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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 36038/J TE was tested for mutagenic effects in bacteria and eucaryontic cells in vitro and for clastogenic effects in eucaryontic cells in vitro. While both Ames tests conducted gave a positive response, an additional HPRT test conducted in vitro gave negative results. Therefore, the test substance is considered to be non-mutagenic in eucaryonic cells. In addition an in vitro chromosomal aberration test according to OECD 473 has been conducted and was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems when tested with CHO cells. Hence, the test substance FAT 36038 may be considered as non-genotoxic and further testing is not considered.

Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
During the conduct of the initial micronucleus assay, due to a dilution error, the three lowest doses tested were 0.25, 0.5, and 1 μg/mL, instead of 0.5, 1, and 2 μg/mL, respectively, as specified in Protocol Amendment 1. Since the dilution error occurred at lower doses and did not affect dose selection for the repeat micronucleus assay or for micronucleus scoring, the Study Director has concluded that this deviation did not adversely impact the integrity of the data or the validity of the study conclusion.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 liver microsomal fraction
Test concentrations with justification for top dose:
dose levels 6, 20, 60, 200 and 2000 μg/mL in the S9-activated 4-hour exposure group, and at doses 0.6 and ≥ 200 μg/mL in the non-activated 20-hour exposure group
Vehicle / solvent:
Dimethyl formamide (DMF)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Chromosome Aberration Assays
Seven to nineteen dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was either based on cytotoxicity (cell growth inhibition relative to the vehicle control) or visible precipitate at the conclusion of the treatment period. Two or three additional dose levels were included in the evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Chromosome Aberration Assay)
The pH at the highest test substance concentration was measured prior to dosing using a pH meter or test strips. Treatment was carried out by re-feeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 50 μL of test substance dosing solution or vehicle alone. Untreated controls were re-fed with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.
After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium were aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium, and returned to the incubator under standard conditions.
For the chromosomal aberration assay only, two hours prior to cell harvest, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts, and then Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL. Thus the treatment time for the precipitating dose levels was 18 hours instead of 20 hours.

Collection of Metaphase Cells (Preliminary Toxicity Test and Chromosome Aberration Assayd)
For the preliminary toxicity test and chromosome aberration assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data was presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity).
Evaluation criteria:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95 % control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations.

The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None

In the initial chromosome aberration assay, the test substance formed workable suspensions in DMF at concentrations ranging from 0.75 to 25 mg/mL and was soluble in DMF at concentrations ranging from 0.05 to 0.5 mg/mL.

The pH of the highest dose level of test substance in treatment medium was 7.36. Toxicity of FAT 36038/J TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 23% at 50 μg/mL, the highest test dose level evaluated for chromosome aberrations.

The mitotic index at the highest dose level evaluated for chromosome aberrations, 50 μg/mL, was 27 % reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 10, 25, and 50 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). Toxicity of FAT 36038/J TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 52 % at 3 μg/mL, the highest test dose level evaluated for chromosome aberrations.

The mitotic index at the highest dose level evaluated for chromosome aberrations, 3 μg/mL, was 17 % reduced relative to the vehicle control. The dose levels initially selected for microscopic analysis were 0.25, 0.5, and 3 μg/mL. In the S9-activated 4-hour exposure group, a statistically significant increase (6.3 %) in structural aberrations was observed at 3 μg/mL (p ≤0.05; Fisher’s Exact test). In order to confirm dose-responsiveness, an additional dose level of 1 μg/mL was included in the microscopic evaluation. However, the Cochran-Armitage test was negative for a dose-response (p >0.05). In addition, the statistically significant increase was within the historical control range of 0.0 % to 9.5 %; but outside the 95 % historical control limit. The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) treatment group (18.0 %) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Conclusions:
FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.
Executive summary:

This study was performed to evaluate the clastogenic potential of FAT 36038/J TE, which was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dimethyl formamide (DMF) was used as the vehicle. Cytotoxicity (≥50 % reduction in cell growth index relative to the vehicle control) was observed at doses ≥200 μg/mL in the non-activated 4-hour exposure group, at dose levels 6, 20, 60, 200 and 2000 μg/mL in the S9-activated 4-hour exposure group, and at doses 0.6 and ≥200 μg/mL in the non-activated 20-hour exposure group.

At the conclusion of the treatment period, visible precipitate was observed at all dose levels in all three treatment conditions. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 250 μg/mL for the non-activated 4 and 20-hour exposure groups, and from 0.25 to 10 μg/mL for the S9-activated 4-hour exposure group.

In the initial chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at dose levels ≥ 3 μg/mL in the S9-activated 4-hour exposure group and at dose levels 100, 175, 200 and 250 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥50 μg/mL in the non-activated 4 and 20-hour exposure groups. The dose levels selected for microscopic analysis were 10, 25, and 50 μg/mL for the non-activated 4 and 20-hour exposure groups; and 0.25, 0.5, 1, and 3 μg/mL for the S9-activated 4-hour exposure group. No significant or dose-dependent increases in structural aberrations were observed in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, a statistically significant increase (6.3 %) in structural aberrations was observed at 3 μg/mL (p ≤0.05; Fisher’s Exact test). In order to confirm dose-responsiveness, an additional dose level of 1 μg/mL was included in the microscopic evaluation. However, the Cochran-Armitage test was negative for a dose-response (p >0.05). In addition, the statistically significant increase was within the historical control range of 0.0 % to 9.5 %; but outside the 95 % historical control limit.

No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the test substance treated groups (p >0.05; Fisher’s Exact and Cochran-Armitage tests). In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL. Due to insufficient cell growth during baseline counts in the vehicle and untreated controls, the assay was repeated again in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL. In the second repeat assay, 55 ± 5 % cytotoxicity was observed at dose levels ≥5 μg/mL in the S9-activated 4-hour exposure group. The dose levels selected for microscopic analysis were 1, 2.5, and 5 μg/mL. No significant or dose-dependent increases in structural or numerical aberrations were observed in the S9-activated 4-hour exposure group (p >0.05; Fisher’s Exact and Cochran-Armitage tests). These results indicated that the statistically significant increase observed in the initial assay at the cytotoxic dose was an isolated event which was not reproducible. Therefore, the test substance was considered to be negative for the induction of structural aberrations in all three exposure groups. All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met. Under the conditions of the assay described in this report, FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Currently no data available to assess this endpoint.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay:

A key study was performed to determine the detection of gene mutations induced by the test material FAT 36038/F or its metabolites in histidine-requiring strains of Salmonella typhimurium. FAT 36038/F was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed.

In the first experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F) led to a slight increase in the number of revertant colonies at the highest concentration. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the number of back-mutants occurred on strains TA 98 and TA 1535 at the highest concentration. No effects were observed with the other strains.

In the confirmatory experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F), led to a slight increase in the number of revertant colonies at the highest concentration. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the number of back-mutants occurred on strains TA 98, TA 1535 and TA 1537 at the highest concentration. No effects were observed with the other strains.

In the mutagenicity tests without metabolic activation performed on strain TA 102, a slight decline in the number of revertant colonies was registered. The test substance exerted a weak inhibiting effect on the growth of this bacterial strain.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Terasil Violet BL roh feucht (FAT 36038/F) exerted a weak mutagenic action on strains S. typhimurium TA 98 and TA 1535. The metabolites of the test material were weakly mutagenic with strain TA 1537.

 

In another supporting study, FAT 36038/C was tested for mutagenicity in selected strains of S. typhimurium both in the presence and absence of in vitro activation by microsomal enzymes from rat liver (Ames test).

FAT 36038/C (a violet dyestuff) was tested in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at eight dose levels: 0.2, 2, 20, 200, 500, 1000, 2000 and 4000 ml per Petri dish. The test substance was diluted in sterile water and every concentration was tested in triplicate.

Positive control was also used to perform the experiment. MNG, 9 -aminoacridine and daunomycine were used as positive control without S9. 2 -anthramine was used as positive control with S9. Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, product FAT 36038/C was found to be mutagenic for S. typhimurium strain TA 1537 without metabolic activation.

In vitro chromosomal aberration assay:

Further, in another key study, FAT 36038/J was tested for chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473.

A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation.

Dimethyl formamide (DMF) was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL.

Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 200 μg/mL in the non-activated 4-hour exposure group, at dose levels 6, 20, 60, 200 and 2000 μg/mL in the S9-activated 4-hour exposure group, and at doses 0.6 and ≥ 200 μg/mL in the non-activated 20-hour exposure group.

Under the conditions of the assay described in this report, FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

HPRT assay:

In another key study, the test substance, FAT 36038/J TE, 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).

Based on the results, FAT 36038/J TE was evaluated in the definitive mutagenicity assay at concentrations of 1.50, 3.00, 6.00, 7.00, 8.00, 9.00, 10.0, 11.0 and 11.7 μg/mL with and 1.25, 2.50, 5.00, 8.00 and 11.7 μg/mL 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 of the cultures. The average adjusted relative survival was 7.58 and 96.44 % at a concentration of 11.7 μg/mL with and without S9, respectively. However, the limit dose was not achieved due to a shift in precipitate profile, and the entire assay was retested with an adjustment in dose levels.

These results indicate FAT 36038/J was negative in the in vitro Mammlian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate cultures, under the conditons and according to the criteria of the test protocol.

Justification for classification or non-classification

Therefore, it can be concluded that the test item FAT 36038 exerted a weak mutagenic action on strains S. typhimurium TA 98 and TA 1535. The metabolites of the test material were weakly mutagenic with strain TA 1537.

FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells as well as in the in vitro Mammlian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate cultures. Hence, the test substance FAT 36038 may be considered as non genotoxic and no classification for mutagenicity is required as per the Regulation (EC) No. 1272/2008.