2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-, coupled with 3-aminophenol, diazotized 5-amino-2-[(4-aminophenyl)amino]benzenesulfonic acid and diazotized benzenamine, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article induced point mutations by frameshifts in the genome of strain TA1538; indication of a possible mutagenic response has also been recorded in strain TA1537. The substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

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 November 17 to December 23, 1989

Reliability:

1 (reliable without restriction)

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26th, 1983

Qualifier:

according to guideline

Guideline:

other: EU Method B.14

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Periodically checked regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory according to Ames et al.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl.
- Incubation: the bacterial culture was incubated in a shaking water bath for 6 hours at 37° C.

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

10.0, 100.0, 333.3, 1000.0 and 5000.0 μg/plate in both experiments

Vehicle / solvent:

- Solvents used: sterile, redistilled water (for the test substance and sodium azide reference substance solution) and DMSO (for 2-aminoanthracene and 4-nitro-o-phenylendiamine reference solutions).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylenediamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121° C in an autoclave.
- Overlay Agar: the overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O, 12.2 mg biotin. Sterilizations were performed at 121° C in an autoclave.

NUMBER OF REPLICATIONS: for each strain and dose level, including the controls, a minimum of three plates were used.

SETTING UP THE PLATES: 0.1 ml of test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control) and 0.5 ml of S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation) and 0.1 ml of bacteria suspension and 2 ml of overlay agar were mixed in a test tube and poured onto the selective agar plates. After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

PRE EXPERIMENT FOR TOXICITY AND DOSE SELECTION: prestudy was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates in the same conditions as for the main experiment. Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DATA RECORDING: the colonies were counted using the BIOTRAN III counter. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

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 5 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.
- S9 mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7. 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.

Evaluation criteria:

For the evaluation of the results the corresponding background growth on both negative vontrol and test plates and the normal range of spontaneous reversion rates are used.
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows: a test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No evaluated statistical procedure was used.

Species / strain:

S. typhimurium TA 1538

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:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1535, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in TA1535, at 100.0 μg/plate (exp 1) without metabolic activation and in TA98 at 1000 and 5000 μg/plate with metabolic activation (exp. I & exp.II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxic effects evidenced by a reduction in the number of spontaneous revertants occurred in strain TA 1535 at 100.0 µg/plate (exp. I), and in strain TA 1537 at 1000.0 µg/plate (exp. Ill) both without S9 mix. Also in strain TA 98 at 5000.0 µg/plate with metabolic activation in both experiments.
The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
A slight increase in revertant colony numbers was observed in strain TA 100 (exp. I) at 333.3 µg/plate. This effect was not reproduced in the independent experiment and therefore it is considered not to be relevant.
In strain TA 1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 µg/plate. This increase was less significant in exp. II. To prove the effects of exp. I and exp. II a third experiment was performed. In this additional experiment no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA 1537 cannot be excluded, however the effect was not reproduced in all three experiments.
In strain TA 1538 (with S9 mix) in both experiments the numbers of revertants were distinctly enhanced at concentrations higher than 10.0 µg/plate. In exp. I a dose-dependent increase was obtained up to 333.3 ug/plate and in exp. II up to 1000.0 µg/plate.
At higher concentrations a decrease in the number of revertants was obtained indicating beginning of toxicity.
Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in the strains TA 1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

The following table presents the revertants/plate mean for the three experiments for each strain.

	Revertants/plate mean from three plates, without S9 mix
Dose μg/plate I/II/III	TA1535			TA1537			TA1538		TA98		TA100
	I	II	III	I	II	III	I	II	I	II	I	II
Neg. Control	9	7	5	4	6	6	15	14	20	17	114	77
Solv. Control	9	6	8	4	4	6	15	13	18	16	110	71
10.0	7	6	9	5	5	5	14	18	18	20	112	60
100.0	4	8	9	4	5	4	14	12	21	22	113	61
333.3	7	6	7	6	5	4	12	18	25	18	100	69
1000.0	5	4	5	4	4	3	14	23	25	16	86	64
5000.0	6	4	5	7	4	6	17	18	21	16	98	57
Sodium azide (10 μg/plate)	697	905	579
4-Nitropophenylene- diamine (50 μg/plate)				171	155	149	1126	1148	1895	1230
	Revertants/plate mean from three plates, with S9 mix
Dose μg/plate I/II/III	TA1535			TA1537			TA1538		TA98		TA100
	I	II	III	I	II	III	I	II	I	II	I	II
Neg. Control	5	7	5	4	4	5	18	17	44	19	116	84
Solv. Control	5	8	7	4	5	6	14	13	44	14	100	75
10.0	7	10	5	5	5	4	23	16	43	17	105	65
100.0	7	11	8	4	7	10	39	37	48	20	123	92
333.3	8	8	10	8	8	7	45	35	47	19	164	98
1000.0	6	9	8	10	5	5	25	55	28	8	101	75
5000.0	7	12	7	12	10	9	22	26	22	7	121	66
2-aminoanthracene (10 μg/plate)	82	97	540	179	319	175	1867	1563	2455	2231	2285	1496

Conclusions:

Under the tested conditions, the test article induced point mutations by frameshifts in the genome of strain TA1538. There was also an indication of a possible mutagenic response in strain TA1537.

Executive summary:

The substance was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation. According to the results of these experiments, a third experiment (III) was performed only with TA1535 and TA1537 with and without metabolic activation. Five concentrations (10.0, 100.0, 333.3, 1000.0 and 5000.0 μg/plate) including the control were inoculated with each of the strains for 72 hours at 37 °C. Each concentration was tested in triplicate.

Toxic effects occurred in strain TA1535 at 100.0 μg/plate (exp. I) and in strain TA1537 at 1000.0 μg/plate (exp. Ill) both without S9 mix. Also in strain TA 98 at 5000.0 μg/plate with metabolic activation in both experiments. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. In strain TA1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 ug/plate. This increase was less significant in exp. II. In the experiment III no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA1537 cannot be excluded, but this the effect was not reproduced in all three experiments. In strain TA1538 (with S9 mix) a dose-dependent increase was obtained up to 333.3 μg/plate (exp. I) and up to 1000.0 μg/plate (exp. ΙI). No indication of mutagenic response occured in strains TA1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists, with and without metabolic activation. The substance induced point mutations by frameshifts in the genome of the strain TA1538 and there is an indication of a possible mutagenic response in strain TA1537.

Conclusion

Under the tested conditions, the test article induced point mutations by frameshifts in the genome of strain TA1538. There was also an indication of a possible mutagenic response in strain TA1537.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November from 16 to 29, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EU Method B.14

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Bacteria growth: several days before the beginning of the study, 4 tials of the bacterial strains were defrosted and grown on Petri dishes with nutrient agar in order to obtain pure cultures.
- Storage: the plates were stored at 4 °C until required.
- Preparation: 16 hours before the study began, an inoculum of each of the four bacterial strains was prepared in 20 ml of nutrient broth and incubated at 37 °C. - Periodically checked: the histidine requirements, the presence or absence of plasmide pKM 101, the rfa factor (sensitivity to crystal violet) and the uvrβ factor (sensitivity to ultraviolet rays) were checked in the strains used to prepare the inocula.

Metabolic activation:

with and without

Metabolic activation system:

homogenate of rat liver (S9), induced by Arochlor 1254, at 10 % in standard cofactors

Test concentrations with justification for top dose:

31.25, 625, 1250, 2500, 5000 and 10000 μg/plate, for both experiments.

Vehicle / solvent:

- Solvents used: sterile, redistilled water (for the test substance and sodium azide reference substance solution) and DMSO (for 9 aminoacridine, 4-nitro-o-phenylendiamin and 2-aminoanthracen reference solutions).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene // 4-nitro-o-phenylendiamin

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The tHo experiments Here carried out on di f ferent days.

METHOD OF APPLICATION: preincubation and plate incorporation.

NUMBER OF REPLICATIONS: three replicates per concentration.

SETTING UP OF THE TEST PLATES
- Plate preparation: 0.1 ml of bacterial culture and 0.1 ml of the diluted test substance were added to 0.5 ml of phosphate buffer (without metabolic activation) or 0.5 ml of S-9 (with metabolic activation). For the controls, 0.1 ml of the corresponding solvents were added to each product.
- First incubation: plates were incubated for 20 minutes at 37 °C and added to 2 ml of top agar melted at 45 °C containing biotin and histidine.
- Incubation: the test tubes were rapidly mixed in a rotamixer, and poured on to plates containing a base layer of minimal agar. The agar added was left to solidify and the plates were incubated at 30 - 35 °C for 48 hours.

Statistics:

The results from the standard products and the control group were compared using the Student's t test. In each group, the statistical comparison between the different concentrations of the test substance was carried out using one-way analysis of the variance.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II.
In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it can not be considered to be mutagenic as the highest revertants count never doubled that of the control.
During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

POSITIVE CONTROLS
In both experiments I and II, all the bacterial strains responded positively and the metabolic activation system responded as expected.
Aminoanthracen, at a concentration of 10 µg/ plate, showed toxicity in strain TA1537 with the addition of S9, in the experiment I. In the experiment II, aminoanthracen, at a concentration of 10 µg/ plate, showed toxicity in strains TA1535 and TA1537 with the addition of S9.

Summary of results obtained in experiment 1

Bacterial  strain	% S-9	Mean no. of revertants per plate (μg/plate)
		0	31.25	62.5	125	250	500	1000
TA-1535	0	6.3	6.0	5.7	5.3	7.7	5.3	6.0
TA-1537	0	10.7	9.7	9.0	10.0	9.7	9.3	10.0
TA-98	0	14.0	12.0	14.3	12.3	14.0	14.3	14.0
TA-100	0	121.0	119.0	119.7	121.7	124.0	122.0	119.0
TA-1535	10	7.7	8.0	8.0	8.3	6.7	8.0	6.7
TA-1537	10	10.7	10.3	10.7	12.0	10.7	9.0	8.3
TA-98	10	21.7	22.7	22.3	25.7	28.0	29.0	20.3
TA-100	10	131.7	132.0	130.7	131.7	132.0	131.3	131.7

Summary of results obtained in experiment 2

Bacterial  strain	% S-9	Mean no. of revertants per plate (μg/plate)
		0	31.25	62.5	125	250	500	1000
TA-1535	0	13.7	12.0	14.0	13.7	13.3	12.7	13.3
TA-1537	0	12.0	13.3	11.3	12.0	13.0	10.7	12.3
TA-98	0	28.0	27.3	27.3	27.0	26.3	26.7	26.3
TA-100	0	150.0	151.0	147.3	148.0	146.0	149.0	148.0
TA-1535	10	13.7	13.0	13.7	13.3	14.0	14.3	14.3
TA-1537	10	18.0	17.0	16.7	18.3	17.7	19.7	16.7
TA-98	10	33.3	30.7	33.3	31.3	31.0	32.7	28.7
TA-100	10	150.7	148.3	151.0	150.0	150.0	147.3	150.3

Summary of statistical analysis for each strain

Experiment 1
Bacterial  strain	% S-9	Statistic F	Degrees of freedom		Significance of F (p)
			N	D
TA-1535	0	0.99	6	14	N.S.
TA-1537	0	0.58	6	14	N.S.
TA-98	0	1.67	6	14	N.S.
TA-100	0	2.23	6	14	N.S.
TA-1535	10	0.37	6	14	N.S.
TA-1537	10	2.44	6	14	N.S.
TA-98	10	4.53	6	14	V.S.
TA-100	10	0.10	6	14	N.S.
Experiment 2
Bacterial  strain	% S-9	Statistic F	Degrees of freedom		Significance of F (p)
			N	D
TA-1535	0	0.72	6	14	N.S.
TA-1537	0	0.41	6	14	N.S.
TA-98	0	0.23	6	14	N.S.
TA-100	0	0.90	6	14	N.S.
TA-1535	10	0.10	6	14	N.S.
TA-1537	10	1.18	6	14	N.S.
TA-98	10	0.92	6	14	N.S.
TA-100	10	0.36	6	14	N.S.

N.S. = Non-significant.

V.S = Significant for p < 0.01.

Conclusions:

The substance is non mutagenic to Salmonella strains, with and without metabolic activation.

Executive summary:

The mutagenic potential of the substance, using four strains of Salmonella typhimurium (TA 98, TA100, TA1535, TA1537), with and without the addition of a metabolic activation system, was assessed according to OECD Guideline 471 and EU Method B.14. Six concentrations of the substance (1000 - 500 - 250 - 125 - 62.5 and 31.25 μg/plate) were inoculated with each of the strains for 48 hours at 30 -35 °C in two experiments. Concurrent positive controls were used in order to assess the metabolic activation system, while solvent controls also run in parallel.

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II.

In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it can not be considered to be mutagenic as the highest revertants count never doubled that of the control.

During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

Conclusion

The substance is non mutagenic to Salmonella strains, with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 31, 1990 to April 12, 1991

Reliability:

1 (reliable without restriction)

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:

other: in vitro chromosome aberration in mammalian cells

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

Description of key information

The test article did not induce micronuclei in the bone marrow cells of the mouse and did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January from 02 to 26, 1990

Reliability:

1 (reliable without restriction)

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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26th, 1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH- 4414 Füllinsdorf/Basel, Switzerland.
- Age at start of acclimatization: minimum 10 weeks.
- Weight at study initiation: approx. 30 g.
- Assigned to test groups randomly: yes.
- Fasting period before study: yes, approximately 18 hours before treatment with the test article. Animals could still have water ad libitum during the fasting period.
- Housing: single in a Makrolon Type I, with wire mesh top and granulated soft wood bedding.
- Diet: pelleted standard diet.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3°C.
- Humidity: not regulated.
- Photoperiod: 12 hrs dark / 12 hrs artificial light (6.00 a.m - 6.00 p.m).

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: destilled water.
- Justification for choice of vehicle: chocen for its non-toxicity to the animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test article was dissolved in distilled water and the animals received orally a single standard dose volume of 20 ml/kg b.w.

Frequency of treatment:

Once at the start of the study.

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

Three groups

No. of animals per sex per dose:

6 males and 6 females per dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)
- Preparation: the substance was dissolved in physiological saline and the solution was prepared on the day of administration.
- Route of administration: orally.
- Dosing: single dose of 40 mg/kg b.w.
- Volume administered: 10 ml/kg b.w.
- Stability: at 20 °C only 1 % of CPA is hydrolysed per day in aqueous solution.

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

PREPARATION OF THE ANIMALS
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 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and
the supernatant was discarded.

DETAILS OF SLIDE PREPARATION
A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. 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. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

CRITERIA FOR DOSE SELECTION
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w of the substance dissolved in aqua dest. The volume administered was 20 ml/kg b.w.
The maximum tolerated dose or the highest dose that can be formulated and administered reproducibly sould be selected. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours. Higher dosing than 5000 mg/kg b.w. was not attainable as appropriate solutions could be obtained only up to 250 mg/ml and application volumes higher than 20 ml/kg b.w. were not justifiable for the animals used.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone 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 non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

slight toxic reactions

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

POSITIVE CONTROL
40 mg/kg b.w. cyclophosphamide showed a distinct increase of induced micronuleus frequency.

PRE-EXPERIMENT FOR TOXICITY
All treated animals expressed slight toxic reactions such as reduction of spontaneous activity. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the substance had no cytotoxic properties.

The number of PCEs (Polychromatic Erythrocytes) with micronuclei and the ratio of PCE to NCE ( Normochromatic Erythrocyte) are given for each test group in the table below.

Summary of results.

Test group	dose (mg/kg bw)	sampling time (h)	PCEs with micronuclei	range	PCE/NCE
solvent	0	24	0.09 %	0-3	1000/935
test article	5000	24	0.09 %	0-3	1000/874
cyclophosphamide	40	24	0.76 %	3-11	1000/1119
solvent	0	48	0.02 %	0-2	1000/842
test article	5000	48	0.04 %	0-1	1000/1000
solvent	0	72	0.07 %	0-2	1000/892
test article	5000	72	0.03 %	0-2	1000/769

Conclusions:

The test article did not induce micronuclei in the bone marrow cells of the mouse.

Executive summary:

The substance was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, according to the OECD Guideline 474 and EU Method B12. Six male and six female mouses were treated orally with a single dose of 5000 mg/kg b.w. This was the maximum attainable dose as estimated by the pre-experiment. Concurrent positive (use of cyclophosphamide) and negative (use of vehicle-distilled water) control groups run. After 24 h, 48 h and 72 hours the bone marrow cells of ten animals were collected for micronuclei analysis. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

Conclusion

The test article did not induce micronuclei in the bone marrow cells of the mouse, under the test conditions.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 22 1990 to April 05, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

The UDS test is an assay for the detection of chemically induced effects on DNA.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

WU

Details on species / strain selection:

The rat is an animals used as suitable experimental animal in genotoxicity investigations (and physiological, pharmacological and toxicological studies) for many years. Many data are available from such investigations that could be helpful in the interpretattion of results and for the design and performance of the UDS test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SAVO-Ivanovas, med. Versuchstierzuchten GmbH.
- Weight at study initiation: appr. 200 g.
- Assigned to test groups randomly: yes.
- Fasting period before receiving test material: starved overnight (4 hours treatment) or appr. 6 hours (16 hours treatment).
- Housing: individually, in Makrolon Type I cages, with wire mesh top and granulated soft wood bedding.
- Diet: pelleted standard diet.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.
- Other: the animals underwent quarantine in the animals house of the laboratory for at least one week after their arrival. During this period the animals did not show signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C.
- Humidity: 30 - 70 %.
- Photoperiod: 12 hrs dark / 12 hrs artificial light (6.00 a.m - 6.00 p.m).

Route of administration:

oral: unspecified

Vehicle:

- Vehicle: bedistilled water.
- Justification for choice of solvent/vehicle: it was chosen according to its relative nontoxicity for the animals.

Details on exposure:

- Preparation of dose solution: the test article was formulated in aqua bidestilled, on the day of the experiment immediately before treatment.
- Volume administered: 10 ml/kg bw

Duration of treatment / exposure:

4 hours and 16 hours treatment.

Frequency of treatment:

Single standard dose.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

4 - hours treatment period.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

16-hours treatment period.

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

16-hours treatment period.

No. of animals per sex per dose:

5 males per test group.

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene
- Preparation: dissolved in DMSO/polyethylene glycol (1+9). The solution was prepared on day of administration.
- Route and frequency of administration: orally, single.
- Dose: 100 mg/kg b.w.
- Volume administered: 10 ml/kg b.w.

Details of tissue and slide preparation:

ISOLATION OF THE PRIMARY HEPATOCYTES
The animals were sacrificied by liver perfusion. After anesthetizing the rats with 1.5 ml/kg b.w. Hypnodil i.p. the liver was perfused through the vena portae with Hank's balanced salt solution (HBSS) supplemented with collagenase (0.05 % w/v) adjusted to pH 7.4 and maintained at 37 °C. The hepatocytes were isolated from the liver and washed twice with HBSS. The crude cell suspension was filtered through a 94 μm stainless steel mesh to yield a single cell suspension. The quality of the actual performed perfusion was determined by the tryptan blue dye exclusion method. The number of the isolated cells was also determined.

CULTURE CONDITIONS
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME) supplemented with 2.38 mg/ml hepes, 100 units/ml penicillin, 0.10 mg/ml streptomycin, 0.29 mg/ml glutamin, 0.50 μg/ml insulin and 100 μl/ml fetal calf serum. The medium without the cells was adjusted to pH 7.6. At least 5 cultures were established for each animal. Aliquotes of 2.5 ml with freshly isolated hepatocytes in complete culture medium (1.0 x 10^5 cells/ml) were added to 35 mm six-well cluster dishes containing one gelatinized 25 mm round plastic coverslip per well. After an attachment period of appr. 1.5 h in a 95 % air / 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then the cell layer was rinsed once with PBS to remove non-adherent cells.

OTHER
Three animals per group were evaluated for UDS. The two remaining animals per test group would be evaluated if an animal dies spontaneously or in case of technical problems concerning the isolation of hepatocytes.

DETERMINATION OF UDS
3HTdR (5 μCi/ml, specific activity 20 Ci/mol) in 2.0 ml culture medium (WME, 1 % FCS) was added to the cultures. After a labelling time of 4 hours the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. The medium was then replaced by a hypotonic solution of 1 % sodium citrate for 10 minutes to swell the nuclei for better grain quantification. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 15 mins each, rinsed with 96 % ethanol and air dried. Two of the cultures were used for determination of cytotoxicity and attachment efficiency with the neutral red assay whereas three cultures were used for the UDS assay.

ANALYSIS-AUTORADIOGRAPHIC PROCESSING
The cover slips were mounted the side carrying the cells up on a glass slides and coated with ILFORD K-2 photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12-14 days at 4 °C. The photographic emulsion was then developed with KODAK D-19 at room temperature, fixed in TETENAL and stained with 0.4 % aceto orcein.

QUANTIFICATION OF UDS
Evaluation was performed microscopically on coded slided using NIKON microscopes with 100 x oil immersion objectives. The number of siver grains above the nucleus was counted automatically using the ARTEK 880 counter. The number of grain counts of one nuclear-sized cytoplasm adjacent to the nucleus was estimated. At least two slides per animal and 50 cells per slide were evaluated. Heavily labelled S-phase cells were excluded from counting.

ASSESSMENT OF ATTACHMENT OF CELLS
Two cultures of each animals were examined by using the neutral red absorption assay. After an attachement period of 1.5 hours the cells were washed and refed with medium containing neutral red (50 μg/ml). After an incubation period of 3 hours the cultures were rinsed and the incorporated dye eluted with 50 % ethanol supplemented with 1 % acetic acid.

CRITERIA FOR DOSE SELECTION
The highest recommended dose level to be used in the assay is 1000 mg/kg b.w. The toxicity data of the test article available indicated that 1000 mg/kg b.w. is the maximum tolerated dose causing toxic reactions without having major effects on the survival of the animals, therefore no pre-experiment was performed.

Evaluation criteria:

A test article is classified as positive if it induces either a statistically significant dose-related increase in radiolabel incorporation expressed as grains per nucleus or a reproducible and statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose related increase in radiolabel incorporation expressed as grain per nucleus nor a statistically significant and reproducible positive response at any one of the test points is considered non-effective in this system.

Statistics:

Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A statistical evaluation of the results of biometry was not necessary to perform as the number of nuclear an net grain counts of the groups treated with the test article were in the range of the corresponding controls.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxic reactions of the animals occurred at any of the tratement periods or dose groups. In addition, neither the viability nor the in vitro attachment of the hepatocytes was dramatically affected due to the in vivo pre-treatment with the test article.
The interindividual variations obtained for the numbers of isolated hepatocytes as well as for the attachment-efficiency (NR-assay) are in the range of the testing laboratory historical laboratory control. In case of hepatotoxicity the NR-vales expected would be approximately zero.

No dose level of the test article revealed UDS induction in the hepatocytes of the trated animals as compared to the current negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the animals with the test article for 4 hours or 16 hours, respectively.

POSITIVE CONTROL
An appropriate reference mutagen (2-AAF, 100 mg/kg bw) was used as positive control. In vivo treatment with 2-AAF revealed disctinct increases in the number of nuclear and net grain counts.

PRE-EXPERIMENT
The highest recommended dose level to be used in the assay resulted to be 1000 mg/kg bw. since the toxicity data of the test article given by the sponsor indicated that 1000 mg/kg bw should have no makor effects on the survival of the animals a pre-experiment was not necessary to perform.

The viability and attachment of the hepatocytes.

Viability and attachment of hepatocytes.

Dose  (mg/kg b.w.)	Tretament period	animal no.	Viability *	Number of isolated cells (x 106)	Attachement  (O.D 540 mm)**
1000	4h	1	71	102	0.367
	4h	2	74	367	0.495
	4h	4	65	152	1.030
Vehicle (aqua bidest.)	16h	6	81	392	0.428
	16h	7	67	326	0.321
	16h	8	81	321	0.554
100	16h	11	82	270	0.226
	16h	12	76	629	0.198
	16h	13	73	424	0.536
1000	16h	18	69#	304	0.270
	16h	19	79#	373	0.355
	16h	20	72#	272	0.366
100 (2AAF)	16h	21	81	376	0.410
	16h	22	77	343	0.449
	16h	23	71	169	0.438

* = viability determined by means of trypan blue dye exclusion assay.

** = mean value of two independent cultures.

# = some of the dead cells elicited black pigmentation indicating an accumulation of the test article in hepatocytes.

The counts of grains per nucleus, grains per cytoplasm area and the net grains per nucleus are summarised for each animal.

Summary of results for individual animals.

Treatment	Animal no.	Grains per nucleus		Grains per cytoplasm area		Net grains per nucleus
		Mean *	± SD	Mean *	± SD	Mean *	± SD
Solvent control (a.dest/16h)	1	4.95	3.42	7.01	3.10	-2.06	2.41
	2	3.57	2.18	5.69	2.28	-2.12	2.61
	3	3.79	2.32	7.86	3.27	-4.07	3.48
Positive control -  (2AAF 100 mg/kg /16h)	1	37.01	19.15	9.68	4.94	27.33	19.10
	2	22.44 #	9.27	11.50	4.61	10.94	8.74
	3	17.93	8.10	12.74	4.56	5.19	8.76
Dose level 1 (100 mg/kg / 16h)	1	5.88	3.50	9.15	4.13	-3.27	3.66
	2	4.93	2.59	7.27	2.19	-2.34	2.89
	3	6.35	3.15	7.51	3.26	-1.16	3.45
Dose level 2 (1000 mg/kg / 4h)	1	4.10	2.27	6.18	2.32	-2.08	2.51
	2	3.92	2.01	6.59	2.63	-2.67	2.22
	3	4.48	2.26	5.73	2.47	-1.25	2.54
Dose level 3 (1000 mg/kg / 16h)	1	4.92	2.66	7.40	3.00	-2.48	2.53
	2	4.08	2.40	6.49	2.88	-2.41	2.73
	3	8.80	6.00	13.69	8.27	-4.89	5.88

* = mean of 100 cells

# = mean of 50 cells; due to technical reasons only one slide was scorable.

The counts of grains per nucleus, grains per cytoplasm area and the net grains per nucleus are summarised for each dose group.

Summary of results for dose groups.

Treatment	Dose level	Grains per nucleus		Grains per cytoplasm area		Net grains per nucleus
		Mean *	± SD	Mean *	± SD	Mean *	± SD
Solvent control	a.dest/16h	4.10	2.75	6.85	3.04	-2.75	3.01
Positive control -  2AAF	100 mg/kg /16h	26.46 #	16.31	11.27	4.90	15.20	17.13
Dose level 1	100 mg/kg / 16h	5.72	3.15	7.98	3.38	-2.26	3.45
Dose level 2	1000 mg/kg / 4h	4.17	2.19	6.17	2.50	-2.00	2.49
Dose level 3	1000 mg/kg / 16h	5.93	4.52	9.19	6.22	-3.26	4.17

* = mean of 3 animals, 100 cells each.

# = mean of 3 animals, two animals 100 cells each, one animal 50 cells.

Conclusions:

The test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats.

Executive summary:

The substance was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The substance was orally administered the substance in concentrations of 1000 mg/kg b.w for the 4 -hours treatment period and in concentrations of 100 and 1000 mg/kg b.w for the 16 -hours treatment period. Bedistilled water was used as vehicle and served as the negative control while an appropriate reference mutagen (2 -AAF) was used as the positive control. Five male rats were used in each dose group. After the treatment period the animals were narcotized and sacrificied by liver perfusion. Primary hepatocyte cultures were established and exposed to 3HTdR which is incorporated if UDS occurs. For each dose level hepatpcytes from three treated animals were assessed for occurence of UDS.

No toxic reactions of the animals occured at any of the treatment periods or dose group. The viability and the in vitro attachment of the hepatocytes was not affected due to the in vivo pre-treatment with the test article. The interindividual varriations obtained for the numbers of isolated hepatocytes as well as for the attachment efficiency were in the range of the historical laboratory control. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment ofthe animals with the test article for 4 or 16 hours.

Conclusion

The test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro and in vivo chromosomal aberration assay and in vivo DNA damage and/or repair study on mammalian cell.

IN VITRO GENE MUTATION ASSAY IN BACTERIA

Two tests have been performed on Direct Black 168 assessing the potential to induce gene mutations in bacteria. Both tests were conducted using Salmonella typhimurium strains.

A plate incorporation test was performed on S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, in two independent experiments, both with and without rat liver microsomal activation. In addition, a third experiment (III) was performed only with TA1535 and TA1537 with and without metabolic activation.

Toxic effects occurred in strain TA1535 at 100.0 μg/plate (exp. I) and in strain TA1537 at 1000.0 μg/plate (exp. III), both without S9 mix. Also in strain TA 98 at 5000.0 μg/plate with metabolic activation in both experiments. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. In strain TA1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 ug/plate. This increase was less significant in exp. II. In the experiment III no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA1537 cannot be excluded, but this the effect was not reproduced in all three experiments. In strain TA1538 (with S9 mix) a dose-dependent increase was obtained up to 333.3 μg/plate (exp. I) and up to 1000.0 μg/plate (exp. ΙI). No indication of mutagenic response occurred in strains TA1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists, with and without metabolic activation. The substance induced point mutations by frameshifts in the genome of the strain TA1538 and there is an indication of a possible mutagenic response in strain TA1537.

The second test was conducted on S. typhimurium strains TA 98, TA100, TA1535, TA1537, with and without the addition of a metabolic activation system; two experiments were carried out.

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II. In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it cannot be considered to be mutagenic as the highest revertants count never doubled that of the control. During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

IN VITRO and IN VIVO CHROMOSOMAL ABERRATION ASSAY

The Direct Black 168 potential to induce structural chromosome aberrations was assayed both in in vitro and in in vivo test systems.

The in vitro test was performed using V79 cells of the Chinese hamster, in the absence and presence of metabolic activation. 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 %.

The in vivo experiment was a micronucleus test, which assessed the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

UNSCHEDULED DNA SYNTHESIS

The substance was assessed in the in vivo UDS assay for the potential to induce DNA repair (UDS) in the hepatocytes of rats. The test procedures followed can be considered as comparable to those described in the OECD guideline 486, which was published later. No toxic reactions of the animals occurred at any of the treatment periods or dose group. The viability and the in vitro attachment of the hepatocytes was not affected due to the in vivo pre-treatment with the test article. The interindividual variations obtained for the numbers of isolated hepatocytes as well as for the attachment efficiency were in the range of the historical laboratory control. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the animals with the test article for 4 or 16 hours.

CONCLUSION

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro and in vivo chromosomal aberration assay and in vivo DNA damage and/or repair study on mammalian cell.

The preliminary assessment of mutagenicity in S. typhimurium bacteria showed that the test article is able to induce point mutations by frameshifts in the genome of strain TA1538; indication of a possible mutagenic response has also been recorded in strain TA1537.

The investigations in vitro of chromosomal aberration in mammalian cells and the in vivo micronucleous assay in bone marrow cells of the mouse did not show any potential for genetic toxicity. Also the in vivo experiment performed to assess the potential for DNA repair synthesis induction in the hepatocyte of rats gave negative results.

In conclusion, the substance did not showed any potential for genetic toxicity in mammalian cells.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity, according to the CLP Regulation (EC) No 1272/2008.