Essential oil of Citrus sinensis (Rutaceae) peel, terpene-free

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:

From 01 July to 06 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 1997 and corrected 26 June 2020

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 2019-07-11.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Citrus sinensis (Rutaceae) peel, terpene-free

EC Number:

951-680-9

IUPAC Name:

Essential oil of Citrus sinensis (Rutaceae) peel, terpene-free

Test material form:

liquid

Details on test material:

Batch No. 0000448007
CAS No. not applicable
EC No. 951-680-9
Form: liquid
Colour: orange-yellow to green-yellow
Date received: 25 May 2020
Production date: 13 January 2020
Expiry date: 12 January 2021
Purity: 100% UVCB
Storage: fridge (6°C+/-3°C), darkness

Method

Target gene:

Histidine and tryptophan.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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% v/v S9 fraction; S9 fraction was prepared from liver homogenates of Sprague Dawley rat induced with Aroclor 1254

Test concentrations with justification for top dose:

First assay: 5, 15, 50, 150 and 500 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Second assay: 5, 15, 50, 150 and 500 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) without S9 and with S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Absolute Ethanol
- Justification for choice of solvent/vehicle: The test item was found soluble in Absolute Ethanol. A stock solution for each assay was prepared at 10 mg/mL in Absolute Ethanol. The highest dose tested was 500 μg/plate because a high toxicity was showed from 1500 to 5000 µg/plate.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: cis-Platinum (II) Diammine Dichloride, 1 µg/plate for Escherichia coli cis WP2 (uvr A-) (pKM101).

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine: 2 and 1 µg/plate for TA98, TA100, TA1537 and TA1535 without and with preincubation respectively. Dimethyl-benzanthracene: 5 and 2.5 µg/plate for Escherichia coli WP2 (uvr A-) (pKM101) without and with preincubation respectively.

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains of Salmonella typhimurium and Escherichia coli were purchased from MOLTOX (TM) and maintained at 2-8°C (lyophilized discs culture).
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period (assay with S9): the test substance was preincubated with the test strain, and 500 μL of S9-mix fraction for 30 min at 37° C prior to mixing with the overlay agar and pouring onto the surface of the minimal agar plate.
- Exposure duration: Plates were incubated at 37 °C over a 48-72 hour period.

NUMBER OF REPLICATIONS: 3 plates/dose for all groups

DETERMINATION OF CYTOTOXICITY
- Method: In a test tube, 0.1 mL of the bacterial suspension and 0.1 mL of the stock solution and dilutions were successively added to 2 mL of top agar at 45°c, containing 10% (v/v) of a solution of L-histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate containing minimal agar (20 mL). 3 plates per concentration were incubated for 24-72 hours at 37°c, and the colonies counted. A negative control containing the blank alone was run in parallel. In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75% or less.

OTHER
- Checking strains: The genotype of bacterial strains was checked for Histidine and tryptophan requirements; Loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains; Ampicillin resistance for the strains which have the pKM 101 plasmide; Δuvr B mutation i.e. U.V.B sensitivity for Salmonella typhimurium and Δuvr A mutation i.e. U.V.A sensitivity for Escherichia coli; Spontaneous revertant rate.

- Sterility tests: The test item and the corresponding dilutions were added to 2 mL of top agar maintained at 45°c and poured agter homogenization on the bottom agar (20 mL) onto a Petri plate (n=3). Plates were incubated for 48-72 hours at 37°c and then examined. There should be no bacterial growth on any plate. S9-mix sterility was checked using the same protocol.

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains with and/or without metabolic activation.

The validity criteria are as follows :
- bacteriostatic activity of the highest concentration shall be equal to or less than 75 %, - the spontaneous reversion rate of the absolute negative control shall comply with the laboratory’s historical control data.
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with laboratory’s historical control data.
- negative and positive values should not show significant difference with the historical values of the laboratory.

The result of the test is considered positive if a dose-response relationship concentration is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into account for a given dilution of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.

Statistics:

Data are presented as the number of revertant colonies (mean +/- standard deviation) per plate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 at 5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 at 5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 at 5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 at 5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 at 5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST
Results show a high toxicity from 1500 to 5000 µg/plates and a toxicity in presence of the test item 500 µg/plat of 72 and 67% which is compatible with the maximum tolerated level of 75%. Therefore, the test item was tested at 5, 15, 50, 150 and 500 µg/plate.


MUTAGENICITY TEST:
- No significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (with and without S9), and the mean of corresponding historical values obtained in the laboratory.
- No evidence of any increase in the number of revertant colonies in the presence of the test item stock solutions and dilutions with and without S9 in all strains in both experiments.
- Evidence of toxicity, demonstrated by a thinning of the background lawn of non-revertant bacteria, was shown at 500 µg/plate in all strains in absence and presence of S9 and using the pre-incubation method.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

OTHERS:
- Sterility test showed absence of any bacterial growth in the presence of the various concentrations of the test item and in the presence of S9-mix.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test condition, doses (5, 15, 50, 150 and 500 µg/plate), prepared from the test item did not induce an increase in revertant colonies of all strains without or with metabolic activation, according to the OECD guideline No. 471.
Therefore, the test material is not mutagenic in the presence and absence of metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 (uvrA-)(pKM101) strains.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test item diluted in DMSO at the following concentrations, 5, 15, 50, 150 and 500 µg/plate, both in the presence and absence of metabolic activation system (10% v/v S9).

 

Negative and positive control groups were also included in mutagenicity tests and showed absolute numbers of revertant colonies comparable to historical data. 

 

Results show a high toxicity from 1500 to 5000 µg/plates and a toxicity in presence of the test item 500 µg/plat of 72 and 67% which is compatible with the maximum tolerated level of 75%. Therefore, the test item was tested at 5, 15, 50, 150 and 500 µg/plate.

There was no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (with and without S9), and the mean of corresponding historical values obtained in the laboratory. No evidence of any increase in the number of revertant colonies in the presence of the test item stock solutions and dilutions with and without S9 in all strains in both experiments. Then, evidence of toxicity, demonstrated by a thinning of the background lawn of non-revertant bacteria, was shown at 500 µg/plate in all strains in absence and presence of S9 and using the pre-incubation method.

 

Under the test condition, doses (5, 15, 50, 150 and 500 µg/plate), prepared from the test item did not induce an increase in revertant colonies of all strains without or with metabolic activation, according to the OECD guideline No. 471.
Therefore, the test material is not mutagenic in the presence and absence of metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 (uvrA-)(pKM101) strains.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.