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
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to the procedure prescribed by Ames et al. (Mut. Res. 21: 347-364, 1975) and was in principle similar to the OECD TG 471, however with following main deviations: neither TA102 nor a DNA repair-proficient E.coli WP2 strain was included as tester strain for detection of cross-linking mutagens. Furthermore, the evaluation of revertants was based on extinction measurement by means of an automatic analyser for microbiological purposes COBAS Bact designed by ROCHE, in conjunction with a Computer Hewlett-Packard 9816. Test considered sufficiently validated.

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:
yes
Remarks:
(No strain for detection of cross-linking mutagens such as TA102 or the DNA repair-proficient E.coli WP2 strain as recommended by the OECD TG was tested.)
Principles of method if other than guideline:
The test was conducted according to the Ames procedure as described by Ames et al. (Mut. Res. 21: 347-364, 1975).
For analysis, the automatic analyser for microbiological purposes COBAS Bact designed by ROCHE was used, in conjunction with a Computer Hewlett-Packard 9816.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4-Amino-2-chlortoluol-5-sulfonsäure
- Analytical purity: No details on purity given in the study report
- Lot/batch No.: BASF

Method

Target gene:
All strains are mutants from histidine auxotrophy (his) to histidine prototrophy (his+).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: defect in the excision repair system (uvrB)
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction from the liver of Sprague-dawley rats treated with Aroclor 1254 mixed with a series of appropriate cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)
Test concentrations with justification for top dose:
Preliminary toxicity test: 3.4 to 2500 µg/mL
Main test, with and without S9 mix: 2.44, 9.77, 39.1, 156, 625, 2500 µg/mL
Vehicle / solvent:
The substance was suspended in DMSO; the solvent alone was used for the negative controls.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(see solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
other: Daunorubicin-HCl 0.5 µg/mL PBS
Remarks:
TA 98
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
sodium azide
Remarks:
TA 1535 and TA 100

Migrated to IUCLID6: 0.083 µg/mL PBS
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
9-aminoacridine
Remarks:
TA 1537

Migrated to IUCLID6: 5µg/mL DMSO
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
other: 2-Aminoanthracene 0.17 µg/mL DMSO
Remarks:
TA 98 and TA 100
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
cyclophosphamide
Remarks:
TA 100 (because of inconsistent findings obtained for this strain when tested with 2-Aminoanthracene, testing with TA 1535 was repeated using Cyclophosphamide for positive control)

Migrated to IUCLID6: 50 µg/mL PBS
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
cyclophosphamide
Remarks:
TA 1535

Migrated to IUCLID6: 133.3 µg/mL PBS
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
other: Acridine orange 3.33 µg/mL DMSO
Remarks:
TA 1537
Details on test system and experimental conditions:
The histidine-auxotrophic bacteria were incubated in minimum medium in measurement chambers with a volume of 0.3 mL together with the test material at 37 °C. In the experiments in which the substance was metabolically activated, 20% of S9 mix was added to the minimum medium. At test initiation the medium contained 1 to 5x10E+6 bacteria/mL.
The minimum medium was Vogel-Bonner medium E, supplemented with 20 g/l glucose, 0.5 mg/l biotin and 0.4% nutrient broth (Difco Laboratories, Detroit, Michigan, U.S.A). The test material in the vehicle was added to the chambers in a volume of 10 µL.
For analysis, the automatic analyser for microbiological purposes COBAS Bact designed by ROCHE was used, in conjunction with a Computer Hewlett-Packard 9816.
Turbidity of the content of the chambers was measured automatically at the wave length of 546 nm at intervals of 20 min over a period of about 46 hours. The cultures were shaken prior to each measurement. Four measurement chambers were incubated per strain and per group (test and control group). In the first experiment for technical reasons the values for the first 6 hours were not registered.
For evaluation, the revertant growth in each of the chamber was determined by measuring the extinction at different time points during incubation (10, 20, 30 and 40 hours).
Evaluation criteria:
A test substance is considered to be mutagenic in this test system if a dose-dependent increase in the number of chambers with revertant growth occurs. The time of the beginning of revertant growth gives an indication of the number of back-mutant bacteria induced. Growth-inhibiting effects can be observed simultaneously. Revertant growth appearing later than after 40 hours was considered to be spontaneous in origin and thus were interpreted as negative result.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(comparison of the number of back-mutant colonies in controls and treated cultures with the various concentrations of test item revealed no marked deviations)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(tested up to 2500 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of main test results expressed in revertant growth (+) or none (-):

Mutagenicity Test with COBAS BACT: Summary of Results in absence of S9 mix

Strain

Test group

Chamber

1

2

3

4

TA 98

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

-

9.77 µg/mL

-

-

-

-

39.1 µg/mL

-

-

+

-

156 µg/mL

-

-

-

-

625 µg/mL

-

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control

+

+

+

+

TA 100

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

+

+

9.77 µg/mL

+

-

-

-

39.1 µg/mL

-

-

-

-

156 µg/mL

+

-

-

-

625 µg/mL

-

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control

+

+

+

+

TA 1535

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

-

9.77 µg/mL

-

-

-

-

39.1 µg/mL

-

-

-

-

156 µg/mL

-

-

-

-

625 µg/mL

-

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control

+

+

+

+

TA 1537

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

-

9.77 µg/mL

-

-

-

+

39.1 µg/mL

-

-

-

+

156 µg/mL

-

+

-

+

625 µg/mL

+

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control

+

+

+

+

Mutagenicity Test with COBAS BACT: Summary of Results in presence of S9 mix

Strain

Test group

Chamber

1

2

3

4

TA 98

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

+

9.77 µg/mL

+

-

-

-

39.1 µg/mL

+

-

-

-

156 µg/mL

-

-

-

+

625 µg/mL

-

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control

+

+

+

+

TA 100

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

+

9.77 µg/mL

-

-

-

-

39.1 µg/mL

-

-

-

-

156 µg/mL

-

-

-

-

625 µg/mL

-

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control (2-Aminoanthracene!)

-

+

-

-

TA 1535

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

+

9.77 µg/mL

-

-

-

-

39.1 µg/mL

-

-

-

-

156 µg/mL

-

-

-

-

625 µg/mL

-

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control

+

+

+

+

TA 1537

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

-

9.77 µg/mL

-

-

-

-

39.1 µg/mL

-

-

-

-

156 µg/mL

-

-

-

+

625 µg/mL

-

-

-

-

2500 µg/mL

-

-

-

-

Pos. Control

+

+

+

+

Additional testing with TA 100, this time using Cyclophosphamide as positive control substance:

Mutagenicity Test with COBAS BACT: Summary of Results in presence of S9 mix

TA 100

Neg. Control

-

-

-

-

2.44 µg/mL

-

-

-

-

9.77 µg/mL

-

-

-

-

39.1 µg/mL

-

-

-

+

156 µg/mL

+

+

-

-

625 µg/mL

-

-

-

+

2500 µg/mL

-

-

-

-

Pos. Control (Cyclophosphamide)

+

+

+

+

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative