Cardamom, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two key studies were available:

- Ames test (OECD 471): non mutagenic (LEMI, 2020, GLP, K, rel. 1).

- Ames test (OECD 471): non mutagenic (Brusick, 1982, GLP, K, rel. 1).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 August to 01 October 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.

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

Target gene:

Histidine and tryptophan.

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, 500 and 1500 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, 500 and 1500 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: DMSO
- Justification for choice of solvent/vehicle: The test item was found soluble in DMSO. A stock solution for each assay was prepared at 100 mg/mL in DMSO. The highest dose tested was 1500 μg/plate because a high toxicity was showed from 1500 to 5000 µg/plate.

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

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.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 to 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 58 and 61% which is compatible with the maximum tolerated level of 75%. A supplementary dose was studied at 1500 µg/plate. Therefore, the test item was tested at 5, 15, 50, 150, 500 and 1500 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 1500 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.

None

Conclusions:

Under the test condition, in presence of doses (5, 15, 50, 150, 500 and 1500 µg/plate), prepared from the test item Cardamom oil, the numbers of revertant colonies in all cases did not exceed the criteria for a positive response for 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, 500 and 1500 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 58 and 61% which is compatible with the maximum tolerated level of 75%. A supplementary dose was studied at 1500 µg/plate. Therefore, the test item was tested at 5, 15, 50, 150, 500 and 1500 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 1500 µg/plate in all strains in absence and presence of S9 and using the pre-incubation method.

 

Under the test condition, in presence of doses (5, 15, 50, 150, 500 and 1500 µg/plate), prepared from the test item Cardamom oil, the numbers of revertant colonies in all cases did not exceed the criteria for a positive response for 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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 June - 12 July 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

minor deviations: one strain is missing.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

one strain is missing

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): B9
- Physical state: Clear colorless liquid
- Date of receipt: 21 June 1982

Target gene:

No data

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.02, 0.04, 0.07, 0.15, 0.29, 0.59, 1.17, 2.34, 4.69, 9.38, 18.75, 37.50, 75.00 and 150.00 µL/plate, without S9 mix in TA100 strain
Main test: 0.04, 0.07, 0.15, 0.29, 0.59, 1.17 and 2.34 µL/plate, with and without S9 mix in all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains of S. typhimurium were obtained from Dr. Bruce Ames, University of California, Berkeley

METHOD OF APPLICATION: In agar (direct plate incorporation)

DURATION
- Incubation period: Plates were incubated at 37 °C for approximately 48 h.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: 2 plates/dose for vehicle control
- Main test: 3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects of the test substance were detected by a substantial reduction in revertant colony counts, decreased or absence of background bacterial lawn.

Evaluation criteria:

- If the solvent control value is within the normal range, a test material producing a positive response equal to three times (TA1535, TA1537 and TA1538) and two times (TA98 and TA100) the solvent control value is considered mutagenic.
- The following normal range of revertants for solvent controls are generally considered acceptable: TA1535: 8-30; TA1537: 4-30; TA1538: 10-35; TA98 : 20- 75; TA100 : 80-250
- Dose-Response phenomena: The demonstration of dose-related increases in mutagen counts is an important criterion in establishing mutagenicity. Since we employ several doses in the actual assay, a dose response would normally be seen with a mutagenic test material. Additional tests may be performed at a narrower dose, if the mutagenic test material fails to exhibit a dose-response in the initial assay. However, occasionally it is difficult to generate a dose-response and the test material will be evaluated based on the available data.
- Reproducibility: If a test material produces a response in a single test which cannot be reproduced in additional runs, the initial positive test data lose significance.
- Control tests: Positive and negative control assays are conducted with each experiment and consist of direct-acting mutagens for nonactivation assays and mutagens requiring metabolic biotransformation in activation assays. Negative controls consist of the test material solvent in the overlay agar together with the other essential components. The negative control plate for each strain gives a reference point to which the test data is compared. The positive control assays is conducted to demonstrate that the test systems are functional with known mutagens.

Statistics:

- No statistical analysis was performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 2.34 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at and above 2.34 µL/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 2.34 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 2.34 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 2.34 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST:
- Toxicity was observed at 2.34 µL/plate and higher doses.

Conclusions:

Interpretation of results
negative with metabolic activation
negative without metabolic activation
Conclusions
Under the test conditions, test item, B9 is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) in the presence or absence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to test item, B9 at the following concentrations:

Preliminary toxicity test: 0.02, 0.04, 0.07, 0.15, 0.29, 0.59, 1.17, 2.34, 4.69, 9.38, 18.75, 37.50, 75.00 and 150.00 µL/plate, without S9 mix in TA100 strain (14 doses)

Main test: 0.04, 0.07, 0.15, 0.29, 0.59, 1.17 and 2.34 µL/plate, with and without S9 mix in all strains

 

Metabolic activation system used in this test was10 % S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

Toxicity was observed at 2.34 µL/plate and higher doses. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at any concentration tested in either the presence or absence of S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Under the test conditions, test item, B9 is not considered as mutagenic in this bacterial system.

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 test

 

Test n°	Test / GuidelineReliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 LEMI, 2020	Ames Test(OECD 471)K, rel. 1	Gene mutation	S. Typhimurium TA 1535,TA 1537,TA 98,TA 100 and E.coli WP (uvrA-)	- S9+ S9	5, 15, 50, 150, 500 and 1500 µg/plateUp to cytotoxic concentration	-S9 : non mutagenic+S9 : non mutagenic
2 Brusick, 1982	Ames Test(OECD 471)K, rel. 1	Gene mutation	TA 1535,TA 1537,TA 98,TA 100,TA 1538	-S9+S9	Up to cytotoxic concentration	-S9 : non mutagenic+S9 : non mutagenic
3 Heck, 1989	Ames Test(OECD 471)K, rel. 4	Gene mutation	TA 1535TA 1537TA 98,TA 100TA 1538	-S9+S9	2340 µg/plate	-S9 : non mutagenic+S9 non mutagenic

 

Two key studies on bacterial gene mutation assay were performed:

- No. 1 - AMES test (LEMI, 2020)

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, 500 and 1500 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 58 and 61% which is compatible with the maximum tolerated level of 75%. A supplementary dose was studied at 1500 µg/plate. Therefore, the test item was tested at 5, 15, 50, 150, 500 and 1500 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 1500 µg/plate in all strains in absence and presence of S9 and using the pre-incubation method.

Under the test condition, in presence of doses (5, 15, 50, 150, 500 and 1500 µg/plate), prepared from the test item Cardamom oil, the numbers of revertant colonies in all cases did not exceed the criteria for a positive response for 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.

 

- No.2 - AMES test (Brusick, 1982)

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

 

The same conclusion was obtained in Test No.3 on Cardamom oil. However the results are from a poorly detailed publication, which cannot be considered as a key study since only one concentration was used and positive controls were not reported.

Justification for classification or non-classification

Harmonized classification:

Cardamom oil has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no additional classification is proposed.