Tagetes minuta, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic ( OECD 471, GLP, rel. 1).

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:

20 October to 4 November 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 471 Guideline without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 26 January 2010 / signed on 22 March 2010)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
-Lot/batch No.of test material: X34121
- Appearance: Yellow-orange liquid
- Date received: 6 October 2010
- Expiration date of the lot/batch: February 2012
- Purity test date: 22 February 2010

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark

Target gene:

His+ for S. typhimurium; trp+ for E. coli

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (10% v/v – test 1; 20% v/v – test 2); S9 fraction prepared from liver homogenates of rats induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

First test (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and Escherichia coli, strain WP2 uvrA (pKM101)
Second test (plate incorporation method): 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and Escherichia coli, strain WP2 uvrA (pKM101)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The Sponsor indicated that test substance was insoluble in water. Its solubility was assessed at 50 mg/mL in DMSO and in ethanol. It was found to be insufficiently soluble in DMSO, but it was found to be soluble in ethanol. Ethanol (analytical reagent grade) was, therefore, used as the vehicle for this study.
The highest concentration of test substance in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with ethanol to produce a series of lower concentrations, separated by approximately half-log10 intervals.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: The strains of S. typhimurium were obtained from the National Collection of Type Cultures, London, England. The strain of E. coli was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland.

METHOD OF APPLICATION: In agar (direct plate incorporation method)

DURATION
- Exposure duration: All plates were incubated at approximately 37 °C for 72 h

NUMBER OF REPLICATIONS:
-3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn.

OTHER: After 72 h of incubation at 37 °C, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

Evaluation criteria:

- If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.

Statistics:

Statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test substance was insoluble in water.
- Precipitation: None

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.
- Positive historical control data: 367-2440 (937 ± 252), 193-1964 (1075 ± 283), 540-5221 (2319 ± 840), 114-792 (302 ± 108), 121-1917 (662 ± 290) for TA 100, TA 1535, WP2 uvr A, TA 98 and TA 1537 (without metabolic activation), respectively; 424-4910 (2257 ± 805), 106-994 (321 ± 102), 269-1286 (550 ± 148), 103-652 (258 ± 72), 76-766 (174 ± 67) for TA 100, TA 1535, WP2 uvr A, TA 98 and TA 1537 (with metabolic activation), respectively.
- Negative (solvent/vehicle) historical control data: 115-202 (163 ± 20), 16-28 (22 ± 3), 109-217 (165 ± 32), 33-55 (41 ± 5), 8-19 (13 ± 3) for TA 100, TA 1535, WP2 uvr A, TA 98 and TA 1537 (without metabolic activation), respectively; 145-220 (182 ± 17), 15-31 (24 ± 4), 110-222 (173 ± 34), 42-71 (54 ± 6), 21-42 (30 ± 4) for TA 100, TA 1535, WP2 uvr A, TA 98 and TA 1537 (with metabolic activation), respectively.

MUTAGENICITY TESTS
First test:
- No evidence of toxicity was obtained following exposure to test substance. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.
Second test
- No evidence of toxicity was obtained following exposure to test substance.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

OTHERS:
- The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.
- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.

None

Conclusions:

Under the test conditions, Tagetes oil is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to Tagetes oil at the following concentrations:

 

First test (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and Escherichia coli, strain WP2 uvrA (pKM101)

Second test (plate incorporation method): 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and Escherichia coli, strain WP2 uvrA (pKM101)

 

Metabolic activation system used in this test was 10% v/v – test 1; 20% v/v – test 2; S9 fraction prepared from liver homogenates of rats induced with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

No signs of toxicity were observed towards the tester strains in either mutation test following exposure to test substance. No evidence of mutagenic activity was seen at any concentration of test substance in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

 

Under the test conditions, Tagetes oil is not considered as mutagenic in these bacterial systems.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to Tagetes oil at the following concentrations:

 

First test (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix inSalmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, andEscherichia coli, strain WP2 uvrA (pKM101)

Second test (plate incorporation method): 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix inSalmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, andEscherichia coli, strain WP2 uvrA (pKM101)

 

Metabolic activation system used in this test was 10% v/v – test 1; 20% v/v – test 2; S9 fraction prepared from liver homogenates of rats induced with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

No signs of toxicity were observed towards the tester strains in either mutation test following exposure to test substance. No evidence of mutagenic activity was seen at any concentration of test substance in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

 

Under the test conditions, Tagetes oil is not considered as mutagenic in these bacterial systems.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No.1272/2008.

Self-classification:

Based on the available information, no classification is proposed.