Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(1-sulphonato-2-naphthyl)azo]naphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 40147 is considered to be not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

equivalent or similar 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.

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

Histidine-auxotrophic strains of Salmonella typhimurium

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from rats

Test concentrations with justification for top dose:

0.2, 2, 20, 200 and 2000 µg (or nl) per Petri dish

Vehicle / solvent:

distilled-water

Untreated negative controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without S9: TA 98 daunomycine (1 µg); TA 100 and TA 1535: MNNG (1.6 µg); and TA 1537: 9-aminoacridine (50 µg). With S9: all strains 2-anthramine.

Details on test system and experimental conditions:

GROWING AND CONSERVATION OF BACTERIAL TEST STRAINS
The bacterial strains were kept as frozen broth cultures, in aliquots of 0.5 ml at -70 °C with 8.0 % dimethylsulfoxide (DMSO). Fresh cultures were prepared by adding 0.1 ml of a thawed stock culture to 15 ml of nutrient broth (normal Difco nutrient broth for strains TA 1535 and TA 100, and double strength Difco nutrient broth for TA 1537 and TA 98, in 0.5 % NaCI). A nutrient agar (2.5 % Difco nutrient broth, 1.2% Difco agar) was also streaked. The broth was incubated in the dark in a shaking water bath at 37 °C for 16 hours, while the plate was incubated at 37 °C overnight. Broths and plates were then transferred
to the refrigerator, for up to one week.

PREPARATION OF MATERIALS
The minimal-glucose agar medium base was prepared from a 1.5 % Bacto-Difco agar in Vogel & Bonner E medium with 2 % glucose. The top agar is a 0.6 % Bacto-Difco agar, 0.6 % NaCI solution. Before use, the agar was melted in a boiling water bath and then left to equilibrate to 45 °C. Ten ml of sterile 0.5 mM L-histidine - 0.5 mM biotin were added per 100 ml of top agar.
The compounds to be tested were prepared fresh daily in DMSO or in sterile water. The appropriate dilutions were made from a 20 mg (or nl)/ml stock. The S-9 mix was prepared fresh daily from sterile stocks. 0.5 ml contains:
4.0 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50.0 µ moles Phosphate Buffer, pH 7.4
150.0 µl liver homogenate.

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

RESULTS

The lack of evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 0.2 to 2000 µg of product per Petri dish.

Conclusions:

FAT 40147/D is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of FAT 40147/D to induce gene mutations according to the bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The test article was tested at the following concentrations:  0.2, 2, 20, 200 and 2000 µg (or nl) per Petri dish. The lack of evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 0.2 to 2000 µg of product per Petri dish. 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. Therefore, FAT 40147/D is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

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 was included

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

Histidine-auxotrophic strains of Salmonella typhimurium

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from rats

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2025 µg/0.1 ml

Vehicle / solvent:

distilled-water

Untreated negative controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without S9: TA 98: daunorubicin- HCl; TA 100: 4-nitroquinoline-N-oxide; TA 1235: N-methyl-N'-nitro-N-nitrosoguanidine; and TA 1537: 9(5)aminoacridine hydrochloride monohydrate. With S9: only TA 1535 with cyclophosphamide

Remarks:

Phosphate buffer (solvent) alone was used for the negative controls

Details on test system and experimental conditions:

The tests were carried out in accordance with the method described by AMES et al..

The bacteria on which the tests were performed were the following histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA 100, TA 1535, TA 1537.

The tests were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. The substance was dissolved in phosphate buffer. Phosphate buffer alone was used for the negative controls (the substances and vehicles used for the positive controls are indicated below). Each Petri dish contained: 1) approx. 20 ml of minimum agar (Agar, Difco Laboratories, Detroit,Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose), 2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6% agar solution with 0.6% NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland).

In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contained: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances: 1) for strain TA 98: daunorubicm-HCl (DAUNOBLASTIN , Farmitalia, Montedison Farmaceutica GmbH, Freiburg i.Br., Germany), 5 and 10 µg/0.1 ml phosphate buffer; 2) for strain TA 100: 4-nitroquinoline-N-oxide (Fluka,Buchs, Switzerland), 0.125 and 0.25 µg/0.1 ml phosphate buffer; 3) for strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (Serva, Heidelberg, Germany), 3 and 5 µg/0.1 ml phosphate buffer; 4) for strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate
(Fluka, Buchs, Switzerland), 50 and 100 µg/0.1 ml DMSO.

The activation mixture was tested with strain TA 1535 and cyclo-phosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 2 50 µg/0.1 ml phosphate buffer.

In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 hours at 37 C in darkness.

Evaluation criteria:

The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

RESULTS

In the experiments performed without and with microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 40 147/D revealed no marked differences.

Conclusions:

FAT 40147/D is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of FAT 40147/D to induce gene mutations according to the bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The test article was tested at the following concentrations:  25, 75, 225, 675 and 2025 µg/0.1 ml. Comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 40 147/D revealed no marked differences in both the experiments performed without and with microsomal activation. 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. Therefore, FAT 40147/D is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-29 to 2010-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline compliant GLP compliant

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

as at 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as at 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males mean value = 36.7 g (SD ± 1.5 g)
- Assigned to test groups randomly: not reported
- Fasting period before study: no
- Housing: singly in Makrolon Type II/III cages with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG,73494 Rosenberg, Germany)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum(Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst, The Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: >= 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 45 - 100
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Source: B. Braun Melsungen AG 34212 Melsungen, Germany
- Catalogue no.: 6724092.00.00
- Justification for choice of solvent/vehicle: chosen for its relative non-toxicity for animals
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in in the vehicle (sterile water).

Duration of treatment / exposure:

single oral gavage

Frequency of treatment:

single oral gavage

Post exposure period:

no

Remarks:

Doses / Concentrations:
main experiment: 500, 1000, 2000 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
toxicity pretest: 2000 mg/kg bw
Basis:

No. of animals per sex per dose:

- main test: 7 males per dose (only males were used in the main study as the toxicity pretest showed comparable susceptibility of males and females)
- toxicity pre-test: 2 males and 2 females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; cyclophosphamide (CPA)
- Supplier: Fisher Scientific GmbH, 61130 Nidderau, Germany
- Justification for choice of positive control(s): recommended as positive control substance in the OECD guideline
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/Kg bw (concentration in vehicle (water): 4 mg/mL)

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
2000 mg/kg bw is the limit dose according to the OECD guideline. Two lower doses with a spacing factor of 2 were selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- single aoral treatment
- Sampling:
- pre-test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration
- main test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h (and 48 h) after administration; animals of all dose levels sacrificed at 24 h post dosing, an additional high dose group sacrificed 48 post treatment

DETAILS OF SLIDE PREPARATION:
- sacrifices of animals using CO2 followed by bleeding
- removal of the femora, cutting off of epiphyses, marrow flushed out with foetal calf serum using a syringe
- centrifugation of the cell suspension at 1500 rpm (390 x g) for 10 minutes
- discarding of supernatant, resuspesion of the remaining cell pellet and spreading of a small drop of the suspension on a slide
- air drying of the slide and staining with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany)
- mounting of cover slips with EUKITT (Kindler, 79110 Freiburg, Germany)
- preparation of at least one slide per each bone marrow sample

METHOD OF ANALYSIS:
- slide analysis using NIKON microscopes with 100x oil immersion objectives
- analysis for micronuclei of 2000 polychromatic erythrocytes (PCE) per animal
- determination of ratio between polychromatic and normochromatic erythrocytes in the same sample for detection of cytotoxicity
- cytotoxicity expressed in polychromatic erythrocytes per 2000 erythrocytes
- slides were coded
- use of samples from all animals per test group

OTHER:

Evaluation criteria:

The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of
micronucleated PCEs compared to the negative control.

Positive result:
If the test item induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Negative result
If the test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes it is considered non-mutagenic in this system.

Statistics:

see above under Evaluation criteria

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

The animals treated with 2000 mg/kg b.w. did not express any toxic reactions. However, discoloured urine was observed (orange) in animals treated with 2000 mg/kg bw both in pre-test and main experiment.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no problems with solubility reported
- Clinical signs of toxicity in test animals:
- no adverse effects
- orange discoloration of the urine, indicating bioavailability of the test item which is a red dye
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: 2000 mg/kg bw is the limit dose recommended in the OECD guideline.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay):
- No micronuclei induced in any of the treatment groups
- Micronuclei induced in the positive control group at the expected rate, indicating the sensitivity of the test system
- for details see Tables 2 and 3
- Ratio of PCE/NCE (for Micronucleus assay):
- not significantly changed in any of the treatment groups
- for details see Tables 2 and 3
- Appropriateness of dose levels and route:
- orange discoloration of the urine in the highest dose group, indicating bioavailability of the test item which is a red dye
- testing up to the limit dose
- Statistical evaluation: non-parametric Mann-Whitney test

- Table 2: Summary of Micronucleus Test Results

test group	Dose mg/kg b.w.	Sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythocytes
vehicle	0	24	0.079	0 - 5	1219
test item	500	24	0.186	0 - 8	1264
test item	1000	24	0.107	1 - 6	1177
test item	2000	24	0.136	2 - 6	1203
positive control	40	24	2.979	23 -82	1118
test item	2000	48	0.057	0 - 2	1233

- Table 3: Statistical significance at the five per cent level (p < 0.05, evaluated by means of the non-parametric Mann-Whitney test).

Vehicle control versus test group	Significance	p
500 mg test material/kg b.w.; 24 h	-	0.0758
1000 mg test material/kg b.w.; 24 h	-	0.2287
2000 mg test material/kg b.w.; 24 h	-	0.0807
40 mg CPA/kg b.w.; 24 h	+	0.0003
2000 mg test material/kg b.w.; 48 h	-	n.t.

-         =         not significant

+         =         significant

n.t. = not tested

- Table 4: Historical controls from 2003 - 2009

	Negative Controls Males	Positive Controls (CPA) Males
Mean* ± SD	0.096 ± 0.040	2.332 ± 0.696
Range**	0.01 - 0.22	0.70 - 4.52
No. of Experiments	318	316

*:        mean value (percent micronucleated cells)

**:      range of the mean group values (percent micronucleated cells)

Conclusions:

Interpretation of results (migrated information): negative
The test material was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474 (single oral doses of 500, 1000 and 2000 mg/kg bw).
No micronuclei were induced in any of the treatment groups while micronuclei were induced in the positive control group at the expected rate, indicating the sensitivity of the test system. Orange discoloration of the urine in the highest dose group, indicating bioavailability of the test item which is a blue dye.
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 in the bone marrow cells of the mouse.

Executive summary:

The test material was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474.

The test item was formulated in sterile water, which was also 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.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw. As estimated by a pre-experiment 2000 mg of the test material per kg b.w. (the maximum guideline-recommended dose) was suitable.

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test material did not have any cytotoxic properties in the bone marrow. However, the animals showed discoloured urine after treatment with 2000 mg/kg b.w. indicating the bioavailability of the test item.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test material were near to the value of the vehicle control group. Additionally all values were within the historical vehicle control database. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which

showed a statistically significant increase of induced micronucleus frequency.

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 in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Two studies to assess the mutagenic potential of FAT 40147 are available.

A study was performed to investigate the potential of FAT 40147/D to induce gene mutations according to the bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The test article was tested at the following concentrations:  0.2, 2, 20, 200 and 2000 µg (or nl) per Petri dish. The lack of evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 0.2 to 2000 µg of product per Petri dish. 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. Therefore, FAT 40147/D is considered to be non-mutagenic in this bacterial reverse mutation assay.

A study was performed to investigate the potential of FAT 40147/D to induce gene mutations according to the bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The test article was tested at the following concentrations:  25, 75, 225, 675 and 2025 µg/0.1 ml. Comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 40 147/D revealed no marked differences in both the experiments performed without and with microsomal activation. 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. Therefore, FAT 40147/D is considered to be non-mutagenic in this bacterial reverse mutation assay.

Data from Read across substance FAT 40850:

This study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD Guideline 471 and EC method B13/14. The test item was tested at the following concentrations: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate.In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and frameshifts in the genome of strains TA 98 and TA 100.

Therefore, the test material is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In a CA test, the test material, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro according to OECD guideline 473 and EU Method B.10. Dose selection for the cytogenetic experiments was performed considering the toxicity data. The evaluation of chromosomal aberration was limited by cytotoxicity.

In Experiment I in the absence of S9 mix and in Experiment II in the presence of S9 mix clear cytotoxicity of about 50 % was observed at the highest evaluated concentration. In Experiment I in the presence of S9 mix higher concentrations could not be evaluated due to strong test item cytotoxicity. In Experiment I and in Experiment II in the presence of S9 mix no biologically relevant clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. The percentages of aberrant cells, excluding gaps were within the total laboratory´s historical solvent control data range (0.0 – 4.0 % aberrant cells, excluding gaps). In Experiment II in the absence of S9 mix statistically significant increases in the number of aberrant cells, excluding gaps were observed at all evaluated concentrations in the range of 180 to 300 µg/mL. All values exceeded the laboratory´s historical solvent control data range (0.0 – 3.5 % aberrant cells, excluding gaps).

Therefore, the occurrence of chromosomal aberrations is considered biologically relevant. No increase in polyploid metaphases was noticed after treatment with the test item as compared to 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 under the experimental conditions reported, the test iteminduced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, test material is considered to be clastogenic in this chromosome aberration test in the absence of metabolic activation.

Further to confirm the results from bacterial reverse mutation assay and CA test a study was performed to investigate the potential of the test material to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476 and EU Method B17. No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximal concentration. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test material is considered to be non-mutagenic in this HPRT assay.

In a in vivo test the test material was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474 (single oral doses of 500, 1000 and 2000 mg/kg bw). No micronuclei were induced in any of the treatment groups while micronuclei were induced in the positive control group at the expected rate, indicating the sensitivity of the test system. Orange discoloration of the urine in the highest dose group, indicating bioavailability of the test item which is a blue dye. 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 in the bone marrow cells of the mouse.

Hence, based on the results both from target and read across substance of above discussed assays, FAT 40147 is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the results of available genetox assays, FAT 40147 does not warrant classification according to the CLP (Regulation 1272/2008) criteria.