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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 92349/B was found to be mutagenic in bacterial reverse mutation assays, while the read across substance was neither mutagenic in bacterial reverse mutation assay and in vitro gene mutation study in mammalian cells nor clastogenic in an in vitro chromosomal aberration assay. Hence, it was considered to be devoid of potential to cause genetic toxicity. Based on the principles of read across, the substance under evaluation, FAT 92349/B was also considered to be not genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Sep, 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar 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 included
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Test material identified as: FAT 40068/B
Target gene:
Histidine-requiring strains of Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Activation mixture (rat liver microsomes and co-factors)
Test concentrations with justification for top dose:
25, 75, 225, 675, and 2025 μg/0.1 mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA 1535
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunoblastin
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation).


DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : One

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Colony counting
Rationale for test conditions:
As per the guidelines
Evaluation criteria:
The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the experiments without and with microsomal activation, treatment with FAT 40068/B led to an increase in the number of backmutant colonies of Strains TA 98, TA 100 and TA 1537. The highest incidence of back-mutant colonies was observed at the following drug-concentrations: Strain TA 98 at 2025 µg/0.1 ml and Strains TA 100 and TA 1537 at 675 µg/0.1 ml (without microsomal activation) and 2025 µg/0.1 ml (with microsomal activation).
Conclusions:
FAT 40068/B was found to have mutagenic potential in this bacterial reverse mutation assay.
Executive summary:

Preparation FAT 40'068/B was tested for mutagenic effects on histidine- auxotrophic mutants of Salmonella typhimurium. This test was conducted in accordance to method similar to OECD TG 471. The investigations were performed with the following concentrations of the test substance with and without microsomal activation: 25, 75, 225, 675, and 2025 μg/0.1 mL. Appropriate positive controls were used for each strains. In the experiments performed without and with microsomal activation, 'the number of back-mutant colonies of strains TA 98, TA 100 and TA 1537 was significantly greater after treatment with FAT 40068/B than in the controls. Hence, it was concluded that FAT 40068/B exerted a clear-cut mutagenic action in this test system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Feb, 1993 to 22 March, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Published on May 26, 1983
Deviations:
yes
Remarks:
A statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Published on September 19, 1984
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
Published on July 13, 1987
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material identified as: Lanasol Blau 3G roh feucht (FAT 92349/A)
- Source and batch No.of test material: 56
- Expiration date of the batch: November 1997

OTHER SPECIFICS:
- Purity: 68 %
Target gene:
Histidine-requiring strains of Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A.
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Rat liver post-mitochondrial fraction and co-factors.

Preparation of the metabolic activation mixture: Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf [SPF]), reared at the Animal Farm of CIBA-GEIGY, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl and the 9000x g supernatant (S9) was stored at approximately -80 °C for no longer than one year. The protein contents of the S9 fractions were 35.8 and 40.1 mg/mL.
Test concentrations with justification for top dose:
Range finding study: 20.5761, 61.7284, 185.1852, 555.5556, 1666.6667 and 5000 µg/plate
Mutagenicity test, original experiment: 61.7284, 185.1852, 555.5556, 1666.6667 and 5000 µg/plate
Mutagenicity test, confirmatory experiment: 61.7284, 185.1852, 555.5556, 1666.6667 and 5000 µg/plate

Justification for high dose: The highest dose (limit concentration) used in range finding study (i.e. 5000 µg/plate) had no inhibitory effect on the growth of the bacteria.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: bidistilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Bi-distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Bi-distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With microsomal activation
Details on test system and experimental conditions:
DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours


METHODS FOR MEASUREMENTS OF GENOTOXICIY : Counting the number of colonies and determining the background lawn.
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. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response:
The test substance is considered to be mutagenic in this test system if the following conditions are met: At least a reproducible meaningful increase of the. mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538.
Generally a concentration-related effect should be demonstrable.
Key result
Species / strain:
other: TA 100, TA 98, TA 1537 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
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:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the experiment without activation Lanasol Blau 3G roh feucht (FAT 92349/A) led to an increase in the number of revertants at the upper concentrations. In the experiment with metabolic activation, this effect occurred at the highest concentrations only. The test material exerted no inhibitory effect on the growth of the bacteria.

Mutagenicity test, original experiment:
In the original experiment performed without metabolic activation, treatment of strains TA 100, TA 98, TA 1537 and TA 1538 with Lanasol Blau 3G roh feucht (FAT 92349/A) led to a distinct and concentration-dependent increase in the incidence of histidine-prototrophic mutants by comparison with the negative controls. No effects were observed with strain TA 1535. In the experiment performed with activation, treatment with Lanasol Blau 3G roh feucht (FAT 92349/A) resulted in an increase in the number of back-mutants with strains TA 100, TA 98 and TA 1537 at the upper concentrations.

Mutagenicity test, confirmatory experiment:
In the confirmatory experiment performed without metabolic activation, after treatment of the cultures with Lanasol Blau 3G roh feucht (FAT 92349/A), again an increase in the number of back-mutant colonies was registered with strains TA 100, TA 98, TA 1537 and TA 1538. In the experiment performed with activation a similar, but less pronounced effect occurred on strains TA 100, TA 98 and TA 1537 at the upper concentrations.
Conclusions:
Lanasol Blau 3G roh feucht (FAT 92349/A) was found to exert mutagenic action in bacterial cells.
Executive summary:

A bacterial cell reverse mutation study was conducted to evaluate the mutagenic potential of the test substance. This test was conducted in accordance with OECD TG 471, EU method B.13/14 and EPA OTS 798.5256 in a GLP certified laboratory. The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were exposed to the test substance without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations. In the original experiment performed without metabolic activation, treatment of strains TA 100, TA 98, TA 1537 and TA 1538 with Lanasol Blau 3G roh feucht (FAT 92349/A) led to a distinct and concentration-dependent increase in the incidence of histidine prototrophic mutants by comparison with the negative controls. No effects were observed with strain TA 1535. In the experiment performed with activation, treatment with Lanasol Blau 3G roh feucht (FAT 92349/A) resulted in an increase in the number of back-mutants with strains TA 100, TA 98 and TA 1537 at the upper concentrations. In the confirmatory experiment performed without metabolic activation, after treatment of the cultures with Lanasol Blau 3G roh feucht (FAT 92349/A), again an increase in the number of back-mutant colonies was registered with strains TA 100, TA 98, TA 1537 and TA 1538. In the experiment performed with activation a similar, but less pronounced effect occurred on strains TA 100, TA 98 and TA 1537 at the upper concentrations. Based on the results of these experiments and on standard evaluation criteria, it is concluded that Lanasol Blau 3G roh feucht (FAT 92349/A) exerted mutagenic action in this test system. This effect was less pronounced, when metabolic activation mixture was added.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer chapter 13 for detailed read across justification.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the osmolality of the cultures.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The source substance, FAT 41001/H TE was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures in the presence and absence of S9.
Executive summary:

The source substance, FAT 41001/H, 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 41001/H was prepared in distilled water and evaluated in a preliminary toxicity assay at concentrations of 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the osmolality of the cultures. pH adjustment with 1N HCl was required at concentrations ≥2500 μg/mL to maintain neutral pH. Adjusted relative survival was 22.37 and 11.39 % at concentrations of 2500 μg/mL with S9 and 1250 μg/mL without S9, respectively. Adjusted relative survival approximated 0 % at all higher concentrations. Based on these results, FAT 41001/H was evaluated in the definitive mutagenicity assay at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000 and 4000 μg/mL with S9, and 29.7, 59.3, 119, 237, 475, 949, 1270 and 1690 μg/mL without S9. No visible precipitate was observed at the beginning or end of treatment. pH adjustment with 1N HCl again was required at concentrations from 29.7 to 1690 μg/mL to maintain neutral pH. pH was measured at 4000 μg/mL after the addition of S9 and was within 7 ± 0.5; therefore, no pH adjustment was performed at concentrations ≥2250 μg/mL. The average adjusted relative survival was 11.10 % at a concentration of 1690 μg/mL without S9. Cultures treated at concentrations of 237, 475, 949, 1270 and 1690 μg/mL without S9 were chosen for mutant selection (cultures treated at concentrations of 29.7, 59.3 and 119 μg/mL without S9 were discarded prior to selection because a sufficient number of higher concentrations was available). No statistically significant increases in mutant frequency were observed without S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01). However, no cultures with S9 exhibited 10 to 20 % adjusted relative survival, and this portion of the assay was repeated using adjusted concentrations. FAT 41001/H was evaluated in the mutagenicity assay retest at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000, 4000 and 5000 μg/mL with S9. No visible precipitate was observed at the beginning or end of treatment. No pH adjustment was performed per the Study Director’s instructions and based on the pH results in the initial mutagenicity assay. The average adjusted relative survival was 14.73 % at a concentration of 4000 μg/mL with S9. Cultures treated at concentrations of 475, 949, 1690, 3000 and 4000 μg/mL with S9 were chosen for mutant selection (cultures treated at concentrations of 237, 1270 and 2250 μg/mL with S9 were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 5000 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). No statistically significant increases in mutant frequency were observed with S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. These results indicate FAT 41001/H was negative in the in vitro mammalian cell forward gene m (CHO/HPRT) assay with duplicate cultures in the presence and absence of S9, under the conditions and according to the criteria of the test protocol.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer chapter 13 for detailed read across justification.
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitate is visible at the beginning and at the end of treatment period at >= 750 μg/mL (exposure time 4 hours)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity of test item in CHO cells when treated for 4 hours in the absence of S9 activation was 37% at 750 μg/mL.Precipitate is visible at the beginning and at the end of treatment period at >= 750 μg/mL (exposure time 4 hours)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity of test item in CHO cells when treated for 20 hours in the absence of S9 activation was 52% at 300 μg/mL.Preciüitate is visible at 750 μg/mL at the beginning of treatment period and at >=550 μg/mL at the end of the treatment period.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
The source substance, FAT 41001/H 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:

The source substance FAT 41001/H 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. 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 the 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. Dose formulations were adjusted for the purity of the test substance (77.3 %), using a correction factor of 1.29. Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, upon sonication for ten minutes at 27 °C, the test substance formed workable suspensions in water at a concentration range of approximately 10 to 50 mg/mL. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively. 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 2000 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 600 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 600 μg/mL in all three treatment groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 50 to 1000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 50 to 750 μg/mL for the non-activated 20-hour exposure group. In the chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 300 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 750 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at dose levels ≥ 550 μg/mL in the non-activated 20-hour exposure group. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 55 ± 5 % reduction in cell growth index relative to the vehicle control, which met the dose limit as recommended by testing guidelines for this assay. No significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). 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 41001/H was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

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

Genetic toxicity in vivo

Description of key information

FAT 92349/B did not induce clastogenicity in a micronucleus assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Nov, 1996 to 19 Dec, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 26, 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
dated December 29, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material identified as: FAT 92349/A
- Source and batch No.of test material: 56
- Expiration date of the batch: November 30, 1997

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

OTHER SPECIFICS:
- Aggregate state at room temperature: Solid
- Colour: Dark-blue
- Purity: 68 %
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
- Strain: NMRI
- Source: BRL, CH-4414 Füllinsdorf
- Number of animals: 72 (36 males/36 females)
- Initial age at start of acclimatization: 8-12 weeks
- Acclimatization: minimum 5 days
- Initial body weight at start of treatment: males mean value 30.9 g (SD ± 2.2 g); females mean value 23.4 g (SD ± 1.4 g)

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Bedding: Granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Feed: Pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Environment: Temperature 21 ± 3 °C
- Relative humidity: 30-70 %
- Artificial light: 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Deionised water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in deionised water.

- All animals received a single standard volume of 20 mL/kg bw orally.
Duration of treatment / exposure:
The animals received the test article, the vehicle or the positive control substance once.
Frequency of treatment:
Once
Post exposure period:
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
670 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
24 and 48 h preparation interval
No. of animals per sex per dose:
6 males and 6 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;
- Justification for choice of positive control(s): Recommended by the guidelines
- Route of administration: oral
- Doses: 40 mg/kg b.w.
- Frequency: once
Tissues and cell types examined:
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-Griinwald (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.
Details of tissue and slide preparation:
Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.
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:
Non parametric Mann-Whitney test
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: A single dose of 2000 mg/kg bw was used
- Clinical signs of toxicity in test animals: The treated animals expressed no toxic reactions. 6 hours after treatment the urine was weak blue.
- Rationale for exposure: Highest (limit dose) was used as recommended by the guideline


RESULTS OF DEFINITIVE STUDY
- The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that FAT 92349/A had no cytotoxic properties in the bone marrow.
- In comparison to the corresponding vehicle controls there was no significant enhancement 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 92'349/A were in the range of the vehicle control group.

Results with positive control: 40 mg/kg bw cyclophosphamide showed a statistically significant increase of induced micronucleus frequency.

Conclusions:
The test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Executive summary:

The test substance FAT 92349/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse as per the methodology described in OECD Guideline 474 and EU Method B.12. The test substance was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 mL/kg bw. 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis. Twelve animals (6 males, 6 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal (exception animal no. 56 and 59 = 2000 PCEs) 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 NCE per 1000 PCE. The following dose levels of the test substance were investigated:

24 h preparation interval: 200, 670, and 2000 mg/kg bw.

48 h preparation interval: 2000 mg/kg bw.

As estimated by a pre-experiment 2000 mg/kg bw. (highest guideline-recommended dose) of the test article were administered as maximum dose and tolerated by the animals. None of the animals expressed toxic reactions. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that FAT 92349/A had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement 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 92349/A were in the range of the vehicle control group. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Study period:
Subject to ECHA approval
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
Hazard endpoint for which vertebrate testing was proposed: Genetic toxicity in vivo with the registered substance.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Reactive Blue 069

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no available GLP studies on the substance or on read-across analogues suitable to fill the endpoint.
- Available non-GLP studies: There are no available non-GLP studies on the substance or on read-across analogues suitable to fill the endpoint.
- Historical human data: There is no historical human data on the substance or on read-across analogues suitable to fill the endpoint.
- (Q)SAR: (Q)SAR analysis is not sufficient to fill the endpoint. There are no adequate models to address this end point.
- In vitro methods: Already available, but further in vivo data needed.

OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Weight of evidence: There is not sufficient data on the substance or read across analogues to be able to establish a weight of evidence argument.
- Grouping and read-across: There is not sufficient data on the substance or read-across analogues to be able to group or propose read-across.
- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: Not applicable
- Other reasons [if applicable]: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- Test proposal is fully in line with ECHA guidance document*, and can neither be replaced by in vitro testing nor by using other data from other substances.

* Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: OECD Guideline 486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

GENETIC TOXICITY OF FAT 92349/B

The genetic toxicity database for FAT 92349/B consists of two Ames assays and a micronucleus assay. No study with investigations into the potential of FAT 92349/B to cause mutations in mammalian cells is available.

Bacterial reverse mutation assays:

A bacterial cell reverse mutation study was conducted to evaluate the mutagenic potential of the test substance. The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were exposed to the test substance without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate, in two stages. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations. In the original experiment performed without metabolic activation, treatment of strains TA 100, TA 98, TA 1537 and TA 1538 with Lanasol Blau 3G roh feucht (FAT 92349/A) led to a distinct and concentration-dependent increase in the incidence of histidine prototrophic mutants by comparison with the negative controls. No effects were observed with strain TA 1535. In the experiment performed with activation, treatment with Lanasol Blau 3G roh feucht (FAT 92349/A) resulted in an increase in the number of back-mutants with strains TA 100, TA 98 and TA 1537 at the upper concentrations. In the confirmatory experiment performed without metabolic activation, after treatment of the cultures with Lanasol Blau 3G roh feucht (FAT 92349/A), again an increase in the number of back-mutant colonies was registered with strains TA 100, TA 98, TA 1537 and TA 1538. In the experiment performed with activation a similar, but less pronounced effect occurred on strains TA 100, TA 98 and TA 1537 at the upper concentrations. Based on the results of these experiments and on standard evaluation criteria, it is concluded that Lanasol Blau 3G roh feucht (FAT 92349/A) exerted a clear-cut mutagenic action in this test system. This effect was less pronounced, when metabolic activation mixture was added.

In a supporting study, preparation FAT 40'068/B was tested for mutagenic effects on histidine- auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the test substance with and without microsomal activation: 25, 75, 225, 675, and 2025 μg/0.1 mL. In the experiments performed without and with microsomal activation, 'the number of back-mutant colonies of Strains TA 98, TA 100 and TA 1537 was significantly greater after treatment with FAT 40'068/B than in the controls. Hence, it was concluded that FAT 40'068/B exerted a clear-cut mutagenic action in this test system.

Micronucleus assay:

The test substance FAT 92'349/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse as per the methodology described in OECD Guideline 474 and EU Method B.12. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that FAT 92'349/A had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement 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 92'349/A were in the range of the vehicle control group. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

FAT 92349/B was found to be mutagenic in bacterial reverse mutation assays, while it was not clastogenic in an in vivo micronucleus assay. However, no study with investigations into the potential of FAT 92349/B to cause mutations in mammalian cells is available. Hence, the genetic toxicity database is considered to be incomplete. To complete this, a test according to 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo) is proposed.

 

GENETIC TOXICITY OF THE SOURCE SUBSTANCE (FAT 41001)

To complete the assessment of the genetic toxicity potential of FAT 92349/B, the genetic toxicity studies with FAT 41001 were used. FAT 92349/B and read across substance, FAT 41001 are both reactive dyes originating from bromamine acid, hence the read across was justified. For further details please refer the read across justification document.

Bacterial reverse mutation assay:

The read across substance, FAT 41001/F was evaluated for mutagenic activity in bacterial test systems in the absence and presence of a rat liver S9 activity system. The bacterial strains used were Salmonella typhimurium Strains: TA 98, TA 100, TA 102, TA 1535 and TA 1537. In the original as well as confirmatory experiments carried out without and with metabolic activation, none of the tested concentrations of FAT 41001/F led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Hence, it was concluded that FAT 41001/F and its metabolites did not induce gene mutations in the strains of S. typhimurium used in this bacterial reverse mutation assay.

In vitro gene mutation study in mammalian cells (HPRT):

The source substance, FAT 41001/H, 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 41001/H TE was evaluated in the definitive mutagenicity assay at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000 and 4000 μg/mL with S9, and 29.7, 59.3, 119, 237, 475, 949, 1270 and 1690 μg/mL without S9. The average adjusted relative survival was 11.10 % at a concentration of 1690 μg/mL without S9. Cultures treated at concentrations of 237, 475, 949, 1270 and 1690 μg/mL without S9 were chosen for mutant selection (cultures treated at concentrations of 29.7, 59.3 and 119 μg/mL without S9 were discarded prior to selection because a sufficient number of higher concentrations was available). No statistically significant increases in mutant frequency were observed without S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01). However, no cultures with S9 exhibited 10 to 20 % adjusted relative survival, and this portion of the assay was repeated using adjusted concentrations.

FAT 41001/H TE was evaluated in the mutagenicity assay retest at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000, 4000 and 5000 μg/mL with S9. The average adjusted relative survival was 14.73 % at a concentration of 4000 μg/mL with S9. Cultures treated at concentrations of 475, 949, 1690, 3000 and 4000 μg/mL with S9 were chosen for mutant selection (cultures treated at concentrations of 237, 1270 and 2250 μg/mL with S9 were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 5000 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). No statistically significant increases in mutant frequency were observed with S9 (p> 0.05). Hence, based on the above results it was concluded that FAT 41001 did not 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).

Chromosomal aberration assay in vitro:

The source substance, FAT 41001/H TE, 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. The 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. The doses chosen for the chromosome aberration assay ranged from 50 to 1000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 50 to 750 μg/mL for the non-activated 20-hour exposure group. No significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p >0.05; Fisher’s Exact and Cochran-Armitage tests). Hence, the read across substance, FAT 41001/H 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.

Based on the above results, it can be concluded that the FAT 41001 did not induce mutagenicity in bacterial or mammalian cells as well as it was not clastogenic in chromosomal aberration assay in vitro. Hence, it can be considered to be devoid of genetic toxicity poetential.

CONCLUSION

FAT 92349/B was found to be mutagenic in bacterial reverse mutation assays, while it was not clastogenic in an in vivo micronucleus assay. However, no study with investigations into the potential of FAT 92349/B to cause mutations in mammalian cells is available. Hence, the genetic toxicity database is considered to be incomplete.

FAT 41001, was considered to be a read across substance for the substance under evaluation, FAT 92349/B, as both are reactive dyes originating from bromamine acid, hence the read across was justified. For further details please refer the read across justification document.

The source substance was neither mutagenic in bacterial reverse mutation assay and in vitro gene mutation study in mammalian cells nor clastogenic in an in vitro chromosomal aberration assay. Hence it was considered to be devoid of potential to cause genetic toxicity. Based on the principles of read across, the substance under evaluation, FAT 92349/B was also considered to be not genotoxic.

However, looking at the high degree of uncertainty involved in read across (the two substances, FAT 92349/B and FAT 41001 have structural dissimilarities that may lead to differerent behaviour in biological systems), a testing proposal for an in vivo unscheduled DNA synthesis assay has been included in the dossier.

Justification for classification or non-classification

Based on the study results with FAT 92349/B and read across substance, FAT 41001, the substance was considered to be not genotoxic, hence does not warrant classification for genetic toxicity as per the CLP (Regulation [EC] 1272/2008) criteria.