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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Red 195 showed weak mutagenic effects in the Salmonella typhimurium reverse mutation assay, however failed to induce gene mutations in mammalian cells in vitro. It also had positive results in an in vitro mammalian chromosome aberrations assay.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Guidelines followed
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: 175
- Expiration date of the batch: June 30, 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
Target gene:
Histidine gene

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Salmonella typhimurium (TA 98, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Original and confirmatory experiment: 61.73, 185.19, 555.56, 1666.67 and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Bidistilled water
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: all strains (except TA 1535) with S9: 2-aminoanthracene; for TA 1535 with S9: cyclophosphamide; for TA 100 and 1535 without S9: sodium azide, for TA 98 without S9: 2 nitrofluorene, for TA1537 without S9: 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: three per test substance concentration and controls

OTHER EXAMINATIONS:
Colony counting and scoring of the plates
Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the Results section.
Rationale for test conditions:
None
Evaluation criteria:
Assay acceptance criteria: A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
Statistics:
A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
other: Weak mutagenic
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:
Range finding test
Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

Mutagenicity test, original experiment
In the experiment performed with metabolic activation, treatment of strain TA 1537 with FAT 20042/C (Neolan Rosa BE roh trocken SFO) led to a marginal increase in the number of revertant counts at the concentration of 5000 µg/plate . In the experiments conducted without activation on strains TA 98 and TA 1537, a similar effect was observed at the same concentration. No effects occurred on the other strains.

Mutagenicity test, confirmatory experiment
In the experiment performed without metabolic activation on strain TA 98, treatment with FAT 20042/C (Neolan Rosa BE roh trocken SFO) led to a slight increase in the number of back-mutants at the concentrations of 555.6 µg/plate and above . No increased revertant counts were observed with the other strains . The marginal effect observed in the original experiment with and without activation on strain TA 1537 could not be reproduced.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
FAT 20042/C (Neolan Rosa BE roh trocken SFO) exerted a weak mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.
Executive summary:

An in vitro bacterial reverse mutation study was performed to investigate the potential of FAT 20042/C (ca. 80 % purity) to induce gene mutations according to OECD Guideline 471, EU Method B.14 and EPA OTS 798.5265 in compliance with GLP. The concentration range to be tested was determined in a preliminary toxicity study. The substance was tested for mutagenic effects without and with metabolic activation at doses ranging from 20.6 to 5000 µg active ingredient/plate on Salmonella typhimurium strains TA 100, TA 1535, TA 98 and TA 1537. An independent repetition of the experiments was performed with the same concentrations.

Mutagenicity test, original experiment: In the experiment performed with metabolic activation, treatment of strain TA 1537 with FAT 20042/C (Neolan Rosa BE roh trocken SFO) led to a marginal increase in the number of revertant counts at the concentration of 5000 µg/plate. In the experiments conducted without activation with strains TA 98 and TA 1537, a similar effect was observed at the same concentration. No effects occurred on the other strains.

Mutagenicity test, confirmatory experiment: In the experiment performed without metabolic activation, treatment with FAT 20042/C (Neolan Rosa BE roh trocken SFO) with strain TA 98 led to a slight increase in the number of back-mutants at the concentrations of 555.6 µg/plate and above. No increased revertant counts were observed with the other strains. The marginal effect observed in the original experiment with and without metabolic activation on strain TA 1537 could not be reproduced. Based on the results of these experiments and using standard evaluation criteria, it is concluded that FAT 20042/C (Neolan Rosa BE roh trocken SFO) exerted a weak mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
1987
Deviations:
no
Principles of method if other than guideline:
Guidelines followed
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test material: FAT 20042/D (Neolan Rosa BE ZP feucht)
Batch No.: 276
Purity: Approx. 50%
Appearance: Black-reddish humid pieces
Expiry date: October 1998
Storage: Room temperature
Material submitted by: CIBA-GEIGY Limited, Dyestuffs Division, Basle, Switzerland
Target gene:
Histidine gene

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Original and confirmatory experiment- 123.46, 370.37, 1111.11, 3333.33 and 10000.00 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: all strains (except TA 1535) with S9: 2-aminoanthracene; for TA 1535 with S9: cyclophosphamide; for TA 100 and 1535 without S9: sodium azide, for TA 102 without S9: mitomycin-C; for TA 98 without S9: 2 nitrofluorene, for TA1537 without S9: 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 per test substance concentration and controls


OTHER EXAMINATIONS:
- Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the Results section.
Rationale for test conditions:
None
Evaluation criteria:
Assay acceptance criteria: A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
Statistics:
A statistical analysis of the test data was not performed.
Key result
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 examined
Positive controls validity:
valid
Key result
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 examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
S. typhimurium TA 102
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
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
without metabolic activation only
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:
Range finding test
Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 10000.0 µg/plate with and without metabolic activation (since the purity of the test substance is about 50%, this corresponds to a concentration of 5000 µg/plate).

Mutagenicity test, original experiment
In the experiment performed without metabolic activation, treatment of strain TA 98 with FAT 20042/D (Neolan Rosa BE ZP feucht) led to a slight increase in the number of back-mutant colonies at the highest concentration. A marginal increase at two intermediate concentrations was also registered with strain TA 102 in both, the experiment with and without metabolic activation. No effects were observed with the other strains

Mutagenicity test, confirmatory experiment
In the experiment performed without metabolic activation, again after treatment of strain TA 98 with FAT 20042/D (Neolan Rosa BE ZP feucht) a slight increase in the incidence of histidineprototrophic mutants was observed in comparison with the negative control. The slight increase in the number of revenants in the experiment without activation on strain TA 1535 at the concentration of 3333.3 µg/plate was not observed in the original experiment conducted on this strain and is attributed to spontaneously occurring back-mutants. No effects at all occurred and in the experiments with activation on strains TA 98 and TA 1535 and with the other strains.

The slight effect observed with strain TA 102 in the original experiment also is attributed to fluctuations in the incidence of spontaneously occurring back-mutant colonies.
In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were occasionally reduced at the upper concentrations.
There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

None

Conclusions:
FAT 20042/D (Neolan Rosa BE ZP feucht) exerted a marginal mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.
Executive summary:

FAT 20042/D, identified as black-reddish humid pieces, purity approx. 50%, batch no. 276, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in the range of 123.5 to 10000.0 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 123.5 to 10000.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. FAT 20042/D led to a slight increase in the incidence of histidine-prototrophic mutants in comparison to the negative control in strain TA 98 at the highest concentration in both original as well as confirmatory experiments when tested in absence of metabolic activation. No relevant effects occurred with the other strains. Based on the results of these experiments and using standard evaluation criteria, it is concluded that FAT 20042/D (Neolan Rosa BE ZP feucht) exerted a marginal mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Principles of method if other than guideline:
Guidelines followed
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation assay
Specific details on test material used for the study:
NoneSOURCE OF TEST MATERIAL
- Source and batch No.of test material: 175
- Expiration date of the batch: June 30, 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
Target gene:
Chinese Hamster Cells V79
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F10 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction)
Test concentrations with justification for top dose:
Pre-experiment:
Cytotoxicity test: with and without metabolic activation: 0.24 to 500 µg/mL

Mutagenicity test:

Original experiment:
Range with metabolic activation: 14.81 to 400.0 µg/ml
Range without metabolic activation: 7.41 to 200.0 µg/ml

Confirmatory experiment:
Range with metabolic activation: 11.11 to 300.0 µg/ml
Range without metabolic activation: 9.26 to 250.0 µg/ml
Vehicle / solvent:
-Vehicle (s)/solvent(s) used: Dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration: Overnight
- Expression time (cells in growth medium): 2.5-5.0E+6 cells

- SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
- STAIN (for cytogenetic assays): Giemsa

DETERMINATION OF CYTOTOXICITY: Cell count
Rationale for test conditions:
None
Evaluation criteria:
All mutant frequencies were normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures was lower than 15%, the corresponding mutant frequency was usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which was defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures,was calculated.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDY
In the preliminary toxicity test conducted with and without metabolic activation, 12 concentrations of FAT 20042/C were tested. The concentrations selected ranged from 0.24 to 500.0 µg/ml and separated
by 2-fold intervals. In the part with metabolic activation, the highest concentration was toxic. The next lower concentration of 250.0 µg/ml produced an acute growth inhibition of 50.55%. In the part without metabolic activation FAT 20042/C exerted a complete growth inhibitory effect at the concentration of 500.0 µg/ml. The next lower concentration of 250.0 µg/ml revealed an acute growth inhibitory effect of 95.12%.

MAIN EXPERIMENTS - Toxicity
Accordingly, four concentrations were selected for the original experiment ranging from 14.81 to 400.0 µg/ml and from 7.41 to 200.0 µg/ml in the presence and absence of metabolic activation, respectively.
In the part with metabolic activation the highest concentration was toxic. At the next lower concentration, growth inhibition determined after treatment and expression revealed values of 19.87% and less than 9.06%.
In the absence of metabolic activation the mean growth inhibitory effect determined after treatment was 45.09% at the highest concentration. After the expression period the determined cytotoxicity revealed a value of less than 10.26%.
Due to the pronounced growth inhibitory response in the part with metabolic activation the concentration range was decreased in the confirmatory experiment ranging from 11.11 to 300.0 µg/ml. In
the part without metabolic activation a concentration range of 9.26 to 250.0 µg/ml was selected in order to reach a complete toxicity at the highest concentration. In the presence of metabolic activation the highest concentration was still fully toxic. At the next lower concentration, the mean growth inhibition determined after treatment and expression revealed values of 74.96 and 24.11%. In the part without activation, the highest concentration proved fully growth inhibiting. Growth inhibition at the next lower concentration of 83.33 µg/ml was less than 15.49% and less than 12.89% after treatment and expression respectively.

MAIN EXPERIMENTS - Mutagenicity
In the presence and absence of metabolic activation, no significant increase in mutant frequency was observed at any concentration level of FAT 20042/C tested in the original or the confirmatory experiment in comparison with the negative control.
The statistical significance obtained with the two higher concentrations of the original experiment without metabolic activation is considered to be of no biological relevance. The respective mutant frequency values of 5.83 and 5.94E+6 are clearly within the range of the historical negative controls.
The positive controls induced a clear increase in mutant frequency.
Remarks on result:
other: all strains/cell types tested

None

Conclusions:
FAT 20042/C and its metabolites did not show any mutagenic activity in this forward mutation system.
Executive summary:

FAT 20042/C, was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours.

Mutagenicity test with metabolic activation: The original experiment was performed at the following concentrations: 14.81, 44.44, 133.33 and 400.0 µg/ml. The highest concentration proved fully growth inhibiting after treatment. At the next lower concentration, the growth inhibiting values found at after treatment and expression were 19.87% and less than 9.06% respectively. In the confirmatory experiment, the concentrations applied were 11.11, 33.33, 100.0 and 300.0 µg/ml. The highest concentration was again fully growth inhibiting after treatment. At the next lower concentration a mean acute growth inhibition of 74.96% was obtained. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation: The original experiment was performed at the following concentrations: 7.41, 22.22, 66.67 and 200.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 45.09%* and less than 10.26%* respectively. In the confirmatory experiment the concentrations applied were 9.26, 27.78, 83.33 and 250.0 µg/ml. The highest concentration proved fully growth inhibiting after treatment. At the next lower concentration, an acute growth inhibitory effect of less than 15.49% was obtained. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Hence, based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 20042/C and its metabolites did not show any mutagenic activity in this forward mutation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Guideline followed
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
NoneSOURCE OF TEST MATERIAL
- Source and lot No.of test material: 0000657250 (India)
- Expiration date of the lot: 04 August 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2 to 8 °C, protected from light
Target gene:
chromosomes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 0.2, 0.6, 2, 6, 20, 60, 200, 600 and 2000 µg/mL. In the chromosome aberration assay, the concentations tested were: 250, 500, 1000, 1600, 1800, 1900, and 2000 µg/mL in the non-activated 4-hour exposure group; 800, 1000, 1200, 1400, 1600 and 1800 µg/mL in the S9-activated 4 h exposure group; and 50, 100, 250, 500, 600, 700, and 800 µg/mL in the non-activated 20 h exposure group.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed. Cells were harvested 20 hours (±30 minutes) after initiation of treatment, which corresponds to 1.5 normal cell cycles.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assay

NUMBER OF CELLS EVALUATED: a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture), whenever possible

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell counts

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Rationale for test conditions:
None
Evaluation criteria:
Toxicity induced by treatment was based upon inhibition of cell growth and was reported for the cytotoxicity and chromosome aberration portions of the study. 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 were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

A test article was considered positive if it induced a statistically significant and dose dependent increase in the frequency of aberrant metaphases (p
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:
Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
For the induction of structural chromosome aberrations in the 20-hour group without S9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at doses ≥ 1800 µg/mL in the non activated 4 hour exposure group; at doses ≥ 1400 µg/mL in the S9 activated 4-hour exposure group. In the non-activated 20-hour exposure group, 48% cytotoxicity was observed at 800 µg/mL.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: Although requisite toxicity (55 ± 5 % reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated 20-hour exposure group, 48 % cytotoxicity was observed at 800 µg/mL, which was within the rounding margin. Therefore, the Study Director selected 800 µg/mL as the top dose to score in the non activated 20-hour exposure group.
Remarks on result:
other: all strains/cell types tested

None

Conclusions:
FAT 20042/E was positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated 20-hour exposure group. FAT 20042/E was negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated 4-hour exposure groups.
Executive summary:

The test substance, FAT 20042/E, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. Water was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was observed at 2000 µg/mL in the non activated and S9-activated 4 hour exposure groups; and at doses 600 µg/mL in the non activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 250 to 2000 µg/mL for the non activated 4-hour exposure group, from 800 to 1800 µg/mL for the S9-activated 4-hour exposure group, and from 50 to 800 µg/mL for the non-activated 20-hour exposure group. In the chromosome aberration assay, cytotoxicity (55 ± 5% reduction in cell growth index relative to the vehicle control) was observed at doses 1800 µg/mL in the non activated 4 hour exposure group; at doses 1400 µg/mL in the S9 activated 4-hour exposure group. Although requisite toxicity (55 ± 5% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated 20-hour exposure group, 48% cytotoxicity was observed at 800 µg/mL, which was within the rounding margin. Therefore, 800 µg/mL was selected as the top dose to score in the non activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 500, 1000, and 1800 µg/mL for the non activated 4 hour exposure group; 800, 1000, and 1400 µg/mL for the S9 activated 4-hour exposure group; and 100, 250, and 800 µg/mL for the non activated 20-hour exposure group. In the non-activated and S9-activated 4-hour exposure groups, no significant or dose dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). In the non-activated 20-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (5.0 and 5.7%) were observed at 250 and 800 µg/mL, respectively (p   0.01; Fisher’s Exact test and p   0.05; Cochran-Armitage test). No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in non-activated 20-hour treatment group. The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. These results indicate FAT 20042/E was positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated 20-hour exposure group. FAT 20042/E was negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9 -activated 4 -hour exposure groups.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Testing proposal

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

An in vitro bacterial reverse mutation assay was performed to investigate the potential of FAT 20042/D to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. In the original and confirmatory experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20042/D (Neolan Rosa BE ZP feucht) led to a slight increase in the number of back-mutant colonies at the highest concentration. No relevant effects were observed with the other strains.

A second bacterial reverse mutation test also conducted according to OECD 471, demonstrated similar results. In the original as well as confirmatory experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20042/C led to a slight increase in the number of back-mutants at the concentrations of 555.6 µg/plate and above. No relevant increased revertant counts were observed with the other strains. In summary, Acid Red 195 was found to be mutagenic in the bacterial reverse mutation assays.

To confirm this, a test on mutagenic effects in an eukaryotic cellular system was conducted. In this in vitro HPRT test conducted according to OECD Guideline 476, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance in the presence and absence of metabolic activation, revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). Thus it was demonstrated that FAT 20042/D did not induce mutagenic effects in this eukaryotic cellular assay, countering the weak mutagenic effects observed in the prokaryotic assays.

To investigate if the test substance is likely to induce changes on a chromosomal level, an in vitro mammalian chromosomal aberration assay was conducted according to OECD Guideline 473 and in accordance with GLP. FAT 20042/E did not lead to the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated 4-hour exposure groups. However, it was found to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated 20-hour exposure group.

Based on the available information, it is clear that Acid Red 195 may have potential to induce mutations in bacterial cells, however it did not induce mutations in the mammalian cells, and hence considered not mutagenic. However, Acid Red 195 was found to be clastogenic, while not being aneugenic, in an in vitro mammalian chromosomal aberration assay. No other data is available to support or negate the clastogenic potential of the substance. Hence, proposal for further testing to clarify if the substance is capable of exerting clasogenic action in mammals is being included in the dossier. To this effect, a testing proposal for conducting micronucleus assay according to OECD Guideline 474 is included in this dossier.

Justification for classification or non-classification

Based on the above mentioned results, a conclusion on the mutagenic and clastogenic activity of Acid Red 195 could not be drawn without the generation of additional data in vivo. Before the results of these tests are available no classification regarding genotoxicity will be applied, since data of comparable chemistry demonstrated in several cases that such molecules are sometimes likely to show false negative results in those test systems.