Reaction mass of Cobaltate(2-), [2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)][2-[[2-hydroxy-5-[(phenylamino)sulfonyl]phenyl]azo]-3-oxo-N-phenylbutanamidato(2-)]-, disodium and Cobaltate(3-), bis[2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)]-, trisodium and sodium bis[2-[[2-hydroxy-5-[(phenylamino)sulphonyl]phenyl]azo]-3-oxo-N-phenylbutyramidato(2-)]cobaltate(1-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August the 09th to October the 02nd, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Principles of method if other than guideline:

Ames test

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yellow 235

IUPAC Name:

Acid Yellow 235

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA 98, TA 1538 and TA 1537

Details on mammalian cell type (if applicable):

- Storage conditions: -70 °C, in liquid nitrogen.
- Culture: 4.5 ml bacterial culture and 0.5 ml dimethylsulfoxide.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Storage conditions: -70 °C, in liquid nitrogen.
- Culture: 4.5 ml bacterial culture and 0.5 ml dimethylsulfoxide.

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolizing system

Test concentrations with justification for top dose:

5, 15.8, 50, 158, 500, 1580, 5000 and 15800 µg/plate

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

benzo(a)pyrene

other: N'-nitro-N-nitrosoguanidine and benzo[a]pyrene 4-,5-oxide // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

TEST CONDITIONS
- Bacterial suspension: 0.1 ml/plate, about 1.7 x 10^9 colony forming units per ml.
- Test solution: 0.01 to 0.1 ml
- S-9 Mix, or a solution of 150 mM KCl buffered with 10 mM Na phosphate at pH 7.4: 0.5 ml/plate
- Top Agar (45 °C): 2.0 ml/plate
- Temperature: 37 °C
- Time: 46 - 55 hours for TA 98 and WPr uvrA; 62 - 68 hours for all other strains.
- Other: in experiments in which epoxide hydratase was inhibited 0.3 µl 1,1,-trichloropropene 2,3-oxide in 10 µl DMSO. Respective plates without epoxide hydratase inhibition only received 10 µl DMSO.

REPLICATES: 2 replicates per dose per strain

PREPARATION OF TEST SOLUTION
- Solvent: H2O, positive controls in dimethylsulfoxide p.a.
- Amount per plate: 100 µl H2O, 10 to 30 µl dimethylsulfoxide, resp.
- Method for suspension of refractory substance: ultrasonication.
- Time of preparation: the day of the expertment.

SOFT AGAR
- Amount per plate: 2 ml
- Temperature: 45 °C
- Composition: 0.55 % Agar, 0.55 % NaCI, 25 mM Na phosphate ph 7.4, 50 µM histidine, 50 µM biotin and 50 µM tryptophan.

TOXICITY TEST
To estimate toxicity, his+ bacteria, about 600 colony forming units, were added as an internal standard to otherwise normal mutagenicity plates. The his+ bacteria used were spontaneous revertants from TA 1537. They were added to plates together with a strain which gives low numbers of revertant colonies (TA 1537). The difference in the number of colonies on plates with and without added his+ bacteria, in the presence of test compound, is compared to the value obtained with solvent controls. The ratio of these two values gives the relative survival rate. In tests where generation ard selection of mutants are not separated, as in the standard Ames test, relative survival rates cannot be used for correcting the number of revertant colonies for toxic effects. However, since toxicity reduces the sensitivity of the test, a measure for toxicity is required for validation of mutagenicity data.
The relative survival rate is more objective and far more sensitive criterion than mere observation of the his- background lawn.

RAT LIVER METABOLIZING SYSTEM
For all experiments fresh tissue preparations from animals sacrificed on the day of the experiment were used.
Male rats (8-13 weeks old, ca 200-300 g), Sprague Dawley, were used. Aroclor 1254 was administrated intraperitoeally. Sunflower oil was used as solvent (4 vol per 1 vol Aroclor 12541). 500 mg Aroclor 1254 per kg body weight was administrated by a sigle application, 6 days before sacrifice.

Composition of S9 mix: S9 0.33 ml, MgCl2 8 µmol, KCl 33 µmol, glucose-6-phosphate 5 µmol, NADP+ 4 µmol, Na-phosphate 50 µmol; pH 7.4 - 7.5.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 98, TA 1538 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No mutagenicity was observed with test item under any experimental condition. Because in the first experiment for direct mutagenicity the number of revertant colonies from TA 98 was slightly (less than two-fold) increased on some plates with test item, this part of the experiment was repeated with a larger number of parallel plates. The negative result of the repeated experiment strongly supported the interpretation that the marginal effect of the first experiment was simply due to dispersion rather than to a real effect. Mutagenicity effects were also not observed when the epoxide hydratase inhibitor and glutathione depletory 1,1,1-trichloropropene 2,3-oxide was added to the S-9 Mix in order to increase the sensitivity of the test towards compounds which are activated to mutagenic epoxides (this experiment was performed with TA 98).

CITOTOXICITY
For determination of toxicity his+ mutants were added to some plates in order to estimate the survival of mutants. In the absence of S-9 Mix the compound started to be noticeably toxic at 500 µg. At this and at the next higher dose (1580 µg) sufficient bacteria survived that there is a good chance that relatively stable mutagens still may be detected. In the presence of S-9 Mix, toxicity was much lower. Only at 15800 µg a substantial decrease in survival occurred. All in all, toxicity and solubility of compound are such that it can be tested for mutagenicity with a good sensitivity.

CONTROLS
In the study, N-methyl-N'-nitro-N-nitrosoguanidine and benzo(a)pyrere 4,5-oxide showed potent mutagenic effects in the absence of S9 Mix. With TA 100 and TA 1537, strains in which the mutagenicity of these compounds is seen well, tests were also performed in the presence of S-9 Mix.
S-9 Mix completely prevented the mutagenic effects indicating the functioning of drug-metabolizing enzymes which play an inactivating role with the compounds urder investigation (but may activate other compounds). Metabolic activity of S-9 Mix is furthermore shown by the strong mutagenic effects of the activation-requiring mutagens in the presence of S-9 Mix, whereas these compounds were inactive or at most marginally active in the direct test (the direct tests were only performed with the most sensitive strains TA 98 and TA 100). Benzo(a)pyrene is activated well by liver preparations from 3-methylcholanthrene-treated rats, but only poorly by preparations from phenobarbital-treated animals. The reverse is true for 3-methylcholanthrene.
Aroclor 1254 induces monooxygenases which are induced by phenobarbital as well as those which are induced by 3-methylcholanthrene. Both benzo(a)pyrene and 3-methylcholanthrene were activated in the experiments. This indicates that both metabolizing systems were functioning. Also 2-aminoanthracene, which requires N-oxidation for activation, was strongly mutagenic. This compound is mutagenic in all strains employed. For simplicity the study director used the same dose with all strains, although the maximal effects occur at different doses due to differences in permeability and toxicity among the strains. Maximal effects with TA 1538 would be seen at lower doses, with WP2 uvrA at higher doses than those used.
Inhibition of epoxide hydratase by 1,1,1-trichloropropene oxide was controlled by studying its effect on the mutagenicity of benzo(a)pyrene. 1,1,1-trichloropropene oxide increased the mutagenicity of benzo(a)pyrene several fold.

Any other information on results incl. tables

AMES test with S-9 mix

Dose µg/plate	Solvent	Relative Survival Rate	TA 100		TA 1535		WP2 uvrA		TA 98		TA 98 TCPO		TA 1538		TA 1537
0	100 µl H2O	1.0	137	125	15	14	39	41	30	22	37	31	21	34	21	19
0	100 µl	1.0	145	141	7	9	37	48	24	33	25	30	27	30	24	23
5		0.9	115	140	10	18	19	19	19	18	27	24	37	22	28	24
Test item 15.8	100µl H2O	1.0	141	109	12	10	30	23	29	28	37	33	26	30	19	16
Test item 50	100 µl	1.0	144	128	12	19	33	32	24	21	34	40	26	27	19	25
Test item 158	100 µl	1.0	152	154	14	17	50	44	21	30	45	41	33	23	29	30
Test item 500	100 µl	1.0	104	117	6	15	40	51	30	20	23	23	26	22	34	23
Test item 1580	100 µl	1.0	102	124	12	7	61	35	27	20	21	7	14	14	18	18
Test item 5000 **	100 µl *	0.8	100	99	9	10	46	48	7	7	2	6	9	6	13	13
Test item 15800 **	100 µl *	0.03	24	14	4	0	58	32	0	0	0	0	0	0	4	1
Benzo(a)pyrene 10	10 µl DMSO	0.7	1050	1140	21	27	78	74	574	601	950	970	270	266	166	144
Benzo(a)pyrene 50	10 µl	1.0	376	391	13	15	42	42	63	72	427	453	50	33	79	77
2-Aminoanthracene 10	10 µl	0.6	4500	4400	242	236	149	143	4050	5100	3600	3700	1136 t	716 t	182	199
3-methylcholanthrene 90	30 µl	0.9	3800	3700	10	5	49	51	1090	1310	1160	1680	255	293	91	109
Benzo(a)pyrene 4,5-oxide	10 µl	1.0	143	139											25	18
N-methyl-N'-nitro-N-nitrosogunidine	10 µl	1.2	152	154											27	19

t: 2-Aminoanthracene showed stronger toxicity with TA 1538 than with all other strains and noticeably reduced at the his- background law in this strain

**macroscopically visible precipitate of test compound on the plate.

* The conpound was dissolved in H2O at 70 °C before being added to the plate.

AMES test withou S-9 mix

Dose µg/plate	Solvent	Relative Survival Rate	TA 100		TA 1535		WP2 uvrA		TA 98 exp.1		TA 98 exp.2			TA 1538		TA 1537
0	100µl H2O	1.0	90	89	14	12	50	49	26	27	32	32	32	13	16	9	8
0	100 µl	1.0	96	108	16	14	49	48	25	18	32	27	26	14	15	12	14
5		0.9	72	81	16	18	32	73	28	38	33	27	30	21	19	11	-*
Test item 15.8	100µl H2O	1.0	103	90	16	18	45	53	31	44	37	27	38	20	24	8	12
Test item 50	100 µl	0.8	88	106	13	17	39	49	41	48	39	29	28	24	18	8	10
Test item 158	100 µl	0.8	92	89	13	21	39	37	19	27	24	28	35	8	14	5	14
Test item 500	100 µl	0.5	43	49	8	13	43	40	25	22	24	20	29	3	7	4	4
Test item 1580	100 µl	0.2	24	29	9	7	50	39	9	16	8	10	11	0	3	0	0
Test item 5000 **	100 µl *	0.0	0	0	3	1	41	54	0	0				0	0	0	0
Test item 15800 **	100 µl *	0.0	0	0	0	0	47	56	0	0				0	0	0	0
Benzo(a)pyrene 10	10 µl DMSO	1.1	76	105					35	26
Benzo(a)pyrene 50	10 µl	1.0	88	82					22	25
2-Aminoanthracene 10	10 µl	1.1	116	123					41	56
3-methylcholanthrene 90	30 µl	1.0	164	161					80	65
Benzo(a)pyrene 4,5-oxide	10 µl	1.0	2200	2250	21	19	63	65	4000	3900	1520	1610	1680	693	720	539	532
N-methyl-N'-nitro-N-nitrosogunidine	10 µl	0.7	19000	13000	36000	33000	418	433	101	95	60	63	58	16	16	129	62

-*: plate contaminated; not tested

**macroscopically visible precipitate of test compound on the plate.

* The conpound was dissolved in H2O at 70 °C before being added to the plate.

Applicant's summary and conclusion

Conclusions:

No mutagenicity was observed with test item under any experimental condition.

Executive summary:

The test item was tested for mutagenicity with Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98 and with Escherichia coli WP2 uvrA, both directly and in the presence of a mammalian metabolizing system. Eight different concentrations from 5 to 15800 µg per plate were used. The compound was dissolved in H₂O at 70 °C. When the solution was added to the plate, part of the test compound precipitated at the two highest doses. Some precipitate was still present at the end of the experiment.

For determination of toxicity his+ mutants were added to some plates in order to estimate the survival of mutants. In the absence of S-9 Mix the compound started to be noticeably toxic at 500 µg. At this and at the next higher dose (1580 µg) sufficient bacteria survived that there is a good chance that relatively stable mutagens still may be detected. In the presence of S-9 Mix, toxicity was much lower. Only at 15800 µg a substantial decrease in survival occurred. All in all, toxicity and solubility of compound are such that it can be tested for mutagenicity with a good sensitivity.

No mutagenicity was observed with test item under any experimental condition. Because in the first experiment for direct mutagenicity the number of revertant colonies from TA 98 was slightly (less than two-fold) increased on some plates with test item, this part of the experiment was repeated with a larger number of parallel plates. The negative result of the repeated experiment strongly supported the interpretation that the marginal effect of the first experiment was simply due to dispersion rather than to a real effect. Mutagenicity effects were also not observed when the epoxide hydratase inhibitor and glutathione depletory 1,1,1-trichloropropene 2,3-oxide was added to the S-9 Mix in order to increase the sensitivity of the test towards compounds which are activated to mutagenic epoxides (this experiment was performed with TA 98).

The positive controls confirmed the suitability of the system.

Conclusion

No mutagenicity was observed with test item under any experimental condition.