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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Brown 49 was found to be non-mutagenic in Ames assay and in-vitro mammalian cell gene mutation test (HPRT). It was however found to be weakly mutagenic in chromosomal aberration assay at cytotoxic concentrations This positive effect was hence considered a false positive effect frequently seen in V79 cells and/or vinyl-sulfone dyes.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
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)
Version / remarks:
February 1998, adopted July 21, 1997
Deviations:
yes
Remarks:
Regarding positive control sun=bstance final concentration and evaluation of results. These deviations had no detrimental impact on the outcome of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
"Kanpoan No. 287 ~ Environment Protection Agency"
"Eisei No. 127 - Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry".
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss GLP
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TVR50
- Expiration date of the lot/batch: September 01, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 h in water, saline, PEG, CMC, vaseline, and FCA
Species / strain / cell type:
mammalian cell line, other: Cells V79 of Chinese hamster
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from rats treated with phenobarbital and ß-Naphthoflavone.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 25, 50, 100, 200, 300, 400 µg/ml
Concentration range in the main test (without metabolic activation): 187.5, 375.0, 750, 1500, 3000, 4000 µg/ml
Vehicle / solvent:
deionised water
Untreated negative controls:
yes
Remarks:
Concurrent negative (culture medium)
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Choice of the Cell Line V79

Exposure period (with and without metabolic activation): 4 h
Recovery period (with and without metabolic activation): 14 h
Preparation interval (with and without metabolic activation): 18 h

Fixation time:
18 h
Evaluation criteria:
Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations.
100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Mitotic index (% cells in mitosis) was determined.
The number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test (10) (p < 0.05).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(>= 312.5 µg/ml in pre-test an at 300 µg/ml in main test)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
only at 4000 µg/ml at cytotoxic conc.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/ml in pre-test and 3000 µg/ml in main test)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test substance is considered to be weakly mutagenic in this chromosome aberration test. Since increased aberration frequencies were observed only in the presence of strong toxicity indicated by reduced cell numbers, it can not be excluded that an indirect, non genotoxic DNA damaging mechanism was involved.

Conclusions:
FAT 40'571/A induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) at a high concentration exhibiting strong toxicity.
Executive summary:

This in-vitro mammalian chromosomal aberration test was carried out with FAT 40'571/A (dissolved in deionised water) was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsteri n vitro in one experiment. The study was carried out as per OECD 473 and B.10 guideline. Exposure period was 4 h with and without S9 mix.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/ml) was chosen with regard to current OECD Guideline 473. Using reduced cell numbers as an indicator for toxicity, clear toxic effects were observed after 4 h treatment with 5000 µg/ml in the absence of S9 mix and 312.5 µg/ml and above in the presence of S9 mix.

Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test. The chosen treatment concentrations.

In the main experiment, clear toxic effects indicated by reduced mitotic indices were observed in the presence of S9 mix after treatment with 300 µg/ml. In addition, reduced cell numbers were observed after treatment with 3000 µg/ml and above in the absence of S9 mix and after treatment with 300 µg/ml and above in the presence of S9 mix. In the absence of S9 mix, the test article did induce a significant increase in the number of cells carrying structural chromosome aberrations after treatment with 4000 µg/ml. This increase beyond our historical control data range was regarded as being relevant in this test system. However, it has to be considered, that the increase was observed at a high concentration inducing strong toxic effects indicated by strongly reduced cell numbers.

In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

 

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 40571 is considered to be weakly mutagenic in this chromosome aberration test. Since increased aberration frequencies were observed only in the presence of strong toxicity indicated by reduced cell numbers, it can not be excluded that an indirect, none genotoxic DNA damaging mechanism was involved.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation HPRT
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-04-25 to 2013-08-21
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)
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
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DER 8107 / BOP 01-12 (Lot: MHC-00160S820)
- Expiration date of the lot/batch: 25 Jan 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (with and without metabolic activation):
50, 100, 250, 500, 1000, 1750, 2500, 3750, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
25, 50, 100, 250, 500, 1000, 2000, 2500, 3000, 5000 µg/mL
Experiment I
without metabolic activation: 50, 100, 200, 400, 600, 800, 1000, 1400, 1800 and 2000 µg/mL
and with metabolic activation: 100, 250, 750, 1500, 1750, 2000, 2250, 2500 and 3000 µg/mL
Experiment II
without metabolic activation: 100, 250, 500, 750, 1000, 1300 and 1600 µg/mL
and with metabolic activation: 50, 100, 200, 400, 800, 1400, 2000, 2300, 2900 and 3200 µg/mL
Experiment III
without metabolic activation: 10, 25, 50, 100, 250, 500, 750, 1000, 1300 and 1600 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; 0.8 and 1.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I without S9: ≥ 400 μg/mL; experiment I with S9: ≥ 250 μg/mL; Experiment II without S9: ≥ 750 μg/mL; Experiment II with S9:≥ 400 μg/mL ; Experiment III without S9: ≥ 750 μg/mL
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-mutagenic
Conclusions:
FAT 40571/C TE is considered to be non-mutagenic in the HPRT assay.
Executive summary:

A mammalian cell gene mutation assay (HPRT locus) was carried out with FAT 40571/C according to OECD 476 and B 17 guidelines. In this assay, V79 cells cultured in vitro were exposed to FAT 40571/C TE dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20 h treatment) at concentrations of

- 50, 100, 200, 400, 600, 800, 1000, 1400, 1800 and 2000 µg/mL (without metabolic activation, Experiment I)

- 100, 250, 750, 1500, 1750, 2000, 2250, 2500 and 3000 µg/mL (with metabolic activation, Experiment I)

- 100, 250, 500, 750, 1000, 1300 and 1600 µg/mL (without metabolic activation, Experiment II)

- 50, 100, 200, 400, 800, 1400, 2000, 2300, 2900 and 3200 µg/mL (with metabolic activation, Experiment II)

- 10, 25, 50, 100, 250, 500, 750, 1000, 1300 and 1600 µg/mL (without metabolic activation, Experiment III).

FAT 40571/C TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 17.2% for the highest concentration (2000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 3000 µg/mL with a relative growth of 13.1%. In experiment II without metabolic activation the relative growth was 11.2% for the highest concentration (1600 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 3200 µg/mL with a relative growth of 14.0%. In experiment III without metabolic activation the relative growth was 7.0% for the highest concentration (1600 µg/mL) evaluated.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 2.68 was found at a concentration of 1800 µg/mL with a relative growth of 18.6%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.26 was found at a concentration of 1750 µg/mL with a relative growth of 35.2%. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.28 was found at a concentration of 500 µg/mL with a relative growth of 89.2%. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.54 was found at a concentration of 400 µg/mL with a relative growth of 64.4%.

In experiment III without metabolic activation the highest mutation rate (compared to the negative control values) of 1.90 was found at a concentration of 1000 µg/mL with a relative growth of 48.9%.

In experiment II without metabolic activation the requirements of testing a maximum concentration resulting in 10 - 20% survival was fulfilled (1600 µg/ml with 11.2% survival). Unfortunately, in this experiment only 6 lower concentrations have been tested and the requirement of the OECD Guideline testing of at least 8 analysable concentrations was not fulfilled. Therefore, a third experiment (as a repetition of the second experiment) was performed without metabolic activation. In this approach the requirements regarding the cytotoxicity criteria haven’t been met, but eight concentrations have been analysable.

Taken together these two experiments, it could be shown, that there is no mutagenic effect in the experiments with long term incubation (without metabolic activation).

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40571/C TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 17, 1998 to December 14, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
"Kanpoan No. 287 ~ Environment Protection Agency"
"Eisei No. 127 -- Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry"
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss GLP
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TVR50
- Expiration date of the lot/batch: September 01, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 hours in water, saline, polyethylene glycol, CMC, vaseline, and FCA
Target gene:
The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from rat treated with phenobarbital and ß-Naphthoflavone.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 33; 100; 333; 1000; 2500; and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
Solvent: deionized water
The solvent was chosen because of its solubility properties.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation- TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabolic activation- TA 1537, TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation- WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation- TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Details on test system and experimental conditions:
When summarised the mutations of the TA strains and the E. coli strain, used in this study can be described as follows:
Salmonella typhimurium
TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 98: his D 3052; rfa-; uvrB-;R-factor: frame shift mutations
TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA 100: his G 46; rfa-; uvrB-;R-factor: base-pair substitutions
Escherichia coli
WP2 uvrA: trp; uvrA: base-pair substitutions and others
Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to Ames et al.. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Dr. B.N. Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen). The bacterial strain WP2 uvrA was obtained from Dr. Heinz Träger, Knoll AG, D-67008 Ludwigshafen.
Evaluation criteria:
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No statistical evaluation of data was required.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and E. Coli WP2 uvrA
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
(5000 µg/plate), slight effects seen with TA 98 at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and E. Coli WP2 uvrA
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
( 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Non mutagenic

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 at 5000 µg/plate with S9 mix in experiment II.

In experiment II, the colony count of the negative control without S9 mix in strain TA 98 did not quite reach the lower limit of our historical range of negative control data. This effect is caused by statistical fluctuations of the rather low numbers of colonies in strain TA 98. Since the results of this study are based on the corresponding solvent control, which remained well within the range of our historical controls, this effect is judged irrelevant.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Conclusions:
FAT 40'571/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of FAT 40'571/A to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. This test was carried out according to OECD 471 and EU B.13 guidelines. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

 

The test article was tested at the 33; 100; 333; 1000; 2500; and 5000 µg/plate concentrations.

 

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 at 5000 µg/plate with S9 mix in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40'571/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 40'571/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Genetic toxicity in vivo

Description of key information

Reactive Brown 49 was found to be not cytogenic in the in-vivo MNT

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: Micronuleus test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Feb 1999 to 30 March 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
but no influence on the integrity and validity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EEC Directive 92/69, L 383, Annex V, B 12, dated December 29, 1992.
GLP compliance:
yes (incl. QA statement)
Remarks:
German GLP
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TVR50
- Expiration date of the lot/batch: September 01, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 hours stable in water, saline, PEG, CMC, vaseline, and FCA
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at start of acclimatization: 8 - 12 weeks
- Weight at study initiation: males mean value 34.4 g (SD ±3.1 g) ; females mean value 28.7 g (SD ± 2.1 g)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Diet : pelleted standard diet, (ALTROMIN 1324, D-32791 Lage/Lippe) ad libitum
- Water : tap water, ad libitum,
- Acclimation period: minimum 5 days
- Fasting period : 18 hrs before treatment, only water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3
- Humidity (%): 21 - 46
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: unspecified
Vehicle:
deionised water.
Details on exposure:
Approximately 18 h before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test article, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 h after treatment, respectively.
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
670 mg/kg bw/day (actual dose received)
Remarks:
Medium dose
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
5/sex/dose.
Additional 5/sex at high dose for 48 h sacrifice
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
Tissues and cell types examined:
polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
Details of tissue and slide preparation:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations in pre-experiment for toxicity in 4 animals (2 males & 2 females): receiving a single oral dose of 2000 mg/kg bw :
The treated animals expressed slight neurologic effects : reduction of spontaneous activity, eyelid closure, apathy during 6 to 48 h.

The mean number of normochromatic erythrocytes was not substantially increased after treatrnent with the test article as compared to the mean value of NCEs of the vehicle control indicating that FAT 40' 571/A had no cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no enhancenmt in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article The mean values of micronuclei observed after treatment with FAT 4057/A were below the value of the vehicle control group.

40 mg/'kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Conclusions:
FAT 40'571/A is considered to be non-mutagenic in this in vivo micronucleus assay.
Executive summary:

This study was performed to investigate the potential of FAT 40"571/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was carried out according to OECD 474 and EU B.12 guidelines. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg b.w. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs.

The following dose levels of the test article were investigated

24 h preparation interval. 200, 670, and 2000 mg/kg b.w..

48 h preparation interval' 2000 mg/kg b.w.

The highest dose (2000 mg/kg, maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test article the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 40371/A had no cytotoxic effectiveness in the bone marrow In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a substantial increase of induced micronucleus frequency

 

In conclusion. it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40' 57/A is considered to non-mutagenic in this micronucleus assay.

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

Mode of Action Analysis / Human Relevance Framework

Kirkland et al (2005) demonstrated an extremely high false-positive rate for in-vitro clastogenicity tests, particularly in mammalian cell tests, when compared to rodent carcinogenicity study results (Poth, A (2008)). “Certain characteristics of the commonly used rodent cell lines (CHO, CHL, V79, L5178Y, etc.), such as their p53 status, their karyotypic instability and their DNA repair deficiencies, are recognized as possibly contributing to the high rate of irrelevant positives. Also the need for exogenous metabolism with the cell systems is expected to contribute to these irrelevant positive findings, as metabolites produced by S9 used as a metabolic source in cell culture may be quite different from those produced by normal human liver metabolism.” Accordingly, “the high false-positive rate of the established in-vitro mammalian cell tests means that an increased number of compounds are subjected to earlier and additional in-vivo genotoxicity testing.”


In addition, it is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, vinyl sulphone moieties are used in fiber-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in plenty of tests with vinyl sulphone dyes. Hence, the in-vivo test produces more reliable data for this kind of substance.


Kirkland et al (2005). Evaluation of the ability of a battery of three in-vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat Res. 2005 July 4;584(1–2):1–256


Poth, A (2008). Challenges in Testing for Genotoxycity. Genetic Toxicology and Cell Biology, RCC Cytotest Cell Research GmbH


Dearfield KL et al. (1991). Genotoxicity in mouse lymphoma cells of chemicals capable of Michael addition. Mutagenesis 1991;6(6):519-525


Warra TJ et al. (1990). Methyl vinyl sulphone: A new class of Michael-type genotoxin. Mutat Res. 1990;245:191-199


MacGregor et at. (1980). Mutagenicity tests of fabric-finishing agents in Salmonella typhimurium: fiber-reactive wool dyes and cotton flame retardants. Environ. Mutagenesis 1980;2:405-418


 

Additional information

Three in vitro tests and one in vivo study were performed. The results were as follows:


Ames test:


This study was performed to investigate the potential of FAT 40'571/A to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. This test was carried out according to OECD 471 and EU B.13 guidelines. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.  The test article was tested at the 33; 100; 333; 1000; 2500; and 5000 µg/plate concentrations. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 at 5000 µg/plate with S9 mix in experiment II.


No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40'571/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.


In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 40'571/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.


 


Chromosome aberration test in vitro:


An in-vitro mammalian chromosomal aberration test was carried out with FAT 40'571/A (dissolved in deionised water) was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment. The study was carried out as per OECD 473 and B.10 guideline. Exposure period was 4 h with and without S9 mix.


In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/ml) was chosen with regard to current OECD Guideline 473. Using reduced cell numbers as an indicator for toxicity, clear toxic effects were observed after 4 h treatment with 5000 µg/ml in the absence of S9 mix and 312.5 µg/ml and above in the presence of S9 mix. Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test. The chosen treatment concentrations.


In the main experiment, clear toxic effects indicated by reduced mitotic indices were observed in the presence of S9 mix after treatment with 300 µg/ml. In addition, reduced cell numbers were observed after treatment with 3000 µg/ml and above in the absence of S9 mix and after treatment with 300 µg/ml and above in the presence of S9 mix. In the absence of S9 mix, the test article did induce a significant increase in the number of cells carrying structural chromosome aberrations after treatment with 4000 µg/ml. This increase beyond our historical control data range was regarded as being relevant in this test system. However, it has to be considered, that the increase was observed at a high concentration inducing strong toxic effects indicated by strongly reduced cell numbers. In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.


In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 40571 is considered to be weakly mutagenic in this chromosome aberration test. Since increased aberration frequencies were observed only in the presence of strong toxicity indicated by reduced cell numbers, it cannot be excluded that an indirect, none genotoxic DNA damaging mechanism was involved.


 


Mammalian gene mutation study in vitro (HPRT):


A mammalian cell gene mutation assay (HPRT locus) was carried out with FAT 40571/C according to OECD 476 and B 17 guidelines. In this assay, V79 cells cultured in vitro were exposed to FAT 40571/C TE dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20 h treatment) at concentrations of


- 50, 100, 200, 400, 600, 800, 1000, 1400, 1800 and 2000 µg/mL (without metabolic activation, Experiment I)


- 100, 250, 750, 1500, 1750, 2000, 2250, 2500 and 3000 µg/mL (with metabolic activation, Experiment I)


- 100, 250, 500, 750, 1000, 1300 and 1600 µg/mL (without metabolic activation, Experiment II)


- 50, 100, 200, 400, 800, 1400, 2000, 2300, 2900 and 3200 µg/mL (with metabolic activation, Experiment II)


- 10, 25, 50, 100, 250, 500, 750, 1000, 1300 and 1600 µg/mL (without metabolic activation, Experiment III).


 


Taken together these two experiments, it could be shown, that there is no mutagenic effect in the experiments with long term incubation (without metabolic activation). The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background.


In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40571/C TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.


 


Micronucleus assay in vivo:


As a result from an in vivo assay performed, to further assess the weakly positive response from the in vitro study, a micronucleus assay in bone marrow cells of mouse was performed.


This study was performed to investigate the potential of Reactive Brown 49 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was carried out according to OECD 474 and EU B.12 guidelines. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg bw 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.


Under the experimental conditions reported, the Reactive Brown 49 did not induce micronuclei. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay in vivo and the weakly positive finding from the in vitro study was not confirmed.


Reactive Brown 49 is found negative in Ames assay and in-vitro mammalian cell gene mutation test however, was found weakly mutagenic in in-vitro chromosomal assay at cytotoxic concentrations; while in an in-vivo micronucleus assay Reactive Brown 49 is found negative. Hence, Reactive Brown 49 considered to be non-mutagenic and non-cytogenic.

Justification for classification or non-classification

Based on the results from three in vitro and one in vivo mutagenicity studies, the Reactive Brown 49 is not considered to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008