Carvacrol

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined Repeated Dose and Reproduction/Developmental Toxicity Screening Test

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Original study in Japanese

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

4. DATA MATRIX

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined Repeated Dose and Reproduction/Developmental Toxicity Screening Test

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the read-across justification.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOEL

Effect level:

8 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

8 mg/kg bw/day (nominal)

System:

other: various

Organ:

adrenal glands

thymus

Treatment related:

yes

Conclusions:

In a combined repeat dose and reproductive/developmental toxicity screening test in Crj:CD (SD) rats, the NOEL for males and females was 8 mg/lkg bw/day.

Executive summary:

In a combined repeated dose and reproduction/developmental toxicity screening test (4L421), Thymol (99.6%) was administered to four groups of Crl:CD (SD) rats (10 animals/sex/group) by gavage in 3% gum arabic solution at dose levels of 0, 8, 40 and 200 mg/kg bw/day, 7 days per week, for 43 days (males) and 14 days before mating to day 3 of lactation (females).

One male in the 200 mg/kg bw/day group died 43 days after the start of administration. Although there was no change in the general condition of this animal, autopsy revealed thickening of the anterior stomach wall, dilation of the atrium, congestion of the liver and lung. Pathological examination revealed mild proliferation of the forestomachial epithelium, mild congestion of the liver, moderate congestive edema in the lung and infiltration of mild inflammatory cells.  One female in the 200 mg/kg bw/day group died due to mis-delivery on the 18th gestation (33 days after starting administration).

There was no significant difference in males in the 200 mg/kg bw/day group, but the body weight and the weight gain increased slightly lower than that of the control group after 14 days from the start of administration and showed a tendency to suppress weight gain. In the 200 mg/kg bw/day group, transient locomotor activity reduction and walking ataxia after administration in a small number of females were observed continuously or intermittently from 1 to 13 days after the start of administration. In addition, transient salivation immediately after administration continued from 13th day after the start of administration in females, intermittently from 0th day of gestation (15th day after starting of administration) in females, and until the end of administration, males In almost all cases, half of the females.

Statistically significant changes in the % of lymphocytes and monocytes at higher doses in males were within the historical data of the lab and are not treatment-related. A statistically significant decrease in triglycerides at 8 mg/kg bw/day was not seen at other doses and was not considered treatment-related. There was no significant difference between the control group and the test substance-administered group in absolute weight and relative weight in either organ in both sexes.

Upon necropsy, thickening of the anterior stomach wall was found in 9 males and 1 female in the 200 mg/kg bw/day group. The surface of the thickened forestomach mucosa became whitened and exhibited roughness. In addition, miniaturization of the thymus was observed in 1 case in females in the 40 and 200 mg/kg bw/day group and in 2 females in the 200 mg/kg bw/day group of whitening of the adrenal glands.

Changes caused by the test substance were observed in the forestomach, thymus and adrenal glands upon histopathological analysis. In the forestomach, changes mainly consisting of mucosal epithelial hyperplasia were observed in both males and females in the 40 and 200 mg/kg bw/day groups, and the forestomach mucosa was thickened by stratified squamous epithelium hyperplasia and hyperkeratosis. A few cases were found in submucosal tissues with invasion of inflammatory cells and edema in males and few in females. Regression in the thymus was observed in each case in 40 and 200 mg/kg bw/day females. These two cases were examples showing the miniaturization of the thymus at necropsy. In the adrenal gland, an increase in lipid droplets in the cortical bundle band was observed in one female in the 200 mg/kg bw/day group. This example was one out of two cases showing macroscopically whitening of the adrenal glands. No change was observed in the other animal of the same group and female adrenal glands of the 8 and 40 mg/kg bw/day groups.

The NOEL for both sexes was 8 mg/kg bw/day.

This combined repeated dose and reproduction/developmental toxicity screening study in the rat is acceptable and satisfies the guideline requirement for this oral study (OECD 422) in rats.

Data source

Reference

Reference Type:

study report

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)

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:

thymol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Thymol, Wako Pure Chemical Industries, Ltd.,Lot No. CAN 1119
- Purity 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, sealed and shielded from light.
- Stability under test conditions: Confirmed by the manufacturer that it was stable during the test period.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co., Ltd
- Age at study initiation:8 weeks
- Weight at study initiation:333 to 371 g for males and 193 to 221 g for females
- Housing: Animals were placed in a polycarbonate cage with an experimental animal floor covering (Beta chip: Nippon Charles River Co., Ltd.) 1 cage per cage, 1 male and 1 female during the mating period
- Diet: autoclaved sterilized solid feed for experimental animals (CRF-1: Oriental Yeast Co., Ltd.)
- Water: UV irradiated tap water ad libitum
- Acclimation period:6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 25 °C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12 times / hour, lighting 12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 3% aqueous solution of arabic gum

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of the administration solution was carried out under yellow lamp lighting and stored under refrigeration and light shielding until application for administration. It was confirmed that the test substance in the administration solution was stable under the preservation conditions and that almost the prescribed amount of the test substance was uniformly contained in the administration solution used.

VEHICLE
- Amount of vehicle (if gavage): 5 ml / kg

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: 43 days
Females:14 days before mating to day 3 of lactation

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

8 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males
10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Malen and female SD rats were dosed at 30, 100 and 300 mg/kg bw/day from 13 days before mating for a total of 17 days for males and 7-9 days for females after mating confirmation. Symptoms noted included body weight loss, slowed breathing, ptosis, salivation nad locomotor activity reduction at 300 mg/kg bw/day. Only salivation was noted at 100 mg/k bw/day. Based on this sighting study, the doses selected for the main study was 8, 40 and 200 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes (appearance and behaviour)
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations:Body weight is measured for the males once a week on the administration start day and once a week thereafter, for the females on the administration start date and once a week until mating, after mating at 0, 7, 14, 20 gestation days and 0 and 4 days of gestation.

HAEMATOLOGY: Yes
Males: All male survivors were fasted for about 21 hours from the day before the dissection day and part of the blood collected from the posterior vena cava under anesthesia by intraperitoneal administration of thiopental sodium.

CLINICAL CHEMISTRY: Yes
Males: For all male surviving animals, blood collected on the day of dissection

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Both sexes were exsanguinated by necropsy of the abdominal aorta under anesthesia by intraperitoneal administration of sodium thiopental for all surviving animals and necropsied for necropsy and the pituitary gland, thymus, liver, kidney, adrenal glands, testis, epididymis and The weight of the prostate was measured. In addition to these organs, brain, heart, spleen and ovaries were collected and fixed with 10% neutral phosphate buffered formalin solution (testis and epididymis was Bouin's solution) and then stored.

HISTOPATHOLOGY: Yes
Histopathological examination was performed in control and 200 mg / kg group of male and female brain, pituitary, heart, liver. Hematoxylin and eosin stained specimens were prepared and examined in the usual manner for the spleen, kidney, adrenal glands, testis, epididymis, prostate and female thymus which changed in autopsy according to a conventional method. As a result, the influence of the test substance on the female thymus and adrenal glands was suspected, and thus these organs in the female 8 and 40 mg / kg group were examined.

Since macroscopic changes due to the test substance were observed in the stomach at the time of necropsy of the males, male stomachs of all groups in each group were examined, and females also received control, 8, 40 and 200 mg / kg each of 2 groups , 5, 5 and 5 cases of stomach were taken as a representative example, and a tissue examination was carried out. For some examples of adrenal glands, oil red O staining was performed.

Statistics:

Parametric data was tested for equal variance by the Bartlett method, and if variance was uniform, one-way analysis of variance was performed. In the case of uneven variance and nonparametric data Kruskal-Wallis's test was done. Multiple comparisons were made by Dunnett method or Dunnett method if the number of cases in each group was fixed when there was a significant difference between groups, Scheff method or Scheff type if unspecified. The counting data was tested by Fisher's direct stochastic method. For the data on newborn babies, the average value calculated for each mother animals was used as the statistical unit.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, transient locomotor activity reduction and walking ataxia after administration in a small number of females were observed continuously or intermittently from 1 to 13 days after the start of administration. One of them developed symptoms on gestation day 19 and nursing day 1, and showed ablation on 1 to 2 days of nursing. In addition, transient salivation immediately after administration continued from 13th day after the start of administration in females, intermittently from 0th day of gestation (15th day after starting of administration) in females, and until the end of administration, males In almost all cases, half of the females. In addition, black mucous leakage from the vagina was observed on the 24th day of pregnancy in 1 non-delivered animal in the 200 mg/kg bw/day group.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male in the 200 mg/kg bw/day group died 43 days after the start of administration. Although there was no change in the general condition of this animal, autopsy revealed thickening of the anterior stomach wall, dilation of the atrium, congestion of the liver and lung. Pathological examination revealed mild proliferation of the forestomachial epithelium, mild congestion of the liver, moderate congestive edema in the lung and infiltration of mild inflammatory cells.

One female in the 200 mg/kg bw/day group died due to misdelivery on the 18th gestation (33 days after starting administration).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no significant difference in males in the 200 mg / kg group, but the body weight and the weight gain increased slightly lower than that of the control group after 14 days from the start of administration and showed a tendency to suppress weight gain.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes in the % of lymphocytes and monocytes at higher doses in males were within the historical data of the lab and are not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in triglycerides at 8 mg/kg bw/day was not seen at other doses and was not considered treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no significant difference between the control group and the test substance-administered group in absolute weight and relative weight in either organ in both sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The thickening of the anterior stomach wall was found in 9 males and 1 female in the 200 mg/kg bw/day group. The surface of the thickened forestomach mucosa became whitened and exhibited roughness. In addition, miniaturization of the thymus was observed in 1 case in females in the 40 and 200 mg/kg bw/day group and in 2 females in the 200 mg/kg bw/day group of whitening of the adrenal glands.

In addition, as an incidental change, expansion of the ventricle was observed in one males in the 200 mg/kg bw/day group.
Since evidence of esophageal perforation was observed in one males in the 8 mg/kg bw/day group, it was judged that there was an error in administration, and all data were excluded from summary.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes caused by the test substance were observed in the forestomach, thymus and adrenal glands.

In the forestomach, changes mainly consisting of mucosal epithelial hyperplasia were observed in both males and females in the 40 and 200 mg/kg bw/day groups, and the forestomach mucosa was thickened by stratified squamous epithelium hyperplasia and hyperkeratosis. A few cases were found in submucosal tissues with invasion of inflammatory cells and edema in males and few in females.

Regression in the thymus was observed in each case in 40 and 200 mg/kg bw/day females. These two cases were examples showing the miniaturization of the thymus at necropsy.

In the adrenal gland, an increase in lipid droplets in the cortical bundle band was observed in one female in the 200 mg/kg bw/day group. This example was one out of two cases showing macroscopically whitening of the adrenal glands. No change was observed in the other group of the same group and female adrenal glands of the 8 and 40 mg/kg bw/day group.

Besides these effects, various other changes observed in the control or test substance administration group were all spontaneously observed and were not judged treatment-related.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

NOEL

Effect level:

8 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

8 mg/kg bw/day (nominal)

System:

other: various

Organ:

adrenal glands

thymus

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

In a combined repeat dose and reproductive/developmental toxicity screening test in Crj:CD (SD) rats, the NOEL for males and females was 8 mg/lkg bw/day.

Executive summary:

In a combined repeated dose and reproduction/developmental toxicity screening test (4L421), Thymol (99.6%) was administered to four groups of Crl:CD (SD) rats (10 animals/sex/group) by gavage in 3% gum arabic solution at dose levels of 0, 8, 40 and 200 mg/kg bw/day, 7 days per week, for 43 days (males) and 14 days before mating to day 3 of lactation (females).

One male in the 200 mg/kg bw/day group died 43 days after the start of administration. Although there was no change in the general condition of this animal, autopsy revealed thickening of the anterior stomach wall, dilation of the atrium, congestion of the liver and lung. Pathological examination revealed mild proliferation of the forestomachial epithelium, mild congestion of the liver, moderate congestive edema in the lung and infiltration of mild inflammatory cells.  One female in the 200 mg/kg bw/day group died due to mis-delivery on the 18th gestation (33 days after starting administration).

There was no significant difference in males in the 200 mg/kg bw/day group, but the body weight and the weight gain increased slightly lower than that of the control group after 14 days from the start of administration and showed a tendency to suppress weight gain. In the 200 mg/kg bw/day group, transient locomotor activity reduction and walking ataxia after administration in a small number of females were observed continuously or intermittently from 1 to 13 days after the start of administration. In addition, transient salivation immediately after administration continued from 13th day after the start of administration in females, intermittently from 0th day of gestation (15th day after starting of administration) in females, and until the end of administration, males In almost all cases, half of the females.

Statistically significant changes in the % of lymphocytes and monocytes at higher doses in males were within the historical data of the lab and are not treatment-related. A statistically significant decrease in triglycerides at 8 mg/kg bw/day was not seen at other doses and was not considered treatment-related. There was no significant difference between the control group and the test substance-administered group in absolute weight and relative weight in either organ in both sexes.

Upon necropsy, thickening of the anterior stomach wall was found in 9 males and 1 female in the 200 mg/kg bw/day group. The surface of the thickened forestomach mucosa became whitened and exhibited roughness. In addition, miniaturization of the thymus was observed in 1 case in females in the 40 and 200 mg/kg bw/day group and in 2 females in the 200 mg/kg bw/day group of whitening of the adrenal glands.

Changes caused by the test substance were observed in the forestomach, thymus and adrenal glands upon histopathological analysis. In the forestomach, changes mainly consisting of mucosal epithelial hyperplasia were observed in both males and females in the 40 and 200 mg/kg bw/day groups, and the forestomach mucosa was thickened by stratified squamous epithelium hyperplasia and hyperkeratosis. A few cases were found in submucosal tissues with invasion of inflammatory cells and edema in males and few in females. Regression in the thymus was observed in each case in 40 and 200 mg/kg bw/day females. These two cases were examples showing the miniaturization of the thymus at necropsy. In the adrenal gland, an increase in lipid droplets in the cortical bundle band was observed in one female in the 200 mg/kg bw/day group. This example was one out of two cases showing macroscopically whitening of the adrenal glands. No change was observed in the other animal of the same group and female adrenal glands of the 8 and 40 mg/kg bw/day groups.

The NOEL for both sexes was 8 mg/kg bw/day.

This combined repeated dose and reproduction/developmental toxicity screening study in the rat is acceptable and satisfies the guideline requirement for this oral study (OECD 422) in rats.