Reaction mass of pentasodium 2-{[4-chloro-6-(ethyl{3-[(2-sulfonatoethyl)sulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate and tetrasodium 2-[(4-chloro-6-{ethyl[3-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in-vitro genotoxicity studies are available among which Ames assay and Chromosomal aberration were showed positive results; while in HPRT assay the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, FAT 40824/A is considered to be non-mutagenic in the HPRT 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:

16 December 2005 to 10 Febuary 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

other: Jananese guideline: Kanpoan No. 287 - Environment Protection Agency;Eisei No. 127 - Ministry of Health & Welfare; Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

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:

Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1 : approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10 %
Expiration date: 01 October 2010
Stability in water: Max. 7 days at room temperature
Solubility in water: >100g/L at 20 °C
Storage: At room temperature at about 20 °C, in a desiccator because test substance is hygroscopic, away from direct sunlight

Target gene:

histidine dependent S. typhimurium and Escherichia coli strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

s9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II and Wa: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for TA 1535, TA 100 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

for TA 1537, TA 98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for WP2 uvrA without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains with S9

Details on test system and experimental conditions:

-Precultures:
From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCI (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

-S9Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8mM MgCI2
33 mM KCl
5mM Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revenants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

HISTORICAL CONTROL DATA

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	19	6	9	35	21	7	7	41
	Negative control	18	5	10	30	21	6	9	38
	Positive control	1681	789	1003	4900	387	126	172	695
TA 1537	Solvent control	12	3	4	29	18	6	6	36
	Negative control	11	3	5	29	19	6	8	33
	Positive control	87	18	52	191	337	191	94	746
TA 98	Solvent control	26	6	14	•58	39	9	21	57
	Negative control	26	6	15	€0	41	9	17	64
	Positive control	361	204	176	1818	2386	1195	296	4854
TA 100	Solvent control	131	24	91	198	147	25	109	281
	Negative control	140	21	101	189	154	23	103	254
	Positive control	2030	340	1178	2872	2629	1326	546	•5230
WP2uvrA	Solvent control	52	8	31	67	55	10	34	75
	Negative control	50	8	36	64	52	8	33	•64
	Positive control	998	515	320	1976	342	134	221	930

 

Mean = mean value of revertants/plate; SD s standard deviation; Min = minimal value; Max = maximal value

Conclusions:

FAT 40824/A did induce gene mutations by frameshifts in the genome of strain TA 1537 in the absence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA with and without liver microsomal activation. 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate were administered in Pre-Experiment/Experiment I; 33; 100; 333; 1000; 2500; and 5000 μg/plate were administrated in Experiment II and VVa test. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. A minor but dose dependent increase in revertant colony numbers was observed following treatment with test substance in strain TA 1537 in the absence of metabolic activation (S9 mix). The number of colonies reached the threshold of thrice (strain TA 1537) the number of the corresponding solvent control at 5000 μg/plate in the pre-experiment. Since the threshold was just reached only at 5000 μg/plate and the values were still in the range of the laboratories historical data, the plate incorporation assay was repeated with strain TA 1537 without metabolic activation (reported as VVa) in parallel to the second experiment (pre-incubation). Both experiments showed a substantial and dose dependent increase in revertant colony numbers. The number of colonies exceeded the threshold of thrice the number of the corresponding solvent control at 1000 μg/plate and above in the pre- incubation assay and at 2500 μg/plate and above in the repeated plate incorporation. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. Thus, it can be concluded that the test item can induce gene mutations by frameshifts in the genome of strain TA 1537 in the absence of metabolic activation.

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:

21 December 2005 to 20 April 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: "Kanpoan No. 287 - Environmental Agency" "Eisei No. 127 - Ministry of Health & Welfare" "Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1: approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10 %
Expiration date: 01 October 2010
Stability in water: Max. 7 days at room temperature
Solubility in water: >100g/L at 20 °C
Storage: At room temperature at about 20 °C, in a desiccator because test substance is hygroscopic, away from direct sunlight

Target gene:

V79 cells of the Chinese hamster

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 μg/ml

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 Mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

cyclophosphamide

Remarks:

with S9 Mix

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 h with S9 Mix and 24 h without S9 Mix

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

- S9Mix:
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCI2
33 mM KCl
5 mM glucose-6-phosphate
4mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Evaluation criteria:

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Fisher's exact test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the mitotic index was reduced

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

FAT 40824/A induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations in the presence of S9 mix under the experimental conditions.

Executive summary:

A study was assessed for the potential of test substance to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix according to OECD TG 473 under GLP test condtion. 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 μg/ml were administrated during 4 h with and without metabolic activation S9 Mix. Toxic effects indicated by reduced cell numbers or mitotic indices of below 50 % of control were observed in all experimental parts in the main experiment after treatment with 625 μg/mL. In detail, in the absence of S9 mix, the mitotic index was reduced to 41.1 % of control, while in the presence of S9 mix, the cell numbers were reduced to 24.6 % of control. In the absence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed. The aberration rates after treatment with the test item (1.5 - 3.5 % aberrant cells, exclusive gaps) were in the range of the solvent control value (3.5 % aberrant cells, exclusive gaps) and within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). In contrast, in the presence of S9 mix, dose-dependent, statistically significant and biologically relevant increases in the number of aberrant cells (6.0 % and 11.5 % of control) were observed at the two highest evaluated concentrations (312.5 and 625.0 μg/mL, respectively), clearly exceeding our laboratory's historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Additionally, the number of cells carrying exchanges (1.0 - 2.5 %) was distinctly increased in a dose-dependent manner after treatment with 312.5 and 625.0 μg/mL, respectively. The observed cells carrying exchanges and the dose-dependency are additional indicators for a clastogenic potential of the test item. The positive controls showed distinct increases in cells with structural chromosome aberrations. Therefore, it can be conculded the test substance induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) up to cytotoxic concentrations 312.5 μg/mL in the presence of S9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Febuary 2006 to 29 June 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1 : approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10 %
Expiration date: 01 October 2010
Stability in water: Max. 7 days at room temperature
Solubility in water: >100g/L at 20 °C
Storage: At room temperature at about 20 °C, in a desiccator because test substance is hygroscopic, away from direct sunlight

Target gene:

Chinese Hamster V79 Cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation S9 Mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/mL
with S9 mix: 75.0; 150.0; 300.0; 450.0; and 600.0 µg/mL
Experiment II:
without S9 mix: 100.0; 200.0; 400.0; 600.0; and 700.0 µg/mL

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation S9 Mix

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation S9 Mix

Details on test system and experimental conditions:

DURATION
- Preincubation period: 12 -16 h in stock cultures
- Exposure duration: 4h with and without S9 Mix; 24h without S9 Mix

NUMBER OF REPLICATIONS: 5 replications per dose.

-S9 Preparation:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8-12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phénobarbital i.p. (Desitin; D-22335 Hamburg) and ß-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules were kept at -20 °C for up to one week. The protein concentration in the S9 preparation was 32.6 mg/mL (Lot No.: 021205) in the pre-experiment and experiment I.

- S9Mix:
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 supernatant to reach following concentrations in the S9 mix:
8 mM MgCI2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath.

Evaluation criteria:

A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test substance can be considered to be non-mutagenic in this HPRT assay.

Executive summary:

A GLP-compliant study was performed to investigate the potential of FAT 40824/A to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The highest concentration (5000 ug/mL) used in the range finding pre-experiment was chosen with respect to the current OECD guideline 476. The dose range of the main experiments was limited by toxicity of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies and thus showed the sensitivity of the test item and the activity of the S9 mix. 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, FAT 40824/A is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item is not genotoxic in 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:

23 Febuary 2006 to 12 October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

See overall remarks attachment section

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1: approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10 %
Expiration date: 01 October 2010
Stability in water: Max. 7 days at room temperature
Solubility in water: >100 g/L at 20 °C
Storage: At room temperature at about 20 °C, in a desiccator because test substance is hygroscopic, away from direct sunlight

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH D-33178 Borchen
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 36.9 g (SD ±2.7 g); females mean value 30.7 g (SD ±1.6 g)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: Single
- Diet: pelleted standard diet, ad libitum, (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3
- Humidity (%): 27 -70
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: unspecified

Vehicle:

Distilled water

Details on exposure:

500, 1000 and 2000 mg/kg bw were administrated to test mice.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

48h

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Middle dose

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

cyclophosphamide

Tissues and cell types examined:

polychromatic erythrocytes in the bone marrow

Details of tissue and slide preparation:

The animals were sacrificed using CO2 following by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal 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 re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test item is classified as mutagenic if it 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.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. FAT 40824/A formulated in deionised water. The volume administered was 10 ml/kg b.w. Few animals treated with 100 mg/kg b.w. expressed toxic reactions such as ruffled fur. In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. FAT 40824/A formulated in deionised waler. The volume administered was 10 ml/kg b.w.. Few animals treated with 1000 mg/kg b.w. expressed toxic reactions such as ruffled fur and reduction of spontaneous activity. In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT40824/A formulated in deionised water. The volume administered was 10 ml/kg b.w.. The animals treated with 2000 mg/kg b.w. expressed toxic reactions such ruffled fur and urine colour. On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

Toxic Symptoms in the Main Experiment

In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 2000 mg/kg b.w. FAT 40824/A formulated in deionised water. The volume administered was 10 mUkg b.w.. The animals treated with 2000 mg/kg b.w. expressed toxic reactions such as reduction of spontaneous activity, ruffed fur and urine colour. For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 1000 mg/kg b.w. FAT 40824/A formulated in deionised water. The volume administered was 10 ml/kg b.w.. The animals treated with 1000 mg/kg b.w. expressed toxic reactions such as ruffed fur. The animals treated with 500 mg/kg b.w. and the vehicle control (deionised water) did not express any toxic reactions. 

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 FAT 40824/A did not have any cytotoxic properties in the bone marrow. The urine of the animals treated with the highest dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. The plasma analysis of the satellite animals treated with the high dose for 1 and 4 h corroborated this finding. 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 FAT 40824/A were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Conclusions:

FAT 40824/A did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse under the experimental conditions.

Executive summary:

In a GLP-compliant study, FAT 40824/A 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 ORCD guideline 474. The test item was formulated in deionised 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. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 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 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 b.w.; 48 h preparation interval: 2000 mg/kg b.w.. 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 FAT 40824/A did not have any cytotoxic properties in the bone marrow. The urine of the animals treated with the highest dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. The plasma analysis of the satellite animals treated with the high dose for 1 and 4 h corroborated this finding. 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 FAT 40824/A were below or near to the value of the vehicle control group. 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

Three in vitro tests and one in vivo test were performed of the test substance, including gene mutation assay, Ames test, chromosome aberration test and micronucleus assay. The gene mutation test (Wollny, 2006) was performed to assess the potential of the test substance to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. A second experiment was performed without metabolic activation and a treatment period of 24 hours. In both experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 0.6 up to 12.1 mutants per 10⁶ cells; the range of the groups treated with the test item was from 0.0 up to 17.7 mutants per 10⁶ cells. Therefore, it can be concluded the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Thus, the test substance is considered to be non-mutagenic in this study. The Ames test (Sokolowski , 2006) was performed to assess the potential of the substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA with and without liver microsomal activation. Both experiments with strain TA 1537 showed a substantial and dose dependent increase in revertant colony numbers, whereas the remaining strains of salmonella typhimurium and Escherichia coli were not significantly affected. The number of TA 1537 colonies exceeded the threshold of thrice the number of the corresponding solvent control at 1000 μg/plate and above in the pre- incubation assay and at 2500 μg/plate and above in the repeated plate incorporation. Thus, it can be concluded that the test substance can induce gene mutations by frameshifts in the genome of strain TA 1537 in the absence of metabolic activation. A chromosome aberration test (Kunz, 2006) was performed to assess the potential of the test substance to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix according to OECD TG 473. In contrast, in the presence of S9 mix, dose-dependent, statistically significant and biologically relevant increases in the number of aberrant cells (6.0 % and 11.5 % of control) were observed at the two highest evaluated concentrations (312.5 and 625.0 μg/mL, respectively), clearly exceeding the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, it can be concluded the test substance induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) up to cytotoxic concentrations 312.5 μg/mL in the presence of S9 mix. The in vivo micronucleus assay (Honarvar, 2006) was performed to assess the test substance in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. 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 the test substance did not have any cytotoxic properties in the bone marrow. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. Therefore, it can be concluded the test substance dose not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. The in vivo test indicated the negative result although Ames test and chromosome aberration test indicated some positive results. The in vivo test results is more reliable for its more similarity to the human metabolic environment. In addition, the gene mutation assay got the negative results as well. Thus, we can conclude that the test substance indicate no genetic toxicity under the experiment condition.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.