Turmeric Leaf Oil

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct, 2018 to 02 Nov, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Curcuma longa, essential oil obtained by steam distillation of the leaves

Molecular formula:

No applicable for UVCB substance

IUPAC Name:

Curcuma longa, essential oil obtained by steam distillation of the leaves

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 31 August 2018 was used in this study.

The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Experiment 1 - Plate incorporation method
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was initially 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. However, results from the second mutation test showed that the toxicity of the test item yielded results that differed significantly from Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100, TA98 and TA1537 in the absence of metabolic activation (S9-mix). Therefore, these strains were repeated employing an amended test item dose range as follows: 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.
Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Vehicle / solvent:

The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other:

Details on test system and experimental conditions:

Test Item Preparation and Analysis
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in pre-dried acetone by mixing on a vortex mixer. The test item was confirmed as a UVCB substance, therefore no correction for purity was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots (Maron et al., 1981).
All formulations were used within four hours of preparation and were assumed to be stable for this period.

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 16 October 2018					To: 19 October 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	137 104 129	(123) 17.2#	17 23 22	(21) 3.2	19 28 28		(25) 5.2	36 30 23	(30) 6.5	20 12 27	(20) 7.5
	1.5 µg	157 116 132	(135) 20.7	20 15 23	(19) 4.0	24 42 37		(34) 9.3	22 23 20	(22) 1.5	19 12 20	(17) 4.4
	5 µg	141 122 134	(132) 9.6	22 21 22	(22) 0.6	17 21 24		(21) 3.5	21 16 17	(18) 2.6	9 7 21	(12) 7.6
	15 µg	130 137 118	(128) 9.6	13 13 16	(14) 1.7	23 22 28		(24) 3.2	12 16 14	(14) 2.0	20 8 24	(17) 8.3
	50 µg	122 116 117	(118) 3.2	19 23 20	(21) 2.1	17 22 29		(23) 6.0	18 16 19	(18) 1.5	9 18 11	(13) 4.7
	150 µg	128 127 119	(125) 4.9	29 13 15	(19) 8.7	34 29 30		(31) 2.6	13 20 27	(20) 7.0	16 11 6	(11) 5.0
	500 µg	125 125 119	(123) 3.5	21 26 23	(23) 2.5	25 23 26		(25) 1.5	23 22 16	(20) 3.8	7 4 4	(5) 1.7
	1500 µg	115 S 116 S 108 S	(113) 4.4	11 S 25 S 25 S	(20) 8.1	19 S 26 S 30 S		(25) 5.6	17 19 21	(19) 2.0	7 S 9 S 6 S	(7) 1.5
	5000 µg	36 SF 32 SF 45 SF	(38) 6.7	11 SF 9 SF 9 SF	(10) 1.2	25 SF 41 SF 29 SF		(32) 8.3	24 SF 17 SF 18 SF	(20) 3.8	0 VF 0 VF 0 VF	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		510 462 487	(486) 24.0	294 276 228	(266) 34.1	501 941 480		(641) 260.3	159 149 130	(146) 14.7	327 274 591	(397) 169.8

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO           4-Nitroquinoline-1-oxide

9AA             9-Aminoacridine

F                 Test Item Film

S                 Sparse bacterial background lawn

V                 Very weak bacterial background lawn

#                 Standard deviation

 

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 16 October 2018						To: 19 October 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains								Frameshift strains
		TA100			TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	119 112 129		(120) 8.5#	16 20 19	(18) 2.1	29 32 34		(32) 2.5	37 38 43	(39) 3.2	34 12 21	(22) 11.1
	1.5 µg	133 132 128		(131) 2.6	30 12 21	(21) 9.0	34 38 38		(37) 2.3	33 28 34	(32) 3.2	18 23 10	(17) 6.6
	5 µg	142 139 134		(138) 4.0	19 20 35	(25) 9.0	39 29 32		(33) 5.1	31 32 27	(30) 2.6	25 12 22	(20) 6.8
	15 µg	134 109 123		(122) 12.5	24 22 11	(19) 7.0	35 45 28		(36) 8.5	33 40 38	(37) 3.6	18 16 14	(16) 2.0
	50 µg	125 133 112		(123) 10.6	21 15 15	(17) 3.5	43 26 39		(36) 8.9	30 31 35	(32) 2.6	24 8 6	(13) 9.9
	150 µg	123 91 112		(109) 16.3	19 15 13	(16) 3.1	35 28 22		(28) 6.5	24 37 29	(30) 6.6	8 5 18	(10) 6.8
	500 µg	109 124 107		(113) 9.3	25 11 10	(15) 8.4	39 34 32		(35) 3.6	26 27 24	(26) 1.5	7 9 15	(10) 4.2
	1500 µg	99 121 132		(117) 16.8	14 9 16	(13) 3.6	31 40 29		(33) 5.9	37 21 28	(29) 8.0	21 14 15	(17) 3.8
	5000 µg	130 SF 127 SF 115 SF		(124) 7.9	17 F 11 F 13 F	(14) 3.1	26 SF 35 SF 31 SF		(31) 4.5	35 F 29 F 38 F	(34) 4.6	11 F 8 F 10 F	(10) 1.5
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA			2AA		2AA			BP		2AA
		1 µg			2 µg		10 µg			5 µg		2 µg
		1776 1794 1909	(1826) 72.2		293 241 244	(259) 29.2	174 208 207		(196) 19.3	182 170 198	(183) 14.0	240 242 176	(219) 37.5

BP            Benzo(a)pyrene

2AA          2-Aminoanthracene

F                     Test Item Film

S              Sparse bacterial background lawn

#              Standard deviation

 

Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 23 October 2018 30 October 2018 †					To: 26 October 2018 02 November 2018 †
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100 †		TA1535		WP2uvrA			TA98 †		TA1537 †
	Solvent Control (Acetone)	134 143 142	(140) 4.9#	12 9 8	(10) 2.1	19 27 21		(22) 4.2	24 26 25	(25) 1.0	25 10 19	(18) 7.5
	0.05 µg	141 136 159	(145) 12.1	N/T		N/T			17 18 32	(22) 8.4	3 10 19	(11) 8.0
	0.15 µg	159 158 123	(147) 20.5	N/T		N/T			22 15 22	(20) 4.0	29 22 8	(20) 10.7
	0.5 µg	140 137 157	(145) 10.8	N/T		N/T			26 31 19	(25) 6.0	26 13 10	(16) 8.5
	1.5 µg	156 143 142	(147) 7.8	16 7 10	(11) 4.6	25 18 21		(21) 3.5	19 30 27	(25) 5.7	21 13 11	(15) 5.3
	5 µg	133 145 140	(139) 6.0	7 8 8	(8) 0.6	21 22 27		(23) 3.2	20 21 30	(24) 5.5	14 15 17	(15) 1.5
	15 µg	157 122 136	(138) 17.6	12 12 12	(12) 0.0	21 24 19		(21) 2.5	30 30 22	(27) 4.6	28 10 11	(16) 10.1
	50 µg	114 S 127 S 103 S	(115) 12.0	8 6 8	(7) 1.2	23 30 23		(25) 4.0	20 S 19 S 20 S	(20) 0.6	5 S 6 S 9 S	(7) 2.1
	150 µg	134 S 117 S 129 S	(127) 8.7	11 S 6 S 9 S	(9) 2.5	12 S 17 S 16 S		(15) 2.6	8 S 17 S 14 S	(13) 4.6	4 S 6 S 3 S	(4) 1.5
	500 µg	N/T		9 S 8 S 11 S	(9) 1.5	21 S 25 S 21 S		(22) 2.3	N/T		N/T
	1500 µg	N/T		8 V 13 V 8 V	(10) 2.9	15 S 17 S 15 S		(16) 1.2	N/T		N/T
	5000 µg	N/T		13 VF 6 VF 6 VF	(8) 4.0	16 SF 10 SF 25 SF		(17) 7.5	N/T		N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		399 453 328	(393) 62.7	467 635 438	(513) 106.4	881 779 792		(817) 55.5	136 172 152	(153) 18.0	131 176 129	(145) 26.6

†                Experimental procedure repeated at a later date due to toxicity in the original test

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO           4-Nitroquinoline-1-oxide

9AA             9-Aminoacridine

F                 Test Item Film

N/T              Not tested at this dose level

S                 Sparse bacterial background lawn

V                 Very weak bacterial background lawn

#                 Standard deviation

 

Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 23 October 2018					To: 26 October 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	76 80 84	(80) 4.0#	9 11 14	(11) 2.5	40 31 37		(36) 4.6	19 30 47	(32) 14.1	8 13 14	(12) 3.2
	1.5 µg	79 79 83	(80) 2.3	13 14 12	(13) 1.0	38 36 35		(36) 1.5	32 41 29	(34) 6.2	9 19 12	(13) 5.1
	5 µg	90 76 78	(81) 7.6	20 7 12	(13) 6.6	27 28 28		(28) 0.6	40 33 32	(35) 4.4	6 9 13	(9) 3.5
	15 µg	93 95 88	(92) 3.6	7 7 8	(7) 0.6	40 21 21		(27) 11.0	35 37 30	(34) 3.6	12 15 9	(12) 3.0
	50 µg	86 103 78	(89) 12.8	8 10 8	(9) 1.2	25 23 35		(28) 6.4	31 34 36	(34) 2.5	14 12 8	(11) 3.1
	150 µg	68 84 79	(77) 8.2	11 12 22	(15) 6.1	31 40 44		(38) 6.7	19 27 25	(24) 4.2	8 12 14	(11) 3.1
	500 µg	80 S 70 S 74 S	(75) 5.0	7 8 11	(9) 2.1	24 27 23		(25) 2.1	34 26 29	(30) 4.0	12 9 17	(13) 4.0
	1500 µg	50 S 74 S 63 S	(62) 12.0	15 11 14	(13) 2.1	17 23 33		(24) 8.1	16 21 42	(26) 13.8	8 4 8	(7) 2.3
	5000 µg	53 VF 79 VF 62 VF	(65) 13.2	10 SF 18 SF 6 SF	(11) 6.1	26 F 34 F 41 F		(34) 7.5	40 F 23 F 16 F	(26) 12.3	11 F 9 F 9 F	(10) 1.2
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1260 1179 1024	(1154) 119.9	245 231 226	(234) 9.8	144 152 150		(149) 4.2	74 87 91	(84) 8.9	208 170 159	(179) 25.7

BP            Benzo(a)pyrene

2AA          2-Aminoanthracene

F                     Test Item Film

S              Sparse bacterial background lawn

V             Very weak bacterial background lawn

#              Standard deviation

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Turmeric leaf oil did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Turmeric leaf oil was considered to be non-mutagenic.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was initially the same as Experiment 1 (1.5 to 5000 μg/plate). However, results from the second mutation test (pre-incubation method) showed that the toxicity of the test item yielded results that differed significantly from Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100, TA98 and TA1537 in the absence of metabolic activation (S9-mix). Therefore, these strains were repeated employing an amended test item dose range of 0.05 to 150 μg/plate. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. In the first experiment (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all the tester strains dosed in the absence of metabolic activation (S9-mix), initially from 500 μg/plate. In the presence of metabolic activation (S9-mix), the test item caused a visible reduction in the growth of the bacterial background lawns or substantial reductions in the revertant colony frequency of tester strains TA100, TA1537 and WP2uvrA at 5000 μg/plate.
Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was initially employed in the second mutation test (pre-incubation method). However, as previously described, the test item induced a much stronger toxic response employing the pre-incubation methodology and three tester strains had to be repeated using the toxic limit as the maximum dose. In the absence of metabolic activation (S9-mix), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate. In the presence of metabolic activation (S9-mix), the test item caused a visible reduction in the growth of the bacterial background lawns of tester strains TA100, and TA1535 from 500 and at 5000 μg/plate, respectively.
A slight test item film was noted by eye at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Turmeric leaf oil was considered to be non-mutagenic under the conditions of this test.