Genetic Toxicology - Mammalian Cell Mutagenicity

Study Information
Study Attribute	Value
CEBS Accession Number:	002-01946-0008-0000-0
Chemical Name:	D-Limonene
CASRN:	5989-27-5
NTP Study Type:	Genetic Toxicology - Mammalian Cell Mutagenicity
NTP Study ID:	861341
Study Result:	Negative

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Table Instructions and Notes:
Studies are indicated to be "Nontoxic" when the observed reductions in relative total growth did not indicate that the maximum tolerated dose was achieved.

Nonactivation Trial: 1 Experiment Call: Inconclusive
		Conc. uL/mL	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
Vehicle Control	Ethanol	0	73	106	63	29	33
			90	94	86	32
			83#	101	86	35
			78	99	89	38
Test Chemical	D-Limonene	0.01	75	108	48	21	19
			75	98	26	12
			69	86	50	24
		0.02	87	99	27	10	15
			80	90	49	21
			89	88	34	13
		0.03	87	92	60	23	19
			102	80	62	20
			109	93	43	13
		0.04	71	108	73	34	32
			84	63	80	32
			84	54	75	30
		0.05	63	32	26	14	14
			82	47	37	15
			82	61	35	14
		0.06	86	80	77	30	36
			73	61	83	38
			73	53	88	40
Positive Control	Methyl Methane Sulfonate	5	72#	55	554	257	208*
			63	55	295	157
			68#	63	427	211

Nonactivation Trial: 2 Experiment Call: Equivocal
		Conc. uL/mL	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
Vehicle Control	Ethanol	0	76	107	69	30	32
			80	89	69	29
			77	103	81	35
			85	101	87	34
Test Chemical	D-Limonene	0.03	104	91	119	38	41
			88	105	117	44
			96	108	117	41
		0.04	101	90	137	45	44
			122r	102	118	32
			70	87	90	43
		0.05	87	76	129	49	47
			98	82	161	55
			87	99	93	36
		0.06	83	111	126	51	56*
			77	75	117	51
			75	62	149	66
		0.08	94	106	160	57	49*
			84	101	107	43
			72	80	106	49
		0.1	83	106	95	38	41
			72	105	115	53
			85	98	78	31
Positive Control	Methyl Methane Sulfonate	5	73	40	443	203	252*
			73#	37	724	332
			74	60	491	221

Nonactivation Trial: 3 Experiment Call: Negative
		Conc. ug/mL	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
Vehicle Control	Ethanol	0	69	66	81	39	31
			70	91	60	29
			81	136	83	34
			72	107	51	24
Test Chemical	D-Limonene	5	64	117	64	34	35
			47	111	56	40
			67	199	64	32
		10	87	134	46	18	23
			74	119	68	31
			77	125	47	20
		20	49	86	67	46	35
			69	118	65	31
			88	157	76	29
		30	64	123	60	31	32
			64	111	56	29
			63	127	66	35
		40	69	172	75	36	33
			88	166	78	30	33
		50

		60


Positive Control	Methyl Methane Sulfonate	5	80	120	218	91	93*
			54	73	169	104
			65	103	164	84

Nonactivation Trial: 4 Experiment Call: Negative
		Conc. ug/mL	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
Vehicle Control	Ethanol	0	118	102	91	26	33
			102	110	105	34
			103	101	110	36
			86	87	97	38
Test Chemical	D-Limonene	5	97	98	81	28	34
			59	53	75	42
			78	79	77	33
		10	80	98	78	33	32
			92	65	86	31	32
		20	62	62	87	47	36
			89	82	79	30
			100	79	90	30
		30	88	41	83	32	74*
			30	18	103	116	74*
		40	108	78	70	22	26
			109	59	95	29
			112	15	94	28
Positive Control	Methyl Methane Sulfonate	5	63	41	523	275	293*
			64	38	462	242
			46	30	494	361

Induced S9 Trial: 1 Experiment Call: Negative
		Conc. ug/mL	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
Vehicle Control	Ethanol	0	88	117	164	62	73
			71	87	180	84
			75	102	153	68
			73	94	171	78
Test Chemical	D-Limonene	10	86	81	74	29	38
			80	87	82	34
			51	47	76	50
		20	69	84	75	36	35
			72	73	76	35
			76	87	75	33
		30	85	91	90	35	37
			91	93	100	37
			79	82	96	41
		40	68	70	137	67	50
			74	28	100	45
			94	92	103	37
		50	1r	0	124	4133	59
			74	53	113	51
			62	50	123	66
Positive Control	3-Methylcholanthrene	2.5	42	27	622	496	410*
			63	46	776	414
			91	54	875	321

Induced S9 Trial: 2 Experiment Call: Negative
		Conc. ug/mL	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
Vehicle Control	Ethanol	0	115	112	182	53	66
			142r	91	168	40
			116#	88	278	80
Test Chemical	D-Limonene	10	93	79	131	47	60
			75	101	92	41
			82	80	228	93
		20	101	94	117	38	56
			70	87	130	62
			79	72	160	68
		30	111	66	173	52	69
			72	73	163	75
			100	86	235	78
		40	62	40	198	106	67
			100	85	110	37
			94	87	163	58
		50	79	16	238	101	87
			79	38	182	77
			78#	39	195	84
Positive Control	3-Methylcholanthrene	2.5	70	21	641	305	304*
			60	20	477	266
			69	24	706	341

Induced S9 Trial: 3 Experiment Call: Inconclusive
		Conc. ug/mL	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
Vehicle Control	Ethanol	0	85	89	178	70	60
			76	102	142	63
			90	118	137	51
			75	91	130	58
Test Chemical	D-Limonene	30	65	65	165	85	59
			71	70	109	51
			119	167	146	41
		40	96	82	136	47	44
			111	89	125	38
			79	87	109	46
		50	62	62	95	51	46
			81	97	117	48
			98	89	114	39
		60	86	105	146	57	78
			78	77	124	53
			39	41	145	123
		80	82	87	98	40	58
			95#	79	161	56
			77	68	178	77
		100	98	121	122	42	46
			92	97	138	50
			120r	115	149	41
Positive Control	3-Methylcholanthrene	2.5	83	47	635	254	212*
			113	75	578	170
			10r	6	644	2183

Footnotes:
Asterisks(*) indicate significant responses.
r = rejected value due to quality control criteria
# = reduced sample size because of the loss of one culture dish due to contamination