Reaction products of diazotised 4,4-diaminodiphenylamine-2-sulfonic acid, subsequently coupled with 6-amino-4-hydroxynaphthalene-2-sulfonic acid, further diazotised and coupled with metaphenylendiamine, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Principles of method if other than guideline:

Pre-incubation test. Screening program: strain TA98 placed in standard plates. Evaluation without metabolic activation.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

without

Metabolic activation system:

No

Test concentrations with justification for top dose:

Range 20-5000 µg/plates: 20, 100, 500, 2500, 5000 µg/plate

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

NPD

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: 2500-5000 µg/plate

	Revertant/plate			Quotient
Dose µg/plate	- S9	M	DS	-S9
Negative control	29 29 18	25	6	1.0
20	19 13 24	19	6	0.7
100	26 26 26	26	0	1.0
500	31 29 28	29	2	1.2
2500	31 30 24	28	4	1.1
5000	32 31 33	32	1	1.3
Positive control	608 572 666	615	47	24.3

Conclusions:

Negative

Non mutagen; no increase of revertants has been observed.

Executive summary:

Non mutagen; no increase of revertants has been observed.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, test procedure in accordance with OECD 476 methods, meets generally accepted scientific principles, acceptable for assessment and GLP compliant.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese Hamster ceU line V79, clone 65/3, Dr. D. Wild, Freiburg, Germany

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

SELECTION OF TEST CONCENTRATIONS
Preliminary test: in the preliminary toxicity test with and without metabolic activation 12 concentrations of the tested substance were tested. The concentrations selected ranged from 0.24 to 500.0 µg/ml and separated by 2-fold intervals.
In the part with metabolic activation the highest concentration produced an acute growth inhibition of 62.70%. In the part without metabolic activation the substance exerted a complete growth inhibitory effect at the highest concentration of 500.0 µg/ml. The next lower concentrations of 250.0 and 125.0 µg/ml revealed an acute growth inhibitory effect of 98.46 and 72.56% respectively.

Main test: accordingly, four concentrations were selected for the original experiment ranging from 18.52 to 500.0 µg/ml and from 4.63 to 125.0 µg/ml in the presence and absence of metabolic activation, respectively.
In the part with metabolic activation, the growth inhibition determined after treatment at the highest concentration of 500.0 µg/ml showed a mean value of 6.91%. After expression growth was inhibited by 13.51%.
In the absence of metabolic activation no significant growth inhibitory effect was seen after treatment and expression.
The highest concentration of 500.0 µg/ml tested in the original experiment with activation was determined to be the highest suitable concentration due to solubility limitations in the vehicle. Therefore the same concentration range was tested in the respective confirmatory experiment, although no severe toxicity was reached. In the part without metabolic activation a concentration range of 9.26 to 250.0 µg/ml was selected for the confirmatory experiment in order to reach a more pronounced toxicity at the highest concentration. In the presence of metabolic activation no significant acute cytotoxicity was obtained at the highest concentration. After expression growth was inhibited by 9.54%. In the part without activation, the mean growth inhibition at the highest concentration of 250.0 µg/ml was 27.91% in spite of the increased concentration.

METHOD OF APPLICATION: in medium

DETERMINATION OF MUTANT FREQUENCY
Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After the incubation time, colonies are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity is determined my measuring the relative cloning efficiency (survival) of the cultures after the treatment period.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Based on the results of the performed experiments and under the given experimental conditions, it is concluded that the tested substance and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

Substance was tested for mutagenicity, in details for the evaluation of properties to induce gene mutations.

The mutagenicity studies were conducted following OECD 476, gene mutation test with Chinese Hamster cell V79.

The studies were performed in the absence and in the presence of metabolic activation. A dose range with different doses was used.

Result

The tested substance shows no mutagenic activity.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

other: read across from similar substance

Adequacy of study:

weight of evidence

Study period:

From October 31, 1990 to April 12, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted May 26th, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: LMP, D-6100 Darmstadt.
- Doubling time: 12-16 hrs in stock culture.
- Plating efficiency: more than 50 %.
- Methods for maintenance in cell culture: stored in liquid nitrogen.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: seeding was done with about 5 x 10^5 cells per flask in 15 ml of MEM (minimal essential medium) supplemented with 10 % fetal calf serum. The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C and 4.5 % CO2 atmosphere.
- Properly maintained: yes. Stored in liquid nitrogen.
- Periodically checked for mycoplasma contamination: yes. Before freezing.
- Periodically checked for karyotype stability: yes. Before freezing.

Metabolic activation:

with and without

Metabolic activation system:

homogenate of rat liver (S9), induced by Arochlor 1254.

Test concentrations with justification for top dose:

Without S9 mix
- 7 h: 0.010; 0.030; 0.100; 0.300 mg/ml.
- 18 h: 0.001; 0.003; 0.010; 0.030; 0.100; 0.300 mg/ml.
- 28 h: 0.010; 0.030; 0.100; 0.300 mg/ml.
With S9 mix
- 7 h: 0.030; 0.100; 0.300; 1.000 mg/ml.
- 18 h: 0.003; 0.010; 0.030; 0.100; 0.300; 1.000 mg/ml.
- 28 h: 0.030; 0.100; 0.300; 1.000 mg/ml.

Vehicle / solvent:

- Solvent used: saline G was used to dissolve the test article. One litre of the solvent was composed of 8000 mg NaCl, 400 mg KCl, 1100 mg Glucose, 290 mg Na2HP04.7H20, 150 mg KH2P04 and the pH was adjusted to 7.2
- Justification for choice of solvent: it was chosen according to its solubility properties and its non-toxicity for the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

PRE EXPERIMENT FOR TOXICITY AND DOSE SELECTION
In order to establish a concentration dependent plating efficiency relationship a pre-experiment was performed in the same conditions as for the main experiment. 8 concentrations were tested (0.001; 0.010; 0.030; 0.100; 0.300; 1.000; 2.000; 4000 mg/ml). Per concentration duplicate cultures were used. The mitotic index was determined in 1000 cells from each of the two slides per test group. Toxicity of the test article was evidenced by a reduction in plating efficiency. The highest dose level used was 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis). If toxic effects were produced the highest dose level should reduce the plating efficiency to approximately 20 - 50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found. According to the results of the pre-experiment six concentrations (18 h interval) were selected for the chromosomal aberration assay.

SEEDING OF THE CULTURES
Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised at 37 °C for approximately 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution. One litre of Ca-Mg-free salt solution was composed as follows of 8000 mg NaCl, 400 mg KCl, 1100 mg glucose, 350 mg NaHC03. Prior to trypsin treatment the cells were rinsed with the Ca-Mg-free salt solution containing 200 mg/ml EDTA (Ethylene diamine tetraacetic acid). Then the cells were seeded into Quadriperm dishes which contained microscopic slides (2 chambers per dish and test group). In each chamber 5 x 10 ^4 - 1 x 10^5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

TREATMENT OF THE CULTURESA
fter 48 h (7 h, 28 h preparation interval) and 55 h (18 h preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 μl/ml S9 mix. Concurrent negative and positive controls (with and without S9 mix) were performed in parallel. After 4 h this medium was replaced with complete medium after rinsing twice with saline G. All incubations were done at 37° C in a humidified atmosphere with 4.5 % CO2.

CHROMOSOME PREPARATION
5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 μg/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3:1 absolute methanol:glacial acetic acid. Both slides per group were prepared. After fixation the cells were stained with giemsa.

ANALYSIS OF METAPHASE CELLS
The evaluation of the slides was performed using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides except in the positive control samples where 25 metaphases were scored. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To determine a cytotoxic effect the mitotic index was determined.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype) was scored.

PREPARATION OF MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9: the S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9.000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 ml and stored at - 70 °C. Small numbers of the ampoules were kept at - 20 °C for only several weeks before use. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml, the S9 used in this study had 30.3 mg/ml protein concentration.
- S9 mix: on the day of the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/ml in the cultures. The composition of the cofactor solution was concentrated to yield the concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

ACCEPTABILITY OF THE ASSAY
1. The number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % - 4.00 %.
2. The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

Rationale for test conditions:

V79 cell line was chosen as it is used for many years in in-vitro experiments with success. The high proliferation rate (doubling timne 12-16 hrs in stock cultures) and the high plating efficiency of untreated cells (more than 50 %), both necessary for the appropriate performance of the study reccomend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points. A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points 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 chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in conc. higher than 1 µg/ml (without S9 mix) and 30 µg/ml (with S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
In the pre-experiment for toxicity the colony forming ability of the V79 cells was distinctly reduced with concentrations higher than 0.001 (without S9 mix) and 0.030 mg/ml (with S9 mix). Based on the results of the pre-experiment and the cytogenic experiment, in the main experiment, in the absence of S9 mix cultures treated with 0.1 mg/ml (7 h interval) and 0.3 mg/ml (18 h and 28 h interval could be evaluated as top dose level. In the presence of S9 mix samples treated with 0.03 (interval 7 h) and 0.3 mg/ml (intervals 18 and 28 h) could be evaluated as top dose level. With higher concentrations not enough scorable cells could be found in the respective intervals. Treatment with concentrations higher than 0.001 mg/ml (without S9 mix) and 0.03 mg/ml (with S9 mix) reduced distinctly the plating efficiency of the V79 cells. Also, in the cytogenetic experiment the mitotic index was distinctly reduced in the samples treated with the highest scorable dose level at each fixation interval except at interval 7 (with S9 mix). However, at this test group at least a slight reduction was observed. One (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.

POLYPLOID METAPHASES
No relevant deviation from the control data was found after treatment with the test article. At fixation interval 18 h (with S9 mix), in the samples treated with 0.10 mg/ml the number of polyploid cells (8.0 %) is increased as compared to the corresponding solvent control (1.5 %). However, taking into account the polyploidy range of all the control groups in this study: 1.5 % - 12.0 % this value falls within that range and also in the range of the laboratory historical controls: 0 - 11 %.

POSITIVE CONTROL
EMS (0.72 mg/ml) and CPA (1.40 µg/ml) used as positive controls, showed distinct increases in cells with structural chromosome aberrations.

Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 5.00 %) were in or near to the range of the control values: 0.00 % - 4.50 %.

The mutagenicity data for each fixation interval are presented in the tables below.

Mutagenicity data at 7 h fixation interval.

Fixation interval 7 h
Article	number of cells analysed	conc. (mg/ ml)	S9 mix	per cent aberrant cells
				incl. Gaps	excl. Gaps	exchanges
Solvent-control	200	0.0	-	3.00	1.00	0.50
Test article	200	0.10	-	4.00	1.50	0.00
Solvent-control	200	0.0	+	2.50	1.00	0.00
Test article	200	0.03	+	6.50	3.00	0.00

Mutagenicity data at 18 h fixation interval.

Fixation interval 18 h after start of experiment
Article	number of cells analysed	conc. (mg/ ml)	S9 mix	per cent aberrant cells
				incl. Gaps	excl. Gaps	exchanges
Negative control	200	0.0	-	10.00	4.00	0.00
Solvent-control	200	0.0	-	6.50	3.50	1.50
Positive control EMS	50	0.72	-	22.00	16.00	12.00
Test article	200	0.01	-	1.50	1.00	0.00
Test article	200	0.03	-	4.00	2.00	0.50
Test article	200	0.10	-	2.50	1.00	0.00
Negative control	200	0.0	+	1.50	1.00	0.00
Solvent-control	200	0.0	+	6.50	4.50	0.50
Positive control EMS	25*	1.4	+	12.00	12.00	8.00
Test article	200	0.01	+	2.50	1.50	0.50
Test article	200	0.03	+	9.00	5.00	1.00
Test article	200	0.10	+	4.00	2.50	1.50

Mutagenicity data at 28 h fixation interval.

Fixation interval 28 h after start of the treatment
Article	number of cells analysed	conc. (mg/ ml)	S9 mix	per cent aberrant cells
				incl. Gaps	excl. Gaps	exchanges
Solvent-control	200	0.0	-	1.00	0.50	0.00
Test article	200	0.30	-	6.50	2.00	0.00
Solvent-control	100*	0.0	+	1.00	0.00	0.00
Test article	200	0.30	+	4.00	2.00	1.00

* one slide was not scorable; technical reasons

Conclusions:

The substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

Executive summary:

The substance was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation, according to the OECD Guideline 473 and EU Method B10 . Prior to the main test the plating efficiency of the cells and the mitotic index were evaluated and the tested concentrations were chosen. The chromosomes were prepared at 7 h , 18 h and 28 h after the start of the treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. 100 metaphases were scored for structural chromosome aberrations per culture; one (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.

Treatment with concentrations higher than 0.001 mg/ml (without S9 mix) and 0.03 mg/ml (with S9 mix) reduced distinctly the plating efficiency of the V79 cells. In the cytogenetic experiment the mitotic index was reduced in the samples treated with the highest scorable dose level at each fixation interval except at interval 7 (with S9 mix). With higher dose levels not enough scorable cells could be found. Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 5.00 %) were in or near the range of the control values: 0.00 % - 4.50 %.

Conclusion

The substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line, under the test conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

other: esperimental data on similar substance

Adequacy of study:

key study

Study period:

From 17 February 1993 to 7 April 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Regarding the hypothesis for analogue approach, refer to the main endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: OECD guidelines for testing of chemicals, section 4: Health Effects, 12.02.1981

Qualifier:

according to guideline

Guideline:

other: Directive 84/449/EEC of 25 04th 1984, published on the Official Journal of the European Communities L 251, 19 09th 1984, pp. 137

Qualifier:

according to guideline

Guideline:

other: Chemicals Act in the version published on 14 03. 1990 (Federal Law Gazette I. No. 13, p 521)

GLP compliance:

yes (incl. QA statement)

Remarks:

And standard operating procedures in their current version of the PFG BIOPHARM GmbH Berlin

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fa. Møllegaard, DE. Certificated
- Age at study initiation: from 6 to 8 weeks
- Weight at study initiation:
male: 27-48 g
female: 23-37 g
- Assigned to test groups randomly: yes, by PC-Program "RANDOM 2.1"
- Housing: box of Macrolon Typ 2. 5 animals per cage, males and female separated
- Bedding: Granular, Fa. Altromin food
- Diet: Standard diet for rat/mouse Altromin N 1326
- Water: ad libitum, tap water. In Glass bottle of 500 ml, with stainless steel lip
- Clinical control: three times at week during the acclimatation period and immediately after the administration of the test substance.
Animals were weighed randomly before the administration of the test substance

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 55 ± 10%
- Air changes: 10 times; automatic system Geringfugugige Abweichungen
- Photoperiod: automatic regulation of 12h/12h (light period from 6:00 a.m to 6:00 pm)

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.7% aqueous amylopectin Ultra with the addition of 2 drops of Tween 80 per 10 ml of preparation

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Pre-test: To determine the maximum tolerated acute dose. LD50 at 48 h was > 1600 mg/Kg, test substance at 80%. Following a single administration animals were observed over a period of 48h. Since none of the treated animals died, this dose was used for the main test.
- Main test: the maximum dose was decreased to 1200 mg/ Kg, because a dose of 1600 mg/kg given to 2 groups of males animals produced some effects. At 800 and 400 mg/Kg no effects were observed on male and female mice.

The substance volume of application has been determined on the basis of body-weight (0.2 ml/10 g bw).

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Single oral administration for the tested item

Post exposure period:

At 24 and 48 h

Remarks:

Doses / Concentrations:
1600, 1200, 800, 400 kg/kg
Basis:
nominal in diet
Males

Remarks:

Doses / Concentrations:
1600, 800, 400 kg/kg
Basis:
nominal in diet
females

No. of animals per sex per dose:

85 mouse (50 male and 35 female), 5 animals for sex and group

Control animals:

yes

Positive control(s):

The preparations of the positive control substance were freshly prepared before administration.
- Substance: Cyclophosphamide
- Source: SIGMA CHEMIE GmbH
- Lot: 19F-0254
- Route of administration: intraperitoneal injection
- Doses / concentrations: 80 mg/kg (0,1 ml/10g bw in aqua)
- Solvent: water for injection

The positive and negative control substances were processing after 24 hours.

Tissues and cell types examined:

For each animal were examined microscopically 2000 polychromatic erithrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
The initial dose of 1600 mg/kg was used, up 80% of the client specified LD50 value of the test substance. After a single oral administration of the initial dose, the animals were observed for the occurrence of intoxication and mortality over a period of 48 hours. Since none of the treated animals had died, this dose was used for the main test. In the main test, however, in the male animal collective group 1 and 11, the tested item in the dose of 1600 mg/kg cause the dead within 24 hours of the animals. The maximum tolerated dose was lowered to 1200 mg/kg and used in the test. The other doses of 800 and 400 mg/kg proved to be non-toxic and have been tested in male and female animals, as provided in the main test.

OBSERVATION
After administration of the maximum tolerated dose, the bone marrow was analysed for 24 and 48 h; for the other two doses the analysis of the bone marrow was performed after 24 hours.

DETAILS OF SLIDE PREPARATION
Animals were killed by cervical dislocation. Removal of the femurs, extraction of the bone marrow and production of slide preparations. The femurs were dissected immediately after killing and removed. 1 ml of foetal calf serum has been warmed to 37°C in a water batch and measured in a syringe of 2ml. The open needle was inserted into the open end of the femur. The marrow has been flushed out.
The resulting cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed using a Pasteur pipette; 4 drops of foetal calf serum Pipette were added to the sediment and resuspended the cells in it.
The cells suspension was treated and the specimens coloured following Pappenheimer's method.
3 slides per animal were prepared.

METHOD OF ANALYSIS
2000 polychromatic erythrocytes per animal were examined microscopically in order to determine the presence and the number o micronuclei.

Evaluation criteria:

The classification of micronuclei was performed using the following criteria:
- Micronuclei are round, rarely oval or crescent shaped.
- They have a sharp contour.
- They are uniformly coloured and are approximately from 1/20 to 1/5 of the diameter of the erythrocytes.
- It is usually present only a micronucleus.
- After treatment with high doses of substances that produce chromosome breaks, in some erythrocytes can also occur several micronuclei, which can also be almond shaped or annular.

The ratio of polychromatic to normochromatic erythrocytes was determined individually for each animal by counting a total of 1000 Erylhrczyten.

Statistics:

The evaluation of micronuclei, the ratio of polychromatic to normochromatic erythrocytes in the experimental groups and comparison groups to which the substance was administered with control groups have been prepared in accordance with the "Statistical analysis of data for micronucleus test".
Polychromatic erythrocytes with micronuclei data (PCE's), total number of micronuclei (MN gas) and ratio of normochromatic to polychromatic erythrocytes (PCE's to NCE's)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Only 2 males died at dose level of 1600 mg/kg

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Clinical signs of toxicity in test animals: no signs in females at 1600, 800 and 400 mg/kg; 2/10 males administrated with a single dose of 1600 mg/kg died

Maximum tolerable dose:
- Males: 1200 mg/kg
- females: 1600 mg/kg

At 1600 mg/kg: no systematic effects observed in females; two dead in males.
At 1200 mg/kg: no systematic effect in males
At 800 and 400 mg/kg: no symptoms for both males and females.

POLYCHROMATIC ERYTHROCYTES

The analysis of polychromatic Erythrocytes in the presence of micronuclei and the subsequent statistical evaluation, justify the following statement:

under the test conditions described, no increased incidence of micronucleated polychromatic containing Erythrozyien from mouse bone marrow was observed. Thus, a potential can be excluded for the induction of chromosomal damage or damage ending of the mitotic apparatus by the test substance in this in vivo short-term test.

In both male and female animals and in any of the doses tested, an increased occurrence of micronuclei in polychromatic erythrocytes from mouse bone marrow was observed.

The calculated frequency of micronucleated polychromatic erythrocytes in groups treated with test substance was 0.17 to 0.42% and thus had no differences in the values ​​of the negative controls at 0.19 to 0.28%.

In almost all animal has been noted a slight increase in the ratio of PCE:NCE (range of 1:1.20 to 1:1.56)

CONTROLS RESULTS

The positive control substance cyclophosphamide resulted in an incidence of 1.78% with a significantly increased incidence of micronucleated PCE-containing. Chance was more than a micronucleus contained in a PCE, which is attributed to the strong mutagenic potential of this substance [2].

Regarding the relationship between polychromatic and normochromatic erythrocytes and the corresponding negative controls, no differences were observed.

Conclusions:

Interpretation of results: negative
Potential for the induction of chromosomal damage or damage ending of the mitotic apparatus by the test substance can be excluded.

Executive summary:

Gene mutation micronucleus assay was performed according to a recognized method: Directive 84/449/EEC of 25 04th 1984, published on the Official Journal of the European Communities L 251, 19 09th 1984, pp. 137.

Substance was administrated by oral gavage to mouse at dose concentrations of 1600, 1200, 800, 400 kg/kg in male and 1600, 800, 400 kg/kg in female.

Conclusion

Potential for the induction of chromosomal damage or damage ending of the mitotic apparatus by the test substance can be excluded.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Mutagenicity of Direct Black 22 (DBk22) has been evaluated considering read across with analogues.

Bacterial reverse mutation assay on Direct Black 22, Ames test showed positive results.

All the available in vitro and in vivo tests on similar substances, does not show any effect.
The in vitro gene mutation test, mammalian cell gene mutation assay and the in vivo micronucleous assay and chromosomal aberration on similar substances did not show any mutagenic effect.

In addiction also a test for unscheduled DNA synthesis, damage and/or repair, for a similar substance showed negative results.

Based on these tests, the Direct Black 22 could be considered as not mutagenic.

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: Substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as:

- In vivo somatic cell mutagenicicty tests such as these indicated in paragraph 3.5.2.3.5:

— mammalian bone marrow chromosome aberration test;

— mouse spot test;

— mammalian erythrocyte micronucleus test.

- In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

In the "Bacterial Reverse Mutation Assay" the test item (Similar Substance 01) shows mutagenic effects. As indicated in the "Flow Chart of the Mutagenicity testing strategy" R.7.7 -1 of ECHA Guidance chapter r.7a (v. 5.0, December 2016), in case of positive response in the gene mutation test in bacteria, it is necessary to fulfil REACH Annex VIII requirements.

An in vivo UDS studyis available on the Target Substance in itself and under test conditions the substance did not induce DNA repair (as measured by unscheduled DNA Synthesis).

As per REACH Regulation requirements, a further in vivo study is considered in which the test substance (Similar Substance 01) didn’t induce increase the number of polychromatic cells with micronuclei.

Therefore, as reported in the Flow chart R.7.7 -1 it is possible to conclude that the test substance is Not Classified for MutagenicIty.