Pepper (Piper), P. nigrum, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April to 26 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

05 March 2015

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pepper (Piper), P. nigrum, ext.

EC Number:

284-524-7

EC Name:

Pepper (Piper), P. nigrum, ext.

Cas Number:

84929-41-9

Molecular formula:

Not available

IUPAC Name:

Essential oil of Piper nigrum (Piperaceae) obtained from the berries by steam distillation

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Essential oil of Piper nigrum (Piperaceae) obtained from the berries by steam distillation
-Other names: Pepper black oil; POIVRE NAT 974518
- Analytical purity: 100% wt UVCB Substance
- Lot/batch No.: 1002657445
- Date or receipt: 06 April 2016
- Expiration date of the lot/batch: 24 May 2017
- Purity test date: 07 November 2016
- Appearance: pale yellow to green liquid
- Storage condition of test material: At room temperature (15°C to 20°C +/- 0°C); protected from light, protected from humidity

Method

Target gene:

Histidine and tryptophan

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

Metabolic activation:

with and without

Metabolic activation system:

10% (v/v) S9 fraction; S9 mix prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate for TA 98, TA 100 and TA 102 strains with and without S9 mix (plate incorporation method)
Mutagenicity experiments
Experiments without S9 mix: 312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in both mutagenicity experiments (plate incorporation method)
Experiments with S9 mix:
312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in the first experiment (plate incorporation method), and for the TA 98, TA 100 and TA 102 strains in the second experiment (pre-incubation method),
62.5, 125, 250, 500, 1000 and 2000 μg/plate for the TA 1535 strain in the second experiment (pre-incubation method),
31.3, 62.5, 125, 250, 500 and 1000 μg/plate for the TA 1537 strain in the second experiment (pre-incubation method).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: According to available solubility data, the vehicle was ethanol.
- Dose formulation preparation: No correction factor was applied. The test item was diluted in the vehicle at a concentration of 200 mg/mL for the preliminary toxicity test and for both mutagenicity experiments. The stock solutions and their dilutions were prepared within 4 h of use, and then kept at room temperature and protected from light until use.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Anthramine

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) or Culture Collections (Public Health England, Porton Down, Salisbury, SP4 0JG, UK).

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes at 37 °C
- Exposure duration: Plates were incubated at 37 °C for 48-72 h

NUMBER OF REPLICATIONS:
Preliminary toxicity test: one plate/dose-level
Mutagenicity experiments: three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed based on the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHERS:
- The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
- After 48 to 72 h of incubation at 37 °C, the number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Sorcerer Automatic Colony Counter for the scoring of colonies and Ames Study Manager for the data management, Perceptive Instruments Ltd, Bury St Edmunds IP33 3TA, UK). Also, the thinning of the bacterial lawn and the presence of precipitate were evaluated.

Rationale for test conditions:

Using a test item concentration of 200 mg/mL in the vehicle and a treatment volume of 25 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable. Thus, the dose-levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
Since the test item was found freely soluble and non-toxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 μg/plate, according to the criteria specified in the international guidelines.

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.
The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level, and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels, nor any evidence of a dose-response relationship is noted.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

PRELIMINARY TOXICITY TEST
- No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any of the tested dose-levels, towards the three strains used, either with or without S9 mix.

MUTAGENICITY EXPERIMENTS
Experiments without S9 mix:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- A moderate toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 5000 μg/plate in the TA 1535, TA 1537 and TA 100 strains, in both experiments. No noteworthy toxicity was noted at any of the tested dose-levels towards the other strains used.
- The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results met the criteria of a negative response.
Experiments with S9 mix:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- In the first experiment using the direct plate incorporation method, no noteworthy toxicity was noted at any of the tested dose-levels in any of the tested strains.
- In the second experiment using the pre-incubation method, a moderate to strong toxicity (thinning of the bacterial lawn) was noted at 1000 μg/plate in the TA 1537 strain, at 2000 μg/plate in the TA 1535 strain, at dose-levels ≥ 2500 μg/plate in the TA 100 strain, and at 5000 μg/plate in the TA 98 strain, whereas no noteworthy toxicity was noted in the TA 102 strain.
- The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results met the criteria of a negative response.

HISTORICAL CONTROL DATA
- Positive historical control data: 710 ± 133.0, 440 ± 401.6, 155 ± 31.9, 787 ± 108.6 and 1699 ± 291.3 for TA 1535 (NaN3), TA 1537 (9AA), TA 98 (2 NF), TA 100 (NaN3) and TA 102 (MMC) strains, respectively (without S9); 249 ± 63.8, 125 ± 32.5, 1594 ± 275.8, 912 ± 250.5 and 1791 ± 467.5 for TA 1535 (2 AM), TA 1537 (2 AM), TA 98 (2 AM), TA 100 (BaP) and TA 102 (2 AM) strains, respectively (with S9)
- Negative (solvent/vehicle) historical control data: 19 ± 4.3, 8 ± 1.9, 24 ± 4.2, 120 ± 15.1 and 340 ± 61.2 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (without S9); 18 ± 2.7, 12 ± 2.1, 36 ± 5.8, 136 ± 12.3 and 449 ± 89.8 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (with S9)
Note: sodium azide (NAN3); 9-Aminoacridine (9 AA); 2-Nitrofluorene (2 NF); Mitomycin C (MMC); 2-Anthramine (2 AM); Benzo(a)pyrene (BaP)

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, test substance is not mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains.

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 (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to test substance at the following concentrations:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate for TA 98, TA 100 and TA 102 strains with and without S9 mix (plate incorporation method)

Mutagenicity experiments

Experiments without S9 mix: 312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in both mutagenicity experiments (plate incorporation method)

Experiments with S9 mix:

312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in the first experiment (plate incorporation method), and for the TA 98, TA 100 and TA 102 strains in the second experiment (pre-incubation method),

62.5, 125, 250, 500, 1000 and 2000 μg/plate for the TA 1535 strain in the second experiment (pre-incubation method),

31.3, 62.5, 125, 250, 500 and 1000 μg/plate for the TA 1537 strain in the second experiment (pre-incubation method).

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 mix prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

Experiments without S9 mix:

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. A moderate toxicity was noted at the highest dose-level of 5000 μg/plate in the TA 1535, TA 1537 and TA 100 strains, in both experiments. No noteworthy toxicity was noted at any of the tested dose-levels towards the other strains used. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results met the criteria of a negative response.

Experiments with S9 mix:

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. In the first experiment using the direct plate incorporation method, no noteworthy toxicity was noted at any of the tested dose-levels in any of the tested strains. In the second experiment using the pre-incubation method, a moderate to strong toxicity was noted at 1000 μg/plate in the TA 1537 strain, at 2000 μg/plate in the TA 1535 strain, at dose-levels ≥ 2500 μg/plate in the TA 100 strain and at 5000 μg/plate in the TA 98 strain, whereas no noteworthy toxicity was noted in the TA 102 strain. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results met the criteria of a negative response.

 

Under the test conditions, test substance is not mutagenic in this bacterial system.