ABSTRACT

With regard to repeat dose toxicity, one male rat died in the 200 mg/kg group. A tendency for suppression of body weight gain in males and decreased locomotor activity and an ataxic gait in females were observed in the 200 mg/kg group. Histopathological examination revealed mucosal hyperplasia, inflammatory cell infiltration and edema in the forestomach of both sexes, involution of the thymus of females and an increase of fatty droplets in the fascicular zone of the adrenals of females in the 200 mg/kg group. In the 40 mg/kg group, similar histopathological changes were observed in the forestomach of both sexes and in the thymus of females. The NOEL for repeat dose toxicity is considered to be 8 mg/kg/day for both sexes. In terms of reproductive/developmental toxicity, the compound had influence on body weight of neonates in the 200 mg/kg group. The NOELs for reproductive/developmental toxicity are considered to be 200 mg/kg/day for parental males and females, and 40 mg/kg/day for offspring.

Thymol was not mutagenic to Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with or without an exogeneous metabolic activation system.

Thymol induced structural chromosomal aberrations in CHL/IU cells in the presence of an exogenous metabolic activation system. Polyploidy was not induced under the present experimental conditions.

Thymol did not induce micronuclei in bone marrow cells of male or female mice under the present experimental conditions.

SUMMARIZED DATA FROM THE STUDIES

1. Repeat Dose and Reproductive/Developmental Toxicity １）

Purity	:	99.6%
Test species/strain	:	Rats/Crj:CD (SD)
Test method	:	OECD Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test
Route	:	Oral (gavage)
Doses	:	0 (Vehicle), 8, 40, 200 mg/kg/day
Number of animals/group	:	Males, 10; females, 10
Vehicle	:	3% gum arabic solution
Administration period	:	Males, 43 days Females, from 14 days before mating to day 3 of lactation
Terminal kill	:	Males, day 44 Females, day 4 of lactation
GLP	:	Yes

　 Test results :

<Repeat dose toxicity>

In the 200 mg/kg group, one animal died and body weight gain tended to be suppressed in male rats. Female rats showed a decrease in locomotor activity and an ataxic gait. Histopathological examination revealed mucosal hyperplasia, inflammatory cell infiltration and edema in the forestomach in both males and females. Involution of the thymus and an increase of fatty droplets in the fascicular zone of the adrenals were also observed in few female rats.

In the 40 mg/kg group, similar histopathological changes were observed in the forestomach in both sexes and in the thymus in female rats. There were no significant differences in food consumption or organ weights among the groups. No effects ascribable to the compound were found on hematological or blood chemical examination in male rats.

The NOEL for repeat dose toxicity is considered to be 8 mg/kg/day for both sexes.

<Reproductive and developmental toxicity>

The compound exerted no effects on reproductive parameters such as the estrous cycle, mating index, fertility index, gestation length, number of corpora lutea or implantations, the implantation index, gestation index, delivery index, parturition or maternal behavior. Birth weight and body weight gain tended to be low in the 200 mg/kg group neonates. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, live birth index or viability index. No abnormal findings ascribable to the compound were found on external examination, or in terms of clinical signs or necropsy finding for the neonates.

The NOELs for reproductive and developmental toxicity are considered to be 200 mg/kg/day for parental males and females, and 40 mg/kg/day for offspring.

2. Genetic Toxicity

2-1. Bacterial test ２）

Purity	:	above 98 %
Test species/strains	:	Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Test methods	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD (471 and 472)
Procedures	:	Plate incorporation method
Solvent	:	DMSO
Positive controls	:	-S9 mix, 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, WP2, TA98), Sodium azide (TA1535) and 9-Aminoacridine (TA1537), +S9 mix, 2-Aminoanthracene (five strains)
Doses	:	-S9 mix, 0, 15.6, 31.3, 62.5, 125, 250 and 500 μg/plate in five strains, +S9 mix, 0, 62.5, 125, 250, 500, 1000 and 2000 μg/plate (TA100, WP2, TA98), 0, 31.3 - 1000 μg/plate (TA1535, TA1537)
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	3
Number of replicates	:	2
GLP	:	Yes

　Test results:

The chemical did not induce gene mutations in the S. typhimurium and E. coli strains. Toxicity was observed at 250 μg/plate (TA1537) and 500 μg/plate (TA100, TA1535, WP2, TA98) without S9 mix, and at 500 μg/plate (TA1535, TA1537) and 1000 μg/plate (TA100, WP2, TA98) with S9 mix.

Genetic effects:
Salmonella typhimurium TA100, TA1535, TA98, TA1537

	+	?	-
Without metabolic activation:	[ ]	[ ]	[*]
With metabolic activation:	[ ]	[ ]	[*]

Escherichia coli WP2 uvrA

	+	?	-
Without metabolic activation:	[ ]	[ ]	[*]
With metabolic activation:	[ ]	[ ]	[*]

2-2. Non-bacterial in vitro test (chromosomal aberration test) ２）

Purity	:	more than 98%
Type of cell used	:	Chinese hamster lung (CHL/IU) cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan)
Solvent	:	Dimethylsulfoxide
Positive controls	:	-S9 mix, Mitomycin C +S9 mix, Cyclophosphamide
Doses	:	-S9 mix (continuous treatment): 0, 0.020, 0.040,0.080 mg/ml -S9 mix (short-term treatment): 0, 0.020, 0.040,0.080 mg/ml +S9 mix (short-term treatment): 0, 0.020, 0.040,0.080 mg/ml
S-9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

Cytogenetic effects were seen as follows.

At the high (0.080 mg/ml) concentration with short-term treatment in the presence of an exogenous metabolic activation system, structural chromosomal aberrations (5.5%) were induced. Thymol did not induce polyploidy under the condition tested.

Lowest concentration producing cytogenetic effects in vitro:

With metabolic activation: 0.080 mg/ml (clastogenicity)

Genotoxic effects:

	clastogenicity			polyploidy
	+	?	-	+	?	-
Without metabolic activation:	[ ]	[ ]	[*]	[ ]	[ ]	[*]
With metabolic activation:	[*]	[ ]	[ ]	[ ]	[ ]	[*]

2-3. Non-bacterial in vivo test (Micronucleus test) ２）

Purity	:	above 98%
Test species/strain	:	Mice/Crj:BDF1, males and females
Test methods	:	Guideline for Screening Mutagenicity Testing of Chemicals (Japan) and OECD (474)
Procedure	:	Bone marrow/acridine orange staining
Solvent	:	Olive oil
Positive control	:	Cyclophosphamide 50 mg/kg
Doses	:	0, 312.5, 625 and 1250 mg/kg
Mice/group	:	Males, 5; Females, 5
GLP	:	Yes

　Test results:

The frequency of micronucleated immature erythrocytes was not significantly increased in males and females up to the dose of 1250 mg/kg oral gavage and animals were teilled 24 h after treatment. Inhibition of bone marrow cell proliferation was not observed under the test conditions.

Lowest dose producing toxicity:

Maximum tolerated dose: 1250 mg/kg in male and female

Genotoxic effect:

	+	?	-
Micronucleus test:	[ ]	[ ]	[*]

1)	The tests were performed by the Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki, 314-02, Japan. Tel +81-479-46-2871 Fax +81-479-46-2874
2)	The tests were performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Tel +81-463-82-4751 Fax +81-463-82-9627