Essential oil of Blue tansy obtained from the flowers and leaves of Tanacetum annuum (Asteraceae) by steam distillation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD TG 471 (Ames): Mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of S9-mix.

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:

19 Apr 2019 - 16 May 2019

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:

July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus (Salmonella typhimurium)
Thryptophan locus (Escherichia coli)

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.7 - 37.7°C). The temperature was continuously monitored throughout the experiment.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix : male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254

Test concentrations with justification for top dose:

Blue Tansy Oil was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence of S9-mix: 5.4, 17, 52, 164, 512 and 1600 μg/plate and in the presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear dark blue solution DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition;
- Any supplementary information relevant to cytotoxicity: defined as a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies

Rationale for test conditions:

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
 
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

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 98

Metabolic activation:

with

Genotoxicity:

positive

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 98

Metabolic activation:

without

Genotoxicity:

negative

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

other: Outside of laboratory historical control data ranges

Remarks:

The mean number of revertant colonies showed a characteristic number of revertants (2 colonies) when compared against historical control data (3 revertants), the validity of the test was considered to be not affected.

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
Precipitation of Blue Tansy Oil on the plates was observed at the start of the incubation period at concentrations of 1600 µg/plate and upwards in tester strain TA100 and WP2uvrA.
Precipitation of Blue Tansy Oil on the plates was observed at the end of the incubation period at the concentrations of 5000 µg/plate in tester strain TA100 in the absence and presence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of Blue Tansy Oil used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item inhibited bacterial growth.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
The negative control values were within the laboratory historical control data ranges, except the response for TA1537 in the absence of S9-mix, first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies
(2 revertant colonies) when compared against relevant historical control data (3 revertant colonies), the validity of the test was considered to be not affected.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : In the presence of S9-mix, in tester strain TA98, the test item induced up to 3.8 and 2.6-fold, dose related increases in the number of revertant colonies compared to the solvent control in the first and second experiment, respectively.

Ames test:
- Signs of toxicity : Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA where no toxicity was observed at any of the dose levels tested.
- Individual plate counts : yes
- Mean number of revertant colonies per plate and standard deviation : refer to tables

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: refer to tables
- Negative (solvent/vehicle) historical control data: refer to tables

Experiment 1

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	592	±	55		1399	±	91
Solvent control	121	±	4		37	±	6
1.7	125	±	6		31	±	9
5.4	113	±	5		32	±	12
17	95	±	7		25	±	4
52	97	±	16	^n	32	±	7
164	91	±	8	^s	33	±	3
512	110	±	13	^m	34	±	5
1600				^e NP MC	32	±	3
5000				^e SP MC	27	±	5	^n NP

 

With S9-mix¹

 

Positive control	1593	±	88		238	±	54
Solvent control	109	±	9		31	±	2
1.7	107	±	16		45	±	7
5.4	97	±	10		31	±	6
17	116	±	21		35	±	11
52	133	±	12		38	±	6
164	124	±	6		37	±	8
512	117	±	17	^n	31	±	7
1600	146	±	22	^s NP	38	±	15
5000				^e SP MC	28	±	11	^n NP

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimurium.
	TA1535	TA1537	TA98

 

Without S9-mix

 

Positive control	1033	±	27		999	±	61		1072	±	264
Solvent control	10	±	5		2	±	1		11	±	3
5.4	8	±	3		4	±	4		13	±	4
17	8	±	2		3	±	2		10	±	3
52	11	±	5	^n	4	±	1	^n	13	±	3
164	7	±	4	^s	2	±	1	^s	16	±	4	^n
512	6	±	5	^m	2	±	1	^m	12	±	2	^s
1600	7	±	3	^m NP	2	±	2	^m NP	16	±	6	^s NP

 

With S9-mix¹

 

Positive control	277	±	17		402	±	21		1329	±	213
Solvent control	14	±	4		3	±	2		14	±	3
17	6	±	4		5	±	4		20	±	8
52	7	±	4		5	±	4		17	±	3
164	7	±	4		4	±	1		23	±	3
512	10	±	2	^n	5	±	3	^n	28	±	3	^n
1600	8	±	6	^s	2	±	3	^m	23	±	6	^s
5000	4	±	4	^m NP	2	±	2	^e NP MC	53	±	14	^m NP

Experiment 2

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	950	±	51		798	±	47		1165	±	62		1049	±	73		1577		±	79
Solvent control	11	±	3		4	±	3		11	±	0		122	±	4			20	±	7
86	7	±	6		4	±	1		14	±	3		106	±	12
154	5	±	4		3	±	2		11	±	5		97	±	7
275	7	±	3	^n	7	±	6		10	±	5	^n	108	±	18	^n		23	±	3
492	6	±	3	^s	3	±	2	^n NP	13	±	4	^s	87	±	7	^m		19	±	4
878	5	±	2	^s NP	3	±	2	^s SP	18	±	9	^s NP	114	±	22	^m NP		17	±	5
1568	4	±	3	^m SP	6	±	5	^s SP	16	±	3	^s SP	93	±	31	^m SP		19	±	5
2800																		16	±	2
5000																		26	±	7	^n NP

 

 

With S9-mix¹

 

Positive control	294	±	16		441	±	20		624	±	19		1755	±	314		333	±	21
Solvent control	11	±	6		6	±	3		16	±	6		112	±	3		28	±	6
154	10	±	1		3	±	2		24	±	2		130	±	21
275	7	±	3		5	±	5		30	±	10		136	±	12		21	±	11
492	9	±	2		4	±	3		30	±	6		120	±	2		30	±	6
878	8	±	7		5	±	2	^n	22	±	8	^n	118	±	13	^n NP	33	±	3
1568	6	±	6	^n NP	5	±	2	^s NP	27	±	4	^s NP	140	±	12	^s SP	20	±	4
2800																	24	±	4
5000	7	±	2	^s SP	2	±	1	^m SP	41	±	6	^m SP				^e SP MC	19	±	4	^n NP

 

 

^1	Plate incorporation assay (10% S9)
MC	Microcolonies
NP	No precipitate
SP	Slight Precipitate
e	Bacterial background lawn extremely reduced
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 Historical positive controls

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 – 27	3 – 20	3 – 23	8 - 61	8 – 60	61 – 176	60 - 176	10 – 61	9 - 68
Mean	10	11	6	6	16	22	112	108	27	33
SD	3	3	2	3	5	7	18	21	8	9
n	3303	3265	3232	3212	3251	3326	3336	3246	3021	2993

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Apr 2016 and Apr 2019. 

 Historical negative controls

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	128 – 1530	73 – 1481	58 – 1422	54 – 1239	365 – 1978	250 – 2018
Mean	919	256	802	328	1305	910
SD	172	122	362	154	236	355
n	3215	3122	2777	3187	3202	3216

 

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1993	408 - 2379	93 – 1999	109 - 1968
Mean	907	1308	1073	437
SD	167	386	537	158
n	3231	3179	2923	2987

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Apr 2016 and Apr 2019. 

Conclusions:

Based on the results of the in vitro gene mutation study in bacteria for Blue tansy oil, it is concluded that the test substance is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. The test item is not mutagenic in the Escherichia coli reverse mutation assay.
 

Executive summary:

The potential of of Blue Tansy Oil and/or its metabolites to induce gene mutations was examined in a OECD TG 471 study, under GLP conditions.

Blue Tansy Oil was examined in 4 Salmonella typhimurium strains; TA98, TA100, TA1535, and TA1537 and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9), in two independent experiments.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay in the tester strains TA1535, TA1537 and TA98 at concentration ranges of 5.4 to 1600 µg/plate and 17 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix respectively.  Blue Tansy Oil did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.  

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay in the tester strains TA1535, TA1537 and TA98 at concentration ranges of 5.4 to 1600 µg/plate and 17 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix respectively.  In the absence of S9-mix, no increases in the number of revertants were observed at any of the tester strains tested. In the presence of S9-mix, in tester strain TA98, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control.  The increases observed were above the laboratory historical control data range and up to 3.8- fold the concurrent control

In a follow up experiment, no increases in the number of revertants were observed at any of the tester strains tested, in the absence of S9-mix. In the presence of S9-mix, in tester strain TA98, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control.  The increases observed were above the laboratory historical control data range and up to 2.6- fold the concurrent control.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Blue Tansy Oil is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. The test item is not mutagenic in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

OECD TG 471

The potential of of Blue Tansy Oil and/or its metabolites to induce gene mutations was examined in a OECD TG 471 study, under GLP conditions.

Blue Tansy Oil was examined in 4 Salmonella typhimurium strains; TA98, TA100, TA1535, and TA1537 and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9), in two independent experiments.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay in the tester strains TA1535, TA1537 and TA98 at concentration ranges of 5.4 to 1600 µg/plate and 17 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix respectively.  Blue Tansy Oil did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.  

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay in the tester strains TA1535, TA1537 and TA98 at concentration ranges of 5.4 to 1600 µg/plate and 17 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix respectively.  In the absence of S9-mix, no increases in the number of revertants were observed at any of the tester strains tested. In the presence of S9-mix, in tester strain TA98, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control.  The increases observed were above the laboratory historical control data range and up to 3.8- fold the concurrent control

In a follow up experiment, no increases in the number of revertants were observed at any of the tester strains tested, in the absence of S9-mix. In the presence of S9-mix, in tester strain TA98, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control.  The increases observed were above the laboratory historical control data range and up to 2.6- fold the concurrent control.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Blue Tansy Oil is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. The test item is not mutagenic in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the results of the in vitro gene mutation study in bacteria for Blue tansy oil, it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. The test item is not mutagenic in the Escherichia coli reverse mutation assay. Further evaluation in an in-vitro mammalian cytogenicity/micronucleus test and a gene mutation study before a (non-)classification can be given to this substance.