Absolute of Dipteryx odorata (Fabaceae) obtained from beans by organic solvents treatment and subsequent ethanol extraction

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test (OECD 471, K, Rel 1): Not mutagenic.

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:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Version 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene for Salmonella

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:

5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: soluble in dimethyl sulfoxide (DMSO) at 108.5 mg/mL

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

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°C
- Exposure duration: 37°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 using limit concentration

Evaluation criteria:

For valid data, the test article will be considered to be mutagenic in this assay if:
1. A concentration related increase in revertant numbers is 2.0-fold (in strains TA98, TA100 or TA102) or 3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response is reproducible under the same treatment conditions

The test article will be regarded positive in this assay if both of the above criteria are met.
The test article will be regarded negative in this assay if neither of the above criteria are met.
A Dunnett’s test will be used to confirm results.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.

Statistics:

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 1535

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

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

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
First exp.: Precipitates were observed on the TA1535 strain without S-9 from 1600 µg/plate and at 5000 µg/plate on all other tester plates following incubation, in both conditions in the presence and the absence of S-9.
Second exp.: Precipitates were observed on all strains in both conditions with and without S-9 from 2500 µg/plate following incubation except in the TA102 strain with S-9 where it was only observed at 5000 µg/plate. However, precipitates interfered with the revertant colonies counting, at 5000 µg/plate in the TA98, TA1535 without S-9 and TA102 strains in both conditions with and without S-9. Consequently, this 5000 µg/plate dose was not scored and not taken into account for these strains and conditions with interfering precipitates.


STUDY RESULTS
- Concurrent vehicle negative and positive control data:
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 (3/2 plates respectively and mean counts) and in the Second Experiment in the TA102 strain without S-9 (1 plate and the mean count). It showed the responsiveness of the strain. Consequently, increases were considered acceptable.
- 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 Tonka bean 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 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), from 1600 µg/plate (TA98 strain) and in the presence of S-9, from 1600 µg/plate (TA100, TA1535 and TA1537 strains) and at 5000 µg/plate in the TA98. No evidence of toxicity was present in the TA102 strain in both conditions with and without S-9.
Second experiment:
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 preincubation method.
The maximum tested dose of 5000 µg/plate of Tonka Bean Absolute was retained for all strains in both conditions the absence and the presence of metabolic activation based on the results from the First Experiment where the observed toxicity differed from no toxicity to moderate or a marked toxicity.
Narrowed dose intervals were employed covering the range 160 – 5000 µg/plate for the TA98 strain with S-9 and TA102 strain in both conditions with and without S-9, plus 80 µg/plate for TA98 strain without S-9. The dose range was extended to 40 - 5000 µg/plate for TA100 and TA1537 strains in both conditions with and without S-9 and for TA1535 strain with S-9 and to 20-5000 µg/plate for the TA1535 strain without S-9 to take into account the observed toxicity in the First Experiment. These narrowed dose intervals were employed in order to examine more closely those concentrations of Tonka bean 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 a toxicity was also observed in the form of a reduction or an absence of the background bacterial lawn with presence of microcolonies and/or diminution of the revertants number, in the absence of S-9, from 160 µg/plate (TA1537 strain), from 625 µg/plate (TA100 strain), from 2500 µg/plate (TA98 and TA1535 strains); in the presence of S-9 from 1250 µg/plate (TA1537 strain), from 2500 µg/plate (TA100 and TA1535 strains) and at 5000 µg/plate (TA98 strain). No evidence of toxicity was present in the TA102 strain in both conditions with and without S-9.
Due to the marked toxicity only four doses were analysable out of the five recommended doses in the TA1537 strains in the absence of S-9. In the presence of S-9, results came from a Repeat Second Experiment as the negative and positive control were not valid in the Second Experiment.

-Mutagenicity:
Regarding the TA1537 strain in the absence of S-9, the analysable doses of the Second Experiment, overlapped and were complemented by the results of the First Experiment in a way that the obtained data were relevant and sufficient to conclude the study. Following Tonka bean 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 Tonka bean 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 Second Experiment in the TA98 strain with S-9 (1250 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 2-fold.
This study was considered therefore to have provided no evidence of any Tonka bean absolute (batch number 2951074) mutagenic activity in this assay system.

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

Conclusions:

It was concluded that Tonka bean 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, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and TA 102 were exposed the test material in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

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 Tonka bean 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 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), from 1600 µg/plate (TA98 strain) and in the presence of S-9, from 1600 µg/plate (TA100, TA1535 and TA1537 strains) and at 5000 µg/plate in the TA98. No evidence of toxicity was present in the TA102 strain in both conditions with and without S-9.

Precipitates were observed on the TA1535 strain without S-9 from 1600 µg/plate and at 5000 µg/plate on all other tester plates following incubation, in both conditions in the presence and the absence of 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 preincubation method.

The maximum tested dose of 5000 µg/plate of Tonka bean absolute was retained for all strains in both conditions the absence and the presence of metabolic activation based on the results from the First Experiment where the observed toxicity differed from no toxicity to moderate or a marked toxicity.

Narrowed dose intervals were employed covering the range 160 – 5000 µg/plate for the TA98 strain with S-9 and TA102 strain in both conditions with and without S-9, plus 80 µg/plate for TA98 strain without S-9. The dose range was extended to 40 - 5000 µg/plate for TA100 and TA1537 strains in both conditions with and without S-9 and for TA1535 strain with S-9 and to 20-5000 µg/plate for the TA1535 strain without S-9 to take into account the observed toxicity in the First Experiment. These narrowed dose intervals were employed in order to examine more closely those concentrations of Tonka bean 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.

Precipitates were observed on all strains in both conditions with and without S-9 from 2500 µg/plate following incubation except in the TA102 strain with S-9 where it was only observed at 5000 µg/plate. However, precipitates interfered with the revertant colonies counting, at 5000 µg/plate in the TA98, TA1535 without S-9 and TA102 strains in both conditions with and without S-9. Consequently, this 5000 µg/plate dose was not scored and not taken into account for these strains and conditions with interfering precipitates.

Following these treatments, evidence of a toxicity was also observed in the form of a reduction or an absence of the background bacterial lawn with presence of microcolonies and/or diminution of the revertants number, in the absence of S-9, from 160 µg/plate (TA1537 strain), from 625 µg/plate (TA100 strain), from 2500 µg/plate (TA98 and TA1535 strains); in the presence of S-9 from 1250 µg/plate (TA1537 strain), from 2500 µg/plate (TA100 and TA1535 strains) and at 5000 µg/plate (TA98 strain). No evidence of toxicity was present in the TA102 strain in both conditions with and without S-9.

Due to the marked toxicity only four doses were analysable out of the five recommended doses in the TA1537 strains in the absence of 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.

 

Mutation:

Regarding the TA1537 strain in the absence of S-9, the analysable doses of the Second Experiment, overlapped and were complemented by the results of the First Experiment in a way that the obtained data were relevant and sufficient to conclude the study.

Following Tonka bean 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 Tonka bean 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 Second Experiment in the TA98 strain with S-9 (1250 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 2-fold.

This study was considered therefore to have provided no evidence of any Tonka bean absolute (batch number 2951074) mutagenic activity in this assay system.

 

It was concluded that Tonka bean 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 at dose up to 5000 µg/plate, the maximum recommended dose according to current regulatory guidelines also a precipitating and cytotoxic dose, in the absence and in the presence of a rat liver metabolic activation system (S-9) with precipitates from 2500 µg/plate with and without S-9 and cytotoxic effect from 300 to 5000 µg/plate depending on the strain.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   GenEvolution, 2020	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, TA102	-S9 +S9	Up to limit concentration	-S9 : non mutagenic +S9 : non mutagenic

 

Gene mutation Assay (Test n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self classification:

Based on the available data (Ames test), no self- classification is proposed regarding genetic toxicity according to the CLP and to the GHS.