Absolute of Nicotiana tabacum (Solanaceae) obtained from leaves by organic solvent treatment and subsequent ethanol extraction

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 13 December 2019 to 03 Janvier 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Version 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Absolute of Nicotiana tabacum (Solanaceae) obtained from leaves by organic solvent treatment and subsequent ethanol extraction

EC Number:

951-779-7

Molecular formula:

Not relevant, UVCB substance

IUPAC Name:

Absolute of Nicotiana tabacum (Solanaceae) obtained from leaves by organic solvent treatment and subsequent ethanol extraction

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: room temperature, darkness (Optimal temperature at 20°C)

Method

Target gene:

Histidine gene for Salmonella

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:

Type and composition of metabolic activation system:
- source of S9: obtained from Molecular Toxicology Incorporated, USA. Prepared from male Sprague Dawley rats induced with Phenobarbital-5,6 Benzoflavon.
- method of preparation of S9 mix : Final Content per mL in 10% S-9 mix
Sodium phosphate buffer pH 7.4 100 µmoles
Glucose-6-phosphate (disodium) (G-6-P) 5 µmoles
β-Nicotinamide adenine dinucleotide phosphate
(NADP) (disodium) 4 µmoles
Magnesium chloride (MgCl2) 8 µmoles
Potassium chloride (KCl) 33 µmoles
S-9 100 µL
Water To volume

Molten agar supplements (Final quantity / plate):
L-histidine HCl (in sterile water for irrigation) 0.1 µmole
D-biotin (in sterile water for irrigation) 0.1 µmole

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL 10% S-9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

A maximum concentration of 50 mg/mL was selected in order that initial treatments were performed up to 5000 μg/plate, the maximum recommended concentration according to current regulatory guidelines (OECD, 1997). Two independent experiments and a Repeat Second Experiment were conducted where the maximum dose of 5000 μg/plate was selected for the First Experiment and reduced to 2500 μg/plate, for the Second Experiment plus the Repeat Second Experiment on the basis of the toxicity and the interfering precipitates seen in the First experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A preliminary solubility assay performed at the test facility indicated that Tobacco absolute was soluble in dimethyl sulfoxide (DMSO) at 263.3 mg/mL and insoluble in sterile water. Consequently the chosen vehicle was dimethyl sulfoxide

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

mitomycin C

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:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

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

DURATION
- Preincubation period: 20 min at 37 +/- 1°C
- Exposure duration: 37 +/- 1°C for 72 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 72 hours at about 37 ºC, the number of colonies per plate was counted.
Data are presented as the number of colonies present per plate (mean ± standard deviation). The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The most concentrated test article treatment formulation, vehicle, S-9 mix and buffer solutions will be checked for sterility by plating the same volume used for treatment onto nutrient agar plates.
- Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility.

Rationale for test conditions:

Standard test

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
• A concentration related increase in revertant numbers was ≥2.0-fold (in strains TA98, TA100 or TA102) or ≥3.0-fold (in strains TA1535 or TA1537) over the concurrent vehicle control values.
• Any observed response is reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
The Dunnett’s test was used to confirm result.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

A Dunnett’s test will be used to confirm the results.

Results and discussion

Test results

Test results 1

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

1st exp

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

1st exp

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

2nd exp

Metabolic activation:

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

2nd exp

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

1st exp

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

1st exp

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

2nd exp

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

2nd exp

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 11

Key result

Species / strain:

S. typhimurium TA 102

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:
First exp.: Precipitates interfering with the observation of the bacterial lawn were present at 5000 μg/plate in all the tester strains in both conditions with and without S-9 following incubation. Consequently, this 5000 μg/plate dose level was not scored and not taken into account. Precipitates without interfering the scoring were observed at 1600 μg/plate in all tester strains in both conditions with and without S-9 except in TA102 strain.
Second exp.: Interfering precipitates were present at 2500 μg/plate in the all tester strains in both conditions with and without S-9 following incubation. Consequently, this 2500 μg/plate dose was not scored and not taken into account. Some precipitates were also observed at 1250 μg/plate in the TA1537 strain in the presence of S-9 without impact on the scoring.


STUDY RESULTS
- Concurrent vehicle negative and positive control data:
In the Second Experiment, negative and positive controls failed in the presence of S-9. Control cultures did not grow. Consequently, the assay with S-9 was not valid and was cancelled. The raw data of toxicity from treated cultures were included in the folder but not reported. A Repeat Second Experiment was conducted and met the acceptance criteria.
From the data it can be seen that mean vehicle control counts fell within the laboratory’s historical ranges. It can be noted an individual negative control plate count was higher than but close to the historical negative control data in the First Experiment without S-9 in the TA1535 strain and with S-9 in the TA98 strain and in particular in the TA1535 strain (three individual plate counts and the mean count); in the Second Experiment without S-9 in the TA1535 and TA102 strains. However, these individual values were largely lower than the minimum historical positive control data and the mean counts were into the historical negative data. Accordingly, results were acceptable.
Regarding the TA1535 strain with S-9 (First Experiment), the mean count was close to the maximum of the historical negative control data (31 vs 29). This result was considered acceptable.
The positive control chemicals all induced increases in revertant numbers of ≥2.0-fold (in strains TA98, TA100 or TA102) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. Some individual positive control plate counts were lower than but close to the historical negative control data without S-9 in the TA100 strain (one plate – First Experiment) and in the TA98 strain (one plate – Second Experiment). However, there were largely higher than the 2-fold rule and the mean counts met the acceptance criteria. Some positive control plate counts were higher than the historical positive control data as follows: in the First Experiment with S-9, TA98 (one plate) TA100, TA1535 and TA1537 strains (3 plates and mean count); in the TA102 strain with and without S-9 (2 or 3 plates respectively and mean counts) and in the Second Experiment without S-9 in the TA102 strain (one plate). It showed the responsiveness of the strain. Consequently, increases were considered acceptable.
The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

- Signs of toxicity:
First experiment:
Treatments of all the tester strains were performed in the absence and in the presence of S-9 using plate incorporation method at final concentrations of Tobacco absolute at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus negative (vehicle) and positive controls.
Following these treatments, evidence of toxicity in the form of a reduction or an absence of the background bacterial lawn with presence of microcolonies or presence of microcolonies without absence of the background bacterial lawn and/or a diminution of the revertants number was observed in the absence of S-9 from 500 μg/plate (TA100, TA1535 and TA1537 strains) and in the presence of S-9, from 500 μg/plate (TA1535 strain) and at 1600 μg/plate (TA1537 on 1 plate and TA100 strains). No evidence of toxicity was present in the TA98 and TA102 strains in both conditions with and without S-9, plus in the TA1537 strain (2 plates out of 3) with S-9.
Second experiment:
Second experiment treatments of all the tester strains were performed in the absenceof S-9 using plate incorporation method and in the presence of S-9 using the pre-incubation method.
The maximum tested dose was reduced to 2500 μg/plate as interfering precipitates did not allow to analyse bacterial lawn at 5000 μg/plate in the First Experiment.
Narrowed dose intervals were employed covering the range 80-2500 μg/plate for TA98 and TA102 strains in both conditions with and without S-9 and for the TA1537 strain with S-9. The dose range was extended to 20 -2500 μg/plate for TA100 and TA1535 strains in both conditions with and without S-9 and for TA1537 strain without S-9 to take into account the observed toxicity in the First Experiment to obtain sufficient analysable doses. These narrowed dose intervals were employed in order to examine more closely those concentrations of Tobacco absolute approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system.
Following these treatments, evidence of toxicity in the form of a reduction and/or a diminution of the revertants number was observed in the absence of S-9 from 625 μg/plate (TA1535 and TA1537 strain), at 1250 μg/plate (TA100 strain with slight toxic effect – ratio value: 0.6) and in the presence of S-9 from 625 μg/plate (TA100 strain) and at 1250 μg/plate (TA1535 and TA1537 strains). No evidence of toxicity was present in TA98 and TA102 strain in both conditions with and without S-9.
It can be noticed that the positive and negative controls of the Second Experiment with S-9, did not met the acceptance criteria and results were not reported. Consequently, results in the presence of S-9 came from a Repeat Second Experiment.

-Mutagenicity:
Following Tobacco absolute treatments of all the tester strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥2.0-fold (in TA98, TA100 or TA102 strains) or ≥3.0-fold (in TA1535 or TA1537 strains) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Tobacco absolute mutagenic activity in this assay system.
These results were confirmed using the Dunnett’s test where no statistically significant test was obtained when the data were analysed at the 1% level except in the First Experiment in the TA1537 strain without S-9 (16 μg/plate) and in the Repeat Second Experiment in the TA100 strain (160 and 300 μg/plate). However, this statistically significance was not considered as clear evidence of mutagenic activity but a biological variability in this assay system as it was below the 3/2-fold rule respectively.

- Individual plate counts
: cf attached document
- Mean number of revertant colonies per plate and standard deviation
: cf. arrached document

Applicant's summary and conclusion

Conclusions:

It was concluded that Tobacco absolute did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, the test item was assessed for mutation in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), both in the absence and in the presence of metabolic activation by a Phenobarbital-5,6 Benzoflavon-induced rat liver post-mitochondrial fraction (S-9), in two independent experiments.
All Tobacco absolute treatments in this study were performed using formulations prepared in dimethyl sulfoxyde (DMSO), and all concentrations are expressed in terms of pure compound considering Tobacco absolute in its entirety liquid form.
Precipitation was observed at the time of treatments from 1600 μg/plate (First Experiment) and from 1250 μg/plate (Second Experiment).

Toxicity:

In the first experiment, treatments of all the tester strains were performed in the absence and in the presence of S-9 using plate incorporation method at final concentrations of Tobacco absolute at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus negative (vehicle) and positive controls.
Precipitates interfering with the observation of the bacterial lawn were present at 5000 μg/plate in all the tester strains in both conditions with and without S-9 following incubation. Consequently, this 5000 μg/plate dose level was not scored and not taken into account. Precipitates without interfering the scoring were observed at 1600 μg/plate in all tester strains in both conditions with and without S-9 except in TA102 strain.
Following these treatments, evidence of toxicity in the form of a reduction or an absence of the background bacterial lawn with presence of microcolonies or presence of microcolonies without absence of the background bacterial lawn and/or a diminution of the revertants number was observed in the absence of S-9 from 500 μg/plate (TA100, TA1535 and TA1537 strains) and in the presence of S-9, from 500 μg/plate (TA1535 strain) and at 1600 μg/plate (TA1537 on 1 plate and TA100 strains). No evidence of toxicity was present in the TA98 and TA102 strains in both conditions with and without S-9, plus in the TA1537 strain (2 plates out of 3) with S-9.

In the second experiment, treatments of all the tester strains were performed in the absence of S-9 using plate incorporation method and in the presence of S-9 using the pre-incubation method.

The maximum tested dose was reduced to 2500 μg/plate as interfering precipitates did not allow to analyse bacterial lawn at 5000 μg/plate in the First Experiment.
Narrowed dose intervals were employed covering the range 80-2500 μg/plate for TA98 and TA102 strains in both conditions with and without S-9 and for the TA1537 strain with S-9. The dose range was extended to 20 -2500 μg/plate for TA100 and TA1535 strains in both conditions with and without S-9 and for TA1537 strain without S-9 to take into account the observed toxicity in the First Experiment to obtain sufficient analysable doses. These narrowed dose intervals were employed in order to examine more closely those concentrations of Tobacco absolute approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system.
Interfering precipitates were present at 2500 μg/plate in the all tester strains in both conditions with and without S-9 following incubation. Consequently, this 2500 μg/plate dose was not scored and not taken into account. Some precipitates were also observed at 1250 μg/plate in the TA1537 strain in the presence of S-9 without impact on the bacterial lawn observation.
Following these treatments, evidence of toxicity in the form of a reduction and/or a diminution of the revertants number was observed in the absence of S-9 from 625 μg/plate (TA1535 and TA1537 strain), at 1250 μg/plate (TA100 strain with slight toxic effect – ratio value: 0.6) and in the presence of S-9 from 625 μg/plate (TA100 strain) and at 1250 μg/plate (TA1535 and TA1537 strains). No evidence of toxicity was present in TA98 and TA102 strain in both conditions with and without S-9.

Controls:

Negative (vehicle) and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were elevated by positive control treatments. These results were confirmed using the Dunnett’s test where no statistically significant test was obtained when the data were analysed at the 1% level except in the First Experiment in the TA1537 strain without S-9 (16 μg/plate) and in the Repeat Second Experiment in the TA100 strain (160 and 300 μg/plate). However, this statistically significance was not considered as clear evidence of mutagenic activity but a biological variability in this assay system as it was below the 3/2-fold rule respectively.

Mutation:

Following Tobacco absolute treatments of all the tester strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥2.0-fold (in strains TA98, TA100 or TA102) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Tobacco absolute mutagenic activity in this assay system.

 

It was concluded that Tobacco absolute did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments using plate incorporation and pre-incubation method. The maximum dose levels were 5000 μg/plate and 2500 μg/plate, these doses were interfering doses with the scoring. Consequently, the maximum analyzed dose level was 1600 μg/plate a precipitating and cytotoxic dose, in the absence and in the presence of a rat liver metabolic activation system (S-9) in accordance with the current regulatory guidelines.