Thymol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 MAR 1996

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crj:CD, SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (8 wks
- Weight at study initiation: Males: 333-371 g; Females: 193-221 g
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 3% gum arabic solution

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

males: 43 days
females: from 14 days before mating to day 3 of lactation

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

8 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males + 10 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on the results of a pre-test performed at doses of 30, 100 and 300 mg/kg bw/d.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: every week and at termination of study

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals 42 d after commencement of treatment
- Maternal animals: All surviving animals at PND 4

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 3 were prepared for microscopic examination and weighed, respectively.

Statistics:

Bartlett, Kruskal-Wallis, Dunnett, Scheffé

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased locomotor activity and an ataxic gait in females were observed in the 200 mg/kg group.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male rat died in the 200 mg/kg group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A tendency for suppression of body weight gain in males was observed in the 200 mg/kg group.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

gavage administration

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

gavage administration

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Decreased locomotor activity and an ataxic gait in females were observed in the 200 mg/kg group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights were not influeced by test item (please refer to table 1).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thickening of the wall in the forestomach was observed in all male rats in the dose group of 200 mg/kg bw/day (table 2).

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

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 (table 3).

Other effects:

no effects observed

Details on results:

In the 200 mg/kg/group, one animal died and body weight gain tend to be suppressed in male rats. Femals rats showed a decrease in locomotor activity an 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 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 based on local irritation effects on the forestomach.

Dose descriptor:

NOEL

Remarks:

local effects

Effect level:

8 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Table 1: Absolute and relative organ weights in rats treated orally with thymol m the combined repeat dose and reproductive/developmental toxicity screening test
Dose (mg/kg/day)	0	8	40	200
Male
Number of animals	10	9	10	9
Body weight (g)	528 ± 23.5	510±24.7	519 ± 46.4	506±27.7
Absolute organ weight
Pituitary (mg)	14.3 ± 1.36	14.4±1.30	14.2±0.99	14.7 ± 1.45
Thymus (mg)	356 ± 54.4	353 ± 82.8	361±108.4	391±67.6
Liver (g)	14.48 ± 1.277	13.37 ± 0.817	14.55 ± 2.130	14.12 ± 1.455
Kidney (g)	3.14±0.186	3.07 ± 0.330	3.34±0.437	3.20 ± 0.211
Adrenal (mg)	70.2 ± 9.05	63.5 ± 11.04	64.9 ± 8.16	65.7±10.00
Testis (g)	3.54 ± 0.210	3.45 ± 0.229	3.48 ± 0.398	3.55 ± 0.175
Prostate (g)	0.71 ±0.209	0.63 ± 0.132	0.65±0.109	0.73±0.251
Epididymis (g)	1.28 ± 0.069	1.28 ± 0.091	1.29 ± 0.129	1.27 ± 0.058
Relative organ weight
Pituitary (mg%)	2.7 ± 0.28	2.8 ± 0.23	2.8 ± 0.19	2.9 ± 0.26
Thymus (mg%)	67±9.9	69 ± 16.2	69 ± 17.0	78 ± 16.3
Liver (g%)	2.74 ± 0.185	2.62±0.174	2.79 ± 0.203	2.79 ± 0.186
Kidney (g%)	0.59±0.032	0.60 ± 0.061	0.64 ± 0.058	0.64 ± 0,060
Adrenal (mg%)	13.3±2.07	12.4 ± 1.88	12.6 ± 2.12	13.0±1.S4
Testis (g%)	0.67±0.052	0.68 ±0.070	0.67 ± 0.095	0.70 ± 0.037
Prostate (g%)	0.13 ± 0.039	0.12 ± 0.028	0.13±0.031	0.14 ± 0.047
Epididymis (g%)	0.24 ± 0.018	0.25 ± 0.022	0.25±0.030	0.25 ± 0.009
Female
Number of animals	10	10	10	8
Body weight (g)	313 ± 12.2	320±13.8	324 ± 11.5.	314±23.3
Absolute organ weight.
Pituitary (mg)	21.3±2.95	21.3±2.90	21.9±1.29	20.3±3.26
Thymus (mg.)	212 ± 56.5	232 ± 39.5	216±67.9	210 ± 79.7
Liver (g)	13.78±0.612	13.50 ± 1.493	15.09 ± 1.021	14.91 ± 1.189
Kidney (g)	1.98 ± 0.163	1.98 ± 0.134	2.02 ± 0.157	2.05 ± 0.180
Adrenal (mg)	71.7±11.83	71.9±9.19	77.7 ± 9.04	70.1 ± 9.05
Relative organ weight
Pituitary (mg%)	6.8 ± 0.94	6.7±0.80	6.8 ± 0.31	6.5±0.75
Thymus (mg%)	68 ± 18.3	73±12.4	67 ±22.6	65±22.8
Liver (g%)	4.41±0.172	4.22 ± 0.422	4.66 ± 0.259	4.75 ± 0.240
Kidney (g%)	0.63 ± 0.034	0.62±0.039	0.63 ± 0.038	0.65±0.073
Adrenal (mg%)	22.9 ± 3.33	22.5 ± 2.93	24.0 ± 2.60	22.3 ± 2.14
Values are expressed as Mean±S.D.

Table 2:Summary of necropsy findings in rats treated orally with thymol in the combined repeat dose and reproductive/developmental toxicity screening test
	Sex	Male					Female
Organ Findings	Fate	Scheduled sacrifice				Dead	Scheduled sacrifice
	Dose (mg/kg/day)	0	8	40	200	200	0	8	40	200
Number of animals		10	9	10	9	1#	10	10	10	9
Thymus
Small		0	0	0	0	0	0	0	1	1
Stomach
Thickening of wall in forestomach		0	0	0	9	1	0	0	0	1
Heart
Dilatation of atrium		0	0	0	0	1	0	0	0	0
Lung
Congestion		0	0	0	0	1	0	0	0	0
Liver
Congestion		0	0	0	0	1	0	0	0	0
Adrenal
Whitish		0	0	0	0	0	0	0	0	2
Brain
Dilatation of cerebral ventricle		0	0	0	1	0	0	0	0	0
#, One animal died at43days after commencement of treatment.

Table 5: Summary of histopathological findings in rats treated orally with thymol in the combined repeat dose and reproductive/developmental toxicity screening test
	Sex		Male					Female
Organ Findings	Fate		Scheduled sacrifice				Dead	Scheduled sacrifice
	Dose (mg/kg/day)		0	8	40	200	200	0	8	40	200
Number of animals			10	9	10	9	1*	10	10	10	9
Thymus
Involution		+	*	*	*	*	*	0	0	1	1
Stomach
Edema, forestomach		+	0	0	4	5	0	0/2&	0/5&	0/5&	0/5&
Erosion, forestomach		+	0	0	0	0	0	0/2	0/5	1/5	0/5
Hyperplasia, mucosa, forestomach		+	0	0	9	4	1	0/2	0/5	2/5	3/5
		++	0	0	0	5	0	0/2	0/5	0/5	1/5
Inflammatory cell infiltration, forestomach		+	0	0	6	9	0	0/2	0/5	0/5	2/5
Heart
Inflammatory cell infiltration, focal		+	6	*	*	1	0	0	*	*	1
Spleen
Extramedullar hematopoiesis		+	0	*	*	0	0	0	*	*	1
Lung
Congestive edema		++	*	*	*	*	1	*	*	*	*
Inflammatory cell infiltration		+	*	*	*	*	1	*	*	*	*
Liver
Congestion		+	0	*	*	0	1	0	*	*	0
Extramedullar hematopoiesis		+	0	*	*	0	0	0	*	*	1
Hemorrhage, focal		+	2	*	*	0	0	0	*	*	0
Microgranuloma		+	9	*	*	6	0	1	*	*	0
Kidney
Basophilic change, tubular epithelium		+	1	*	*	0	0	3	*	*	2
Calcification, corticomedullar junction		+	2	*	*	1	0	1	*	*	1
Cyst		+	1	*	*	0	0	1	*	*	0
Fibrosis, focal		+	0	*	*	0	0	1	*	*	1
Hyaline droplet, tubular elithelium		+	2	*	*	1	0	0	*	*	0
Testis
Atrophy, seminiferous tubule		+	1	*	*	0	0
Epididymis
Inflammatory cell infiltration		+	1	*	*	1	0
Prostate
Inflammatory cell infiltration		+	10	*	*	4	0
Pituitary
Cyst		+	0	*	*	0	0	0	*	*	1
Hyperplasia, rathke's pouch		+	1	*	*	0	0	0	*	*	0
Adrenal
Increase of fatty droplet, fascicular zone		+	0	*	*	0	0	0	0	0	1
Brain
Dilatation of cerebral ventricle		+	0	*	*	1	0	0	*	*	0
+, Slight; ++, Moderate; *, Not examined.
&, Number of animals showing lesion / number of animals examined.
#, One animal died at 43 days after commencement of treatment.

Conclusions:

The NOEL for repeat dose toxicity is 8 mg/kg bw/day for both sexes based on local irritation effects on the forestomach.

Executive summary:

Thymol was studied for oral toxicity in rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test according to OECD TG 422 at doses of 0 (vehicle), 8, 40, 200 mg/kg day. Clinical signs, behaviour, weight was recorded and hematological and biochemical parameters were determined. After termination of the study a pathological and histopathological examination was conducted. The NOEL for repeat dose toxicity is 8 mg/kg bw/day for both sexes based on local irritation effects on the forestomach.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 MAR 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thymol

EC Number:

201-944-8

EC Name:

Thymol

Cas Number:

89-83-8

Molecular formula:

C10H14O

IUPAC Name:

5-methyl-2-(propan-2-yl)phenol

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crj:CD, SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) 8 wks
- Weight at study initiation: (P) Males: 333-371 g; Females: 193-221 g
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 3% gum arabic solution

Details on mating procedure:

no data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

males: 43 days
females: from 14 days before mating to day 3 of lactation

Frequency of treatment:

daily

Details on study schedule:

terminal kill:
males: 44 days
females: day 4 of lactation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

8 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males + 10 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on the results of a pre-test performed at doses of 30, 100 and 300 mg/kg bw/d.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: every week and at termination of study

Oestrous cyclicity (parental animals):

estrous cycle in days was monitored

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals 42 d after commencement of treatment
- Maternal animals: All surviving animals at PND 4

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 3 were prepared for microscopic examination and weighed, respectively.

Statistics:

Bartlett, Kruskal-Wallis, Dunnett, Scheffé

Reproductive indices:

Mating index (%) = (Number of pairs with successful mating/number of pairs examined) x 100
Fertility index (%) = (Number of pregnant animals/number of pairs with successful mating) x 100
Gestation index (%) = (Number of females with live pups/number of pregnant females) x 100
Implantation index (%) = (Number of implantation sites/number of corpora lutea) x 100

Offspring viability indices:

Delivery index (%) = (Number of pups delivered/number of implantation sites) x 100
Live birth index (%) = (Number of live pups on day 0/number of pups delivered) x 100
Viability index (%) = (Number of live pups on day 4/number of live pups on day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased locomotor activity and an ataxic gait in females were observed in the 200 mg/kg group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One male rat died in the 200 mg/kg group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A tendency for suppression of body weight gain in males was observed in the 200 mg/kg group.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

gavage administration

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

gavage administration

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Decreased locomotor activity and an ataxic gait in females were observed in the 200 mg/kg group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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 (table 3).

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The duration of the oestrous cycle was unchanged by treatment (table 4).

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The compound exerted no effects on reproductive parameters such as the estrous cycle, mating index, fertility index, gestation length, number of corpora lutea or implatations, the implantation index, gestation index, delivery index, parturition or maternal behaviour. Birth weight and body weight gain tended to be low in the 200 mg/kg group neonates. This is considered to be caused by the higher litter size. 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 (please refer to table 4 and 5).

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The slightly reduced viability index in the 200 mg/kg bw dose group on PND 4 (table 5) is considered to be caused by the higher litter size.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The slightly reduced body weight of the pups on PND 0 and PND 4 in the 200 mg/kg bw dose group (table 5) is considered to be caused by the higher litter size.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Marginally reduced birth weight at 200 mg/kg bw is considered to be related to higher litter size.

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Remarks on result:

not measured/tested

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Absolute and relative organ weights in rats treated orally with thymol m the combined repeat dose and reproductive/developmental toxicity screening test
Dose (mg/kg/day)	0	8	40	200
Male
Number of animals	10	9	10	9
Body weight (g)	528 ± 23.5	510±24.7	519 ± 46.4	506±27.7
Absolute organ weight
Pituitary (mg)	14.3 ± 1.36	14.4±1.30	14.2±0.99	14.7 ± 1.45
Thymus (mg)	356 ± 54.4	353 ± 82.8	361±108.4	391±67.6
Liver (g)	14.48 ± 1.277	13.37 ± 0.817	14.55 ± 2.130	14.12 ± 1.455
Kidney (g)	3.14±0.186	3.07 ± 0.330	3.34±0.437	3.20 ± 0.211
Adrenal (mg)	70.2 ± 9.05	63.5 ± 11.04	64.9 ± 8.16	65.7±10.00
Testis (g)	3.54 ± 0.210	3.45 ± 0.229	3.48 ± 0.398	3.55 ± 0.175
Prostate (g)	0.71 ±0.209	0.63 ± 0.132	0.65±0.109	0.73±0.251
Epididymis (g)	1.28 ± 0.069	1.28 ± 0.091	1.29 ± 0.129	1.27 ± 0.058
Relative organ weight
Pituitary (mg%)	2.7 ± 0.28	2.8 ± 0.23	2.8 ± 0.19	2.9 ± 0.26
Thymus (mg%)	67±9.9	69 ± 16.2	69 ± 17.0	78 ± 16.3
Liver (g%)	2.74 ± 0.185	2.62±0.174	2.79 ± 0.203	2.79 ± 0.186
Kidney (g%)	0.59±0.032	0.60 ± 0.061	0.64 ± 0.058	0.64 ± 0,060
Adrenal (mg%)	13.3±2.07	12.4 ± 1.88	12.6 ± 2.12	13.0±1.S4
Testis (g%)	0.67±0.052	0.68 ±0.070	0.67 ± 0.095	0.70 ± 0.037
Prostate (g%)	0.13 ± 0.039	0.12 ± 0.028	0.13±0.031	0.14 ± 0.047
Epididymis (g%)	0.24 ± 0.018	0.25 ± 0.022	0.25±0.030	0.25 ± 0.009
Female
Number of animals	10	10	10	8
Body weight (g)	313 ± 12.2	320±13.8	324 ± 11.5.	314±23.3
Absolute organ weight.
Pituitary (mg)	21.3±2.95	21.3±2.90	21.9±1.29	20.3±3.26
Thymus (mg.)	212 ± 56.5	232 ± 39.5	216±67.9	210 ± 79.7
Liver (g)	13.78±0.612	13.50 ± 1.493	15.09 ± 1.021	14.91 ± 1.189
Kidney (g)	1.98 ± 0.163	1.98 ± 0.134	2.02 ± 0.157	2.05 ± 0.180
Adrenal (mg)	71.7±11.83	71.9±9.19	77.7 ± 9.04	70.1 ± 9.05
Relative organ weight
Pituitary (mg%)	6.8 ± 0.94	6.7±0.80	6.8 ± 0.31	6.5±0.75
Thymus (mg%)	68 ± 18.3	73±12.4	67 ±22.6	65±22.8
Liver (g%)	4.41±0.172	4.22 ± 0.422	4.66 ± 0.259	4.75 ± 0.240
Kidney (g%)	0.63 ± 0.034	0.62±0.039	0.63 ± 0.038	0.65±0.073
Adrenal (mg%)	22.9 ± 3.33	22.5 ± 2.93	24.0 ± 2.60	22.3 ± 2.14
Values are expressed as Mean±S.D.

Table 2: Summary of necropsy findings in rats treated orally with thymol in the combined repeat dose and reproductive/developmental toxicity screening test
	Sex	Male					Female
Organ Findings	Fate	Scheduled sacrifice				Dead	Scheduled sacrifice
	Dose (mg/kg/day)	0	8	40	200	200	0	8	40	200
Number of animals		10	9	10	9	1#	10	10	10	9
Thymus
Small		0	0	0	0	0	0	0	1	1
Stomach
Thickening of wall in forestomach		0	0	0	9	1	0	0	0	1
Heart
Dilatation of atrium		0	0	0	0	1	0	0	0	0
Lung
Congestion		0	0	0	0	1	0	0	0	0
Liver
Congestion		0	0	0	0	1	0	0	0	0
Adrenal
Whitish		0	0	0	0	0	0	0	0	2
Brain
Dilatation of cerebral ventricle		0	0	0	1	0	0	0	0	0
#, One animal died at43days after commencement of treatment.

Table 3: Summary of histopathological findings in rats treated orally with thymol in the combined repeat dose and reproductive/developmental toxicity screening test
	Sex		Male					Female
Organ Findings	Fate		Scheduled sacrifice				Dead	Scheduled sacrifice
	Dose (mg/kg/day)		0	8	40	200	200	0	8	40	200
Number of animals			10	9	10	9	1*	10	10	10	9
Thymus
Involution		+	*	*	*	*	*	0	0	1	1
Stomach
Edema, forestomach		+	0	0	4	5	0	0/2&	0/5&	0/5&	0/5&
Erosion, forestomach		+	0	0	0	0	0	0/2	0/5	1/5	0/5
Hyperplasia, mucosa, forestomach		+	0	0	9	4	1	0/2	0/5	2/5	3/5
		++	0	0	0	5	0	0/2	0/5	0/5	1/5
Inflammatory cell infiltration, forestomach		+	0	0	6	9	0	0/2	0/5	0/5	2/5
Heart
Inflammatory cell infiltration, focal		+	6	*	*	1	0	0	*	*	1
Spleen
Extramedullar hematopoiesis		+	0	*	*	0	0	0	*	*	1
Lung
Congestive edema		++	*	*	*	*	1	*	*	*	*
Inflammatory cell infiltration		+	*	*	*	*	1	*	*	*	*
Liver
Congestion		+	0	*	*	0	1	0	*	*	0
Extramedullar hematopoiesis		+	0	*	*	0	0	0	*	*	1
Hemorrhage, focal		+	2	*	*	0	0	0	*	*	0
Microgranuloma		+	9	*	*	6	0	1	*	*	0
Kidney
Basophilic change, tubular epithelium		+	1	*	*	0	0	3	*	*	2
Calcification, corticomedullar junction		+	2	*	*	1	0	1	*	*	1
Cyst		+	1	*	*	0	0	1	*	*	0
Fibrosis, focal		+	0	*	*	0	0	1	*	*	1
Hyaline droplet, tubular elithelium		+	2	*	*	1	0	0	*	*	0
Testis
Atrophy, seminiferous tubule		+	1	*	*	0	0
Epididymis
Inflammatory cell infiltration		+	1	*	*	1	0
Prostate
Inflammatory cell infiltration		+	10	*	*	4	0
Pituitary
Cyst		+	0	*	*	0	0	0	*	*	1
Hyperplasia, rathke's pouch		+	1	*	*	0	0	0	*	*	0
Adrenal
Increase of fatty droplet, fascicular zone		+	0	*	*	0	0	0	0	0	1
Brain
Dilatation of cerebral ventricle		+	0	*	*	1	0	0	*	*	0
+, Slight; ++, Moderate; *, Not examined.
&, Number of animals showing lesion / number of animals examined.
#, One animal died at 43 days after commencement of treatment.

Table 4: Fertility and pregnancy data in rats treated orally with thymol in the combined repeat dose and reproductive/developmental toxicity screening test
Dose (mg/kg/day)	0	8	40	200
Estrous cycle (days)	4.3±0.41^c)	4.3 ± 0.35	4.6 ± 0.60	4.3 ± 0.42
Number of pairs examined	10	10	10	10
Number of pairs with successful mating	10	10	10	10
Mating index (%)^a)	100.0	100.0	100.0	100.0
Number of pregnant females	10	10	10	10
Fertility index (%)^b)	100.0	100.0	100.0	100.0
Pairing days until mating	2.3 ± 1.06	2.7 ± 1.16	3.0 ± 0.94	3.6 ± 3.89
Number of estrous stages without mating	0.0 ± 0.00	0.0 ± 0.00	0.0 ± 0.00	0.0 ± 0.00
a)  Mating index (%) = (Number of pairs with successful mating/number of pairs examined) x 100 b)  Fertility index (%) = (Number of pregnant animals/number of pairs with successful mating) x 100 c)  Values are expressed as Mean±S.D.

Table 5: Delivery and litter data in rats treated orally with thymol in the combined repeat dose and reproductive/developmental toxicity screening test
Dose (mg/kg/day)	0	8	40	200
Number of females examined	10	10	10	9
Number of females with live pups	10	10	10	8
Gestation index (%)^a)	100.0	100.0	100.0	88.9
Gestation length (days)	22.3 ± 0.48^f)	22.5 ± 0.53	22.4 ± 0.52	22.4 ± 0.52
Number of corpora lutea	17.2 ± 1.81	17.5±1.72	18.3 ± 2.26	17.9 ± 1.97
Number of implantation sites	15.9 ± 1.37	16.5 ± 1.95	17.2 ± 2.57	15.6 ± 4.58
Implantation index (%)^b)	92.9	94.4	93.9	86.9
Delivery index (%)^c)	96.7	90.7	93.5	84.9
Number of pups delivered	15.4 ± 1.78	15.0 ± 2.16	16.0 + 2.05	16.4 ± 1.77
Number of live pups on day 0	15.3 ± 1.70	14.8 ± 2.10	15.8 ± 1.81	16.1 ±1.73
Live birth index (%)^d)	99.4	98.7	99.0	98.5
Sex ratio (male/female)	0.83 (70/84)	0.74 (64/86)	1.05 (82/78)	0.87 (61/70)
Number of live pups on day 4	15.3 ± 1.70	14.6 ± 2.46	15.6 ± 1.90	15.3±1.98
Viability index on day 4 (%)^e)	100.0	98.3	98.7	94.9
Body weight of pups (g)
on day 0 male	6.8 ± 0.66	6.7 ± 0.53	6.5 ± 0.85	6.1 ± 0.56
female	6.4 ± 0.62	6.3 ± 0.52	6.1 ± 0.78	5.8 ± 0.46
on day 4 male	10.8 ±1.06	10.7 ± 1.07	10.5 ±1.09	9.7±1.29
female	10.2 ± 1.06	10.2 ±1.06	10.2 ± 1.11	9.3 ± 1.16
Body weight gain of pups (g)
day 0 to 4 male	4.1 ± 0.61	4.0 ± 0.59	4.0 ± 0.48	3.5 ± 0.79
female	3.8 ± 0.63	3.9 ± 0.57	4.1 ± 0.53	3.4 ± 0.78
a)   Gestation index (%) = (Number of females with live pups/number of pregnant females) x 100 b)   Implantation index (%) = (Number of implantation sites/number of corpora lutea) x 100 c)   Delivery index (%) = (Number of pups delivered/number of implantation sites) x 100 d)   Live birth index (%) = (Number of live pups on day0/number of pups delivered) x 100 e)   Viability index (%) = (Number of live pups on day4/number of live pups on day0) x 100 f)    Values are expressed as Mean + S.D.

Applicant's summary and conclusion

Conclusions:

The NOELs for reproductive and developmental toxicity are considered to be 200 mg/kg/day for parental males and females, and 200 mg/kg/day for offspring. The marginally reduced birth weight at 200 mg/kg bw was related to higher litter size.

Executive summary:

Thymol was studied for oral toxicity in rats in an OECD combined repeated dose and reproductive/developmental toxicity screening test according to OECD TG 422 at doses of 0 (vehicle), 8, 40, 200 mg/kg day.

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 behaviour. Birth weight and body weight gain tended to be low in the 200 mg/kg group neonates. This is considered to be related to the higher litter size. 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 200 mg/kg/day for offspring (marginaly reduced birth weight at 200 mg/kg bw was related to higher litter size).