5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity of Basic Blue 159 was assessed in a bacteria reverse mutation assay (OECD 471)and an in vitro mammalian mutagenicity test (HPRT) (OECD 476) and an in-vivo mouse micronucleus test (OECD 474).

Basic Blue 159 was not genotoxic in the in-vitro mammalian mutagenicity test, but was tested positive in a bacterial reverse mutation assay (OECD 471) in Salmonella typhimurium TA 98 and 1538 at probably cytotoxic concentrations. Based on the result of the higher tier mammalian cell assays and the overall evaluation of the available data in a weight-of-evidence approach Basic Blue 159 is considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from June 8 to 25, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Based on B.N. Ames et al., Mutation Research, 31, 347, 1975; Tajima et al., Determination methods for mutagenicity of chemicals substances (Kodansha), P. 37-39, 1973.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0, 1, 5, 10, 50, 100, 500 µg/plate without S-9 mix
0, 5, 10, 50, 100, 500, 1000 µg/plate with S-9 mix
Both with and without S-9 mix, at top dose, growth inhibition was noted in all S. typhimurium strains. No effect on E. coli strain.

Vehicle / solvent:

Water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all S.typhimurium strains with S-9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

TA 98, TA 100, WP2 uvrA without S-9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 without S-9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

TA 1538 without S-9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S-9 mix

Details on test system and experimental conditions:

In agar (plate incorporation): 0.1 ml bacterial suspension, 0.1 ml test material solution and 0.5 ml phosphoric acid solution (0.5 ml S-9 mix solution in case of the metabolic activation test) were added to 2 ml top agar in a small test tube. The contents were mixed well, poured on a spread out evenly over the base agar plate.

After incubation for 48 hours at 37 °C, the number of transmuted colonies were counted.

Species / strain:

S. typhimurium, other: TA 98 and TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 1535, TA 100, TA 1537; E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 98 and TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Mean number of revertants/plate with test substance

µg/plate	S-9	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA 1538
500	-	0*	0*	16	0*	0*	0*
100	-	62	9	27	4	3	9
50	-	148	21	25	26	10	13
10	-	154	29	23	31	8	12
5	-	151	27	29	25	12	16
1	-	154	25	32	21	10	13
0	-	155	27	28	36	6	18
1000	+	0*	0*	15	0*	0*	0*
500	+	21	14	23	84	22	82
100	+	175	16	25	183	19	132
50	+	185	15	28	76	17	83
10	+	191	27	34	55	18	49
5	+	170	26	28	54	15	32
0	+	173	27	23	45	17	31

*growth of tester strain was inhibited

Mean number of revertants/plate in positive controls

	S-9	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA 1538
positive control	-	AF2 0.01 µg 564	NaN3 0.5 µg 179	AF2 0.04 µg 495	AF2 0.1 µg 476	9AA 80 µg 1445	4NOPD 1 µg 179
	+	2AA 0.5 µg 420	2AA 2 µg 199	2AA 10 µg 538	2AA 0.5 µg 227	2AA 2 µg 110	2AA 0.5 µg 173

AF2: 2 -(2 -furyl)-3 -(5 -nitro-2 -furyl)acrylamide

2AA: 2 -aminoanthracene

NaN3: sodium azide

9AA: 9 -aminoacridine

4NOPD: 4 -nitro-o-phenylenediamine

Conclusions:

Test substance showed a mutagenic potential in bacteria reverse mutation assay in presence of metabolic activation.

Executive summary:

The mutagenic effect of Basic Blue 159 was investigated in the Bacteria reverse mutation assay according to Ames (equivalent to OECD test guideline 471) using 5 strains of Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (requiring L-histidine) and Escherichia coli WP2 uvrA (requiring L-tryptophane).

Test was performed by plate incorporation method with and without metabolic activation with S-9 mix. With S-9 mix, concentrations tested were: 0, 5, 10, 50, 100, 500, 1000 µg/plate; without S-9 mix, concentrations tested were: 0, 1, 5, 10, 50, 100, 500 µg/plate. The test solution was poured over an agar plate and incubated for 48 h at 37°C. After incubation, the number of transmuted colonies was counted.

The test item had a distinct cytotoxic effects on all Salmonella strains in the presence and in the absence of the metabolising system at higher concentrations. No bacterial growth was seen in all top concentrations. In E.coli, only the highest concentration showed a slight cytotoxic effect with and without S9-mix.

There was an increase in the number of transmuted colonies with Salmonella typhimurium TA 98 and TA 1538 in the presence of the metabolic activation system. Unfortunately, the report does not contain information whether these concentrations caused cytotoxic effects or not. Therefore, the substance was considered as mutagen in this assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

HPRT method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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:

2015

Deviations:

yes

Remarks:

detailed below

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: frozen permananet cell culture from European Collection of cells cultures; cells kept at -196 °C under liquid nitrogen. After activation, cells were grown in Dulbecco's modified Eagles's medium with 4 mM L-glutamine and 10 % FBS in humidified incubator (5 % CO2, 37 ± 1 °C).
Cells underwent maximum 5 passages after thawing the original culture delivered
from cell collection before using for mutagenicity testing.
Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed in experiment without metabolic activation, because of bad growth of cells in HAT medium.

MEDIA USED
- DMEM (Dulbecco's minimal essential medium): minimal medium, part of complete growth medium
- HAT supplement: liquid mixture of sodium hypoxanthine (5 mM), aminopterin (20 µM) and thymidine (0.8 mM). HAT-supplemented medium is suitable for post-fusion selection against unfused or self-fused HGPRT–myeloma cells. Hypoxanthine and thymidine supply preformed purines and pyrimidines for DNA synthesis by hybridomas via the salvage pathway that utilizes HGPRT- contributed by the fused spleen cell. Aminopterin, a folic acid antagonist, inhibits the de novo nucleoside biosynthesis pathway. For HAT medium is diluted 50×. It is used as cleansing medium for reduction of mutants at the start of experiment
- FBS: fetal bovine serum, part of complete growth medium
- trypsin-EDTA (0.5%) solution: for release of cells from the bottom of dishes
- Atb - penicillin (10000 U/ml) streptomycin (10000 µg/ml): for prevention of contamination, part of complete growth medium
Complete growth medium DMEM : FBS : Atb = 500 : 55 : 5.5, prepared in laboratory.

- Periodically checked for mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: HPRT deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and a mixture of cofactors

Test concentrations with justification for top dose:

Test substance is soluble in water till 25.22 g/l. Test substance was dissolved in assay medium (DMEM). The highest recommended concentration is 2 mg/ml.
On the basis of cytotoxicity test results, the mutagenicity test with metabolic activation was arranged with range of concentrations 0.02-0.2 mg/ml. Concentrations for the mutagenicity test without metabolic activation were 0.01-0.04 mg/ml.

Vehicle / solvent:

- Solvent used: DMEM

Untreated negative controls:

yes

Remarks:

DMEN

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Rinsing of cells after treatment: PBS prepared in laboratory
Selective agent: 6-thioguanine 98 % diluted in 0.5 % Na2CO3, 5 µg/ml (final concentration) for selection of mutants
Dye for staining of colonies: methylene blue, 0.1 % solution

Cytotoxicity
The first cytotoxicity experiment was performed with as well as without metabolic activation. The concentrations for cytotoxicity were in range 0.01-2.0 mg/ml.
The first experiment failed in determination of doses for the mutagenicity tests, because doses with low, medium and high cytotoxicity were not possible to determine. Toxicity test had to be repeated for two more times.
On the basis of cytotoxicity test results, the mutagenicity test with metabolic activation was arranged with range of concentrations 0.02-0.2 mg/ml. Concentrations for the mutagenicity test without metabolic activation were 0.01-0.04 mg/ml.

Treatment: at least 2E+06 cells will be treated for each concentration/control for 3 hours. After treatment, every dish will be trypsinised and 300 cells will be seeded to 3 Petri dishes. Relative viability will be counted by comparison of viability in single concentration to viability in solvent control.

Mutation assay procedure
Each concentration was tested in two simultaneously performed independent runs (duplicates).
Experimental design
Concentrations for mutagenicity experiments were chosen on the basis of cytotoxicity testing. No precipitation was observed in any concentration in experiment with as well as without metabolic activation.
Test substance was dosed to dishes with cells in volume of 100 μl.
Cells were treated for 3 hours (with as well as without metabolic activation; day 1). After treatment, approximately 2E+06 cells were transferred to suitable number of dishes to seed enough cells. At the same time, cells were seeded for detection of number of cells (PE estimation).
On the 3rd, 6th and 8th day, approximately 2E+06 cells from every culture were transferred and 10th day, extractions of mutants was performed with using selective medium together with PE estimation again.

Experimental design (3 hours treatment)
Day Activity
1 treatment, passage of 1E+06 cells, plating of an aliquot (300 cells) for estimating of viability
3 passage of approx. 2E+06 cells
6 passage of approx. 2E+06 cells
8 passage of approx. 2E+06 cells
10 extraction of mutant cells, plating of an aliquot (300 cells) for estimating of viability

Fresh solutions of test substance were prepared for every experiment; solutions were prepared on a weight/volume in volumetric vials.
Neither assay of test substance stability, nor assays of its concentration and homogeneity in vehicle was undertaken.

Determination of survival
After treatment period, the cultures were trypsinised and an aliquot (0.3 ml of 1000/ml cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells.
A number of cells were then replaced in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give two millions of viable cells. Cells were grown in 10 cm Petri dishes.

Subculturing
On day 3, 6 and 8, cell populations were subcultured in order to maintain them in exponential growth. The number of cells taken forward was adjusted according to the expected viability, to give two millions viable cells seeded in 10 cm Petri dishes.

Incubation, staining and scoring
Survival and plating efficiency plates were incubated for at least six days (37±1 ºC, 5 % CO2, moistened) prior to scoring. Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.

Determination of mutant frequency
At expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. Then, an adequate number of cells were subcultured to maintain the treated populations of cells. This step is not performed on day 10.
After dilution, an estimated 220000 cells were plated in each of ten 100 mm tissue culture Petri dishes (together 2200000 cells). After about 1 hour, 6-thioguanine was added to each the Petri dish to final concentration of 5 µg/ml. Only HPRT mutant colonies are able to grow in the presence of 6-thioguanine; these plates were subsequently scored for the presence of mutants.
After dilution, an estimated 300 cells were plated in each of three 60 mm tissue culture Petri dishes. These plates were used to estimate plating efficiency.

Evaluation criteria:

Each experiment will be evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987.
The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive than a repeated experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions [i.e. S9 concentration or S9 origin]) could be performed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility
Precipitation occurred in the first toxicity test starting from 1.0 mg/ml. In the end, due to cytotoxicity of test substance, lower concentrations were used for mutagenicity testing in which no precipitation occurred.

Cytotoxicity experiments
In the first cytotoxicity test (concentration range 0.01 - 2 mg/ml) with as well as without metabolic activation, cytotoxicity was seen at concentration of 0.01 (without metabolic activation) and 0.1 (with metabolic activation). A second experiment was necessary to be performed for determination of doses with high, medium and low cytotoxicity.

Concentrations between 0.0005 - 0.01 mg/ml were used for second cytotoxicity experiment without metabolic activation. Concentrations 0.0025 - 0.05 mg/ml were used for the second cytotoxicity experiment with metabolic activation.
In this experiment doses appropriate for mutagenicity testing were not found because of no or small toxicity.
The third experiment had to be done for clarification. Concentrations 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 were used without metabolic activation; concentrations 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.50 and 0.75 mg/ml were used with metabolic activation.
Based on all the cytotoxicity tests, the following concentrations were determined for mutagenicity testing:
- without metabolic activation: 0.01; 0.015; 0.02; 0.025, 0.030 and 0.040 mg/ml
- with metabolic activation: 0.02, 0.05, 0.1 and 0.2 mg/ml.

Cytotoxicity in mutagenicity experiments
Experiment without metabolic activation was started with 6 concentrations, with the idea to choose the 4 best concentrations (OECD 476).
Cytotoxicity immediately after treatment and after the first plating for cultivation to allow expression of the mutant phenotype was recorded. During further passages the influenced cells either were growing slower than control or some cells died. Overall, decrease of cells in all of the three passages was seen.
Concentration of 0.02 mg/ml was similarly toxic as the surrounding concentrations 0.015 and 0.025 mg/ml; so it was not kept untill the end of experiment.

Experiment with metabolic activation was started at concentrations of 0.02, 0.05, 0.1 and 0.2 mg/ml; concentration of 0.4 mg/ml was added during the mutagenicity because no toxicity was observed immediately after treatment in the lower concentrations.
Concentrations of 0.2 and 0.4 mg/ml were completely cytotoxic after plating for other growing so they were not continued. Toxicity of concentration 0.1 mg/ml was in accordance with OECD 476 but less than 2 millions of cells were transferred for other growing.
Additional experiment was performed with concentrations 0.075 and 0.1 mg/ml to get another suitable concentration for evaluation. In the end, four concentrations were available for 0.02, 0.05, 0.075, 0.1 mg/ml.

pH determination
conc. mg/ml measured pH
DMEM 7.62
0.02 7.64
0.05 7.65
0.1 7.65
0.2 7.65

Addition of test substance to cultivation medium changed pH of treatment solutions by 0.03. No pH adjustment was needed.

Mycoplasma determination
Three withdrawals of medium were performed: experiment without metabolic activation, experiment with metabolic activation and additional experiment with metabolic activation, after minimum of 14 days of growing of cells.
Result of test sample was negative so all media after cultivation of cells were free of mycoplasma.

Plating efficiency
For the assessment of number of plated cells, PE was determined always after treatment.
In all concentrations used in all experiments more than 2 millions cells were influenced in single replicates. In both experiments with metabolic activation and concentration of 0.1 mg/ml less than 2 millions of cells were seeded for further cultivation. In the additional experiment in both replicates together, condition of 2 millions cells was fulfilled.

Mutagenicity
Mutation frequency of cells treated with test substance in both experiments was low in all concentrations; Mt/Msc ratios generally never exceeded 3 fold limits (in fact all of them were below 2 fold vs solvent control). At the same time, no dose dependence was observed in any experiment.

Assay acceptance criteria compliance
Mutation frequencies of negative control (DMEM) were 1.07-2.12 mutants per 1E+05 plated cells. Historical control range (97.5 % confidence interval) is 0.78-2.28 mutants per 1E+05cells (123 entries).
Mutation frequency of positive controls was sufficiently high:
EMS 50 µl: 18.97; historical control range (95 % confidence interval) is 10.07 – 18.22 mutants per 1E+05cells (40 entries);
EMS 100 µl: 44.14; historical control range (95 % confidence interval) is 19.23 – 37.41 mutants per 1E+05cells (41 entries);
DMBA: 30.69-38.06 what is an evidence of good function of the test system. Historical control range (95 % confidence interval) is 19.23 – 37.41 mutants per 1E+05cells (54 entries).

Mutagenicity without metabolic activation, 3h treatment

Conc. mg/ml	viability (number of colonies)			avg	PE %	Mutants (number of colonies)										∑M	NPC	MF/10^5cells	Mt/Msc
NC (1)	335	402	394	377	102.8	2	2	2	6	4	5	4	4	6	1	36	2,764,667	1.30	1.09
NC (2)	337	364	368	356	97.2	1	1	6	1	6	5	3	2	1	2	28	2,613,111	1.07	0.90
0.01 (1)	298	311	328	312	85.2	2	4	3	3	1	4	3	3	2	3	28	2,290,444	1.22	1.03
0.01 (2)	304	312	387	334	91.2	3	2	2	4	3	4	3	1	5	4	31	2,451,778	1.26	1.06
0.015 (1)	379	378	376	378	103.0	3	4	2	6	2	2	4	1	4	4	32	2,769,556	1.16	0.97
0.015(2)	320	311	303	311	84.9	6	5	4	5	1	3	1	4	2	2	33	2,283,111	1.45	1.21
0.025 (1)	310	266	301	292	79.7	4	6	2	0	5	5	3	5	2	4	36	2,143,778	1.68	1.41
0.025 (2)	381	386	375	381	103.8	1	4	3	5	2	4	2	2	3	5	31	2,791,556	1.11	0.93
0.03 (1)	259	302	303	288	78.5	3	5	4	1	4	4	2	5	4	5	37	2,112,000	1.75	1.47
0.03 (2)	302	380	389	357	97.4	4	3	4	4	2	5	3	4	6	4	39	2,618,000	1.49	1.25
0.04 (1)	346	324	351	340	92.8	4	5	4	2	4	2	3	3	2	4	33	2,495,778	1.32	1.11
0.04 (2)	346	342	358	349	95.1	3	3	2	7	4	2	2	4	4	9	40	2,556,889	1.56	1.31
EMS50	320	328	286	311	84.9	40	46	45	40	51	41	38	46	39	47	433	2,283,111	18.97	15.94
EMS100	336	326	301	321	87.5	107	104	104	114	120	90	105	105	100	90	1 039	2,354,000	44.14	37.09

∑M = sum of mutants in all 5 dishes

NPC = no. of planted cells

MF/1E+05 cells = mutation frequency

M_t/M_sc = no. of mutants in test conc. vs no. of mutants in solvent control

Mutagenicity with metabolic activation, 3h treatment

Conc. mg/ml	Viability (number of colonies)			avg	PE %	Mutants (number of colonies)										∑M	NPC	MF/1E+05 cells	Mt/Msc
NC (1)	371	385	385	380	112.5	9	1	8	8	7	7	4	7	1	7	59	2,789,111	2.12	1.12
NC (2)	283	295	310	296	87.5	1	4	2	5	3	2	5	4	2	7	35	2,170,667	1.61	0.85
0.02 (1)	399	402	363	388	114.7	4	6	5	5	5	6	6	4	7	5	53	2,845,333	1.86	0.98
0.02 (2)	257	291	277	275	81.3	2	3	2	2	5	2	6	3	2	4	31	2,016,667	1.54	0.81
0.05 (1)	362	331	367	353	104.5	1	2	2	5	3	2	5	4	1	2	27	2,591,111	1.04	0.55
0.05 (2)	395	382	360	379	112.1	5	6	6	4	4	4	4	2	3	4	42	2,779,333	1.51	0.80
0.1 (1)	452	442	409	434	128.4	6	3	5	3	4	1	2	6	4	3	37	3,185,111	1.16	0.61
0.1 (2)	376	345	358	360	106.4	3	9	5	6	5	4	1	4	2	9	48	2,637,556	1.82	0.96
DMBA (1)	338	327	340	335	99.1	77	85	98	93	112	95	85	101	82	107	935	2,456,667	38.06	20.08
DMBA (2)	376	345	358	360	106.4	80	93	115	110	100	91	102	83	89	96	959	2,637,556	36.36	19.18

Mutagenicity with metabolic activation, 3h treatment

Conc. mg/ml	Viability (number of colonies)			avg	PE %	Mutants (number of colonies)										∑M	NPC	MF/1E+05 cells	Mt/Msc
NC (1)	340	302	295	312	97.6	1	2	7	4	2	6	3	3	6	1	35	2,290,444	1.53	1.02
NC (2)	327	315	341	328	102.4	4	3	4	3	3	3	3	2	5	5	35	2,402,889	1.46	0.98
0.075 (1)	273	288	282	281	87.8	7	8	13	2	8	4	4	4	4	2	56	2,060,667	2.72	1.43
0.075 (2)	258	275	276	270	84.3	2	2	3	8	1	5	4	4	5	4	38	1,977,556	1.92	1.01
0.1 (1)	400	465	470	445	139.1	5	8	2	3	3	2	5	5	3	4	40	3,263,333	1.23	0.65
0.1 (2)	255	272	300	276	86.1	4	2	8	8	5	6	6	9	6	5	59	2,021,556	2.92	1.54
DMBA (1)	412	326	343	360	112.6	82	81	82	72	89	81	75	81	84	84	811	2,642,444	30.69	16.19
DMBA (2)	342	375	324	347	108.4	70	82	80	87	85	80	74	92	68	64	782	2,544,667	30.73	16.21

Conclusions:

Test substance Basic Blue 159 trichlorozincate was not mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test item was assayed for mutagenicity in in vitro mammalian cell gene mutation test, according to OECD guideline 476. Chinese hamster V79 lung fibroblasts were used for testing; test substance was dissolved in DMEM. Cytotoxicity tests without as well as with metabolic activation were performed to provide the optimal dose range for the mutagenicity study. Concentrations used for cytotoxicity testing were in the range of 0.0005 – 2.0 mg/mL.

On the basis of cytotoxicity testing results, six concentrations were chosen for the mutagenicity experiment without metabolic activation, i.e. 0.01, 0.015, 0.020, 0.025, 0.03 and 0.04 mg/mL. Four concentrations 0.02, 0.05, 0.1 and 0.2 mg/mL were used for the experiment with metabolic activation. The concentration of 0.2 mg/mL was completely toxic and in the concentration 0.1 mg/mL insufficient number of cells was transferred for further cultivation. An additional experiment was performed with the concentration of 0.075 mg/mL and the concentration 0.1 mg/mL was repeated to get four analysable concentrations according to guidelines.

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. Concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average mutant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the experimental conditions, test substance was non-mutagenic for V79 cells with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

HPRT method

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Read-across

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached justification document

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Basic Blue 159 trichlorozincate / CAS 93783-70-1 / EC 298-265-2
Target: Basic Blue 159 methyl sulfate / CAS 83969-12-4 / EC 281-589-3

3. ANALOGUE APPROACH JUSTIFICATION
see attached justification document

Reason / purpose for cross-reference:

read-across source

Type of assay:

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

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The structural analogue test substance Basic Blue 159 trichlorozincate was not mutagenic for V79 cells with as well as without metabolic activation. The same is considered for the other salt form of Basic Blue 159, the methyl sulfate.

Executive summary:

The test item was assayed for mutagenicity in in vitro mammalian cell gene mutation test, according to OECD guideline 476. Chinese hamster V79 lung fibroblasts were used for testing; test substance was dissolved in DMEM. Cytotoxicity tests without as well as with metabolic activation were performed to provide the optimal dose range for the mutagenicity study. Concentrations used for cytotoxicity testing were in the range of 0.0005 – 2.0 mg/mL.

On the basis of cytotoxicity testing results, six concentrations were chosen for the mutagenicity experiment without metabolic activation, i.e. 0.01, 0.015, 0.020, 0.025, 0.03 and 0.04 mg/mL. Four concentrations 0.02, 0.05, 0.1 and 0.2 mg/mL were used for the experiment with metabolic activation. The concentration of 0.2 mg/mL was completely toxic and in the concentration 0.1 mg/mL insufficient number of cells was transferred for further cultivation. An additional experiment was performed with the concentration of 0.075 mg/mL and the concentration 0.1 mg/mL was repeated to get four analysable concentrations according to guidelines.

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. Concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average mutant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the experimental conditions, test substance was non-mutagenic for V79 cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genotoxicity of Basic Blue 159 was assessed in an in-vivo mouse micronucleus test (OECD 474). Basic Blue 159 was not genotoxic in the in-vivo mouse micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From February 20 to March 31 and from October 9 to December 14, 1992

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:

(1983)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1984)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

SPF Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8-12 weeks of age
- Weight at study initiation: 28-44 g (first trial), 28-43 g (second trial)
- Assigned to test groups randomly: yes
- Housing: Makrolon type I cages; females 3 per gorpu, males singly
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 22.5-23 °C
- Humidity: 44-47 % (first trial), 38-45 % (second trial)
- Air changes: 10 times per hour
- Photoperiod: 12 hours light

Route of administration:

intraperitoneal

Vehicle:

Physiological saline solution.

Details on exposure:

Administered volume: 10 ml/kg body weight

Duration of treatment / exposure:

Negative control: 0 mg/kg, 24 h
Test substance: 8.5 mg/kg, 16 h
Test substance: 8.5 mg/kg, 24 h
Test substance: 8.5 mg/kg, 48 h
Test substance: 8.5 mg/kg, replacement group
Positive control, cyclophosphamide: 20 mg/kg, 24 h

Frequency of treatment:

Single exposure.

Dose / conc.:

8.5 mg/kg bw/day (actual dose received)

Remarks:

based on results of a pilot study

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

yes, cyclophosphamide dissolved in deionized water.

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

Schmid's method was q,etto produce the smears.
At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor).

A suitable tube was filled with sufficient fetal calf serum.
A small amount of serum was drawn from the tube into suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow cavity.
The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.
The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension.
Finally, the flushing might be repeated from the other end, after it had been opened.
The tube containing the serum a4 bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette, leaving only a small remainder.
The sediment was mixed to produce a homogeneous suspension.
One drop of the viscous suspension was placed on a well-cleaned slide and spread with a suitable object, to allow proper evaluation of the smear.
The labeled slides were dried overnight. If fresh smears needed td-be stained, they needed to be dried with heat for a short period.

Staining of smears
Smears were stained automatically with an Ames Hema-Tek Slide Stainer from the Miles Company. Slides were then "destained" with methanol, rinsed with deionized water, and left to dry.

Covering of Smears
Following this treatment, the smears were transferred to a holder. A cuvette was filled with xylene, into which the holder was immersed for approximately ten minutes. Slides were removed singly (e.g. with tweezers) to be covered.
A small amount of covering agent was taken from a bottle with a suitable object (e.g. glass rod) and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. Slides were not evaluated until the covering agent had dried.

Evaluation
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
It is expedient to establish the ratio of polychromatic to normochroztic erythrocytes for two reasons:
1.Individual animals with pathological bone-marrow depressions may be identified and excluded from the evaluation.
2. An alteration of this ratio may show that the test compound actually reaches the target.

Evaluation criteria:

The number of normochromatic erythrocytes per 1000 polychromatic ones was noted. If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation.
A relevant, treatment related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority ot the animals in the treated group than in the negative control.
In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established.
This information is useful in two ways. Firstly, it permits the detection of individuals already subject to damage before the start of the test. Secondly, combined with the number of micronucleated pychromatic erythrocytes, it permits a representation of the time-effect curve for positive substances.
An increase in the number of micronucleated normochromatic erythrocytes, without a preceding increase in micronucleated polychromatic erythrocytes, is irrelevant to the assessment of a clastogenic effect, since normochromatic erythrocytes originate from polychromatic ones. Before an effect can be observed in normochromatic erythrocytes, there must be a much greater increase in micronucleated polychromatic erythrocytes, duo to the "dilution effect" of the "old" cells, i.e. normochromatic erythrocytes already present at the start of the test, and this effect would have been observed previously.

Statistics:

Test groups with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochronatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (ls ranges) were calculated for all the means.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- No. of animals: 5/sex
- Dose range: 5, 7.5, 8.5, 9, 9.5 and 10 mg/kg
- Clinical signs of toxicity in test animals: symptoms were recorded for, up to 48 hours, starting at 5 mg/kg: apathy, roughened4 far, staggering gait, sternal recumbency, spasm, shivering and difficulty in breathing. In addition, 2 of 5 animals died in the 9.5 mg/kg group and 4 of 5 animals dd in the 10 mg/kg group. Based on these results, 8.5 mg/kg was chosen as MTD for this test. Due to the use of another batch in the second trial a toxicity test was performed in advance, in which 3 males and 2 females received 8.5 mg/kg. The following symptoms were recorded for up to 4 days: apathy, reduced motility, roughened fur, staggering gait, spasm and difficulty in breaing. In addition, one male died.

Toleration by the Animals
After single intraperitoneal administration of 8.5 mg/kg, treated animals of both trials, showed the following compound-related symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm shivering and difficulty in breathing. In addition, reduced motility was observed during the first trial. The feedlng behavior of the animals was normal. Two of 40 treated animals died during the test period of the first trial, due to the acute toxicity of 8.5 mg/kg the substance. However, there were no substance-induced mortalities in the second trial. No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic evaluation
Evaluation of trial 1
Concerning the assessment of the clastogenic potential of the substance, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment, being 1000: 918 (ls=331) in the negative control, 1000: 1893 (ls=586) in the 16 hours group, 1060: 1864 (ls=838) in the 24 hours group and 1000:147 (ls=2194) in the 48 hours group. Relevant variations were noted.

MAIN STUDY

Toleration by the Animals

After single intraperitoneal administration of 8.5 mg/kg, treated animals of both trials, showed the following compound-related symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm shivering and difficulty in breathing. In addition, reduced motility was observed during the first trial. The feedlng behavior of the animals was normal. Two of 40 treated animals died during the test period of the first trial, due to the acute toxicity of 8.5 mg/kg the substance. However, there were no substance-induced mortalities in the second trial. No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic evaluation: trial 1

Concerning the assessment of the clastogenic potential of the substance, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly. The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment, being 1000: 918 (ls=331) in the negative control, 1000: 1893 (ls=586) in the 16 hours group, 1060: 1864 (ls=838) in the 24 hours group and 1000:147 (ls=2194) in the 48 hours group. Relevant variations were noted.

No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 8.5 mg/kg dose, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.0/1000 (ls=2.3) in the negative control, and 3.3/1000 (ls=2.9), 2.5/1000 (ls=2.0) and 3.5/1000 (I=2.8) in the treated groups.

Similarly, there could be no biologically significant variation between the negative control and treated groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed.

The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 18.6/1000 (ls=7.8), which represents a biologically relevant increase in comparison to the negative control. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.

No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree (1000: 1070 (1s=385), as against 1000: 918 in the negative control). This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

The means of micronucleated polychromatic erythrocytes of the 16 and 48 hours groups treated with 8.5 mg/kg test substance exceeded the range of historical negative control means. Despite the negative results of this trial but due to the assay assessment criteria, the results of the test must be rated as equivocal and a second test had to be performed.

Microscopic evaluation: trial 2

Concerning the assessment of the clastogenic potential of test substance, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly. The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with test substance, being 1000: 556 (ls=110) in the negative control, 1000: 1742 (ls=522) in the 16 hours group, 1000: 1096 (ls=428) in the 24 hours group and 1000: 1155 (ls=489) in the 48 hours group. Relevant variations were thus noted.

No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with dose of 8.5 mg/kg, with respect to the incidence of micronucleated polychromatic erythrocytes. The negative result of the first trial was reproduced by the second trial and no mean exceeded the range of historical negative controls. The incidence of micronucleated cells was 1.6/1000 (ls=1.6) in the negative control, and 1.3/1000 (ls=0.8), 1.9/1000 (ls=2.0) and 2.9/10b0 (ls=3.0) in treated groups.

Similarly, there could be no biologically significant variation between the negative control and test groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated ce1ls was 24.3/1000 (ls=10.8), which represents a biologically relevant increase in comparison to the negative contro1. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.

No further effect of cyc1ophosphide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary a biologically relevant degree (1000: 578 (ls=192N as against 1000: 556 in the negative control). This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

Assessment

Normally, cells with micronuclei (Howell-Jolly bodies) occur in polychromatic erythrocytes with an incidence of up to approximately 3.0/1000 (5, 6, and own experience). The increase in micronucleated polychromatic erythrocytes, due, for example, to chromosome breaks or spindle disorders, is the criterion for clastogenic effects in this test model.

The combined results of both trials gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 8.5 mg/kg. The known mutagen and clastogen, cyclophosphamide, had in both trials a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg body weight. The number of micronucleated polychromatic erythroytes increased to a biologically relevant degree.

The number of micronucleabed normochromatic erythrocytes did not increase relevantly in any of the groups.

It is of further interest to establish the number of normochromatic cells, to learn whether the ratio of polychromatic to normochromatic erythrocytes was altered by treatment. In both trials this ratio did vary to a biologically relevant degree in treated groups in comparison to the negative control. Cyclophosphamide did not change this ratio.

Conclusions:

There was no indication of a clastogenic effect of an intraperitoneal dose of 8.5 mg/kg test substance in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

Executive summary:

Method

The micronucleus test was carried out on male and female mice to assess a possible clastogenic effect of test substance on chromosomes of bone-marrow erythroblasts. Cyclophosphamide, i.e. a known clastogen and cytostatic agent, served as positive control.

Two independent trials with identical setup were performed. Treated animals of both trials received a single intraperitoneal administration of either test substance or cyclophosphamide. The femoral marrow of groups treated with test substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours.

Test substance and positive control, cyclophosphamide, were administered at doses of 8.5 and 20 mg/ bw, respectively.

Results

After administration both trials, test substance caused symptoms of toxicity, in terms of apathy, roughned fur, staggering gait, spasm, reduced motility,....

Two of forty animals of the first trial died before the end of the test due to acute intraperitoneal toxicity of the 8.5 mg/kg dose, while all animals of the second trial survived until the end of this test.

In both trials there was an altered ratio between polychromatic and normochromatic erythrocytes. Combined results of both trials gave no indications of relevant clastogenic effect of test substance after a single intraperitoneal treatment with 8.5 mg/kg.

Positive control had in both trials a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei; however, the ratio of polychromatic to normochromatic erythrocytes was not altered.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic effect of Basic Blue 159 was investigated in the Bacteria reverse mutation assay according to Ames (equivalent to OECD test guideline 471) using 5 strains of Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (requiring L-histidine) and Escherichia coli WP2 uvrA (requiring L-tryptophane).

Test was performed by plate incorporation method with and without metabolic activation with S-9 mix. With S-9 mix, concentrations tested were: 0, 5, 10, 50, 100, 500, 1000 µg/plate; without S-9 mix, concentrations tested were: 0, 1, 5, 10, 50, 100, 500 µg/plate. The test solution was poured over an agar plate and incubated for 48 h at 37°C. After incubation, the number of transmuted colonies was counted.

The test item had a distinct cytotoxic effects on all Salmonella strains in the presence and in the absence of the metabolising system at higher concentrations. No bacterial growth was seen in all top concentrations. In E.coli, only the highest concentration showed a slight cytotoxic effect with and without S9-mix.

There was an increase in the number of transmuted colonies with Salmonella typhimurium TA 98 and TA 1538 in the presence of the metabolic activation system. Unfortunately, the report does not contain information whether these concentrations caused cytotoxic effects or not. Therefore, the substance was considered as mutagen in this assay.

The test item was assayed for mutagenicity in in vitro mammalian cell gene mutation test, according to OECD guideline 476. Chinese hamster V79 lung fibroblasts were used for testing; test substance was dissolved in DMEM. Cytotoxicity tests without as well as with metabolic activation were performed to provide the optimal dose range for the mutagenicity study. Concentrations used for cytotoxicity testing were in the range of 0.0005 – 2.0 mg/mL.

On the basis of cytotoxicity testing results, six concentrations were chosen for the mutagenicity experiment without metabolic activation, i.e. 0.01, 0.015, 0.020, 0.025, 0.03 and 0.04 mg/mL. Four concentrations 0.02, 0.05, 0.1 and 0.2 mg/mL were used for the experiment with metabolic activation. The concentration of 0.2 mg/mL was completely toxic and in the concentration 0.1 mg/mL insufficient number of cells was transferred for further cultivation. An additional experiment was performed with the concentration of 0.075 mg/mL and the concentration 0.1 mg/mL was repeated to get four analysable concentrations according to guidelines.

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. Concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average mutant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the experimental conditions, test substance was non-mutagenic for V79 cells with and without metabolic activation.

The micronucleus test was carried out in male and female mice to assess a possible clastogenic or aneugenic effect of test substance on chromosomes of bone-marrow erythroblasts. Cyclophosphamide, a known clastogen and cytostatic agent, served as positive control. Two independent trials with an identical setup were performed. Treated animals of both trials received a single intraperitoneal administration of either test substance or cyclophosphamide. The femoral marrow of groups treated with test substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. Test substance and positive control, cyclophosphamide, were administered at dose levels of 8.5 and 20 mg/kg bw, respectively.

After administration of the test item, in both trials clinical sign of toxicity consisting of apathy, piloerection, staggering gait, spasm, and reduced motility were observed. Two of forty animals of the first trial died before the end of the test due to acute intraperitoneal toxicity of the 8.5 mg/kg bw dose, while all animals of the second trial survived until the end of this test. In both trials there was an altered ratio between polychromatic and normochromatic erythrocytes. Combined results of both trials gave no indications of relevant clastogenic or anougenic effect of test substance after a single intraperitoneal treatment with 8.5 mg/kg bw.

The positive control had a clear clastogenic effect in both trials, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei; however, the ratio of polychromatic to normochromatic erythrocytes was not altered.