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Acute Toxicity: inhalation

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acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles.
Reason / purpose:
reference to same study

Data source

Reference Type:
The physiological response of animals to cyclohexane, methylcyclohexane, and certain derivatives of these compounds. II Inhalation
Treon, J. et al.
Bibliographic source:
J. Indust. Hygiene Toxicol. 25(8), 323-346

Materials and methods

Principles of method if other than guideline:
Determination of the minimum lethal concentration after repeated inhalation exposure
GLP compliance:
Test type:
other: minimum lethal concentration
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): methylcyclohexane
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: 97%
- Impurities (identity and concentrations): toluene, 3%

Test animals

not specified
not specified

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:
- Exposure apparatus: at higher exposure concentrations (within the inflammable range), experiments were performed in a 223 L metal cage as described in Machle et al. (1940, J. Indust. Hygiene Toxicol. 22:315 pp.). Long-term inhalation exposure experiments at lower concentrations were carried out in a battery of nine insulated 600 L plywood cages provided with a 24 x 18 inches front glass door.
- Source and rate of air: air was drawn from an inside corridor, passed through a steel filter and then through a Westinghouse electric "Precipiton" to remove particulates.
- Method of conditioning air: air was passed over a cooling coil and then over a steam reheating coil before it entered individual tubes which lead from the main duct to each individual cage.
- System of generating vapours: a Bosch fuel injection pump with an 11 mm plunger was used for measuring the liquid introduced and vaporised into the air stream of the metal cage. The liquid test material was stored in a 2-gallon stainless steel reservoir provided with an exit tube which led to a three-way stopcock allowing delivery i) directly from the reservoir, or ii) from the reservoir into a calibrated pipette used for daily check of liquid flow rate, or iii) directly from the pipette. The liquid test material either from the reservoir or the pipette was passed through a Cuno filter into a Viking supply pump and then further into the Bosch pump, where the required volume was measured by a micrometer setting, the excess liquid being recycled. The volatile and readily vaporised test material was then sprayed directly into the air stream through a Bosch injection nozzle.
- Temperature, humidity, pressure in air chamber: 23.9 ± 1.7 °C, ≤ 45%
- Air flow rate: 350-800 L/min
- Air change rate: ca. 0.6-1.3 per min
- Treatment of exhaust air: air was removed from the chamber through a hole at the rear side, beneath a metal screen supporting the animals, which entered a duct leading to the exhaust stack.

- Brief description of analytical method used: the test material concentrations were determined by different methods, depending on the concentration level. At concentrations levels from 4.57 to 59.9 mg/L, measurements were conducted by passing the sample over a 10-inch platinum star in a 1.25-inch fused silica tube for 10-60 min at 100 mL/min and 850°C, and weighing the resulting CO2 collected on ascarite. Concentration levels from 0.95 to 1.46 mg/L were determined by a combustible gas indicator having two electrically balanced circuits which passed through platinum filaments. One of these filaments is unbalanced by the burning of the test material. The degree of circuit unbalance is used as parameter to measure concentration after calibration of the instrument with known concentrations.
Analyses were performed every 15-30 min when using the combustible gas indicator method was used. In all other 6-hour exposure periods, daily measurements were conducted at the end of the first, third and fifth hour, respectively.
- Samples taken from breathing zone: yes
Analytical verification of test atmosphere concentrations:
Mean concentrations over the whole experimental exposure period were calculated from the daily mean values recorded. The mean values with their probable errors were: 0.948 ± 0.007, 4.57 ± 0.05, 11.35 ± 0.08, 21.90 ± 0.39, 28.75 ± 0.29, 39.55 ± 0.51 and 59
Duration of exposure:
6 h
0.948, 4.57, 11.35, 21.90, 28.75, 39.55 and 59.9 mg/L (241, 1162, 2886, 5567, 7308, 10054 and 15227 ppm)
No. of animals per sex per dose:
Control animals:
Details on study design:
- Control animals: five control groups (No. 1-5) of 4 rabbits each were exposed to clean air under the same experimental conditions as the treatment group. These animals were tested at various seasons of the year in order to determine the potential effects of housing in the air-conditioned cages.
Group No. 1 was kept in the chamber for 6 h/day, 5 days/week for 10 weeks. An average body weight loss of 59 g was noted, which was mainly due to a considerable body weight decrease of one animal having an intercurrent infection. The rate of body weight gain of the remaining animals was, however, lower than that of other groups. A mean group daily decrease of ca. 0.62 ± 0.04 °C in rectal temperature during the caging period.
Group No. 2 was kept in the air-conditioned chamber for 6 h/day, 5 days/week for 5 weeks. The mean group body weight gain was 285 g and the mean group daily rectal temperature was decreased by 0.36 ± 0.04 °C during the exposure period.
Groups No. 3 and 4 were caged for 6 h/day, 5 days/week for 11 and 10 weeks, respectively. In this time the mean group body weight gain was 626 and 483 g, respectively. Mean group daily rectal temperature decreases of 0.29 ± 0.04 and 0.27 ± 0.04 °C were observed in the respective groups. The same animals showed a mean group daily increase of about 0.24 ± 0.04 °C during the following two months when not caged in the inhalation chambers.
Group No. 5 was caged in an air-conditioned chamber for 8 h/day, 5 days/week for 26 weeks. The mean group body weight gain was 1580 g and showed a mean group daily decrease in rectal temperature of 0.21 ± 0.02 °C.
The authors pointed out that normal rabbits housed in the usual way would show a substantially uniform tendency to increase their body temperature during the day, and suggested a relationship between the decrease in body temperature and the fact that the animals were not fed during the exposure period.
- Experimental animals: rabbits selected for exposure were caged in the air-conditioned chamber for 6-8 h/day for 2-6 weeks prior to exposure to the test material. Body weight gain was normal during this period of time.

Results and discussion

Effect levelsopen allclose all
not specified
Dose descriptor:
Effect level:
59.9 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
70 min
not specified
Dose descriptor:
other: mortality after repeated inhalation exposure
Effect level:
28.75 - 39.55 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
6 h
Remarks on result:
other: 1/4 and 4/4 rabbits died after 2 weeks of repeated exposure (6 h/day, 5 days per week) at 28.75 and 39.55 mg/L, respectively
not specified
Dose descriptor:
other: minimum narcotic concentration
Effect level:
21.9 mg/L air
Based on:
test mat.
Exp. duration:
6 h
Remarks on result:
other: slight lethargy observed
0.948 to 21.90 mg/L: no mortality occurred.
28.75 and 39.55 mg/L: mortality occurred in 1/4 and 4/4 animals, respectively, after 2 weeks of exposure.
59.9 mg/L: mortality occurred in 4/4 within 70 min after exposure initiation.
Clinical signs:
0.948 to 11.35 mg/L: no effects.
21.90 mg/L: slight lethargy.
28.75 mg/L: lethargy and impaired coordination of legs.
39.55 mg/L: convulsions, light narcosis, laboured breathing, salivation, conjunctival congestion.
59.55 mg/L: severe convulsions, rapid narcosis, laboured breathing, salivation, conjunctival congestion.
Body weight:
Animals exposed to non-lethal concentrations gained weight. Mean group body weight gains were:
0.948 mg/L: 752 g (after 10 weeks)
4.57 mg/L: 956g (after 10 weeks)
11.35 mg/L: 328 g (after 3 weeks)
21.90 mg/L: 201 g (after 4 weeks)

Animals exposed to lethal concentrations lost weight. Mean group body weight gains were:
28.75 mg/L: -39 g (after 2 weeks)
39.55 mg/L: -390 g (after 2 weeks)
Other findings:
- Histopathology: After a 3-week exposure to 11.35 mg/L barely demonstrable evidence of cellular liver and kidney injury (not further specified) was reported.
- Potential target organs: CNS at high concentrations

Applicant's summary and conclusion

Interpretation of results:
other: STOT-SE 3 H336 May cause drowsiness and dizziness
Criteria used for interpretation of results: EU
The minimum lethal concentration of methylcyclohexane after repeated inhalation exposure was determined in rabbits. 4 animals per concentration level were whole body-exposed to methylcyclohexane vapours at 0.948, 4.57, 11.35, 21.90, 28.75, 39.55 and 59.9 mg/L, 6 h/day, 5 days/week for up to 10 weeks. Different groups of animals each exposed to clean air for different periods of time (6-8 h/day, 5 days/week for 10-26 weeks) served as control.
No mortality occurred in animals exposed to 0.948-21.90 mg/L. At 28.75 and 39.55 mg/L: mortality occurred in 1/4 and 4/4 animals, respectively, after 2 weeks of exposure. All 4 animals died within 70 min after exposure initiation at 59.9 mg/L. Therefore, 59.9 mg/L was the LC100 following single inhalation exposure to methylcyclohexane. An LC50 was not determined in the study. However, since no animals died after the first (and up to the ninth) exposure to 39.55 mg/L, the LC50 value is likely to be higher.
During the course of the study, no clinical signs of toxicity were observed in animals exposed to 0.948-11.35 mg/L. At higher concentrations, narcotic effects were noted and included lethargy, impaired leg coordination, convulsions, narcosis, laboured breathing, salivation and conjunctival congestion. The number and severity of observed effects increased with concentration and indicated that the central nervous system is a potential target at high concentrations. The minimum narcotic concentration was 21.90 mg/L based on the reported slight lethargy.
Body weight gain was not affected in animals exposed to non-lethal concentrations (0.948-21.90 mg/L), while animals exposed to lethal concentrations lost weight: 39 and 390 g after a 2 week exposure to 28.75 and 39.55 mg/L, respectively.
After a 3-week exposure to 11.35 mg/L barely demonstrable evidence of cellular liver and kidney injury (not further specified) was reported.

In this study, mortality after single and repeated exposure occurred only at high to very high concentrations of methylcyclohexane, which are above the currently valid LC50 ranges used for classification. Therefore, based on expert judgement, the study results do not fulfil the classification criteria for acute toxicity by inhalation according to Regulation (EC) No 1272/2008 and Directive 67/548/EEC. However, based on the narcotic effects consistently observed at high concentrations in this and further studies (Weight of Evidence), methylcyclohexane fulfils the classification criteria for Specific target organ toxicity-single exposure (STOT-SE) Category 3 (narcotic effects) according to Regulation (EC) No 1272/2008 and R67 (vapours may cause drowsiness and dizziness) according to Directive 67/548/EEC.

CLP: STOT-SE 3, H336
DSD: R67