Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
modified version of the Ames test (OECD 471), designated Ames II Assay

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Principles of method if other than guideline:
The test method is used to evaluate the mutagenic potential of the test item and based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium (TA 98, TA Mix (TA 7001- 7006)) in a modified version of the Ames test (OECD 471), designated Ames II Assay (microtiter version), both with and without the addition of a metabolizing system (S9 mix) obtained from liver from induced rats. This method shows a good accuracy concerning the prediction of the results in the regular Ames test.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Santalene oil: fermentation products of glucose with santalene synthase modified Rhodobacter sphaeroides, distilled
Molecular formula:
not applicable
IUPAC Name:
Santalene oil: fermentation products of glucose with santalene synthase modified Rhodobacter sphaeroides, distilled

Method

Target gene:
his
Species / strainopen allclose all
Species / strain / cell type:
bacteria, other: S. typhimurium TA Mix (TA 7001 - TA 7006)
Remarks:
different strains of S. typhimurium
Species / strain / cell type:
S. typhimurium TA 98
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- liver S9 mix from induced male Wistar rats (not further specified)
- 40 µL S9 mix in 200 µL bacteria suspension and 10 µL test substance or vehicle or positive control
Test concentrations with justification for top dose:
0; 4; 20; 100; 500; 2500 and 5000 µg/mL
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
other: 2-aminoanthracene (with S9 mix)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
Deep-frozen bacterial cultures (Salmonella typh. TA 98 or Salmonella typh. TA 7001 - 7006 [=TA Mix]) were thawed at room temperature. From this bacterial suspension a volume of 28 µL was inoculated in 20 mL growth medium and was subsequently incubated at 37°C with shaking at about 60 rpm for 14 - 17 hours (overnight cultures, showing an optical density of about 1.0 (measured at a wavelength of 500 nm)).
5 mL of the overnight cultures were added to tubes containing 19 mL Ames II Exposure medium and were gently mixed. After thorough pipetting the following components were added in 24-well plates:
- without S9 mix: 240 µL bacteria suspension (tester strain + exposure medium) + 10 µL test substance, vehicle or positive control
- with S9 mix: 200 µL bacteria suspension + 40 µL S9 mix (liver homogenate to buffer ratio: 3:7) + 10 µL test substance, vehicle or positive control
The 24-well plates were incubated at 37°C with shaking at 60 rpm for about 90 minutes.
After this incubation period, 2.8 mL Ames II Reversion indicator medium (containing bromocresol purple) was pipetted to each well of the 24-well plate. The contents of each well of the 24-well plates were distributed in 50 µL aliquots over 48 wells of a 348-well Revertant Colony Selection plate (RCSP). The plates were sealed in plastic bags and incubated at 37°C in the dark. After 48 hours incubation, each 48-well section of the RCSP were scored and the number of positive wells (yellow = high number of his' revertants) were counted.
Evaluation criteria:
Evaluation was performed by the following comparisons/calculation:

- An increase in the mean number of positive wells in dose groups was compared to the mean value of the concurrent negative control (Evaluation factor 1 F).
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of the actual experiment (Evaluation factor 2 F). The baseline was derived from the mean spontaneous revertant number plus the value of standard deviation (mean + SD) from the distribution of spontaneous data.
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of an experimental run (Evaluation factor 3 F). A run consists of a variable number of experiments generally testing different test substances together each using the same vehicle control. This leads to an accumulation of replicates for negative controls which was used to calculate the mean spontaneous reversion number for each run.

A test substance is considered mutagenic in this test system, if more than a doubling of Evaluation factor 3 F is observed in at least one test group. This finding should be dose dependent and/or reproducible.
Statistics:
Not specified

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typimurium, TA Mix (TA 7001 - 7006)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: the precipitation in plates was at 500 µg/mL and above

Ames test:
- Signs of toxicity: No bacteriotoxic effect (clearing of the background lawn, decrease in the number of yellow wells) was observed.
- Mean number of revertant colonies per plate and standard deviation: An increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system (see table 1 and 2 in section "Attached background material").

Applicant's summary and conclusion

Conclusions:
According to the results of the present study, the test item is not mutagenic in the Ames II Assay (S. typhimurium reverse mutation assay) under the conditions of the test.
Executive summary:

A modified version of the Ames test (OECD 471), i.e. a Salmonella typhimurium reverse mutation assay (Liquid fluctuation test - microtiter version; Ames II Assay), was carried out to evaluate the mutagenic potential of Santalene Oil. The test material was tested at 4, 20, 100, 500, 2500 and 5000 µg/mL in DMSO using several strains of Salmonella typhimurium (TA 98 or TA 7001 - 7006 [=TA Mix]) both with and without the addition of a metabolizing system (S9 mix) from livers of induced male Wistar rats. This method shows a good accuracy concerning the prediction of the results in the regular Ames test. A bacteria suspension (240 µL) was incubated with 10 µL test substance, vehicle or positive controls with or without S9 mix (40 µL) in 24-well plates at 37°C for about 90 minutes. After this incubation period, bacteria were transferred to indicator medium (containing bromocresol purple) and respective 50 µL aliquots were transferred to and incubated in 348-well Revertant Colony Selection plates (RCSP) at 37°C for 48 hours. The wells of the RCSP were scored and the number of positive wells (yellow = high number of his' revertants) were counted. Each experiment included negative controls (vehicle control) and positive controls (2-aminoanthracene (with S9 mix), 2-nitrofluorene + 4-nitroquinoline-N-oxide without S9 mix)).

 

No bacteriotoxic effect (clearing of the background lawn, decrease in the number of yellow wells) but precipitation starting at 500 µg/ml was observed. An increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance Santalene Oil

is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation.

Categories Display