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Toxicological information

Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
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 mg/m³ 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:
other: acute inhalation toxicity
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hsd:Cpb:WU
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 2 months old
- Weight at study initiation: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex.
- Housing: singly in conventional Makrolon® Type III H cages.
- Diet: ad libitum (ssniff® R/M-H pellets maintenance diet for rats and mice)
- Water: ad libitum (tap water)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-60
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h; artificial light from 6.00 a.m. to 6.00 p.m. Central European Time; approximately 14 watt/m2 floor area (nominal)

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.1 µm
Geometric standard deviation (GSD):
1.8
Remark on MMAD/GSD:
MMAD for 4225 mg/m³ group: 3.16 µm, GSD: 1.63
for 5038 mg/m³ group: 3.07 µm, GSD: 1.97
Mass <3 μm: 46.0% for 4225 mg/m³ group and 50.1% for 5038 mg/m³ group
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).

Mode of exposure:
Animals were exposed to the aerosolized test substance in Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animals’ size. These restrainers were designed so that the rat's tail remained outside the restrainer, thus restrained-induced hyperthermia can be avoided. This type of exposure principle is comparable with a directed-flow exposure design and is preferable to whole-body exposure on scientific and technical reasons. Moreover, contamination of the haircoat can largely be avoided and confounding effects as a result of uptake of test substance by non-inhalation routes are minimized.

Description of apparatus:
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.

Atmosphere generation by nebulization:
Under dynamic conditions, the targeted concentrations were achieved by nebulization using the nozzle-baffle-systems and inhalation chamber. For aerosol generation a modified BGI 1- or 2-nozzle Collison nebulizer (Type CN-25 MRE, BGI Inc., Waltham MA, USA) was used in order to attain a temporally stable concentration of exposure atmospheres.
Temperature control used digitally controlled thermostat. Targeted concentrations were attained by secondary dilution air flow.

Characterization of Aerodynamic Particle-Size Distribution
The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor.

Control Animals
To identify exposure-related effects, comparisons with an appropriate historical control were performed. This control was exposed to an atmosphere using essentially similar exposure conditions as were used for the test substance (15 L air/min; conditioned air; duration of exposure = 1 x 4 h; 5 rats/sex/group);
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
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter. Individual weights are also recorded at death, if applicable. The period of observation was for 2 weeks. The appearance and behavior of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were made once a day (morning). Assessments from restraining tubes were made only if
unequivocal signs occurred (e.g. spasms, abnormal movements, and severe respiratory signs). Following exposure, observations are made and recorded systematically; individual records are maintained for each animal.
- Necropsy of survivors performed: yes
- Other examinations performed: The rectal temperatures were measured shortly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats.
Statistics:
Body weights: Means and single standard deviations of body weights are calculated. Mean body weights are also depicted graphically as a function of time. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.
Physiological data: Data of rectal temperature measurements are statistically evaluated using the ANOVA procedure (vide infra).
Calculation of the LC50: If calculation of a median lethal concentration (LC50) is possible, it is performed by computer (PC) according to the method of Rosiello et al. (1977) as modified by Pauluhn (1983). This method is based on the maximumlikelihood
method of Bliss (1938). If only 2 pairs of values with greater than 0% lethality and less than 100% are available then the first linear approximation is based on these values and a chi²-homogeneity test is not performed. In this case the interpolated concentration at 50% lethality is designated the approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.

Results and discussion

Effect levels
Key result
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
Mortality:
2/3 male rats and 1/3 female rats died in the highest dose group. None died in the 4225 mg/m³ dose group.
Clinical signs:
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;
CNS-related effects were rapidly reversible. Bradypnea and labored breathing patterns were observed up to postexposure day 10.
Body weight:
transient decrease in body weights
Gross pathology:
Animals succumbing during the observation period: 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.

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:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
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/m³

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.