p-mentha-1,3-diene

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:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Salmonella/microsome assay performed by the plate incorporation procedure as described by Maron and Ames, 1983, and Gomes-Carneiro et al., 1998

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

p-mentha-1,3-diene

EC Number:

202-795-1

EC Name:

p-mentha-1,3-diene

Cas Number:

99-86-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,3-diene

Test material form:

liquid

Details on test material:

Commercial Name: Terpinene Alpha
Chemical name: 1-methyl-4-(1-methylethyl-1,3-cyclohexadiene)
CAS No.: 99-86-5
Lot No.: VE00294368
Purity: 94.1%
Expiration Date: 2015-04-06

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material: alpha terpinene
- Source of test material: Sigma Chemical Co. (St Louis, MO, USA)
- Purity: 95% or higher

Method

Target gene:

The histidine-dependent Salmonella typhimurium strains were used.

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA100, TA98, TA97a and TA1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Extrinsic metabolic system (rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

Concentration range 0-5000 µg/plate: 0, 100, 250, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 2000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle / solvent used: ethanol

Controls

Negative solvent / vehicle controls:

yes

Remarks:

100 µl

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

BACTERIAL STRAINS:
- TA100, TA98, TA97a and TA1535 strains of S. typhimurium
- Source: University of California, Berkeley, USA
- An inoculum (200 µl) of a thawed permanent culture was added to 20 ml of Nutrient Broth No.2 and incubated at 37°C with shaking until a concentration of approximately 1-2x10^9 bacteria per ml was obtained.

METABOLIC ACTIVATION SYSTEM (S9 mixture):
- Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox (Molecular Toxicology Inc., Boone, NC, USA).
- S9 mixture: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate bu¿er pH7.4; 0.84ml of 100mM NADP solution; 0.105ml of 1M glucose-6-phosphate; 0.42ml of 1.65M KCl + 0.4M MgCl2 salt solution; and 2.1ml of lyophilized S9 fraction reconstituted with water provided by a MilliQ water puri¿cation system.

SALMONELLA/MICROSOME ASSAY:
The following was mixed with 2 ml of top agar which poured onto a minimal glucose plate:
- 100 µl of an overnight grown culture
- 100 µl test substance (diluted in analytical grade ethanol, Vetec)
- the negative (solvent) control or positive control
- 500 µl sodium-phosphate buffer or S9 mix

- The plates were incubated at 37°C for 72-hr in the dark and then scored for revertant his+ bacterial colonies
- Each determination was made in triplicate and at least two independent experiments were carried out

POSITIVE CONTROLS:
- tested in concentration range 0-1500 µg/plate: 0, 1, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 and 1000 µg/plate

Rationale for test conditions:

The assay is specifically designed to determine the ability of alpha terpinene to produce genetic damage that leads to gene mutations.

Evaluation criteria:

Toxicity was apparent either as a reduction in the number of his+ revertant bacterial colonies and/or as a change in the auxotrophic background growth (i.e. the background lawn). The criteria for a positive mutagenic response was a clear dose-dependent increase in the number of revertants within the non-toxic range.

Statistics:

Values recorded are means ± SD of three plates.

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium TA100, TA98, TA97a and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Addition of the S9-mixture seemed to have reduced toxicity.

Remarks on result:

other: not mutagenic

Any other information on results incl. tables

Table1: First assay testing of alpha terpinene (1-isopropyl-4-methyl-1,3-cyclohexadiene) in the Salmonella/microsome assay
Dose (µg/plate)		Number of revertants (Mean ± SD)
		TA100		TA98		TA97a		TA1535
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
a-terpinene	5000	-	-	-	43 ± 36*	-	45 ± 17*	2 ± 3*	6 ± 4*
	2500	-	-	-	65 ± 16*	-	108 ± 22*	0 ± 0*	14 ± 5*
	2000	-	104 ± 28*	13 ± 13*	-	-	-	-	-
	1500	-	-	23 ± 12*	-	-	-	-	-
	1250	-	73 ± 35*	19 ± 14*	-	-	-	-	-
	1000	-	51 ± 35*	9 ± 12*	62 ± 12	-	144 ± 17*	12 ± 1*	15 ± 6*
	750	-	-	35 ± 8*	-	-	-	-	-
	500	-	84 ± 18	20 ± 17	59 ± 2	110 ± 16*	142 ± 5	8 ± 4*	17 ± 2
	400	-	-	-	-	79 ± 2*	-	-	-
	300	-	-	-	-	83 ± 10*	-	-	-
	250	-	-	-	67 ± 4	-	146 ± 19	-	-
	200	-	-	-	-	101 ± 8*	-	-	-
	100	102 ± 8*	116 ± 35*	-	57 ± 4	97 ± 17	185 ± 28	16 ± 2	17 ± 4
	75	128 ± 19	-	-	-	-	-	-	-
	50	118 ± 2	-	-	-	141 ± 6	-	-	-
	35	147 ± 19	-	-	-	-	-	-	-
	20	139 ± 8	-	-	-	-	-	-	-
	10	152 ± 36	-	-	-	-	-	-	-
Negative control (100 µl)		167 ± 10	206	43 ± 12	58 ± 8	150 ± 2	202 ± 28	18 ± 4	27 ± 5
Positive control (1 µg/plate)		SA	2-AA	2-NF	2AA	4-NQNO	2AF	SA	2AA
		804 ± 59	614 ± 97	173 ± 35	430 ± 15	845 ± 17	928 ± 24	506 ± 8	154 ± 4

Negative control: 100 µl ethanol. Positive controls: sodium azide (SA), 2-aminoanthracene (2-AA), 2-nitrofluorene (2-NF), 4-nitroquinoline-N-oxide (4-NQNO), 2-aminofluorene (2-AF). Dose not tested: (-). Toxicity apparent as an alteration of the background lawn: (*). Values are means ± SD of three plates.

Table 2. Second assay testing of alpha terpinene in the Salmonella/microsome assay
Dose (µg/plate)		Number of revertants (Mean±SD)
		TA100		TA98		TA97a		TA1535
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Alpha terpinene	1500	-	-	-	50 ± 2*	-	-	-	-
	750	-	-	-	58 ± 14	-	-	-	19 ± 6*
	500	-	-	34 ± 6*	58 ± 8	-	172 ± 4	0 ± 0*	21 ± 4
	250	-	-	25 ± 5*	58 ± 4	-	220 ± 8	2 ± 3*	16 ± 2
	200	-	-	-	-	107 ± 20*	-	-	-
	100	-	-	36 ± 12	56 ± 8	109 ± 13	267 ± 8	4 ± 3	19 ± 5
	75	149 ± 7	120 ± 3	-	-	138 ± 20	-	-	-
	50	133	130 ± 12	41 ± 6	60 ± 3	123 ± 16	226 ± 14	20 ± 4	26 ± 2
	35	170 ± 15	146 ± 19	-	-	-	-	-	-
	25	-	-	37± 2	-	141 ± 14	194 ± 4	22 ± 5	22 ± 7
	20	162 ± 12	132 ± 39	-	-	-	-	-	-
	10	170 ± 6	140 ± 1	-	-	158 ± 2	-	21 ± 5	-
	5	154 ± 15	145 ± 18	-	-	158 ± 8	-	28 ± 7	-
Negative control (100 µl)		158 ± 26	148 ± 19	37 ± 7	47 ± 1	152 ± 13	190 ± 4	30 ± 4	23 ± 5
Positive control (1 µg/plate)		SA	2-AA	2-NF	2AA	4-NQNO	2AF	SA	2AA
		449 ± 32	774 ± 54	88 ± 8	183 ± 22	752 ± 87	841 ± 24	696 ± 81	182 ± 10

Negative control: 100 µl ethanol. Positive controls: sodium azide (SA), 2-aminoanthracene (2-AA), 2-nitrofluorene (2-NF), 4-nitroquinoline-N-oxide (4-NQNO), 2-aminofluorene (2-AF). Dose not tested: (-). Toxicity apparent as an alteration of the background lawn: (*). Values are means ± SD of three plates.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, alpha terpinene was shown to be not mutagenic in the Ames assay in the four Salmonella typhimurium strains tested, either in the presence or absence of the metabolic activation system.

Executive summary:

The aim of this study performed using the plate incorporation procedure as described by Maron and Ames, 1983, and Gomes-Carneiro et al., 1998, was to investigate the genotoxicity of alpha terpinene when tested at different concentrations in four Salmonella typhimurium strains in the Salmonella/microsome assay, with and without the addition of an extrinsic metabolic activation system.

200 µl of four S. typhimurium strains (TA100, TA98, TA97a and TA1535) were incubated separately with 20 ml Nutrient Broth 2 at 37 °C with shaking until approximately 1-2x10^9 bacteria per ml were obtained. 100 µl of overnight culture, 100 µl alpha- terpinene (diluted in ethanol)/ negative control /positive control and 500 µl sodium phosphate buffer or S9-mix were mixed with 2ml top agar and then poured into a minimal glucose plate. The plates were incubated in the dark at 37°C for 72-h.

The negative control used was ethanol and the positive controls were TA100/-S9 and TA1535/-S9, SA (1 µg/plate); TA100/+S9 and TA1535/-S9, 2AA (1µg/plate); TA98/-S9, 2-NF (1,5lµ/plate); TA98/+S9; 2AA (0,5µg/plate); TA97a/-S9, 4-NQNO (1µg/plate); TA97a/+S9, 2AF (10µg/plate).

Alpha terpinene was tested at several concentrations in the range of 0-5000 µg/plate and the positive controls were tested in the range of 0-1500 µg/plate.

Based on the results of this study, alpha terpinene did not induce any increase in the number of his+ revertant colonies over the negative control values in the four S. typhimurium strains tested, either in the presence or absence of the metabolic activation system. The results also suggest that the addition of the S9-mixture seemed to have induced a reduced toxicity.

Based on the results of this study, it was concluded that alpha terpinene was not mutagenic in the Salmonella/microsome assay.