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Diss Factsheets

Toxicological information

Repeated dose toxicity: inhalation

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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

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 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 / concentrationsopen allclose all
Dose / conc.:
0.009 mg/L air (analytical)
Remarks:
0.1% thymol (Article‐1)
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

open allclose all
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
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.