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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was not conducted under GLP (adopted from OECD SIDS; additionally, a strain to detect crosslinking/ oxidizing agents is missing)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989
Reference Type:
secondary source
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strain missing to detect crosslinking/ oxidizing agents
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The purity of the test material was > 99%.

Method

Target gene:
his-
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
liver S-9 mix from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
20 - 5000 micrograms/plate (Standard Plate test; TA 100, TA 98); 4 - 2500 micrograms/plate (Standard plate test; TA 1535, TA 1537 and preincubation test on all strains)
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
S-9 was prepared from at least 5 male Sprague-Dawley rats (200 - 300 g) that received an i.p. injection of 500 mg Aroclor 1254 (as a 20% solution in peanut oil) five days before liver collection. S-9 was stored at -70 to -80 degrees C for up to 2 months. S-9 mix was prepared freshly prior to each experiment. Three volumes of S-9 were mixed with 7 volumes of a cofactor mix containing 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phsophate, 4 mM NADP and 100 mM sodium phosphate buffer (pH 7.4).
The Salmonella strains (TA98, TA100, TA1535, and TA1537) were checked periodically for deep rough character (rfa), UV sensitivity (uvrB), and ampicillin resistance (R factor plasmid). Deep-frozen bacterial cultures were thawed, and 0.1 ml of the bacterial suspension was inoculated in nutrient broth and incubated in a shaking water bath at 37 degrees C for about 16 hours (to an approximate density of > = 10E8 bacteria/ml). The cultures were then placed in ice water to prevent further growth.
For the standard plate test, test solution (0.1 ml), bacterial suspension (0.1 ml), and phosphate buffer or S-9 mix (0.5 ml) were added to tubes containing 2 ml soft agar. For the preincubation test, the test solution, bacterial suspension and S-9 mix were incubated at 37 degrees C for 20 minutes before being added to the soft agar. After mixing, the samples for either test were poured onto minimal glucose agar plates. Cells were incubated at 37 degrees C for 48 hours in the dark, and the number of colonies was counted. All tests were performed in triplicate.
The concentrations tested varied according to experiment: (standard incubation experiment 1 (with and without S-9 mix): 0, 20, 100, 500, 2500 and 5000 micrograms/plate in strains TA98 and TA100; standard incubation experiment 2 (with and without S-9 mix): 0, 4, 20, 100, 500, and 2500 micrograms/plate in strains TA1535 and 1537; preincubation experiment (with and without S-9): 0, 4, 20, 100, 500 and 2500 micrograms/plate in all strains.
A solvent control (DMSO) and the positive controls N-methyl-N'-nitro-N-nitroso-guanidine (5 micrograms/plate for strains TA100 and TA1535), 4-nitro-o-phenylenediamine (10 micrograms/plate, strain TA98), 9-aminoacridine (100 micrograms/plate for strain TA1537), and 2-aminoanthracene (10 micrograms/plate for all strains with S-9) were tested in each experiment. The positive control chemicals were dissolved in DMSO.
Precipitation of the test material was recorded (if present).
Toxicity was detected by a decrease in the number of revertants, a clearing or dimunition of the background lawn or a reduction in the titer.
Evaluation criteria:
A substance was considered mutagenic if it caused a doubling in the spontaneous (control) mutation rate, and the effect was dose-dependent and reproducible.
The experiment was considered valid if the number of colonies in the negative controls was within the normal range of the historical data for the strain, sterility controls had no evidence of contamination, the positive controls induced a significant increase in the number of revertants and the titer of viable bacteria was >/= 10E9/ml.

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
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 micrograms/plate (Standard Plate test; TA100 and TA98), >= 500 micrograms/plate (preincubation test, all strains)
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

Standard plate test: There was no mutagenic effect of test material in the presence or absence of S-9. In the absence of S-9, the average numbers of mutants in controls in strains TA98, TA100, TA1535 and TA1537 were 21, 108, 18 and 8. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 ranged from 9-22, 123-141, 15-21 and 8-12. In the presence of S-9, the average numbers of mutants in controls in strains TA98, TA100, TA1535 and TA1537 were 34, 113, 17 and 9. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 with S-9 ranged from 30-36, 107-122, 19-26 and 8-14.
Preincubation test:
 There was no mutagenic effect of test material in the presence or absence of S-9. In the absence of S-9, the average numbers of mutants in controls in strains TA98, TA100, TA1535 and TA1537 were 18, 106, 20 and 7. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 ranged from 0-25, 0-133, 0-23 and 0-10. In the presence of S-9, the average numbers of mutants in controls in strains TA98, TA100, TA1535 and TA1537 were 20, 109, 13 and 6. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 with S-9 ranged from 13-34, 80-115, 2-19 and 3-10.
All positive controls induced at least a 11-fold increase in mutants (with respect to the solvent control).

Applicant's summary and conclusion