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Toxicological information

Genetic toxicity: in vitro

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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Remarks:
Conducted according to guideline dated 1998
Qualifier:
according to
Guideline:
other: US FDA Redbook 2000 (Short-term Tests for Genetic Toxicity, 07 July 2000)
Deviations:
no
Remarks:
Conducted according to the guideline dated 2000
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Colorless gas
Details on test material:
- Purity: 99.971% (mole %)

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 urvA
Metabolic activation:
with and without
Metabolic activation system:
supplemented liver fraction (S9 mix)
Test concentrations with justification for top dose:
Initial Test: 0, 0.032, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100% v/v.
Confirmatory Test: 0, 3.2, 6.4, 12.8, 25.6, 51.2, 100% v/v.
Vehicle / solvent:
- Vehicle used: Air
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Air
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar, standard plate incorporation

The study employed the standard plate incorporation method. In the initial test, a 0.5 mL aliquot of S9 mix (+S9) or phosphate buffer 0.2 M pH 7.4 (0S9) was combined with 0.1 mL bacterial culture in a sterile container. Where appropriate, a standard volume (0.1 mL) of the test positive control was added. Approximately 2 mL of molten top agar supplemented with 0.05 mM biotin and minimal (0.05 mM) histidine and minimal (0.05 mM) tryptophan was added immediately afterward. The solution was mixed and overlaid onto a minimal glucose plate (1.5% agar, Vogel-Bonner medium E, 2% glucose). After the overlay solidified, the plates were inverted and incubated at ca. 37°C for 48 to 72 h. After the overlay solidified, the plates were inverted, stacked and placed in Tedlar bags that had been slit on one side (one bag per treatment). The bags were sealed with adhesive tape and the excess air inside the bags was evacuated.

Appropriate volumes of ambient laboratory air and stock test gas were added via the septum/valve into each bag to achieve the final concentrations indicated in the Study design. For the highest dose of the test gas, the gas was added to the bag, then purged prior to the addition of a second volume of gas.

The plates in the bags were transferred to incubators at ca. 37°C for 48 to 72 h. The sterility plates for the buffer and S9 mix as well as the plates treated with the routine liquid positive controls were not placed in Tedlar bags. They were inverted and incubated at ca. 37°C for the same duration as the test atmosphere-treated plates. After the treatment period, the bags were opened in a fume hood or extracted laminar flow cabinet and the plates allowed to equilibrate for ca. 20 mins before counting colonies.

The initial test used a range of dose levels separated by a half-log10 dose interval up to the maximum practical limit (100% test substance). Based on the results of the initial test, a confirmatory test was performed using the same methodology and the same upper exposure level but, in this case, a narrow (approximately 2-fold) concentration interval was employed.

After the incubation/equilibration period plates were examined visually and, if needed, with aid of inverted microscope. Plates were evaluated for the quality of the background lawn and number of revertant colonies. Revertant colony counts were routinely collected and saved directly into a database. Colony numbers were enumerated visually if precipitation or other artifacts interfered with the colony counter or at the discretion of the Study Director. Visual counts and the presence of visible precipitate were manually recorded in the raw data.

If available, plates from at least five non-toxic dose levels were assessed in each experiment. Toxic effects of the test substance are normally indicated by the partial or complete absence of a background lawn or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle controls taking into account the laboratory historical control range. Where five (relatively non-toxic) dose levels were analyzed, the remaining plates from lower dose levels may not have been evaluated and are not reported. The mean number of revertant colonies for all treatment groups was compared with those obtained for the concurrent vehicle control group.
Evaluation criteria:
The mutagenic activity is assessed using the following criteria:

1. Results were considered positive if these 2 criteria were met:
a) results for test substance showed a substantial increase in revertant colony counts, i.e. response ≥ 2 times the concurrent vehicle control values for TA98, TA1535, TA1537 and WP2 uvrA, or ≥ 1.5 times for TA100 with mean value(s) outside the laboratory historical control range. Otherwise results are considered negative;
b) the above increase must be dose related and/or reproducible, i.e. increases must be obtained at more than one experimental point (more than one strain or dose level, more than one occasion or with different methodologies).

2. If the second criterion is not met, further testing might be appropriate to clarify equivocal results using an appropriately modified study design, e.g., a narrower dose interval with the appropriate strain. In such cases, if no substantial increase is obtained in the confirmatory test, the results would be considered negative.
Statistics:
Means and standard deviations for appropriate dose levels were calculated using Microsoft® Excel. The mean number of revertant colonies for all treatment groups was compared with those obtained for the concurrent vehicle control group.

Results and discussion

Test results
Species / strain:
other: 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:
cytotoxicity
Remarks:
occasional reductions in the revertant colony count were obtained with some bacterial strains at the highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
No substantial increases in the revertant colony counts were obtained with any strain following exposure to the test substance in either the initial or confirmatory assays in the absence or presence of S9 mix. It is concluded that the test substance did not show any evidence of genotoxic activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.
Executive summary:

The purpose of this study was to evaluate the genotoxicity of the test substance using the bacterial reverse mutation test.

 

Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia coli  strain WP2uvrA were treated with the gaseous test substance at a range of concentrations separated by a half-log10 dose interval up to 100%, i.e., the maximum practical level) in the presence and absence of a supplemented liver fraction (S9 mix) using the plate incorporation version of the bacterial mutation test. Since no evidence of genotoxicity and only slight toxicity was seen in this initial test, the study was repeated using a narrower (approximately 2-fold) dose interval to confirm the results. Bacteria were incubated with standard positive control agents, and the response of the various bacterial strains to these agents confirmed the sensitivity of the test system and the activity of the S9 mix.

 

No substantial increases in the revertant colony counts were obtained with any strain following exposure to the test substance in either the initial or confirmatory assays in the absence or presence of S9 mix. It is concluded that the test substance did not show any evidence of genotoxic activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.