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Toxicity to reproduction

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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

Constituent 1
Chemical structure
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 / concentrationsopen allclose all
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

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±0.41c)

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 ± 0.48f)

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).