Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
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): Lucirin LR 8728
- Analytical purity: not reported
- Physical state: pale yellow powder
- Lot/batch No.: 010213
- Storage condition of test material: stored in brown glass screw-top jars at approximately 4°C in the dark (handling time restricted to 15 minutes under fluorescent light)

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500 and 5000 µg/plate (in the preliminary toxicity study);
0, 8, 40, 200, 1000, and 5000) µg/plate (first experiment);
0, 312.5, 625, 1250, 2500, and 5000) µg/plate (second experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: N-methyl-n-nitro-n-nitrosoguanidine
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37°C

SELECTION AGENT (mutation assays): histidine (or tryptophan in the case of WP2uvrA-)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: 0.1 ml of bacterial suspension (TA100 or WP2uvrA-) and 0.1 ml of test solution were added to 2 ml of molten, trace histidine or tryptophan supplemented media (histidine/biotin or tryptophan & top agar) and overlayed onto sterile plates of Vogel-Bonner agar (minimal agar -25 ml/ plate). Five doses of the test substance and a solvent control were tested in duplicate. After 48-72 hours incubation the plates were scored for revertant colonies and examined for a thinning of the background lawn.
Evaluation criteria:
- The test chemical is considered positive in this assay if it induces a dose-related and statistatistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S-9 microsomal enzymes in both experiments.
- The test substance is generally considered non-mutagenic in this test if the number of induced revertants compared to spontaneous revertants is less than 2-fold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 µg/plate.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at the highest tested dose (5000 µg/plate)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mean maximum revertants/plate and the corresponding concentration with/without S-9 mix are resumed below:

- First experiment:

                   Mean maximum revetants observed/plate [corresponding test concentration (µg/plate)]
      Solvent control   Test substance     Positive control (substance)
 Strains  with  without  with  without  with  without
 TA100  78  87  90 [40]  83 [40]  732 [3.3] (2-AA)   472 [2.0] (MNNG)
 TA1535  18  9  19 [40, 5000]  12 [40, 1000]  49 [3.3] (2-AA)   139 [2.0] (MNNG)
WP2uvrA-  24  30  31 [40]  31 [8]  126 [10.0] (2-AA)   734 [3.3] (4NQO)
 TA98  25  18  27 [200, 1000]  22 [200]  501 [3.3] (2-AA)   803 [10.0] (4NOPD)
 TA1537  6  4  6 [8]  4 [8, 200, 1000]  48 [3.3] (2-AA)   202 [50.0] (9-AA)
             

The spontaneous reversion counts/plate was 17.7, 8.3, 29, 107.7 and 33 respectively for TA1535, TA1537, TA 98, TA100 and WP2uvrA-.

- Second experiment

                   Mean maximum revetants observed/plate [corresponding test concentration (µg/plate)]
      Solvent control   Test substance     Positive control (substance)
 Strains  with  without  with  without  with  without
 TA100  70  75  78 [5000]  79 [312.5]  1060 [3.3] (2-AA)   599 [2.0] (MNNG)
 TA1535  14  11  14 [312.5]  12 [312.5, 2500]  74 [3.3] (2-AA)   289 [2.0] (MNNG)
WP2uvrA-  24  21  27 [312.5]  22 [625]  127 [10.0] (2-AA)   823 [3.3] (4NQO)
 TA98  23  19  24 [2500]  19 [5000]  444 [3.3] (2-AA)   741 [10.0] (4NOPD)
 TA1537  7  6  7 [625]  6 [5000]  59 [3.3] (2-AA)   134 [50.0] (9-AA)
             

The spontaneous reversion counts/plate was 15.5, 5, 23.7, 83.3 and 148.3 respectively for TA1535, TA1537, TA 98, TA100 and WP2uvrA-.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions in this study, the test substance is not a mutagenic agent in a bacterial reverse mutation test in vitro.