Registration Dossier

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:
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

Reference
Name:
Unnamed
Type:
Constituent
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
Controlsopen allclose all
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
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.