Almond, sweet, ext.

Genetic toxicity: in vitro

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

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Almond, sweet, ext.

EC Number:

291-063-5

EC Name:

Almond, sweet, ext.

Cas Number:

90320-37-9

IUPAC Name:

Almond, sweet, ext.

Test material form:

liquid

Details on test material:

Amber liquid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of rats

Test concentrations with justification for top dose:

Main study - Direct plate incorporation method: 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix in TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains
Confirmatory assay - pre-incubation method: 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix in TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains

Vehicle / solvent:

Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test substance was soluble in water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 37 °C for 20-30 minutes
- Exposure duration: Plates were incubated at 37 ± 1 °C for 48-72 h
Each test item dilution and each reference item are tested on 3 Petri plates

Evaluation criteria
For each assay the following observations were performed and reported:
- Observation of the reagent mix before Petri plates pouring: reporting of any abnormal sign (precipitate, trouble, etc.),
- Petri plates observation and reporting of any cytotoxicity sign (bottom bacterial layer reduction). The cytotoxicity intensity on the bottom bacterial layer is evaluated qualitatively on each plate by naked eyes: o total destruction of the bottom bacterial layer (the revertants development does not occurs in this
case), this one is noted in tables of results as “A”. o moderated destruction of the bottom bacterial layer. This one is noted in tables of results as “S”.

Acquisition and storage of raw data were managed by the following electronic system:
- Reading of plates: Sorcerer, version 2.2.
- Transfer and storage of raw data: Ames Study Manager, version 1.22.
The result tables edition was managed by the Ames Report Generator, version 1.

Rationale for test conditions:

The solubility test showed no insolubility of the test item. Therefore, the maximale concentration retaines is 5000 µg/plate.
According to OECD fuideline, 5 concentrations of test item have been studied approximatively half log interval). These doses (rounded to the higer value) used for the preliminary cytotoxicity test are therefore: 5000, 1600, 500, 160 and 50 µg/plate.
As the preliminary experiment revealed no cytotoxicity of the test item, this range of concentration has been conserved for the test 1.

Evaluation criteria:

The test is considered valid if the following criteria are fulfilled:
- The sterility tests are conform,
- The mean negative controls are within the historical data,The solvent used (negative control) must not show genotoxic nor cytotoxic activity,The revertants rate obtained for the positive controls must be in agreement with the historical data,
- The positive controls must show a revertants number equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 (R superior or egual to 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 (R superior or egual to 3),
- No more than 5% of the plates of the test are lost through contamination or any other unforeseen event,
- At least 3 concentrations are available for mutagenicity assessment

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA98, TA100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No cytotoxic effect was observed,
No concentration of the test item showed ratio R higher or equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 strains and to the triple of the spontaneous rate of
reversion for TA1535 and TA1537 strains, with and without metabolic activation, No dose response was observed, whatever the test system or conditions of the test.
In addition, no sign of precipitate was observed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
At the light of the results obtained during this study, we can conclude that the test item does not show any mutagenic nor pro-mutagenic activity, under the test conditions used.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to test item at the following concentrations: 50, 160, 500, 1600 and 5000μg/plate.

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of rats. Vehicle and positive control groups were also included in mutagenicity tests.

The preliminary study showed no cytotoxicity of the test item; therefore this concentrations range was used for the genotoxicity Test 1.

According to the result obtained for the Test 1, the Study Director decided to use the same dilution range for the test 2.

The revertant analysis shows that:

# No cytotoxic effect was observed,

# No concentration of the test item showed ratio R higher or equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 and to the triple of the spontaneous rate of reversion for TA1535 and TA1537, with and without metabolic activation,

# No dose response was observed, whatever the test system or conditions of the test.

At the light of the results obtained during this study, we can conclude that the test item does not show any mutagenic nor pro-mutagenic activity, under the test conditions used.