Mentha citrata, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

The test chemical did not induce chromosomal aberration in the cell lines used in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA1535 and TA102

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The liver microsome fraction (S-9) was prepared from the liver of Fischer rats

Test concentrations with justification for top dose:

1. 6 different concentrations were used; 1.0 mg/plate was the maximum concentration
2. 0, 5, 15, 50, 150 and 500 μg/plate or tube

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: Without S9: ICR191 (TA97), and and 2-aminoanthracene (all strains) With activation: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: Plate incorporation and preincubation
Initial number of cells: (0.37-1.65E08)

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

1. No data

Evaluation criteria:

1. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).
2. The plates were observed for an increase in the number of revertants/plate

Statistics:

1. No data

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Remarks:

1.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA1535 and TA102

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity in the plate incorporation assay at ≥ 500 μg/plate, in the preincubation test ≥ 50 μg/plate (without S9) and ≥ 100 μg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Toxicity experiments were performed to determine the doses for the main study

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of Mentha citrata, ext. The studies are as mentioned below:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 1.0 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemica did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Another gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the plate incorporation and preincubation assay at dose upto 0, 5, 15, 50, 150 and 500 μg/plate or tube. The chemical was dissolved in DMSO. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce an increase in the number of revertant colonies over the control using S. typhimurium strains TA97, TA98, TA100, TA1535 and TA102 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar test chemicals

GLP compliance:

not specified

Type of assay:

other: Chromosome aberration assay

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum
Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
- Properly maintained: yes by 4 day passages
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

lymphocytes: Human

Remarks:

2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

1. At three different doses with 0.25 mg/mL being the maximum dose concentration
2. 10-180 μg/ml
3 hrs: 33, 100, 130 μg/ml
24 hrs: 33, 100, 130 μg/ml
48 hrs: 56, 100, 130 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

Untreated negative controls:

yes

Remarks:

Untreated cells served as negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 24 and 48 hrs
- Expression time (cells in growth medium): 24 and 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data

- Exposure duration:
-S9: 3 h exposure + 24 h fixation
24 and 48 h exposure + 24 and 48 fixation
+S9: 3 h exposure + 24 h fixation
3 h exposure + 48 h fixation

- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells):
-S9: 24 h fixation
24 and 48 h exposure: 24 and 48 fixation
+S9: 24 h fixation
3 h exposure : 48 h fixation

SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.

2. The cells were observed for aberrations

Statistics:

No data

Species / strain:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

1

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Species / strain:

lymphocytes: Human

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The maximum dose of each sample was selected
by a preliminary test in which the dose needed
for 50% cell-growth inhibition was estimated using a cell densitometer

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The doses for the main study were based on precipitation

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosomal aberration in the cell lines used in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of Mentha citrata, ext. The studies are as mentioned below:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.25 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

The study was performed to evaluate the ability of linalyl acetate to induce chromosome aberrrations in cultured peripheral human lymphocytes. The test chemical was dissolved in DMSO and used at dose levels of 10-180 μg/ml. The study was performed in the presence and absence of S9 metabolic activation system and for a short duration of 3 hrs and continuous duration of 24 and 48 hrs. The 3.5% aberrations seen at the lowest examined dose (48 h treatment, 48 h fixation) was statistically significant. Since no increase was seen at the higher concentrations, it was considered to be without biological significance. Based on the observations made, the test chemical linalyl acetate did not induce chromosomal aberration in Human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, test chemical did not induce chromosomal aberration in the cell lines used in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the test chemicals was reviewed to determine the mutagenic nature of Mentha citrata, ext. The studies are as mentioned below:

Ames test:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 1.0 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemica did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Another gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the plate incorporation and preincubation assay at dose upto 0, 5, 15, 50, 150 and 500 μg/plate or tube. The chemical was dissolved in DMSO. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce an increase in the number of revertant colonies over the control using S. typhimurium strains TA97, TA98, TA100, TA1535 and TA102 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.25 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

The study was performed to evaluate the ability of linalyl acetate to induce chromosome aberrrations in cultured peripheral human lymphocytes. The test chemical was dissolved in DMSO and used at dose levels of 10-180 μg/ml. The study was performed in the presence and absence of S9 metabolic activation system and for a short duration of 3 hrs and continuous duration of 24 and 48 hrs. The 3.5% aberrations seen at the lowest examined dose (48 h treatment, 48 h fixation) was statistically significant. Since no increase was seen at the higher concentrations, it was considered to be without biological significance. Based on the observations made, the test chemical linalyl acetate did not induce chromosomal aberration in Human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, test chemical did not induce chromosomal aberration in the cell lines used in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available, the test chemical Mentha citrata, ext does not exhibit gene mutation in vitro. Hence the test chemical is not likely to to be mutagenic as per the criteria mentioned in CLP regulation.