Carvacrol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeated Dose and Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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

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: 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: % gum arabic solution

Details on 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

Details on mating procedure:

For females, vaginal plaques were collected in the morning for 15 days from the start of administration, and Giemsa staining was performed to examine the sex cycle. After the administration before mating, pairs of males and females 1 were established within each group, living together for a maximum of 14 days for day and night, female vaginal plaques were collected every morning and examined in the morning. In cases where vaginal plug formation or sperm was found in vaginal plaque specimens, mating was established, and that day was taken as the 0th day of pregnancy. The mated pairs were separated from male and female and submitted for further examination.

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

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

The number of estrus stages missed by mating was calculated.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
Testis, epididymis and the weight of the prostate was measured

Litter observations:

On day 0 of nursing, the number of pups born, the number of stillborn pups, the sex and the presence or absence of abnormal outer table were inspected. Thereafter, the general condition, the presence or absence of death was observed every day. On the 0 and 4th day of nursing, measurements were made in each sex with each sex, and the average value of each was calculated. Also, the body weight gain was calculated based on the body weight on day 0 of nursing.

Postmortem examinations (parental animals):

All females confirmed copulation were allowed to spontaneously deliver, and the delivery status was observed. The animals whose delivery was completed at 9:00 a.m. were made on that date, and that day was designated as day 0 of nursing. After that, the newborn was allowed to nurture until 4th day of birth (4th day of nursing), and the nursing condition such as general condition, lactation, nesting, presence or absence of feeding was observed every day.

At dissection on 4th day of nursing, the ovaries and uterus were removed and the number of corpus luteums and the number of landings were examined. A female who did not deliver after 25 days after the copulation was confirmed was necropsied, and the uterus of an animal to which implantation was not observed macroscopically was immersed in a 2% KOH aqueous solution and the presence or absence of implantation was confirmed.

Postmortem examinations (offspring):

Dead animals were immersed in 10% neutral phosphate buffered formalin solution and fixed, except for those that could not withstand the examination by killing, and then autopsied under a stereoscopic microscope. For all surviving pups, on the 4 th day of nursing, the external surface including the oral cavity was examined, laparotomized under anesthesia by intraperitoneal administration of sodium thiopental, aborted by abdominal aortic dissection and necropsied by exsanguination.

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 pups, the average value calculated for each mother animals was used as the statistical unit.

Reproductive indices:

Number of estrus stages missed by mating,
Mating rate ([number of copulatory animals / number of living animals] × 100),
Conception rate ([ Number of conceived animals / number of copulatory animals] × 100)
Pregnancy period (the period from the 0th day of gestation to the day when birth was confirmed),
Birth rate ([live birthing females / female conception number] × 100),
Implantation rate ([ Number of corpus luteum] × 100),
Delivery rate ([total number of births infant / landing number] × 100)

Offspring viability indices:

The number of births (number of births / total number of babies) × 100) and 4-day survival rate ([number of nursing 4 day births / number of births births] × 100) were calculated from the number of surviving children on nursing 0 and 4 .

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

Dermal irritation (if dermal study):

not examined

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/kw bw/ 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:

no effects observed

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Most of each group showed a normal sexual cycle of 4 to 5 days, and no significant difference was observed between the control group and the test substance administration group for both the copulation rate and the conception rate. In addition, most females mated within 4 to 5 days after the start of mating, and there was no significant difference between the number of days required for copulation and the number of estrus periods missed by copulation.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Most of each group showed a normal sexual cycle of 4 to 5 days, and no significant difference was observed between the control group and the test substance administration group for both the copulation rate and the conception rate. In addition, most females mated within 4 to 5 days after the start of mating, and there was no significant difference between the number of days required for copulation and the number of estrus periods missed by copulation.

Details on results (P0)

Childbirth and feeding status
One case of non-delivered animals was observed in the 200 mg/kg bw/day group, but all other mother animals showed normal labor. There was no significant difference between the control group and the test substance administered group in gestation period, luteinum count, implantation number, implantation rate, birth rate and delivery rate.
Three dead fetuses in the uterus were observed in non-delivering animals at necropsy. In addition, although the implantation number, implantation rate and delivery rate of the 200 mg/kg bw/day group showed somewhat lower values than the other groups, those that reflected the fact that the implantation number of non-delivered animals was three , And no abnormality was found in the test values of other animals of the same group.

In the observation period, in the 200 mg/kg bw/day group maternal animals that showed symptoms and scarring after 1 to 2 days of nursing, the intragastric milk volume was small on the first day of nursing and 5 of the 17 littermates died. No abnormality was found in any of the mother animals of any group.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormality was found in other newborn babies although abnormalities on the outer table, bulges of the auricular and umbilicus were observed in each case in the 40 mg/kg bw/day group. In addition, no abnormalities were found in neonates in each group in the general condition.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no significant difference between the control group and the test substance administered group for both the number of births, the number of births, sex ratio, birth rate and neonatal survival rate.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no significant difference in the 200 mg/kg bw/day group, but the body weights at 0 and 4 days of nursing and the body weight gain during that were slightly lower in both males and females than in the control group. In the 8 and 40 mg/kg bw/day group, both males and females showed almost the same values as the control group.

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 examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

In the surviving animals, the cervical residuals of the thymus were observed in 2 cases in the 8 mg/kg bw/day group and in the case where the umbilical hernia showed an abdominal bulge in the group of 40 mg/kg bw/day, but no abnormality was found in other neonates.
For stillborn pups, patent foramen oval holes were found in 8, 40 and 200 mg/kg bw/day groups, respectively, in 1, 2 and 1 cases. No abnormalities were found in deaths after 1 day of nursing.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Applicant's summary and conclusion

Conclusions:

In a combined repeated dose and reproductiond/evelopmental toxicity screening test with Thymol in Crj:CD (SD) rats, the The NOAEL for reproductive and developmental toxicity are considered to be 200 mg/kg bw/day for parental males and females, and 40 mg/kg bw/day for offspring.

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

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.