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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:
key study
Study period:
February 23- May 26, 1994
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methylal
- Physical state: Colourless liquid
- Analytical purity: 99.9%
- Lot/batch No.: B-383-3-327, Drum #109
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Wistar Hoe: WISKf(SPF71)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: approximatively 5 - 6 weeks at beginning of the prestudy period
- Weight at study initiation: 149-166g for males; 151-166g for females
- Fasting period before study: not specified
- Housing: Animals were maintained individually in plastic tubes during exposure. They were maintained in groups of 5 animals between exposures in fully air-conditioned rooms in Makrolon cages (Type 4) on soft wood granulate.
- Diet (e.g. ad libitum): Altromin 1324 rat diet (Altrornin GmbH, LagelLippe), ad libitum, except for the exposures and the period in which the
animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum, except for the exposures and the period in which the animals were kept in diuresis cages
- Acclimation period: approximatively 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light (artificial light OSRAM Weiss-Universal white)

IN-LIFE DATES: From:February 23, 1994 To:May 25, 1994 (for males); From:February 24, 1994 To:May 26, 1994 (for females).

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not specified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The rats were placed individually in cylindrical plastic cages and exposed to test substance. The plastic tubes leading into the exposure chamber were arranged in such a way that only the noses of the animals were inside the chamber. The exposure chamber itself consisted of a stainless-steel and glass cylinder with a volume of 80 I, standing in a vent pipe with a volume of approx. 4 m3. The chambers have been tested and were shown to yield a uniform distribution of dusts in the different breathing zones of the animals.
- Method of holding animals in test chamber: Not specified
- Source and rate of air: Not specified
- Method of conditioning air: A suction device at the bottom of the exposure chamber drew off the dust through a washing flasks filled with water, a cotton-wool filter, a Buehler filter, and a calcium chloride flask.
Particles of test substance escaping from the exposure chamber into the vent pipe were drawn off and neutralised by gas-cleaning equipment.

- System of generating particulates/aerosols: Not relevant
- Temperature, humidity, pressure in air chamber: In order to ensure the atmospheric humidity required by the current guidelines, air was passed via a porous stone at a rate of 100 I/h through a 1000 ml vacuum filter flask filled with water. The moisturised air was then conducted straight into the exposure chamber.
Temperature is 20.5-23.6, 20.5-24.7, 21.5-24.2, 20.2-24.9°C for control group, animals exposed to 400, 2000, 10000ppm of test substance, respectively.
Humidity is 24.8-52.7, 27.1-53.0, 24.2-55.8, 20.9-49.6% for control group, animalsexposed to 400, 2000, 10000ppm of test substance, respectively.
- Air flow rate: The inhalation chambers operated under dynamic conditions. The different concentrations were achieved by evaporation of defined amounts of Methylal and subsequent diluting with an appropriate quantity of air. The total amount of air introduced into the chamber was 800 I per hour. Airflow through the chambers was held constant by means of air-calibrated rotameters.
- Air change rate: Air was drawn off from the chambers at a rate of 1100 I/h. The difference in rate between the introduction of air at 800 Vh and its extraction at 1100 l/h is necessary to maintain the kinetic energy of the aerosol particles and to ensure a largely laminar flow of the aerosol from the top to the bottom of the exposure chamber. A minimal quantity of additional air enters via the animal cages, but causes no significant dilution ofthe aerosol.
- Method of particle size determination: Not specified
- Treatment of exhaust air: Not specified

TEST ATMOSPHERE
- Brief description of analytical method used:
During the exposures, methylal concentration, CO, CO2, O2 in all exposure chambers were measured continuously by means of air-monitoring equipment manufactured by Hartmann & Braun (CO and CO2: Uras 2 T IR-gas-analyser, 02: Magnos 3 Oxygen-analyser, operating paramagnetically). Atmospheric humidity and temperature in the exposed groups were checked continuously by alternatively measuring the individual chambers. Measurement of temperature was performed via the CMR-transformer TEU 320 and humidity was checked by means of the transmitter HMT 12 manufactured by VAISALA.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: The test substance is volatile
- Composition of vehicle: not specified
- Type and concentration of dispersant aid (if powder): not applicable
- Concentration of test material in vehicle: Nominal concentrations of test material in vehicle are 0, 400, 2000, 10000 ppm. Measured mean concentrationsof test materialin vehicle are 0, 377, 1908, 9652 ppm.
- Lot/batch no. of vehicle (if required): not applicable
- Purity of vehicle: A suction device at the bottom of the exposure chamber drew off the dust through a washing flasks filled with water, a cotton-wool filter, a Buehler filter, and a calcium chloride flask.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Methylal in the exposure chambers was measured using IR-analysis via
one-beam photometers (Miran lA and Miran 80, Foxboro Analytical, South Norwalk, USA), which were calibrated for the test compound. Measurements were carried out at intervals of approximately 30 minutes.
Additionally, analytical examinations were performed. For this purpose, the test atmosphere was drawn through a gas sample vessel and the latter closed after an approx. 20fold gas exchange. The substance present in the gas sample vessel was analysed using gas chromatography. These measurements were carried out weekly for each concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week, 65 exposures within 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
400 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations of the present study were based on the results of earlier inhalation studies with the test compound.
- Rationale for animal assignment (if not random): Not applicable (random)
- Rationale for selecting satellite groups:Not applicable (no satellite group)
- Post-exposure recovery period in satellite groups: Not applicable (no satellite group)
- Section schedule rationale (if not random): Not applicable (random)
Positive control:
No positive control was used

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
The behaviour and general health condition of the animals were observed twice daily (before and after the exposure). During the exposures, observation of the animals was performed continuously. The animals were taken out of the tubes for a very short time for this purpose. At weekends and during the recovery period, the animals were observed once daily. The animals were examined weekly for neurological disturbances, opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of all animals were determined weekly throughout the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
Food consumption was determined continuously (one measurement per week). The values on the printouts refer to the interval between one measurement and the next. They are converted to the food consumption per 100 g body weight over a 24 hour period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was determined once weekly over a period of 16 hours and is given as water consumption / 100 g body weight / 16 h.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were carried out five days before the start of the study (males),
six days before the start (females), six days before termination (males) and seven days before termination (females), with the aid of a slit lamp.
- Dose groups that were examined: All dose groups were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study, haematological examinations were performed on all animals without previous withdrawal of food.
- Anaesthetic used for blood collection: Yes (Ketamin)
- Animals fasted: No
- How many animals: All animals of each group (expect for reticulocyte count and Heinz bodies that were scored only in the animals of the control group and the high concentration group.
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not specified
- Animals fasted: No data
- How many animals: All animals of each group
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine analysis was performed a few days before termination of the study (overnight from day 80 to 81 in females and day 81 to 82 in males).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 5)
Main organ weight (see table 6)
Statistics:
Body weights at the designated measurement times, haematological data, clinical chemistry parameters, urine data and absolute organ weights and organ to body weight ratios were compared statistically with the control group values at the level of significance p = 0.05.
Evaluation was performed by Pharma Research and Development Informatics with the aid of a program package for the evaluation of toxicological studies. One-way analysis of variance with sequentially rejective multiple comparisons (p<0.05) was used as calculation method for body weights at the designated measurement times, haematological data, clinical chemistry parameters, urine data. One-way analysis of variance based on ranks with sequentially rejective multiple comparisons (p<0.05) was used as calculation method for absolute and relative organ weights.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Animals exposed to 10 000 ppm Methylal showed disequilibrium, uncoordinated gait, ataxic gait and decreased spontaneous activity activity due to anesthetic effects previously reported in animals. These symptoms were reversible at the beginning of the next exposure. Behaviour and general health condition remained unaffected by exposure to the test substance in the other groups. Blood-coloured encrusted noses were observed in all groups including the control group. Therefore, this finding is considered not to be compound-related.
Compound-related neurological disturbances, opacity of the refracting media of the eyes, impairment of dental growth or changes in the oral mucosa were not observed.
No deaths occurred during the study.

BODY WEIGHT AND WEIGHT GAIN
The body weight gains were not impaired by the administration of the test substance. Slightly
higher body weights were observed in males of the low concentration group, which were
statistically significant from the control group from days 8 - 43 and 57 - 92. Additionally,
statistical evaluation revealed higher body weights in males of the intermediate concentration
group on day 22 of the study.

FOOD CONSUMPTION
The absolute and relative food consumption remained unaffected by the treatment throughout the study and was comparable in all groups.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Water consumption was slightly increased in both sexes of the high concentration group, the effect being more pronounced in females. The administration of the test compound did not alter the relative water consumption in the animals of the other groups.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic examination of the animals revealed no abnormalities.

HAEMATOLOGY
Haematological examinations revealed statistically significant decreases in erythrocyte counts and haematocrit values in males of the high concentration group. However, these changes were observed in one sex only and the values were well within the physiological range of rats. Furthermore, there were no signs indicative for anaemia observable by other parameters and by histopathological examination. Therefore, a compound-related effect is not evident.
Additionally, coagulation times were slightly, but statistically significantly decreased in females of the intermediate and high concentration group. As there was no concentration dependency, a compound-related effect is unlikely.
Evaluation of differential leucocyte count and red cell morphology revealed no abnormalities.
Heinz bodies were not detected.

CLINICAL CHEMISTRY
Statistically significant decreases in inorganic phosphate levels were found in males of the high concentration group. However, the changes were only slight and observed in one sex only. Additionally; increases in urea and GPT values were observed in females of the high dose group. These changes were also minor and occurred in one sex only. Furthermore, no changes were detected in kidney and liver by histopathological examination. Therefore, a compound-related effect is questionable.
In all other cases, where statistically significant changes occurred (decreases in potassium and bilirubin values in males of all concentration groups, increases in total lipid levels in males of the low concentration group, decreases in phosphate values in females of the low and high concentration group, decreases in calcium levels in females of the low concentration group) there was no concentration dependency. Therefore, a compound-related effect is unlikely.

URINALYSIS
Specific gravity of the urine was slightly, but statistically significantly decreased in females of the high concentration group and in males of the intermediate concentration group. In females, this finding was accompanied by an increased urine volume and may be a consequence of the increased water consumption observed in these animals. The sediments were inconspicuous.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Absolute and relative liver weights were increased to a statistically significant degree in females of the high concentration group. Liver to body weight ratios were also increased in males of the high concentration group. As both sexes were affected, a compound-related effect cannot be ruled out. However, there were no changes in this organ observable by histopathological examination.
Absolute and relative spleen weights were statistically significantly decreased in females of the high dose group. As this finding was observed in one sex only and no microscopically visible changes were present in this organ, a compound-related effect is not evident. The same applies to the increases in relative kindey weights observed in males of the high dose group, which were only minor and occurred in one sex only.
Furthermore, statistical evaluation revealed elevated weights oflung, liver, kidneys and spleen in males ofthe low concentration group. These changes are considered to be a consequence of the higher body weights in this group and not to be compound-related .

GROSS PATHOLOGY
No compound-related macroscopically visible changes were found at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination did not reveal any compound-related changes.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not examined

HISTORICAL CONTROL DATA (if applicable)
Not examined

OTHER FINDINGS

Effect levels

Dose descriptor:
NOEL
Effect level:
ca. 2 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Sixty-five exposures to Methylal at the concentration of 10 000 ppm caused slight transient symptomatology. Additionally, slightly increased liver weights were recorded in both sexes. However, no changes were observed in this organ by histopathological examinations.
No compound-related effects were detected after repeated exposure to 2 000 ppm Methylal.
According to the present study the 'no observed effect level (NOEL)' is 2 000 ppm Methylal, corresponding to 6.3 mgl/L air.
Executive summary:

Groups of male and female Wistar rats were exposed to Methylal by (nose only) inhalation at concentrations of 0, 400, 2000 or 10000 ppm (6 hours/day, 5 days/week; 13 weeks) with 10 animals per sex per dose and were necropsied one day after the last exposure.

Behaviour and state of health were observed daily in all groups. Body weights, food consumption and water consumption were recorded once weekly. Ophthalmoscopic examinations were performed at the start and at the termination of the study.

Haematological examinations, clinical chemistry and urine analysis were carried out at the termination of the study.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes. Body weights, haematological and clinical chemistry data, urine data, absolute and relative organ weights were analysed with the aid of a statistical program to show differences compared with the controls .

Mean analytical concentrations were 377, 1908 and 9652 ppm Methylal and thus close to the target values. Animals exposed to 10 000 ppm Methylal showed disequilibrium, uncoordinated gait, ataxic gait and decreased spontaneous activity due to anesthetic effects. These symptoms were reversible at the beginning of the next exposure. Behaviour and general health condition remained unaffected by exposure to the test substance in the other groups. No signs of neurological disturbances, opacity of the refracting media of the eyes, damage to the oral mucosa or impairment of dental growth were observed in the control or treatment groups.

Likewise, ophthalmoscopic examination of the animals showed no abnormalities. Body weight development and food consumption remained unaffected by the application of the test substance. Water consumption was slightly increased in both sexes of the high concentration group. The administration of the test compound did not alter the relative water consumption in the animals of the other groups. Haematological examinations and clinical chemistry revealed no .abnormalities . Urine volume was slightly increased in females of the high concentration group, and specific urine weight (specific gravity) was slightly decreased. No treatment-related changes were detected by urine analysis in the other groups. Liver weights were slightly increased in both sexes of the high concentration group. At necropsy, no macroscopically visible compound-related changes were found. Histopathological examinations did not reveal any compound-related changes.

In conclusion, 65 exposures to Methylal at the concentration of 10 000 ppm caused slight transient symptomatology. Additionally, slightly increased liver weights were recorded in both sexes. However, no changes were observed in this organ by histopathological examinations. No compound-related effects were detected after repeated exposure to 2 000 ppm Methylal. According to the present study the 'no observed effect level (NOEL)' is 2 000 ppm Methylal, corresponding to 6.3 mgl/air .