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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC-20/2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

Method

Target gene:
The test substance, FAT 40061/F, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80% (Li et al., 1987). The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN).
Frozen stock cultures were tested to confirm the absence of mycoplasma contamination and for karyotpye stability.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
- Preliminary toxicity assay: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9.
- Definitive mutagenicity: 17.8, 35.6, 71.2, 142, 190, 253, 338 and 450 μg/mL with S9 and 235.6, 71.2, 142, 190, 253, 338, 450 and 600 μg/mL without S9.
Test substance dilutions were prepared immediately before use and delivered to the test system.
Vehicle / solvent:
SOLUBILITY TEST:
Water was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water at a concentration of ~50 mg/mL, the maximum tested.
The vehicle used to prepare the test substance dose formulations, and also used as the vehicle control was distilled water, as indicated below:
- Vehicle: distilled water
- CAS number: 7732-18-5
- Supplier: Gibco
- Lot number: 1662088
- Purity/Grade: Sterile, distilled
- Expiration date: 30 December 2016
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Remarks:
EMS is the positive control for the mutagenicity assay without S9. Benzo(a)pyrene is the positive control for themutagenicity assay with S9.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cells were treated for 5 ± 0.5 hours in the presence and absence of S9, by addition of the test and control substance formulations to the treatment medium (with or without S9, as appropriate). This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981; Li et al., 1987).

Preliminary Toxicity Test for Selection of Dose Levels
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated (5000 μg/mL). Lower concentrations were prepared by 2-fold dilutions. The pH of the cultures was within pH 7 ± 0.5. Therefore, no pH adjustment was necessary to maintain neutral pH in the treatment medium. Osmolality of the vehicle control and the highest concentration also was measured at the beginning of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival.

NUMBER OF REPLICATIONS: Duplicate
Evaluation criteria:
See any other information on materials and methods section
Statistics:
See any other information on materials and methods section

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Results:

Definitive mutagenicity assay:

Based on the results of the preliminary toxicity assay, FAT 40061/F was evaluated in the definitive mutagenicity assay at concentrations of 17.8, 35.6, 71.2, 142, 190, 253, 338 and 450 μg/mL with S9, and 35.6, 71.2, 142, 190, 253, 338, 450 and 600 μg/mL without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. However, due to unacceptable vehicle cloning efficiency values at the time of selection this trial was not scored for mutagenicity (not shown), and the assay was repeated.

A re-test was conducted under identical conditions. No visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. The average adjusted relative survival was 18.11 and 38.84 % at concentrations of 338 μg/mL with S9 and 600 μg/mL without S9, respectively. Cultures treated at concentrations of 71.2, 142, 190, 253 and 338 μg/mL with S9, and 142, 253, 338, 450 and 600 μg/mL without S9, were chosen for mutant selection (cultures treated at concentrations of 17.8 and 35.6 μg/mL with S9 and 35.6, 71.2 and 190 μg/mL without S9 were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 450 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). Statistically significant* increases in mutant frequency were observed with and without S9 (*: p <0.05 and **: p <0.01, respectively). With S9, the increases in mutant frequency observed were 5.30, 7.82*, 5.90, 7.36* and 7.35* at 71.2, 142, 190, 253 and 338 μg/mL concentrations, respectively. Thus, it was evident that the increases observed with S9 were not dose dependent (p >0.05) and were within the historical negative control 95 % confidence limit. In contrast, the increases observed (i.e. 8.96*, 10.33*, 14.89**, 15.44** and 19.80** at the test concentrations of 142, 253, 338, 450 and 600 μg/mL, respectively) without S9 were dose dependent (p <0.01) and exceeded this limit. The positive controls also induced significant increases in mutant frequency (p< 0.01).

All criteria for a valid assay ultimately were met.

CHO/HPRT Assay Historical Control Data (2011 -2013)

      Non-activated     S9 -activated
   Solvent control  0.2 microL/mL EMS  Solvent control  4.0 microL/mL B(a)P
 Mean MF  4.2 239.2  4.4  143.1 
 SD  3.6 144  3.9  76.2 
 Maximum  15.7 764.2  16.9  314.5 
 Minimum  0.0 12  0.0  5.8 

Solvent control (culture medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor). It has been demonstrated that all of the above solvents exhibit the same mutant frequency range.

EMS Ethyl methanesulfonate

B(a)P Benzo(a)pyrene MF Mutant frequency per 106 clonable cells

SD Standard deviation

Applicant's summary and conclusion

Conclusions:
FAT 40061/F TE was positive in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures in the absence of S9. The results observed in the presence of S9 were considered to be equivocal.
Executive summary:

The test substance, FAT 40061/F, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). FAT 40061/F was prepared in distilled water and evaluated in a preliminary toxicity assay at concentrations of 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures. Adjusted relative survival was 8.49 and 25.47 % at a concentration of 313 μg/mL with and without S9, respectively. Adjusted relative survival approximated 0 % at all higher concentrations. Based on these results, FAT 40061/F was evaluated in the definitive mutagenicity assay at concentrations of 17.8, 35.6, 71.2, 142, 190, 253, 338 and 450 μg/mL with S9, and 35.6, 71.2, 142, 190, 253, 338, 450 and 600 μg/mL without S9 (an earlier trial, performed under identical conditions, was not scored for mutagenicity due to unacceptable vehicle cloning efficiency values at the time of selection; not shown). No visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. The average adjusted relative survival was 18.11 and 38.84 % at concentrations of 338 μg/mL with S9 and 600 μg/mL without S9, respectively. Cultures treated at concentrations of 71.2, 142, 190, 253 and 338 μg/mL with S9, and 142, 253, 338, 450 and 600 μg/mL without S9, were chosen for mutant selection (cultures treated at concentrations of 17.8 and 35.6 μg/mL with S9, and 35.6, 71.2 and 190 μg/mL without S9, were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 450 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). Statistically significant increases in mutant frequency were observed with and without S9 (*: p <0.05 and **: p <0.01, respectively). With S9, the increases in mutant frequency observed were 5.30, 7.82*, 5.90, 7.36* and 7.35* at 71.2, 142, 190, 253 and 338 μg/mL concentrations, respectively. Thus, it was evident that the increases observed with S9 were not dose dependent (p >0.05) and were within the historical negative control 95 % confidence limit. In contrast, the increases observed (i.e. 8.96*, 10.33*, 14.89**, 15.44** and 19.80** at the test concentrations of 142, 253, 338, 450 and 600 μg/mL, respectively) without S9 were dose dependent (p <0.01) and exceeded this limit. The positive controls also induced significant increases in mutant frequency (p <0.01). Thus, based on the above findings, FAT 40061/F was considered to be positive in the CHO/HPRT Assay with Duplicate Cultures in the absence of S9. The results observed in the presence of S9 were considered to be equivocal.