Thymol

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two sequential 6‐month studies of identical design were conducted to assess the chronic toxicity of inhaled thymol in mice. Four treatment groups, (a) Air; (b) vehicle control; (c) Article‐1 (thymol 0.1%); and (d) Article‐2 (thymol 0.5%) were assessed in 128 mice for 26 weeks. The mice were sacrificed at the end of the treatment period and a histopathologic evaluation was performed with respect to lungs, bronchial lymph nodes, nasal passages/nasopharynx, and trachea.

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 animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Indianapolis, IN
- Age at study initiation: 7 weeks
- Weight at study initiation: approx. 30 g
- Housing: housed in a clean environment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

DETAILS OF FOOD AND WATER QUALITY: The routine diet for mice was 2016 Teklad Global 16% Protein Rodent Diet, purchased from Harlan Laboratories. No dietary contaminants were expected to interfere with the outcome of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26 (indicated as 64 ‐ 79°F)
- Photoperiod (hrs dark / hrs light): 12/12

All standard animal housing and handling procedures including sacrifice of the animals conformed to the lab facility standard operating procedures (SOPs), NIH Guide for the Care and Use of Laboratory Animals, and GLP guidelines. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC).

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

other: composition of metered dose inhaler (not further specified) but without active ingredients

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: In the studies, the test article was sprayed into a specially designed stainless steel 21.5‐L breathing tank. The tank size is designed such that the total breath volume of all eight mice in the 10‐minute study time (1.8 L) is less than 10% of the tank size (21.5 L). The internal wall of the tank is electrically polished so that the thymol adsorption will be reduced to minimal. Eight mice were mounted to the tank with four mice on each side.
- Method of holding animals in test chamber: small animal restraints were used
- System of generating particulates/aerosols: Metered dose inhaler; At the start of each treatment session, 15 sprays of test article were sprayed into the precleaned tank. In order to ensure consistent concentration of test article, a stirring fan installed inside the tank was set at 400 RPM and was started before the first spray of the test articles. Thirty seconds after the last spray (t = 0 minute), eight mice were mounted to the inhalation chamber to breathe the air from inside the breathing tank.

TEST ATMOSPHERE
- Brief description of analytical method used: validated LC‐MS method (please refer to 'Details on analytical verification of doses or concentrations')
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After spraying the study article into the tank, the actual concentration of thymol in the air of the breathing tank was determined by a validated LC‐MS method. First, the air sampling pump was connected to the Sorbent Sample Tube using Tygon tubing. The pump was preadjusted to 100 mL/min. The stirring fan was set at 400 RPM and started before the first spray of the test articles. The air sampling unit was then inserted into the tank. Thirty seconds after the last spray, the air sampling pump was turned on for 10 minutes. This drew 1000 mL of air through the sampling tube and thymol was captured on a coconut charcoal sorbent. Both the front and back ends of charcoal were desorbed into 1 mL ethyl acetate. Then, 0.1 mL of the ethyl acetate was transferred to 0.9 mL of the HPLC mobile phase (50/50 v/v Methanol/H2O with 0.1% formic acid) to analyze thymol concentration by LC‐MS. For each of the two test articles (Article‐1 0.1% thymol, and Article‐2 0.5% thymol), three replicates of determinations were performed. Between any two tests of these three replicates, a method blank was run to assure the data quality. Tank air was also sampled and tested before the sprays of thymol, after the sprays of thymol, as well as after washing/cleaning of the tank between any two treatment sessions in order to ensure removal of residual thymol.

Duration of treatment / exposure:

10 min

Frequency of treatment:

treatment sessions per week for 26 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.009 mg/L air (analytical)

Remarks:

0.1% thymol (Article‐1)

Doses / concentrations 2

Dose / conc.:

0.069 mg/L air (analytical)

Remarks:

0.5% thymol (Article‐2)

No. of animals per sex per dose:

16

Control animals:

yes, concurrent vehicle

yes, sham-exposed

Details on study design:

- Dose selection rationale: The amount of thymol in the two test articles is 10 and 50 times the amount of thymol normally used in the metered dose inhaler formulation.
- Rationale for animal assignment (if not random): random

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
After the last treatment, each mouse was sacrificed and four organs: (a) lungs, (b) bronchial lymph nodes, (c) nasal passages/nasopharynx, and (d) trachea were taken out and preserved in a labeled histology container prefilled with 10% Neutral Buffered Formalin. The organs were sent to Experimental Pathology Laboratories, Inc (EPL) for histopathologic evaluation. 45 pathologic assessment parameters (PAP) were assessed. Hematoxylin and eosin stained (H&E) slides of lung lobes, trachea, four levels of the nasal turbinates, and bronchial lymph nodes were prepared by EPL for microscopic evaluation by a board‐certified veterinary pathologist. The EPL rated the pathologic assessment parameters (PAPs) as Grade‐0 to 5, where
• GRADE‐0 = “Nothing Abnormal Discovered” (NAD);
• GRADE‐1 = minimal/very few/very small;
• GRADE‐2 = slight/mild/few/small;
• GRADE‐3 = moderate/moderate number/moderate size;
• GRADE‐4 = marked/many/large/moderately severe; and
• GRADE‐5 = massive/extensive number/extensive size/severe

Statistics:

Fisher's Exact Test (FET) and one‐sided t test for the occurence of pathologic assessment parameters (PAP)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

single treatment

Effect level:

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

Based on:

act. ingr. (total fraction)

Sex:

male/female

2

Dose descriptor:

NOAEL

Remarks:

3 treatments of 10 min per week

Effect level:

0.42 other: mg/kg bw/week

Based on:

act. ingr. (total fraction)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on this chronic toxicity study of mouse model, the no observed‐adverse‐effect level (NOAEL) of thymol by inhalation is 0.42 mg/kg bw/d for a single treatment and 1.26 mg/kg bw/week for a long term treatment.

Executive summary:

Two sequential 6‐month studies of identical design were conducted to assess the chronic toxicity of inhaled thymol in mice. Four treatment groups, (a) Air; (b) vehicle control; (c) Article‐1 (thymol 0.1%); and (d) Article‐2 (thymol 0.5%) were assessed in 128 mice for 26 weeks. The mice were sacrificed at the end of the treatment period and a histopathologic evaluation was performed with respect to lungs, bronchial lymph nodes, nasal passages/nasopharynx, and trachea. 45 pathologic assessment parameters (PAPs) were evaluated. No significant differences for chronic toxicity were found when comparing mice under long‐term repeated exposure of high doses of inhaled thymol and mice that inhaled no thymol. Based on this chronic toxicity study of mouse model, the no observed‐adverse‐effect level (NOAEL) of thymol by inhalation is 0.42 mg/kg bw/d for a single treatment and 1.26 mg/kg bw/week for a long term treatment.