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Genetic toxicity in vitro

Description of key information

The genetic toxicity in vitro was tested for test article TFS plantation-grown Indian sandalwood (santalum album) oil according to OECD 471 method and guideline EPA OPPTS 870.5100.

The test item did not have mutagenicity potential in the Bacterial Reverse Mutation Test.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
E. coli WP2 uvrA (cat #72-188L) T/PE
S. typh. TA-97a (cat #71-097L) his D 6610
S. typh. TA-1535 (cat #71-1535L) his G 46
S. typh. TA-98 (cat #71-098L) his D 3052
S. typh. TA-100 (cat #71-100L) his G 46
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Test concentrations with justification for top dose:
Five concentrations (0.005, 0.01, 0.05, 0.1 and 0.5 µL/plate) of the test article were tested in each of five bacterial tester strains.
Two sets of culture plates were dosed per concentration (+S9 and -S9).
A vehicle control and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO), Lot #112509 and 133317
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Remarks:
without S9
Positive control substance:
methylmethanesulfonate
other: Acridine 6-chloro-9-(3-((2-chloroethyl)amino) propyl)amino-2-methoxy dihydrochloride ( S. typh. TA-97a Sodium azide (S. typh. TA-1535 and S. typh. TA-100) Daunomycin hydrochloride (S. typh. TA-98 )
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Remarks:
with S9
Positive control substance:
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Tester strains were dosed in triplicate with five concentrations of the test article. Six plates each were dosed with the vehicle control and the positive control specific for each tester strain.
Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. #26-555). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37 °C ±2 °C with agitation. The cultures were used after they reached the late exponential growth phase as determined by absorbance readings at 600 nm.
Evaluation criteria:
Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account.
In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose-related increases approaching a 2-fold increase are deemed equivocal.
A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.
Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
no significant increase or dose-dependent increase of the number of revertants in the tester strain treated with the test article in the presence or absence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
no significant increase or dose-dependent increase of the number of revertants in the tester strain treated with the test article in the presence or absence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
no significant increase or dose-dependent increase of the number of revertants in the tester strain treated with the test article in the presence or absence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
no significant increase or dose-dependent increase of the number of revertants in the tester strain treated with the test article in the presence or absence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the main test, the test article even at the highest dosing concentration (0.5 µL/plate) did not cause severe or moderate cytotoxicity to tester strain WP2 uvrA.
In the Independent Repeat Assay including the Guideline-recommended maximum test concentration for soluble non-cytotoxic test article (5 µL/plate),no reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in WP2 uvrA treated with the test article in the presence or absence of S9.
All Positive and Negative Control values were within acceptable ranges, and all criteria for a valid study were met. The results confirmed that the test article did not cause mutation to WP2 uvrA.
Conclusions:
Under test conditions, test article TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APIS0-130608SD/SA, did not have mutagenicity potential in the Bacterial Reverse Mutation Test.
Executive summary:

Test article TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APISO-130608SD/SA, in the vehicle, Dimethyl sulfoxide, was tested in a Bacterial Reverse Mutation Assay.

The test article showed severe cytotoxicity to tester strain TA-100 at a dose range of 1 to 5µL/plate with S9 and at a dose range of 0.5 to 5µL /plate without S9. In the main test, the test article showed severe to moderate/mild cytotoxicity to all S. typhimurium strains (TA 97a, TA1535, TA98, TA100) at several top concentrations, but not to E Coli WP2 uvrA. The test article at 0.005 to 0.5µL /plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

In the independent repeat assay (confirmatory test), the test article at 0.05 to 5µL/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of tester strain WP2 uvrA, and showed no/minimal cytotoxicity to WP2 uvrA, indicating that the test article is negative for mutagenicity to this strain.

Conclusion: Under test conditions, test article TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# AP1S0-130608SD/SA, did not have mutagenicity potential in the Bacterial Reverse Mutation Test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

So far only an in vitro Ames test is available for the substance showing negative results for mutagenicity. Thus, based on currently available data the substance is not subject to classification for mutagenicity according to GHS and/or CLP (Regulation EC No 1272/2008).

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