[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Blue 050 had positive outcomes in the bacterial reverse mutation assay and in vitro mammalian chromosomal aberration assay, while was found to be not mutagenic in an in vitro mammalian cell gene mutation assay.

Link to relevant study records

Reference

Reference 1

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:

1996

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:

yes

Remarks:

only four tester strains tested; no tester strain to detect cross-linking mutagens was included.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not Applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

- Confirmatory experiment: 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

The starin cultures were stored as stock cultures in ampoules with nutrient broth +5 % DMSO in liquid nitrogen.

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 - strains 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, 4-NOPD

Remarks:

WIthout metabolic activation - Strains TA 1537, TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation - Starins TA 1535, TA 1537, TA 98, TA 100

Details on test system and experimental conditions:

Selection Agar:
The plates with the minimal agar were obtained.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
-corresponding background growth on both negative control and test plates.
-normal range of spontaneous reversion rates.

Range of spontaneous reversion frequencies
1535 1537 98 100
10-29 5-28 15-57 77-189

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 atleast one test concentration in induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non mutagenic in this system. A biologically relevant response is described as follows:
A test article is considered mutagenic if the number of reversions is atleast twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.

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 criteria described above or not.

Statistics:

The colonies were counted using the AUTOCOUNT. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the intense colour of the test article the colonies were counted manually from 2500 µg/plate upto 5000 µg/plate.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The generally accepted conditions for the evaluation of the results are:

- corresponding background growth on both negative control and test plates

- normal range of spontaneous reversion rates.

 

Range of spontaneous reversion frequencies • (3)
1535	1537	98	100
10 - 29	5-28	15-57	77 - 189

  

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 dose related and reproducible increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A biologically relevant response is described as follows:

A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. 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 criteria described above or not. No toxic effects evident as a reduction in the number of revertants occurred in any of the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

In experiment I a dose dependent increase in revertant colony numbers exceeding the recommended threshold for a mutagenic response was observed in the absence of metabolic activation in strains TA 1537 (recommended factor 3.0, cf. page 17), TA 98 (recommended factor 2.0) and TA 100 (recommended factor 2.0). [n the presence of metabolic activation only strain TA reached this threshold. A dose dependent increase was also observed in strains TA 1535 (without S9 rnix) and TA 1537 (with S9 mix) but the recommended factor of 3.0 was not reached. In experiment II a dose dependent increase in revertant colony numbers was observed in strains TA 1535, TA 1537 (with and without S9 mix), TA 98 (without S9 mix) and TA 100 (with and without S9 mix). In the absence of metabolic activation, the threshold for a mutagenic response (factor 3.0 in strains TA 1535 and TA 1537 and 2.0 in strains TA 98 and TA 100) was exceeded in all strains used, whereas, in the presence of metabolic activation the recommended factor could only be reached in strain TA 100.

 

In both experiments a dose dependent and reproducible increase in revertant colony numbers was observed following treatment with Lanasol Blue 3R with and without S9 mix in all strains used. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

 

Conclusions:

FAT 40069 induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 98, TA 100, TA 1535 and TA 1537.

Executive summary:

The study was performed to investigate the potential of Lanasol Blue 3R to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. 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 following concentrations:

33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate

No toxic effects, evidenced by a reduction in the number of revertants, occured in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth upto 5000.0 µg/plate with and without S9 mix in all strains used. In both experiments, a dose dependent and reproducible increase in revertant colony numbers was observed following treatment with the test article in the absence of metabolic activation in all strains used. In the presence of metabolic activation a relevant increase of the factor exceeding the threshold of 2.0 occured in strain TA 100 exclusively. 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 mutagenecity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98 and TA 100. Therefore, FAT 40069 is considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

Deviations:

yes

Remarks:

The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml. In the pre-test the protein concentration was 50.4 mg/ml and 30.0 mg/ml in the main experiment.

GLP compliance:

yes (incl. QA statement)

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: 018631.A8
- Expiration date of the lot/batch: August 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: at least 24 h
- Solubility and stability of the test substance in the solvent/vehicle: 100g/l (at 20 °C) in water

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from the livers of 8-12 weeks old male rats, strain Wistar Hanlbm.

Test concentrations with justification for top dose:

Pre-test on toxicity (without S9 mix, exposure time 24 hours): 15.6, 31.3, 62.5, 125, 250, 500, 750, 1000 µg/L.
Pre-test on toxicity (with S9 mix, exposure time 4 hours): 62.5, 125, 250, 500, 750, 1000, 1500, 2000 µg/L.
Main experiment (without S9 mix): 31.3, 62.5, 125, 250, 375, 500 µg/L.
Main experiment (with S9 mix): 62.5, 125, 250, 500, 750, 1000 µg/L.

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test article was dissolved in culture medium MEM. The solvent was chosen according to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent controls (MEM) were performed.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Positive control substance with metabolic activation part.

Details on test system and experimental conditions:

Seeding of the Cultures
- Exponentially growing stock cultures more than 50 % confluent were treated with trypsin at 37 °C for approximately 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg.
- Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA.
- The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 104 - 6 x 104 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

Treatment
Exposure period 4 hours (with S9 mix):
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing different concentrations of the test article and 50 ul/ml S9 mix. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
Exposure period 18 (without S9 mix):
The culture medium of exponentially growing cell cultures was replaced with complete medium (10 % FCS) containing different concentrations of the test article without S9 mix. The medium was not changed until preparation of the cells. All cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO: (95.5 % air). 16 h after the start of the treatment colcemid was added (0.2 ug/ml culture medium) to the cultures. 2 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt). Additionally, two cultures V79 cells per treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the substance is given as reduction of % cells as compared to the solvent control.

Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik". Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Statistics:

Statistical significance at the five per cent level (p <0.05) was evaluated by means of the Fischer's-exact-test. Evaluation was performed only for cells carrying aberrations exclusive.
Statistical significance was confirmed by means of the Fischer's exact test (10). However, both biological and statistical significance should be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The evaluation of the treatment concentration (375 microgr/ml without S9 mix and 750 microgr with S9 mix was not possible due to the test article induced toxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Range Finding/Screening Studies: The highest applied concentration in the pre-test on toxicity (1000 µg/ml without and 2000 ug/ml with S9 mix) was chosen with regard to the results of RCC project 619312 (HPRT-Assay).

Comparision with historical control data:
Additional information on Cytotoxicity: Test article concentrations between 15.6 and 1000 µg/ml (without S9 mix) and 62.5 and 2000 µg/ml(with S9 mix) were applied for the assessment of the cytotoxic potential.

Comparision with historical control data: In the absence of S9 mix, the number of cells carrying structural chromosome (at 250 µg/ml 2.0 % aberrant cells exclusive gaps) was within our historical control data range (0 - 4 %).

Summary of results of the chromosome aberration study with FAT 40069/C:

Exposure period	Conc. of FAT 40069/C in ug/ml	Polyploid cells in %	Mitotic index in % of control	incl. gaps	Aberrant cells in % excl. gaps *	Exchanges
Without metabolic activation
18 h	solvent control	3.6	100.0	1.5	0.5	0.0
18 h	positive control	4.1	51.4	16.5	15.0s	11.5
18 h	250	2.8	82.1	3.0	2.0	1.0
With metabolic activation
4 h	solvent control	4.6	100.0	1.5	0.5	0.5
4 h	positive control	3.1	67.7	21.5	18.5 s	8.0
4 h	500	3.3	80.5	12.5	12.0 s	8.0

*Inclusive cells carrying exchanges

s Aberration frequency statistically significant higher than solvent control values.

Conclusions:

FAT 40069/C is considered to be clastogenic in the presence of S9 mix in chromosome aberration in V79 cells.

Executive summary:

The test article FAT 40069/C, dissolved in medium, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. This experiment was done according to the OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test). The exposure period was 4 h with and 18 h without metabolic activation. The chromosomes were prepared 18 h after start of treatment with the test article. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In the absence of S9 mix the mitotic index and the cell growth was not reduced after treatment with 250 µg/ml (82.1 % and 77 % of solvent control). In the presence of S9 mix the mitotic index and the cell growth was not reduced after treatment with 500 µg/ml (80.5 % and 100 % of solvent control). However, the evaluation of the next higher treatment concentration (375 µg/ml without S9 mix; 750 µg/ml with S9 mix) was not possible due to test article induced toxicity. In the presence of S9 mix, a biologically relevant and significant increase in the number of cells carrying structural chromosome aberrations was observed (at 500 µg/ml 12.0 % versus 0.5 % aberrant cells in the solvent control). In conclusion, FAT 40069/C is considered to be clastogenic in the presence of S9 mix in chromosome aberration in V79 cells.

Genetic toxicity in vivo

Description of key information

FAT 40069/C was considered to be non- clastogenic in a micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

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 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: 018631.A8
- Expiration date of the lot/batch: August 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: at least 24 hours
- Solubility and stability of the test substance in the solvent/vehicle: 100g/l (at 20 °C) in water

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at study initiation: 8-12 weeks
- Weight at study initiation:
*males mean value 33.8 g (SD ± 3.0 g)
*females mean value 27.3 g (SD ± 1.4 g)


- Housing: single
- Cage type: Makrolon Type I, with wire mesh top
- Feed: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Water : tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): relative humidity 30-70 %
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

The animals were distributed into the test groups at random and identified by cage number.

Route of administration:

oral: gavage

Vehicle:

Deionised water

Details on exposure:

Approximately 18 hours 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 animals body weight. The animals received the test item, 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 hours after treatment, respectively.

Duration of treatment / exposure:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Frequency of treatment:

One treatment

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

Sampling time: 24 h

Dose / conc.:

670 mg/kg bw (total dose)

Remarks:

Sampling time: 24 h

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

Sampling time: 24 h

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

Sampling time: 48 h

No. of animals per sex per dose:

Twelve animals, six males and six females, were treated per dose group and sampling time.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: CPA; Cyclophosphamide
- Route of administration: Once / Orally
- Doses / concentrations: 10 ml/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 25 °C only 3.5 % of its potency is lost after 24 hours.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

The epiphyses of femur 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 air dried smear then stained with May-Grünwald /Giemsa. At least one slide was made from each bone marrow sample.

Evaluation criteria:

Analysis of Cells:
Evaluation of the slides was performed with 100x oil immersion objectives. 2000 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 2000 the PCEs. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.

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:

RESULTS OF RANGE-FINDING STUDY:
Pre-Experiment for Toxicity
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 40'069/C formulated in deionised water.
The volume administered was 10 ml/kg b.w..
Reduction of spontaneous activity:
-1/1 (after 1 hour post treatment male/female).
-1/1 (after 6 hours post treatment male/female).
-1/1 (after 24 hours post treatment male/female).
-0/0 (after 48 hours post treatment male/female).

Additionally, the urine of the treated animals was coloured: after 1 hour = blue; after 6 hours = green; after 24 hours = light green.
On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

RESULTS OF DEFINITIVE STUDY:
Summary of micronucleus test results:
- Test group / dose mg/kg b.w. / Sampling time (h)/ PCEs with micronuclei (%) / range / PCE/ NCE.
- vehicle / 0 mg/kg b.w. / 24 hours /0.150 % /0-7 /2000/ 1784.
- test article / 200 mg/kg b.w. / 24 hours /0.075 % /0-3 /2000/ 1885.
- test article / 670 mg/kg b.w. / 24 hours /0.045 % /0-2 /2000/ 1710.
- test article / 2000 mg/kg b..w / 24 hours /0.070 % /1-3 /2000/ 1983.
- Cyclo-phosphamide / 40 mg/kg b.w. / 24 hours /0.070 % /12-5 /2000/ 2064.
- test article / 2000 mg/kg b.w. / 48 hours /0.055 % /0-4 /2000/ 1828.

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle contro vs test group	Significance	P
200 mg FAT 40'069/C/kg b.w.; 24 h	n.t.	-
670 mg FAT 40'069/C/kg b.w.; 24 h	n.t.	-
2000 mg FAT 40'069/C/kg b.w.; 24 h	n.t.	-
40mgCPA/kgb.\v.;24h	+	<0.0001
2000 mg FAT 40'069/C/kg b.w.; 48 h	n.t.	-

+ = significant;

n.t = not tested, as the mean micronucleus frequency was not above the vehicle control value

Conclusions:

FAT 40069/C is considered to be non-mutagenic in the micronucleus assay.

Executive summary:

An in vivo study was performed to investigate the potential of FAT 40069/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This test was performed according to the OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and follows GLP methodology. The test item 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 item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 /sex) 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 item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. 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 item and with any dose level used (Up to 2000 mg/kg/d). However the positive control at 40 mg/kg b.w. cyclophosphamide administered showed a substantial increase of induced micronucleus frequency. In conclusion under the experimental conditions reported, the test item did not induce micronuclei by the in-vivo mouse micronucleus test. So, FAT 40069/C is considered to be non- clastogenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The study was performed as the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 in two independent experiments both with and without liver microsomal activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. In both experiments, a dose dependent and reproducible increase in revertant colony numbers was observed following treatment with the test article in the absence of metabolic activation in all strains used. In the presence of metabolic activation a relevant increase of the factor exceeding the threshold of 2.0 occurred in strain TA 100 exclusively. Under the study conditions, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro chromosomal aberration assay:

In another key in vitro study, the test article FAT 40069/C, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. This experiment was performed according to the OECD Guideline 473. In the absence of S9 mix the mitotic index and the cell growth was not reduced after treatment with 250 µg/ml. In the presence of S9 mix the mitotic index and the cell growth was not reduced after treatment with 500 µg/ml. However, the evaluation of the next higher treatment concentration (375 µg/ml without S9 mix; 750 µg/ml with S9 mix) was not possible due to test article induced toxicity. In the presence of S9 mix, a biologically relevant and significant increase in the number of cells carrying structural chromosome aberrations was observed (at 500 µg/ml 12.0 % versus 0.5 % aberrant cells in the solvent control). In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article FAT 40069/C induced structural chromosome aberrations in V79 cells (Chinese hamster cell line).

In vitro mammalian cell gene mutation assay:

Another in vitro assay was performed to assess the potential of the test article to induce gene mutations by means of a HPRT experiment using the Chinese hamster cell line V79. The experiment was done according to the OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test) and following GLP. The assay was performed with and without liver microsomal activation. The test article was tested at the concentrations up to 1000 µg/ml. Up to the highest investigated concentration no relevant increase in mutant colony numbers was observed in this experiment. An isolated increase at 500 µg/ml in the absence of metabolic activation was judged to be based upon toxic effects rather than indicating a mutagenic effect. 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 at the HPRT locus in V79 cells. Therefore, FAT 40069/C is considered to be non-mutagenic in this HPRT assay.

In vivo micronucleus assay:

A key in vivo study was performed to investigate the potential of FAT 40069/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This test was performed according to the OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and follows GLP methodology. The test item was formulated in deionised water. The volume administered orally was 10 ml/kg b.w. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 40069/C 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 item and with any dose level used. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40069/C is considered to be non-clastogenic in this micronucleus assay.

Conclusion:

Reactive Blue 050 had positive outcomes in the bacterial reverse mutation assay and in vitro mammalian chromosomal aberration assay, while was found to be not mutagenic in an in vitro mammalian cell gene mutation assay. Further the clastogenic effect observed in the in vitro study could not be seen in the the in vivo micronucleus assay. Hence, considering all the above-mentioned studies, FAT 40069 can be considered as non-genotoxic in nature.

Justification for classification or non-classification

FAT 40069 can be considered as non-genotoxic in nature and hence does not need to be classified as per the Regulation (EC) No. 1272/2008.