2,9,11,13,15,22,24,26,27,28- decaazatricyclo[21.3.1.1^(10,14)]octacosa- 1(27),10,12,14(28),23,25-hexaene-12,25-diamine, N,N'-bis(1,1,3,3-tetramethylbutyl)-2,9,15,22-tetrakis(2,2,6,6-tetramethyl-4-piperidinyl)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-03-05 to 1985-04-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study close to Guideline with acceptable restrictions (pre-incubation method not performed)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

no

Remarks:

QAU statement included

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: solid
- Analytical purity: commercial grade

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

0.08 - 5000 µg / 0.1 mL range in the toxicity test
20 - 5000 µg / 0.1 mL range in the mutagenicity test

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 98: daunorubicin-HCl; TA 100: 4-nitroquinoline-N-oxide; TA 102: mitomycin-C; TA 1535: sodium acide; TA 1537: 9(5)-aminoacridine hydrochloride monohydrate

Remarks:

without activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

with activation

Migrated to IUCLID6: TA 98, TA 100, TA 1537: 2-aminoanthracene;TA 102: 2-aminoanthracene; TA 1535: cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: incubated for about 48 hours at 37 ± 1.5 °C in darkness

NUMBER OF REPLICATIONS: without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In order to confirm the results the experiments were repeated.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the colony count

OTHER:
The test substance was suspended in acetone. Acetone alone was used for the negative controls (the substances and vehicles used for the positive controls are indicated below). Each Petri dish contained:
1) approx. 20 mL of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 mL of a solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8 % plus 0.5 % NaCl) in 2.0 mL of soft agar. The soft agar was composed of: 100 mL of 0.6 % agar solution with 0.6 % NaCl and 10 mL of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland).
In the experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture was added also. 1 mL activation mixture contained: 0.3 mL S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 mL of a solution of co-factors.
Positive control experiments were carried out simultaneously with the following substances:
1) for strain TA 98: daunorubicin-HCl (DAUNOBLASTIN®, Farmitalia, Montedison Farmaceutica GmbH, Freiburg i. Br., Germany), 5 and 10 µg/0.1 mL phosphate buffer
2) for strain TA 100: 4-nitroquinoline-N-oxide (Fluka, Buchs, Switzerland), 0.125 and 0.25 µg/0.1 mL phosphate buffer
3) for strain TA 102 : mitomycin-C (SYNTEX PHARM AG, Allschwil/Basle, Switzerland), 0.5 and 1.0 µg/0.1 mL bidistilled water
4) for strain TA 1535: sodium acide (Fluka, Buchs, Switzerland), 2.5 and 5.0 µg/0.1 mL bidistilled water
5) for strain TA 1537: 9(5)- aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland), 50 and 100 µg/0.1 mL DMSO.
The activation mixture was tested with strains TA 98, TA 100, TA 1537: 2-aminoanthracene (EGA-Chemie, Steinheim, Germany), 5 µg/0.1 mL DMSO; 2) with strain TA 102: 2-aminoanthracene, 20 µg/0.1 mL DMSO; 3) with strain TA 1535: cyclophosphamide (ENDOXAN-ASTA® , Asta-Werke, Bielefeld, Germany), 250 µg/0.1 mL phosphate buffer.

Evaluation criteria:

The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth-inhibiting effect occurred in the experiments without microsomal activation at the highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At concentrations of 1250 µg/0.1 mL and above precipitation of the test substance was perceptible in soft agar.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg / 0.1 mL. Thereupon, the concentration of 5000 µg / 0.1 mL was used as the highest in the mutagenicity test and the tests were performed with the following concentrations of the trial substance without and with microsomal activation: 20, 78, 313, 1250 and 5000 µg / 0.1 mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Owing to a growth-inhibiting effect of the substance a reduction in the colony count was observed in the experiments without microsomal activation at the highest concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment I

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	28	50	140	152	172	317	13	20	5	16
20	26	51	170	145	201	285	15	22	7	17
78	26	60	147	130	230	305	13	17	8	13
313	28	48	134	127	207	276	8	22	5	15
1250	23	43	161	133	211	230	17	20	8	19
5000	8	39	111	114	95	264	14	12	6	12
solvent control	24	43	152	122	215	259	10	16	7	18
positive control A	370	291	1001	492	792	2352	852	391	19	54
positive control B	714		1456		1631		1165		372

Experiment II

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	23	34	126	123	256	296	17	14	12	19
20	26	43	133	114	293	325	12	15	10	19
78	23	39	120	118	332	330	13	12	13	19
313	26	48	120	124	318	307	16	20	6	16
1250	24	37	100	106	286	290	14	17	9	20
5000	24	42	95	112	193	283	16	17	5	20
solvent control	21	39	112	117	258	335	18	16	8	14
positive control A	659	922	725	674	851	1227	836	396	39	129
positive control B	874		1194		1073		1104		635

Positive Controls

Without S9 mix:

TA 98: daunorubicin-HCl, A: 5 and B: 10 µg/0.1 mL phosphate buffer

TA 100: 4-nitroquinoline-N-oxide, A: 0.125 and B: 0.25 µg/0.1 mL phosphate buffer

TA 102: mitomycin-C, A: 0.5 and B: 1.0 µg/0.1 mL bidistilled water

TA 1535: sodium azide, A: 2.5 and B: 5.0 µg/0.1 mL bidistilled water

TA 1537: 9(5)- aminoacridine hydrochloride monohydrate, A: 50 and B: 100 µg/0.1 mL DMSO

With S9-Mix:

TA 98, TA 100, TA 1537: 2-aminoanthracene, 5 µg/0.1 mL DMSO

TA 102: 2-aminoanthracene, 20 µg/0.1 mL DMSO

TA 1535: cyclophosphamide, 250 µg/0.1 mL phosphate buffer

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the presented results and under the conditions employed, the test article did not induce point mutations in presence or absence of a metabolic activation system and is therefore regarded as not mutagenic in the Ames test.

Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES test similar in design to the OECD guideline No. 471 was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test article was applied by the plate incorporation method at concentrations of 20, 78, 313, 1250 and 5000 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates and repeated once for confirmation. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. A growth-inhibiting effect occurred in the experiments without microsomal activation at the highest concentration. At the concentrations of 1250 µg/0.1 ml and above the substance precipitated in soft agar. In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the substance is considered as not mutagenic in this assay.