Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 36038/J was tested for mutagenic effects in bacteria and eucaryotic cells in vitro and for clastogenic effects in eucaryotic cells in vitro. There was only a weak mutagenic action on strains S. typhimurium TA 98 and TA 1535 observed, while the metabolites of the test material were weakly mutagenic with strain TA 1537 in a test conducted in 1994 according to OECD 471 under GLP and in strain 1537 without metabolic activation in a test conducted according to OECD 471 (previous guideline) in 1979. Despite these results all confirmatory experiments in vitro, that include an in vitro mammalian cell gene mutation assay and an in vitro mammalian chromosomal aberration assay, did not result in any concerns regarding mutagenicity and/or clastogenicity.

The debate if negative data generated on mammalian cells in vitro are sufficient to exclude a potential risk of mutagenic behavior of a test substance proposed by a positive Ames test result is lasting for ages now. Already in 2013, a workshop was organized and sponsored by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) to investigate this further. An initial analysis of the data presented at this workshop suggested that the association of negative in vitro mammalian cell test results with lack of in vivo genotoxic or carcinogenic activity could have some significance. A review of the ‘Carcinogenicity and Genotoxicity eXperience (CGX)’ database of rodent carcinogens and in vivo genotoxin databases resulted in the observation, that almost all of 318 rodent carcinogens identified were found to have positive results in the Ames test also had at least one positive result in either the in vitro chromosomal aberration test or the in vitro micronucleus test. Thus, it appears that almost all Ames-positive rodent carcinogens are also positive in at least one of the in vitro mammalian cell tests. Where the mammalian cell test results are not clearly positive there maybe justifiable explanations in terms of metabolic conditions or study design. Thus, there are no clear examples of Ames-positive carcinogens giving negative results in two mammalian cell tests covering different endpoints.

Thus, taking above arguments as well as the negative results from the in vitro mammalian cell gene mutation assay and the in vitro mammalian chromosomal aberrationa assay into account, Disperse Violet 057 is considered to be not genotoxic.

Link to relevant study records

Referenceopen allclose all

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:
28 October, 2015 to 29 April, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Test Substance
Identification: FAT 36038/J TE
Commercial Name: Terasil Violet BL Crude Moist
Batch/Lot No.: 1404301 (China)
Purity: 99.1% (provided by Sponsor)
Expiration Date: 13 August 2019
Description by BioReliance: Very dark blackish violet powder
Storage Conditions: Room temperature, protected from light
Receipt Date: 29 May 2015
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:
Experimental Design
The in vitro mammalian chromosome aberration assay was conducted using standard procedures (Galloway et al, 1994; Preston et al, 1981; Swierenga et al, 1991) by exposing Chinese hamster ovary (CHO) cells to appropriate concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.

Preliminary Toxicity Test for Selection of Dose Levels
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose level, and the lowest precipitating dose level was measured. Dose levels for the definitive assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control) or visible precipitate at the conclusion of the treatment period.

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).

Scoring for Metaphase Chromosome Aberrations (Chromosome Aberration Assays)
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 (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture were reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 150 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure will not be scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level).
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.

Statistical Analysis
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Criteria for Determination of a Valid Test
Vehicle Controls
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).

Positive Controls
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).

Cell Proliferation
The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.

Test Conditions
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 20-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

Analyzable Concentrations
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored were reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.
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 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. 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.

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:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June, 2015 to 02 February, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Test Substance
Identification: FAT 36038/J TE
Commercial Name: Terasil Violet BL Crude Moist
Batch/Lot No.: 1404301 (China)
Purity: 99.1% (provided by Sponsor)
Expiration Date: 13 August 2019
Description by BioReliance: Very dark blackish violet powder
Storage Conditions: Room temperature, protected from light
Receipt Date: 29 May 2015
Target gene:
None
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
FAT 36038/J TE was evaluated at concentrations of 1.00, 3.00, 4.50, 6.00 and 12.0 μg/mL with S9 and
3.00, 6.00, 9.00, 10.0, 12.0, 25.0, 50.0, 100, 500 and 1500 μg/mL without S9.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
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 %. The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories.
Evaluation criteria:
The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p≤ 0.05) that exceeded the 95 % confidence limit of the historical vehicle control data from this laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Statistics:
The primary computer or electronic systems used for the collection of data or analysis included, but were not limited to, the following:

LIMS Labware System - Test Substance tracking
Excel 2007 (Microsoft Corporation) - Calculations
Kaye Lab Watch Monitoring system (Kaye GE) - Environmental monitoring
BRIQS - Deviation and audit reporting
ProtoCOL Colony Counter- Data collection
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

Solubility Test

DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. Following sonication at 35.0°C for 10 minutes, the test substance formed a workable suspension in DMSO at a maximum concentration of ~50 mg/mL in the solubility test conducted at BioReliance.

Preliminary Toxicity Assay

FAT  36038/J  TE  was prepared  in  DMSO  and evaluated  in  a  preliminary toxicity assay at  concentrations  of  2.93, 5.86,  11.7,  23.4,  46.9,  93.8,  188,  375,  750  and  1500  µg/mL  with  and  without  S9.   The maximum dose evaluated was based on the solubility limitations of the test substance in the vehicle.    The  test  substance  formed  a  clear  solution  in  DMSO  at  a  concentration  of 0.293 mg/mL   and   workable   suspensions   in   DMSO   at   concentrations   from   0.586   to 150 mg/mL.     Visible  precipitate  was  observed   at  concentrations  ≥11.7  μg/mL  at  the beginning and end of treatment.   The pH of the cultures was adjusted at a concentration of 1500  μg/mL  to  maintain  neutral  pH,  and  the  test  substance  had  no  adverse  impact  on  the osmolality  of  the  cultures  [417,  414,  418  and  417  mmol/kg  for  the  vehicle  control,  the highest concentration (1500 μg/mL), the lowest precipitating concentration (11.7 μg/mL) and the highest soluble concentration (5.86 μg/mL), respectively].  Adjusted relative survival was

27.70 and 11.61% at a concentration of 1500 µg/mL with and without S9, respectively.

Definitive Mutagenicity Assays

Based on the results of the preliminary toxicity assay, 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 S9 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.

In the mutagenicity assay retest, FAT 36038/J TE was evaluated at concentrations of 1.00, 3.00, 4.50, 6.00 and 12.0 µg/mL with S9 (see Deviations) and 3.00, 6.00, 9.00, 10.0, 12.0, 25.0, 50.0, 100,  500  and  1500  µg/mL  without  S9.   Visible  precipitate  was  observed  at  concentrations ≥10.0 μg/mL at the beginning and end of treatment, and the pH of the cultures was adjusted at  a  concentration  of  1500  μg/mL  to  maintain  neutral  pH.   The  average  adjusted  relative survival  was  25.39  and  24.56%  at  concentrations  of  6.00 µg/mL  with  S9  and  1500  µg/mL without S9, respectively.  Cultures treated at concentrations of 1.00, 3.00, 4.50 and 6.00 µg/mL with S9 and 9.00, 12.0, 100, 500 and 1500 µg/mL without S9 were chosen for mutant selection

(cultures treated at a concentration of 6.00 µg/mL without S9 were excluded from evaluation of mutagenicity  because  a  sufficient  number  of  higher  concentrations  was  available;  cultures treated at other concentrations were discarded prior to selection because a sufficient number of higher concentrations was available, or due to excessive toxicity).   No significant increases in mutant  frequency,  as  compared  to  the  concurrent  vehicle  controls,  were  observed  at  any concentration evaluated without S9 (p> 0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p< 0.01).

Conclusions:
FAT 36038/J TE was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures.
Executive summary:

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).

FAT 36038/J TE was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 2.93, 5.86, 11.7, 23.4, 46.9, 93.8, 188, 375, 750 and 1500 μg/mL with and without S9. The maximum dose evaluated was based on the solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥11.7 μg/mL at the beginning and end of treatment. The pH of the cultures was adjusted at a concentration of 1500 μg/mL to maintain neutral pH, and the test substance had no adverse impact on the osmolality of the cultures. Adjusted relative survival was 27.70 and 11.61% at a concentration of 1500 μg/mL with and without S9, respectively.

Based on these 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 S9 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.

In the mutagenicity assay retest, FAT 36038/J TE was evaluated at concentrations of 1.00, 3.00, 4.50, 6.00 and 12.0 μg/mL with S9 and 3.00, 6.00, 9.00, 10.0, 12.0, 25.0, 50.0, 100, 500 and 1500 μg/mL without S9. Visible precipitate was observed at concentrations ≥10.0 μg/mL at the beginning and end of treatment, and the pH of the cultures was adjusted at a concentration of 1500 μg/mL to maintain neutral pH. The average adjusted relative survival was 25.39 and 24.56% at concentrations of 6.00 μg/mL with S9 and 1500 μg/mL without S9, respectively. Cultures treated at concentrations of 1.00, 3.00, 4.50 and 6.00 μg/mL with S9 and 9.00, 12.0, 100, 500 and 1500 μg/mL without S9 were chosen for mutant selection (cultures treated at a concentration of 6.00 μg/mL without S9 were excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available; cultures treated at other concentrations were discarded prior to selection because a sufficient number of higher concentrations was available, or due to excessive toxicity). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p >0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p <0.01)

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.

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

Genetic toxicity in vivo

Description of key information

Currently no is 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

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.