disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The 3 following test have been performed with the following results:

• OECD Guideline 471 (Bacterial Reverse Mutation Assay): positive
• OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test): positive
• OECD Guideline 476 (HPRT Assay): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Date of Study Plan: April 11, 2002 - Date of Final Report: November 04, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted July 21, 1997

Deviations:

yes

Remarks:

concerning the acceptability of the test and more precisely Aberrant cells in % (excl. gaps). Reason for the deviation : updating. Please refer to the study report for more details.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 19, 2000

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines: "Kanpoan No. 287 -- Environmental Protection Agency", "Eisei No. 127 -- Ministry of Health & Welfare", "Heisei 09110131 Kikyoku No. 2 - Ministry of lnternational rrade & lndustry".

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, Ð-64287 Darmstadt)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The highest applied concentration in the pre-test on toxicity (5310 pg/ml) was chosen respect to the current OECD Guideline 473.
Dose selection of the cytogenetic experiments was performed considering the toxicity data (for details, please refer to the study report).

Main experiment I : 175.0 µg/ml with S9 mix and 350.0 µg/ml without S9 mix
Main experiment II: 100 µg/ml (18h interval & 28h interval) without S9 mix; 262.5 µg/ml with S9 mix

Vehicle / solvent:

- Solvent used: deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).
- Justification for choice of solvent: the solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

EXPERIMENTAL PERFORMANCE
SCHEDULE: please refer to section "any other information on materials and methods incl. tables" here below.

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 enzymat¡c digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCl 200 mg, KH2PO4 200 mg and NaHPO4 150 mg.

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (Ethylene diamine tetraacetic acid).
The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). ln each chamber 1 x 10EXP4 - 6 x 10EXP4 cells were seeded with regard to preparation time. The medium was MEM with 10 % FCS (complete medium).

TREATMENT:
Exposure period 4 hours:
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with 39 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µl S9 mix per ml culture medium were added. Concurrent negative, solvent, and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.

The "saline G" solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HPO4.7H2O 290 mg, KH2PO4 150 mg. pH was adjusted to 7.2.

Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item 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 % CO2 (95.5 % air).

Preparation of the Cultures:
15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 37°C. After incubation in the hypotonic solution the cells were fìxed with a mixture of methanol and glacial acetic acid (3 parts + 1 part). Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).

Evaluation of Cell Numbers:
For evaluation of cytotoxicity indicated by reduced cell numbers additional two cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Analysis of Metaphase Cells:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der lndustrie, Cytogenetik" [9]) using NIKON microscopes with 100x oil immersion objectives.
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 metaphase plates 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. ln addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Acceptability of the Test
The chromosome aberration test performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.0 - 4.0 %
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data:

Evaluation criteria:

A test item is classifìed as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 %aberrant cells exclusive gaps).
and/or
-no significant increase of the number of structural chromosome aberrations is observed.

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

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:

Statistical significance was confirmed by means of the Fisher's exact test (10) (p < 0.05).
However, both biological and statistical significance should be considered together. lf the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Ref: Richardson, C., et al (1989). Analysis of data from in vitro cytogenetic tests. Kirkland, D.J. (ed.). "Statistical evaluation of mutagenicity test data", Cambridge University Press, Cambridge, 141-154

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II (Preparation interval of 28h)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II (Preparation interval of 28h)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II and II (Preparation interval of 18h)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Strong toxic effects in exp I at 40 without and at 320 µg/ml with m. activation. ln exp. II strong toxicity at 240 µg/ml and above. Results acceptable as cell density at 1st subcultivation after treatment remained above 20% at the highest evaluated conc.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ln a range fìnding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 41.5and 5310 pg/ml were applied.
Clear toxic effects were observed after 4 hrs treatment with 663.8 µg/ml and above in the absence of S9 mix and with 331.9 µg/ml and above in the presence of S9 mix. ln addition, 24 hrs continuous treatment with 663.8 µg/ml and above in the absence of S9 mix induced strong toxic effects.

ln the pre-experiment, neither test item precipitation in culture medium 4 hrs after start of treatment nor relevant influence of the test item on the pH value or osmolarity was observed (solvent control 292 mOsm, pH 7.3 versus 305 mOsm and pH 7.4 at 5310 µg/ml.

Clearly reduced cell numbers of below 50 % were observed in experiment I in the absence of S9 mix after 4 hrs treatment at 18 hrs preparation interval with 350 µg/ml (47 % of control) and in experiment ll in the presence of S9 mix after 4 hrs treatment at 28 hrs preparation interval with 262.5 µg/ml (42% of control). No strong toxic effects indicated by reduced mitotic indices of below 50 % were observed after treatment at the evaluated test item concentrations. However, concentrations showing clearly reduced mitotic indices were not evaluable for cytogenetic damage.

ln experiment I and ll in the absence of S9 mix and in experiment I in the presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberratíon rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. A single statistically significant (p < 0.05) increase was observed in the absence of S9 mix after treatment with 262.5 µg/ml . Although this increase of 3.0% aberrant cells, exclusive gaps, was statistically signifìcant compared to the low response (0.5 % aberrant cells, exclusive gaps) in the corresponding solvent control, the response is within the historical control data range (0.0 % - 4.0 % aberrant cells, exclusive gaps). Therefore, this statistical signifìcance has to be regarded as being biologically irrelevant. ln experiment ll, in the presence of S9 mix at preparation interval 28 hrs the aberration rate (7.0 % aberrant cells, exclusive gaps) was statistically signifìcant and biologically relevant increased after 4 hrs treatment with 262.5 µg/ml as compared to the corresponding solvent control (1.5% aberrant cells, exclusive gaps). ln addition, a dose related increase of the number of cells carrying structural chromosomal aberrations (1.0, 2.5, and 7.0 %) was observed at the concentration range evaluated (87.5 to 2625 µg/ml ). The observation of distinct increased numbers of micronucleated cells and of cells containing fragmented nuclei after 4hrs treatment with 262.5 µg/ml give additional evidence for a genotoxic potential of the test item. Therefore, these findings have to be regarded as being biologically relevant.

ln both experiments, EMS (100 and 200 µg/ml, respectively) and CPA (0.7 and 1.0 µg/ml, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Experiment	Fixation interval	Test item concentration in µg/ml	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control	Aberrant cells
						Inc. gaps*	Excl. gaps*	With exchanges
Exposure period 4 hrs without S9 mix
I	18h	negative control	2.2	n.t.	100	2.0	1.5	0.5
		solvent control^1	2.3	100	100	1.0	0.5	0.0
		positive control^3	2.3	n.t.	98	17.5	13.5 ^S	7.5
		175.0	2.6	76	100	1.5	1.5	0.0
		262.5	1.8	59	63	5.0	3.0 ^S	2.5
		350.0	2.0	47	81	2.0	0.5	0.0
Exposure period 18 hrs without S9 mix
I	18h	negative control	1.8	n.t.	100	2.0	0.5	0.0
		solvent control^1	2.2	100	100	1.0	0.0	0.0
		positive control^2	3.4	n.t.	101	11.0	9.0 ^S	4.0
		175.0	4.0	87	103	0.5	0.0	0.0
		262.5	2.0	83	106	1.5	0.5	0.5
		350.0	2.7	67	83	1.5	0.5	0.0
Exposure period 28 hrs without S9 mix
II	28h	negative control	1.7	n.t.	100	1.0	1.0	0.5
		solvent control^1	1.7	100	100	3.5	2.5	0.5
		positive control^2	1.4	n.t.	102	11.5	10.0 ^S	2.0
		100.0	2.1	77	71	3.0	2.0	0.0
Exposure period 4hrs with S9 mix
I	18h	negative control	2.1	n.t.	100	1.5	1.0	0.5
		solvent control^1	1.6	100	100	2.0	1.5	0.5
		positive control^2	1.5	n.t.	101	12.0	9.0 ^S	4.0
		43.8	2.9	112	75	1.0	1.0	0.5
		87.5	2.1	119	96	2.0	1.0	0.0
		175.0	1.7	82	58	2.0	1.5	0.0
II	28h	negative control	1.9	n.t.	100	1.5	1.5	0.0
		solvent control^1	2.2	100	100	1.5	1.5	0.0
		positive control^3	1.2	n.t.	98	11.5	9.5 ^S	4.5
		87.5	2.4	113	87	2.0	1.0	0.0
		175.0	2.6	64	86	4.5	2.5	0.5
		262.5	3.3	42	52	11.5	7.0 ^S	1.0

*inclusive cells carrying exchanges

n.t. not tested

^Saberration frequencystatistically significant higher than corresponding control values

¹deionised water 10% (v/v)

²EMS 100 µg/ml

³EMS 200 µg/ml

Conclusions:

It can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, RED Rwa 4565 is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix at high toxicity level.

Executive summary:

The test item RED Rwa 4565, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

	Without S9 mix			With S9 mix
	Exp. I	Exp. II		Exp. I	Exp. II
Exposure period	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	-	14 hrs	24 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

ln each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (5310 pg/ml) was chosen respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data. The chosen treatment concentrations are described in Table 2, Page 17 of the study report. The evaluated experimental points and the results are summarised in Table 1, page 10 of the study report.

Toxic effects indicated by reduced cell numbers of below 50 % of control were observed in experiment I in the absence of S9 mix and in experiment ll in the presence of S9 mix. No toxic effects indicated by reduced mitotic indices were observed at the test item concentrations evaluated. However, concentrations showing clearly reduced mitotic indices were not evaluable for cytogenetic damage.

ln experiment ll, in the presence of S9 mix, at the 28 hrs preparation interval a statistically significant and biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed at the highest test item concentration evaluated.

No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: April 29, 2002 - Date of Final Report: August 14, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 08, 2000

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 -- Environment Protection Agency
Eisei No. 127 -- Ministry of Health, Labour and Welfare, (JMHLW)
(former Ministry of Health and Welfare, MHW)
Heisei 09/10/31 Kikyoku No. 2 -- Ministry of Economy, Trade and lndustry, (METI),
(former Ministry of lnternational Trade and lndustry, MlTl)
JMAFF (February 24,2Q00): Japanese Ministry of Agriculture, Forestry and Fisheries, Test
Data for Registration of Agricultural Chemicals, 12 Nohsan No 8147, Agricultural Production
Bureau, November 24,200. English translation by Huntingdon Life Sciences Co., Ltd. Tokyo,
Japan,2000.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Target gene:

S. typhimurium TA1537: hisC3076; rfa-; uvrB-;
S. typhimurium TA 98: hisD3052; rfa-; uvrB-; R-factor
S. typhimurium TA1535: hisG46; rfa-; uvrB-;
S. typhimurium TA100: hisG46; rfa-; uvrB-; R-factor;
E. coli WP2 uvrA: trp-; uvrA-

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB deficient

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA deficient

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation (S9 mix)

Test concentrations with justification for top dose:

Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay.
Since no relevant toxic effects were observed, 5000 µg/plate were chosen as maximal concentration. The following concentrations were tested in the first and second experiments with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/plate.
Experiment I - TA98, TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment II - TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment II - TA 98 at 33, 100, 333, 1000, 2500 and 5000 µg/plate with S9 mix
Experiment II - TA 98 at 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: On day of experiment Red Rwa 4565 was dissolved in bidistilled water at room temperature.
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.The test item was soluble up to the concentration of 50 mg/ml.

Untreated negative controls:

yes

Remarks:

Concurrent solvent control was performed

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent control was performed

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I was performed as a plate incorporation assay. Experiment ll was performed as a pre-incubation assay.

DURATION
- Preincubation period: 30 minutes
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37 ±2° C in the dark.

NUMBER OF REPLICATIONS: Each concentration and the controls were tested in triplicate.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations of the test item. The concentrations applied were 20.6, 61 .7, 185.2, 555.6, 1666.7 and 5000 µg/plate. One plate was prepared per test item concentration and negative control. The plates were inverted and incubated for about 48 hours at 37°C in darkness.
Thereafter, they were evaluated by counting the colonies and determining the background lawn.
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The range finding test is reported separately.

Evaluation criteria:

Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants 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.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis was not required. At present the use of statistical methods concerning this particular test system is not generally recommended.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without 59 mix in both experiments. Occasionally, a weak growth inhibiting effect of the test item occurred in the experiments with and without metabolic activation at the concentration of 5000 µg/plate.

A dose dependent increase in revertant colony numbers following treatment with Red Rwa 4565 on strain E. coli without metabolic activation occurred at concentrations of 1250 to 5000 µg/plate and with activation at the concentrations of 2500 to 5000 µg/plate in the first experiment. ln the second experiment again an increase in the number of revertant colonies was visible on strain E. coli without metabolic activation at the concentrations of 2500 and 5000 µg/plate and with activation at the concentrations of 1250 to 5000 µg/plate. The number of revertant colonies exceeded the threshold of twice.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revefiant colonies.

Summary of the First Mutagenicity Test

Results with S9 mix (number of revertants/plate – mean from 3 plates)

Concentration [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Bidist. water	136	26	36	40	15
313.0	140	18	36	45	14
625.0	139	17	47	30	13
1250.0	139	16	49	32	14
2500.0	159	14	82	27	12
5000.0	138	20	102	24	5
2-A-anthracene	1724			1393	192
2-A-anthracene			572
CPA		382

 

Results without S9 mix (number of revertants/plate – mean from 3 plates)

Concentration [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Bidist. water	119	22	28	23	17
313.0	131	23	35	19	12
625.0	132	17	42	18	8
1250.0	122	13	54	21	8
2500.0	129	18	71	7	8
5000.0	122	12	88	20	5
2-Nitrofluorene				704
4-Nitroquinoline			283
9-A-acridine					1285
Sodium azide	253	467

Summary of the Second Mutagenicity Test

Results with S9 mix (number of revertants/plate – mean from 3 plates)

Concentration [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Bidist. water	115	13	27	28	14
313.0	119	13	33	22	14
625.0	126	12	51	20	8
1250.0	135	13	63	24	10
2500.0	150	12	95	22	10
5000.0	149	8	138	24	8
2-A-anthracene	1144			1071	194
2-A-anthracene			341
CPA		288

 

Results without S9 mix (number of revertants/plate – mean from 3 plates)

Concentration [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Bidist. water	116	15	25	19	10
313.0	106	19	33	15	11
625.0	144	19	43	18	7
1250.0	123	20	43	14	6
2500.0	144	16	70	17	8
5000.0	121	11	92	13	5
2-Nitrofluorene				158
4-Nitroquinoline			405
9-A-acridine					851
Sodium azide	724	406

 

Conclusions:

ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by base-pair changes in the genome of the strain Escherichia coli WP2 uvrA with and without metabolic activation.
Therefore, Red Rwa 4565 is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay performed with and without metabolic activation.

Executive summary:

Red Rwa 4565, identified as dark red powder, purity 94.3%, batch no REG, was tested for mutagenic effects in vitro in histidine-requir¡ng strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used:

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

The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The test item was dissolved in bidistilled water and tested at five concentrations: 313, 625, 1250,2500 and 5000 µg/plate.

ln order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61 .7, 185.2, 555.6, 1666.7 and 5000 µg/plate. On strain E. coli WP2 uvrA an increase in the number of revertant colonies was observed in the experiment without metabolic activation at the concentration of 5000 µg/plate and in the experiment with activation at the concentrations of 185.2 to 5000 µg/plate.

Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentration tested. The test item did not precipitate on the surface of the agar plates.

From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.

 

ln the first mutagenicity test perfomed without metabolic activation, treatment of strain E. coli WP2 uvrA with Red Rwa 4565 led to an increase in the number of revertant colonies at the concentrations of 1250 to 5000 µg/plate. ln the experiment with metabolic activation an increase in the number of revertant colonies occurred at the concentrations of 2500 and 5000 µg/plate. No similar effect was visible on strains TA 100, TA 1535, TA 98 and TA 1537 in the experiment with and without metabolic activation.

 

ln the second mutagenicity test perfomed without metabolic activation, again an increase in the number of revertant colonies was visible on strain E. coli after treatment with Red Rwa 4565 at the concentrations of 2500 and 5000 µg/plate and in the experiment with activation at the concentrations of 1250 to 5000 µg/plate. No similar effect was visible on strains TA 100, TA 1535, TA 98 and TA 1537 in the experiment with and without metabolic activation .

 

ln the mutagenicity tests normal background growth was observed with all strains at all concentrations. Occasionally, a weak growth inhibiting effect of the test item occurred in the experiments with and without metabolic activation at the concentration of 5000 µg/plate. The test item did not precipitate on the surface of the agar plates.

 

ln the experiments negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Date of the Study Plan : August 21, 2002 - Date of Final Report: January 09, 2003

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)

Version / remarks:

Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test"

Qualifier:

according to guideline

Guideline:

other: commission Directive2000/32/EC, L1302000, Annex 4E, dated May 19, 2000.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; D-64287 Darmstadt) are stored in liquid nitrogen in the cell bank of RCC-CCR allowing the repeated use of the same cell culture batch in experiments.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/p-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I :
without S9 mix: 5.0;10.0; 20.0; 30.0 and 40.0 µg/ml
with S9 mix: 20.0; 40.0; 80.0; 160.0 and 240.0 µg/ml
Experiment II:
without S9 mix at 20.0; 40.0; 80.0; 160.0 and 240.0 µg/ml

DOSE SELECTION:
The concentration range of the main experiments was selected according to toxicity data generated in the range finding pre-experiment.
According to the recommendations of the guidelines, several concentrations (usually at least four) of the test item should be used. These should yield a concentration-related toxic effect. The highest concentration should produce a low level of survival and the survival in the lowest concentration should approximate the negative control. Relatively insoluble substances should be tested up to their limit of solubility under culture conditions.
For freely-soluble non-toxic substances the maximum concentration should be 5 mg/ml or 10 mM. lf the maximal concentration is based on cytotoxicity the cloning efficiency should be reduced to less than 50 % andlor culture growth at sub-cultivation should be at least 20 % of the corresponding solvent control.
The pre-experiment was performed with eight concentrations ranging from 39.1 to 5000.0 g/ml without metabolic activation (4 h and 24 h treatment) and with metabolic activation (4 h treatment).
Toxic effects occurred at 39.1 µg/ml without and at 312.5 µg/ml with metabolic activation (4 h treatment), and without metabolic activation (24 h treatment). At higher concentrations the cell growth was completely inhibited.
No precipitation of the test item visible to the unaided eye occurred in any of the experiments up to the maximum concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
the final concentration of deionised water in the culture medium did not exceed 10 % v/v
the osmolarity and PH-value were determined in a pre-experiment without metabolic activation in the solvent control (osmolarity 281 and pH-value 7.34) and at the maximum concentration of 5000 µg/ml (osmolarity 303 and pH-value 7.37)

Untreated negative controls:

yes

Remarks:

Untreated cells used and cells used throughout the assay without addition of test item

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

CELL CULTURES

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; D-64287 Darmstadt) are stored in liquid nitrogen in the cell bank of RCC-CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Thawed stock cultures are propagated at 37 'C in 80 cm2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5x10s cells are seeded into each flask with 15 ml of MEM (minimal essential medium; SEROMED, D-12247 Berlin) supplemented with 10% fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells are sub-cultured twice weekly.
The cell cultures are incubated at 37°C in a 4.5 % carbon dioxide atmosphere (95.5% air).
For the selection of mutant cells the medium is supplemented with 11 µg/ml thioguanine (6TG, SIGMA GmbH, D-82041 Deisenhofen).

MAMMALIAN MICROSOMAL FRACTION S9 MIX PREPARATION

Metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with mammalian liver microsome preparations (S9 mix).

S9 Preparation :
Phenobarbital/p-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 applícations of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D- 22335 Hamburg) and p-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 KCI 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 35.7 mg/ml (Lot No.: 120702) in the preexperiment and 31.8 mg/ml (Lot No.: 220802) in experiment l.

S9 mix Preparation :
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mgiml in the cultures. Cofactors were added to the 59 mix to reach following concentrations:
MgCl2 8 mM, KCl 33 mM, glucose-6-phosphate 5 mM and NADP 4 mM in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the 59 mix was stored in an ice bath.


SEEDING

Three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37°C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin: Disco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution had the following constituents (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg and NaHCO3 350 mg.
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (ethylene diamine tetraacetic acid).
The cell suspension was seeded into plastic culture flasks (Greiner, D-72632Frickenhausen).
Approximately 1.5x10EXP6 (single culture) and 5x10EXP2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.

TREATMENT

After 24 h the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/ml S9 mix. Concurrent negative and positive controls were treated in parallel.
After 4 h this medium was replaced with complete medium following two washing steps with "saline G". ln the second experiment the cells were exposed to the test item for 24 h in complete medium in the absence of metabolic activation.
The "saline G" solution had the following constituents (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HPO4.7H2O 290 mg and KH2PO4 150 mg.

The pH was adjusted to 7.2.
The colonies used to determine the cloning efficíency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three days after treatment 1.5x10EXP6 cells per experimental point were sub-cultivated in 175 cm2 flasks containing 30 ml medium. Following the expression time of approx. 7 days five 80 cm2 cell culture flasks were seeded with about 3 - 5x10EXP5 cells each in medium contaíning 6-TG. Two additional 25 cm2 flasks are seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures are incubated at 37°C in a humidified atmosphere with 4.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution (E. MERCK, D-64293 Darmstadt).
The stained colonies with more than 50 cells were counted. ln doubt the colony size were checked with a preparation microscope (Nikon, D4o4o7 Düsserdorf).
Subculturing of a log-phase culture showed an initial spontaneous mutation rate at the beginning of the experiments of 5.5 mutant colonies per 10EXP6 cells.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY: for details, please refer to "Any other information on materials and methods incl. tables" hereafter.

EVALUATION OF RESULTS:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
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.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 40.2 mutants per 10EXP6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.

Statistics:

BIOMETRY: Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item visible to the unaided eye was observed up to the maximal concentration in all experiments.

Strong toxic effects were observed in the first experiment at 40 µg/ml without - and at 320 µg/ml with metabolic activation. ln the second experiment strong toxicity occurred at 240 µg/ml and above. Although the values of the relative cloning efficiency fall below 10 % of the corresponding solvent control the results are judged as acceptable since the cell density at the first sub-cultivation after treatment remained well above 20 % at the highest evaluated concentrations.

No relevant and reproducible increase in mutant colony numbers/10EXP6 cells was observed in the main experiments up to the maximal evaluated concentration. All mutant frequencies remained well within the historical range of negative and solvent controls.

ln both experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 1.6 up to 121 mutants per 10EXP6 cells; the range of the groups treated with the test item was from 1.7 up to 12.7 mutants per 10EXP6 cells.

EMS (0.15 mg/ml) and DMBA (2.7 µg/ml ) were used as positive controls and showed a distinct increase in induced mutant colonies.

SUMMARY OF RESULTS

	Conc. µg per ml	S9 mix	Relative cloning efficiency I %	Relatvie cloning efficiency II	Mutant colonies/10EXP6 cells	Induction factor	Relative cloning efficiency I		Relative cloning efficiency II	Mutant colonies/10EXP6 cells	Induction factor
ExperimentI			Culture I					Culture II
Negative control		-	100.0	100.0	8.0		100.0		100.0	1.6
Solvent control with water		-	100.0	100.0	4.5	1.0	100.0		100.0	3.8	1.0
Positive control with EMS	150.0	-	119.6	91.7	188.1	23.5	92.6		79.3	249.7	152.0
Test item	2.5	-	111.1	Culture was not continued #			108.7		Culture was not continued #
Test item	5.0	-	110.0	124.4	2.5	0.5	121.7		88.4	11.4	3.0
Test item	10.0	-	110.4	106.2	6.7	1.5	11.0		76.6	4.9	1.3
Test item	20.0	-	98.0	103.6	6.9	1.5	100.8		78.4	5.6	1.5
Test item	30.0	-	69.1	109.0	9.5	2.1	52.4		83.2	8.4	2.2
Test item	40.0	-	4.1	81.9	12.5	2.8	2.5		91.7	4.2	1.1
Negative control		+	100.0	100.0	2.8		100.0		100.0	12.7
Solvent control with water		+	100.0	100.0	4.4	1.0	100.0		100.0	8.3	1.0
Solvent control with DMSO		+	100.0	100.0	4.1	1.0	100.0		100.0	9.8	1.0
Positive control with DMBA	2.7	+	50.3	79.3	244.2	59.3	63.1		119.7	460.7	46.9
Test item	20.0	+	89.0	69.2	6.1	1.4	89.0		97.1	9.6	1.2
Test item	40.0	+	96.3	88.3	4.9	1.1	92.1		92.0	5.6	0.7
Test item	80.0	+	99.0	90.9	2.0	0.5	90.5		97.4	6.8	0.8
Test item	160.0	+	89.7	72.8	2.6	0.6	101.2		149.4	6.0	0.7
Test item	240.0	+	45.9	81.3	2.7	0.6	57.1		152.4	5.0	0.6
Test item	320.0	+	0.2	Culture was not continued ##			3.0		Culture was not continued ##
Experiment II		-
Negative control		-	100.0	100.0	6.8		100.0		100.0	2.2
Solvent control with water		-	100.0	100.0	5.9	1.0	100.0		100.0	2.1	1.0
Positive control with EMS	150.0	-	50.5	81.7	331.0	48.8	52.8		92.0	296.1	134.5
Test item	20.0	-	90.2	66.5	3.0	0.5	98.9		89.5	4.8	2.3
Test item	40.0	-	101.5	99.1	5.8	1.0	108.4		92.5	2.0	0.9
Test item	80.0	-	95.1	73.2	6.1	1.0	102.1		83.0	3.2	1.5
Test item	160.0	-	86.7	90.2	4.0	0.7	92.5		95.7	1.7	0.8
Test item	240.0	-	0.7	46.6	12.7	2.1	0.8		97.8	2.3	1.1
Test item	320.0	-	0.0	Culture was not continued ##			0.5		Culture was not continued ##

#concerning concentration range five concentrations were selected to be evaluated at the end of the experiment

## culture was not continued due to strong toxic effects

Conclusions:

ln conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Red Rwa 4565 is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Red Rwa 4565 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

Experiment I was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The test item was evaluated at the following concentrations:

Experiment I :

      without S9 mix: 5.0;10.0; 20.0; 30.0 and 40.0 µg/ml

      with S9 mix: 20.0; 40.0; 80.0; 160.0 and 240.0 µg/ml

Experiment II:

      without S9 mix at 20.0; 40.0; 80.0; 160.0 and 240.0 µg/ml

No precipitation of the test item visible to the unaided eye occurred in any of the experiments up to the maximum concentration.

The concentration range of the main experiments was selected according to toxicity data generated in the range fìnding pre-experiment. Strong toxic effects were observed in both main experiments at the maximum concentration.

Up to the highest investigated concentration no relevant increase of the mutation frequency was observed in both independent experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo est performed according to OECD Guideline 474 shows negative results.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Date of Study Plan: October 29, 2002 - Date of Final Report: February 07, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

9th addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted July 21, 1997, "Mammalian Erythrocyte Micronucleus Test".

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: micronucleus assay in bone marrow cells of the mouse

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation (at start of the treatment): males mean value 32.7 g (SD ± 2.7 g); females mean value 26.4 g (SD ± 1.8 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single. Cage type: Makrolon Type 1, with wire mesh top (EHRET GmbH, D-79302 Emmendingen). Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Rof1dorf)
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 25-82 *
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

* ln the present study, the relative humidity under which the experiment was conducted ranged between 25 - 82 % and not between 30 -70 % as described in the study plan. This deviation, however, does not affect the validity of the experiment.

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: deionised water
- Justification for choice of solvent/vehicle: the vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle: 10 ml/kg b.w.

Details on exposure:

Pre-experiment for toxicity
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Dose Selection
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Treatment
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 positíve control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of around t h, 24h, 6 h and 24 h after administration of the test item.

Duration of treatment / exposure:

acute exposure

Frequency of treatment:

1 treatment per animal

Post exposure period:

24 and 48 h

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

24h preparation interval

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

24h preparation interval

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24h and 48h preparation intervals
maximum guideline-recommended dose and estimated by a pre-experiment to be suitable

No. of animals per sex per dose:

10 animals (5 males, 5 females) per test group were evaluated for the occurence of micronuclei.

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide
- Justification for choice of positive control: The stability of CPA at room temperature is sufficient. At 25°C only 3.5 % of its potency is lost after 24 hours.
- Route of administration: oral
- Doses / concentrations: 40 mg/kg b.w.

Tissues and cell types examined:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively (to cover the intervals in which maximum frequencies of micronuclei occur).

Details of tissue and slide preparation:

Preparation of the Animais:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.

Data Recording
The data generated are recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups, vehicle, and positive control. The micronucleated cells per 2000 PCEs and the ratio of polychromatic to total erythrocytes are presented for each animal.

Evaluation criteria:

Acceptance criteria:
The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.01 - 0.15 %; mean = 0.066 x 0.032 PCEs with micronuclei).
- the positive controls are in the range of our historical control data (0.91 - 2.975 %imean = 1.644 + 0.446 PCEs with micronuclei).
- at least 80 % of animals are evaluable

Evaluation of Results:
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 (8)) will be used as an aid in evaluating the results. However, the primary point of consideratrion 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:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Ref.: Krauth, J. (1971). Locally most powerful tied rank test in a Wilcoxon situation Annals of Mathematical Statistics, 42, 1949 - 1956

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg b.w. (maximum guideline-recommended dose)
- Clinical signs of toxicity in test animals: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): significant with positive control (substantial increase); not significant with test item (no substantial decrease)
- PCE per 2000 erythrocytes (for Micronucleus assay): 1090 for vehicle; 1050 for test item (500 mg/kg b.w.; 24h sampling); 1094 for test item (1000 mg/kg b.w.; 24h sampling); 1152for test item (1000 mg/kg b.w.; 24h sampling); 1040 for Positive control (40 mg/kg b.w.; 24h sampling); 1022 for test item (2000 mg/kg b.w.; 48h sampling)
- Appropriateness of dose levels and route: yes
- Statistical evaluation: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Not significant with P = 0.0518 for test item at 2000 mg/kg bw; 24h
Not significant with P = 0.5000 for test item at 2000 mg/kg bw; 48h
Significant with P<0.0001 for positive control at 40 mg/kg bw; 24h

PRE-EXPERIMENT FOR TOXICITY

ln a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. RED RWA 4565 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 as shown in the table:

	Hours post-treatment male/female
Toxic reactions	1h	2-4h	6h	24h	30h	48h
Reduction of spontaneous activity	1/2	1/2	2/1	0/0	0/0	0/0
Abdominal position	1/0	0/0	0/0	0/0	0/0	0/0
Eyelid closure	1/1	1/1	0/1	0/0	0/0	0/0
Ruffled fur	2/2	2/2	2/1	0/0	0/0	0/0
Urine colour	-	-	Red-orange			-

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

TOXICITY SYMPTOMS IN THE MAIN EXPERIMENT

ln 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. RED RWA 4565 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 as shown in the table:

	Hours post-treatment male/female
Toxic reactions	1h	2-4h	6h	24h
Reduction of spontaneous activity	8/11	5/6	5/5	1/0
Abdominal position	3/5	0/0	0/0	0/0
Eyelid closure	5/5	0/1	0/0	0/0
Ruffled fur	12/12	12/10	12/9	1/0
Urine colour	-	-	-	red

ln the main experiment for the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 1000 mg /kg b.w. RED RWA 4565 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 as shown in the table:

	Hours post-treatment male/female
Toxic reactions	1h	2-4h	6h	24h
Reduction of spontaneous activity	2/2	2/3	2/3	0/0
Abdominal position	0/1	0/0	0/0	0/0
Eyelid closure	0/1	0/1	0/1	0/0
Ruffled fur	3/4	4/4	2/4	0/0

ln the main experiment for the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 500 mg /kg b.w. RED RWA 4565 formulated in deionised water.

The volume administered was 10 ml/kg b.w.. The animals treated with 500 mg /kg b.w. expressed toxic reactions as shown in the table:

	Hours post-treatment male/female
Toxic reactions	1h	2-4h	6h	24h
Reduction of spontaneous activity	1/1	1/2	1/2	1/1
Eyelid closure	0/0	0/0	0/0	1/0
Ruffled fur	1/1	1/1	1/2	1/1
Urine colour	-	-	-	Red/pink

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	0.025	0-2	1090
Test item	500	24	0.045	0-4	1050
Test item	1000	24	0.015	0-1	1094
Test item	2000	24	0.065	0-3	1152
Positive control	40	24	1.315	10-51	1040
Test item	2000	48	0.030	0-3	1022

 

BIOMETRY

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

Vehicle control versus test group	Significance	p
500 mg RED RWA 4565/kg b.w.;24 h	-	0.3214
1000 mg RED RWA4565/kg b.w.;24h	n.t.	-
2000 mg RED RWA 4565/kg b.w.;24 h	-	0.0518
40 mg CPA/kg b.w.;24 h	+	< 0.0001
2000 mg RED RWA 4565/kg b.w.; 48 h	-	0.5000

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

- = not significant

+ = significant

Conclusions:

ln 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, RED RWA 4565 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of RED RWA 4565 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

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 total 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:

- 24h preparation interval: 500, 1000, and 2000 mg/kg b.w.

- 48 h preparation interval: 2000 mg/kg b.w.

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

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that RED RWA 4565 d¡d not exert any cytotoxic effects in the bone marrow. However, the urine of the treated animals had taken over the colour of the test item indicating its systemic distribution and thus its bioavailability.

ln comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

As specified in line 13 of table R.7.7 -5 of ECHA guidance R7a (v6.0 -July 2017), with a positive result in in vitro gene mutation test in bacteria (OECD 471), a positive result in in vitro cytogenicity assay in mammalian cells (OECD 473) and a negative result in in vivo cytogenicity assay in experimental animals (OECD 474), no conclusion on genotoxicity is provided. However, the positive result observed in in vitro gene mutation test in bacteria - Ames test (OECD 471) - that is used as an initial screening step is counterbalanced by a negative result in in vitro gene mutation test in mammalian cells - HPRT test (OECD 476) - that is regarded as a higher tier test.

The test item is identified as not genotoxic and no further test are therefore not required according to Annexes VII to X of the REACH Regulation (EC) No 1907/2006.