a direct (short-term, 6 hours) or a continuous (24 hours) treatment method. In the direct method, CHL cells were exposed to 40, 60, 80 or 100 µg/ml of test chemical in the absence of S9 mix or to 80, 110, 140 or 170 µg/ml in the presence of S9 mix for 6 hours, then the cells were washed and further cultured for 18 hours. In the 24-hours continuous treatment, the cells were incubated with 40, 60, 80 or 100 µg/ml test chemical for 24 hours in the absence of metabolic activation. Vehicle (DMSO) and positive control (mitomycin C without S9 and cyclophosphamide with S9) substances were also included in the assay. Chromosome specimens were prepared using the conventional method and the cells were stained with Giemsa. Three-hundred metaphases (150 metaphases/dish) were examined for structural aberrations (excluding gaps) and polyploidy. The significant differences in the frequencies of structural aberrations and polyploid cells were tested using Fisher’s exact test and the Cochran-Armitage tests. The frequencies of chromosomal aberration in the negative and positive control groups were within the 95% probability distribution of background data, consequently those in the positive control groups were significantly increased. All the treatment groups had ≤ 1.0% structural aberrations and ≤ 1.3% polyploidy, and exhibited no significant differences in the frequencies of chromosomal aberrations from the negative controls or dose-dependency. The RPDs of the 6-h and 24-h treatments were 21% and 23%, respectively. However, since no significant increases in chromosomal aberrations were seen at these doses, it was considered that the test chemical does not induce chromosomal aberrations in vitro.

Based on the available results and applying the weight of evidence approach, the test chemical can failed to induce chromosomal aberrations when tested in vitro in the presence and absence of metabolic activation study.

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:

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:

8. 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.
9. Thymidine kinase (Tk)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

8

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:

9

Details on mammalian cell type (if applicable):

For cell lines:
- Methods for maintenance in cell culture:
The TK+/- -3 .7 .2C heterozygote of the L5178Y mouse lymphoma cell line was maintained in Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate, and Pluronic F68 .

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

8.Type and composition of metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
9. Type and composition of metabolic activation system:
Aroclor 1254-induced rat liver S9 activation system.

Test concentrations with justification for top dose:

8. 0, 0.5, 1.0, 2.5 or 5.0 mM
9. 1000, 1500 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)]: Ethanol
Justification for choice of solvent/ vehicle: Methyl phenylacetate was easily dissolved in ethanol.
9. No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

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

Remarks:

8

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:

9

Details on test system and experimental conditions:

8. 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.
9. METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3x10^6 cells
- Test substance added in medium - in agar

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:No data
- Exposure duration/duration of treatment: 4 hour exposure period
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells):No data
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used - the cells were washed and incubated at 37° C for 48 hours to allow phenotypic expression before cloning 3x10^6 cells in Noble agar containing the selective agent trifluorothymidine or bromodeoxyuridine.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Colonies were counted after 10-14 days' growth using an automatic colony counter
- Criteria for small (slow growing) and large (fast growing) colonies: No data

Rationale for test conditions:

No data

Evaluation criteria:

8. The plates were scored for total number of colonies
9. Mutant frequency was determined by calculating the ratio of mutant to viable colonies cloned without selective medium .

Statistics:

No data available

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

8

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

9

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

8. 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
9. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned 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.

 

In another study, the Mouse Lymphoma cell mutagenesis (MLY) assay was performed by using the given test chemical on TK+/- -3.7.2C heterozygote of the L5178Y mouse lymphoma cell line maintained in Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate, and Pluronic F68 with and without Aroclor 1254-induced rat liver S9 activation system at 1000, 1500 μg/ml. In a typical assay procedure, the thymidine kinase competent heterozygote was exposed to the test chemical in both the presence and absence of an induced rat liver S9 and cofactors (CORE). After a 4 hour exposure period, the cells were washed and incubated at 37° C for 48 hours to allow phenotypic expression before cloning 3x10^6 cells in Noble agar containing the selective agent trifluorothymidine or bromodeoxyuridine. Colonies were counted after 10-14 days growth using an automatic colony counter. Mutant frequency was determined by calculating the ratio of mutant to viable colonies cloned without selective medium. The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic in nature.

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro bacterial gene mutation study

Various studies have been reviewed to determine the mutagenic potential of the test chemical. The results are mentioned as follows:

A bacterial cell gene mutation assay was performed using Salmonella strains TA1535, TA1537, TA98, and TA100 with and without Aroclor 1254-induced rat and hamster metabolic activation systems to assess the mutagenic potential of the test chemical. Since the test chemical was insoluble in water, DMSO was used as the solvent.Salmonella strains TA1535, TA1537, TA97, TA98, and TA100 were obtained by the individual laboratories from Dr. Bruce Ames and were stored according to the protocol described in AMES et.al, 1975. The test chemical was assayed for mutagenicity in the preincubation assay [Haworth et al, 1983]. To each of 13 X 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.5 ml or 2.0 ml of molten (45°C) top agar supplementedwith 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubeswere mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel andBonner, 19561 in 15 X 100-mm plastic petri dishes. When the top agar had solidified, theplates were inverted and incubated at 37°C for 48 hr. The doses of the test chemical tested were 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate. The following mutagens were used as concurrent positive controls: sodium

azide for TA1535 and TA 100,4-nitro-o-phenylenediaminef or TA98, and 9-aminoacridine for TA97 and TA1537; 2-aminoanthracene was used with all strains with hamster and rat liver metabolic activation systems. The criteria for a positive response was as described: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

This result is supported by another similar AMES assay performed according to OECD 471 Guidelines(1997) to determine the mutagenic potential of the test chemical.Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were used for the study.Doses tested in the mutagenicity assay were selected based on results of the dose range finding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 ug per plate were tested.Cytotoxicity was observed in the dose range finding study, and the highest dose level of test article used in the subsequent mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn which in this study was 5000 microgram/plate. S9 Homogenate (Aroclor) in S9 Mix was used as the metabolic activation system.Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls and positive controls were plated in triplicate.The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.Revertant colonies were counted by automated colony counter or by hand.The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of Aroclor™ induced rat liver (S9).

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 both in the presence and absence of exogenous metabollic activation system.

In vitro cytogenicity study in mammalian cells

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

 

The test chemical was tested for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells.Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc- Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The S9 mix consisted of 15 microliter/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium. Doses were chosen for the aberration assay based on a preliminary test of cell survival 24 hours after treatment or on observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of aberrations. The chromosome aberration assay was performed using both short and long-term incubation periods and test chemical was tested at doses up to the cytotoxic level. In the presence of S9 mix ,cells were exposed to the test chemical at doses ranging from 500-5000 µg/mlfor 2 hours at the beginning of the test period.In tests without S9 mix,the test chemical was added at concentration range 160-1600 µg/ml and was left in culture until colcemid addition. The cells were harvested 14 hours after the beginning of treatment.Cells were collected by mitotic shake-off and slides were stained with Giemsa. Hundred cells were scored from each of the three highest dose groups and from positive (mitomycin C without S9, or cyclophosphamide with S9) and solvent controls. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaksand terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. Gaps and endore duplications were recorded but were not included in the totals. In the aberration test, slight increases were seen, but these were not statistically significant. Hence, the test chemical can be considered to be non-mutagenic when tested in-vitro in CHO cells in the presence or absence of S9 metabolic activation system.

This result is supported by another similarin vitro chromosome aberration assay conducted according to the OECD test guideline (OECD TG 473) to assess the clastogenic potential of the test chemical either with or without metabolic activation (S9 mix) in Chinese hamster lung cells. The cytotoxic nature of the test chemical was assessed in a preliminary test, and the highest test dose was selected as the one which caused 50% cytotoxicity. A cytotoxic index was produced by calculating the relative population doubling (RPD) at each dose compared with the PDs in negative control groups. The preliminary test indicated that the concentration at which approximately 50% cell growth was inhibited was 92 μg/ml for the 6-hours treatment without metabolic activation, 178 μg/ml for the metabolic activation method, and 97 μg/ml for the continuous treatment. Consequently, the main test was carried out at doses of 0–170 μg/ml. The aberration assay was performed employing either a direct (short-term, 6 hours) or a continuous (24 hours) treatment method. In the direct method, CHL cells were exposed to 40, 60, 80 or 100 µg/ml of test chemical in the absence of S9 mix or to 80, 110, 140 or 170 µg/ml in the presence of S9 mix for 6 hours, then the cells were washed and further cultured for 18 hours. In the 24-hours continuous treatment, the cells were incubated with 40, 60, 80 or 100 µg/ml test chemical for 24 hours in the absence of metabolic activation. Vehicle (DMSO) and positive control (mitomycin C without S9 and cyclophosphamide with S9) substances were also included in the assay. Chromosome specimens were prepared using the conventional method and the cells were stained with Giemsa. Three-hundred metaphases (150 metaphases/dish) were examined for structural aberrations (excluding gaps) and polyploidy. The significant differences in the frequencies of structural aberrations and polyploid cells were tested using Fisher’s exact test and the Cochran-Armitage tests. The frequencies of chromosomal aberration in the negative and positive control groups were within the 95% probability distribution of background data, consequently those in the positive control groups were significantly increased. All the treatment groups had ≤ 1.0% structural aberrations and ≤ 1.3% polyploidy, and exhibited no significant differences in the frequencies of chromosomal aberrations from the negative controls or dose-dependency. The RPDs of the 6-h and 24-h treatments were 21% and 23%, respectively. However, since no significant increases in chromosomal aberrations were seen at these doses, it was considered that the test chemical does not induce chromosomal aberrations in vitro.

Based on the available results and applying the weight of evidence approach, the test chemical can failed to induce chromosomal aberrations when tested in vitro in the presence and absence of metabolic activation study.

In vitro mammalian cell gene mutation study

 

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned 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.

 

In another study, the Mouse Lymphoma cell mutagenesis (MLY) assay was performed by using the given test chemical on TK+/- -3.7.2C heterozygote of the L5178Y mouse lymphoma cell line maintained in Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate, and Pluronic F68 with and without Aroclor 1254-induced rat liver S9 activation system at 1000, 1500 μg/ml. In a typical assay procedure, the thymidine kinase competent heterozygote was exposed to the test chemical in both the presence and absence of an induced rat liver S9 and cofactors (CORE). After a 4 hour exposure period, the cells were washed and incubated at 37° C for 48 hours to allow phenotypic expression before cloning 3x10^6 cells in Noble agar containing the selective agent trifluorothymidine or bromodeoxyuridine. Colonies were counted after 10-14 days growth using an automatic colony counter. Mutant frequency was determined by calculating the ratio of mutant to viable colonies cloned without selective medium. The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic in nature.

 

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the available results, the test chemical can be considered to be non-genotoxic when tested in vitro. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.