Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to ECVAM ToxRTool reliability 1. Acute Inhalation Toxicity study, OECD 403, EU B.2 performed under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Principles of method if other than guideline:
One group 5 male and 5 female rats at aerosol concentration of 4225 and one group 3 male and 3 female rats at 5038 mg/m³ were nose-only exposed. The aerosol was generated neat without any vehicle (aerosolization of the molten crystalline, solid substance above the melting point). The animals were observed for mortality, weight and clinical signs untill day 14. A gross necropsy was performed.
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no

Test material

Constituent 1
Reference substance name:
Menthol
EC Number:
201-939-0
EC Name:
Menthol
Cas Number:
89-78-1
IUPAC Name:
2-isopropyl-5-methylcyclohexanol
Test material form:
aerosol dispenser: not specified
Remarks:
migrated information: aerosol
Details on test material:
Test substance: DL-MENTHOL
Batch/sample no.: CHHYDN0130
Identity/stability: 99.6%. Stability certified for the duration of study
Appearance: White crystalline solid
Characterization of chamber atmosphere - Mean values
Group 1 Group 2 Group 3
Target Conc. (mg/m³) 0 4500 5000
Gravimetric Conc. (mg/m³) -- 4225 5037.5

Cascade Impactor:
MMAD (μm) -- 3.16 3.07
GSD 1.63 1.97
Mass <3 μm (%) 46.0 50.1

Test animals

Species:
rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animal room environment was as follows:
Room temperature: 22 ± 3 C
Relative humidity: 40-60%
Dark/light cycle: 12 h/12 h; artificial light from 6.00 a.m. to 6.00 p.m. Central European Time
Light intensity: approximately 14 watt/m2 floor area (nominal)
Ventilation: approximately 10 air changes per hour

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Dry conditioned air was used to generate the test substance atmospheres as described below. The test atmosphere was conveyed through openings in the inner concentric cylinder of the chamber, directly towards the rats' breathing zone. This directed-flow arrangement minimizes re-breathing of exhaled test atmosphere (for details see Pauluhn and Thiel, 2007). Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. All air flows were monitored and adjusted continuously by means of calibrated and computer controlled mass-flow-controllers. A digitally controlled calibration flow meter was used to monitor the accuracy of mass-flow-controller. As demonstrated in Table 1, the ratio between supply and exhaust air was selected so that 90% of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Gas/aerosol scrubbing devices were used for exhaust air clean-up. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers. The pressure difference between the inner inhalation chamber and the exposure zone was 0.02 cm H20 (Pauluhn, 1994). The exposure system was accommodated in an adequately ventilated enclosure. The control/management of the inhalation chamber data, including the current calibration data, was performed using a computerized and validated data acquisition and control system (DAS).
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
0, 4225, or 5038 mg/m³
No. of animals per sex per dose:
5 male and 5 female animals per each control and low dose;
3 male and 3 female per high dose
Control animals:
yes

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
ca. 5 289 mg/m³ air
Based on:
act. ingr.
Exp. duration:
4 h
Remarks on result:
other: LC50 inhalation aerosol

Any other information on results incl. tables

Summary of acute inhalation toxicity - 4 hour exposure - Mean values

NGroup /sex       Target Concentration (mg/m³)       Toxicological Result       Onset and Duration of Signs       Onset of Mortality

1 / m                                   0                                          0 / 0 / 5                             --                                           --

2 / m                                    4500                                    0 / 5 / 5                             0d– 10d                                  --

3 / m                                    5000                                    2 / 1 / 3                             0d – 14d                             3h

1 / f                                    0                                           0 / 0 / 5                               --                                         --

2 / f                                    4500                                    0 / 5 / 5                             0d – 8d                             --

3 / f                                    5000                                    1 / 2 / 3                             0d – 8d                                  2h

N = group assignment, m = males, f = females, animals found dead 2-3h after onset of exposure (0h-

3h), * = p < 0.05, ** = p < 0.01.

Values given in the 'Toxicological results' column are: 1st = number of dead animals, 2nd = number of

animals with signs after cessation of exposure, 3rd = number of animals exposed.

Necropsy

The qualitative description given below focuses on key-findings only.

Animals succumbing during the observation period: The most salient findings are characterized by colorless discharge from nose and a viscous, white content in the nostrils; less collapsed lung; stomach: bloated; small intestine: reddened mucosa and red mucous content; parenchymatous organs: pallor.

Animals sacrificed at the end of the observation period: The macroscopic findings in surviving rats were essentially indistinguishable from the control.

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
DL-menthol was shown to be of low acute toxicity when applied as aerosol to rats. The LC50 (rat, aerosol, 4h) found was 5289 mg/m3 and therefore above the threshold for classification being 5 mg/l according to CLP (Regulation (EC) No 1272/2008).
Executive summary:

A study on the acute inhalation toxicity of DL-Menthol on rats has been conducted in accordance with OECD TG#403 (2009). Two groups of rats were exposed nose-only at actual aerosol concentration of 4225 and 5038 mg/m³. The aerosol was generated

neat without any vehicle (aerosolization of the molten crystalline, solid substance above the melting point).

The results can be summarized as follows:

LC50 (inhalation aerosol, 4 h) Approximate LC50-males&females: 5289 mg/m³1

NO(A)EL Males&females: <4225 mg/m³

The following clinical signs were observed: bradypnea, labored breathing pattern, dyspnea, motility reduced, atony, tremor, high-legged gait, staggering gait, movements uncoordinated, piloerection, haircoat ungroomed, nasal discharge (serous), nose and/or muzzle: red encrustations, nostrils: red encrustations, stridor, breathing sounds, apathy, narcosis, prostration, miosis, hypothermia, decreased reflexes, and transient decrease in body weights. The lead pathodiagnostic effects were suggestive of a narcotic condition associated with increased airway secretions/mucous membrane irritation. Consistent with this mode of action, mortality occurred at 5038 mg/m³ during the course of the exposure period. CNS-related effects were rapidly reversible. Bradypnea and labored breathing patterns were observed up to postexposure day 10.

The respirability of the aerosol was adequate to achieve the objective of study, i.e. the average mass median aerodynamic diameter (MMAD) was 3.1 μm, the average geometric standard deviation (GSD) was 1.8.

The aerosolized test substance proved to has a low acute inhalation toxicity in rats and a classification is not justified.