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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 4, 2019 - October 2, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was performed for worker safety requirements.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methoxy-3-methylbutan-1-ol
EC Number:
260-252-4
EC Name:
3-methoxy-3-methylbutan-1-ol
Cas Number:
56539-66-3
Molecular formula:
C6H14O2
IUPAC Name:
3-methoxy-3-methylbutan-1-ol
Test material form:
liquid
Details on test material:
Specific gravity: 0.928 (20°C)
Appearance: Colorless liquid
Specific details on test material used for the study:
Storage conditions: Room temperature (actual range: 14.9°C to 22.8°C), dark place, airtight container, filled with nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
This strain is widely used in toxicity studies using rodents, and there are abundant historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at start of exposure: 8 weeks old.
- Body weight range at start of exposure: Males: 269.0 to 311.3 g, females: 181.5 to 219.7 g.
- Housing: Two animals (same sex) /cage.
Cages: Stainless-steel wire mesh hanging cage (W 390 × D 285 × H 180 mm).
Racks: Stainless steel racks.

- Diet: Pellet feed for experimental animals (ad libitum) except for the following periods:
· During exposure.
· During fresh urine collection.
· Leading up to scheduled necropsy from the evening on the day before necropsy.

- Water: Tap water filtered through a 5-µm pore size filter followed by irradiation with ultraviolet
light. Provided ad libitum, except no water was provided during inhalation exposure or fresh urine collection.

- Acclimation period: 6 days.

DETAILS OF FOOD AND WATER QUALITY: Analysis results were obtained from the food supplier, and then levels of contaminants such as residual pesticides in the used lots of food were confirmed to meet the criteria specified in the SOP of the test facility. Water quality was periodically (twice a year) analyzed by an external agency. The values obtained from the analysis were confirmed to meet the criteria specified in the SOP of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1°C to 23.5°C.
- Humidity: 37.3% to 69.5%
- Air changes: Six to 20 times/hr, all fresh air.
- Photoperiod: Twelve hr/day.

Administration / exposure

Route of administration:
inhalation: mist
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A flow past nose-only inhalation exposure chamber (hereinafter referred to as “chamber”). The chamber is constructed from stackable tiers, which had 16 exposure ports per tier. This study used a chamber constructed from two tiers.

- Method of holding animals in test chamber: Animals individually held in the restraint tube.

- System of generating particulates/aerosols: The test substance was converted into a mist containing the test substance in vapor-form using a double-fluid nebulizer (NB-2N: Shibata Scientific Technology Ltd.) by supplying pressurized air and the test substance at a prescribed flow rate.

- Temperature in air chamber: The generated mist was passed through a heat exchanger with a set temperature of 40°C to prepare a test atmosphere containing the vaporized test substance at a specified concentration. The test atmosphere was continuously supplied to the chamber for exposure to animals.

- Air flow rate: The flow rate of the test atmosphere supply to the chamber was set at 16 L/min/tier (permissible range: 16.0 to 22.0 L/min/tier). The flow rate through the chamber exhaust was set at approximately 10% lower than the test atmosphere supply, because the pressure inside the chamber must be positive to ensure reliable exposure.

TEST ATMOSPHERE
- Brief description of analytical method used:
* Syringe: 1 mL gas tight syringe (#1001)
* Sampling bag: 1.56 L inner volume (actual inner volume, Smart bag)
* Vacuum sampling container: A container for sampling the test atmosphere into the constituent sampling bag by reducing the pressure inside (made in-house).
* Air pump: General purpose pump.

- Samples taken from: Exposure port of the chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal concentration was calculated from the amount of test substance supplied to the chamber and the air volume passed through the chamber. Measurement of exposure concentration and determination of concentration stability was done using a validated analytical method (gas chromatography).

Sampling position: Exposure port of the chamber.
Sampling time point:
30 minutes, 3 hours, and 5 hours 30 minutes after the start of exposure on the day of the start of exposure (males), and at 7-days intervals thereafter 30 minutes after the start of exposure on other days.
Number of sample: 1 sample/time point.
Sampling method: The sampling bag was placed into the vacuum sampling container, and connected to the exposure port. The test atmosphere as an analytical sample was sampled into the sampling bag by reducing the pressure inside the container using an air pump.

One analysis per sample was performed. The concentration of the test substance was calculated from the peak area derived from the test substance and calibration curve (minimum unit: 0.01 mg/L).

Time of measurement:
Start of test atmosphere generation, start of exposure, and once an hour thereafter until completion of exposure, and termination of test atmosphere generation.
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
Inhalation exposure was conducted once a day for 6 hours, 5 days a week, for 13 weeks.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/L air
Remarks:
Control
Dose / conc.:
0.25 mg/L air (nominal)
Remarks:
The measured concentration was 0.27 mg/L.
Dose / conc.:
1 mg/L air (nominal)
Remarks:
The measured concentration was 1.04 mg/L.
Dose / conc.:
2 mg/L air (nominal)
Remarks:
The measured concentration was 2.07 mg/L.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The exposure concentration was set to obtain information to determine whether or not the test substance falls into Category 2 of the GHS classification criteria for specific target organ toxicity (repeated exposure). The guidance values for concentrations that cause toxic effects equivalent to GHS Category 2 are greater than 0.02 mg/L and less than 0.2 mg/L for mist or dust, and greater than 0.2 mg/L and less than 1.0 mg/L for vapor.

In a preliminary subacute inhalation toxicity
study (28-day study, study No. B180761), high levels of plasma inorganic phosphorus were observed in males in groups exposed to 1 mg/L or more of the tests substance and high relative kidney to body weight ratio was observed in males and females of the 2 mg/L group. No toxicological changes related to the test substance were observed in the 0.5 mg/L group; however, the possibility of toxic effect could not be eliminated in a 90-day study with a longer exposure period. Based on the above observations, target exposure concentrations needed to obtain information to determine whether or not the test substance falls into the GHS Category 2 classification for specific target organ toxicity (repeated exposure) were set at 0.25 mg/L as the low exposure concentration, and 1 and 2 mg/L as the middle and high exposure concentrations, respectively.


- Animals assignment: The animals were allocated to study groups so as to obtain approximately equivalent mean body weights among the groups using the stratified-by-weight randomization method based on body weight on the allocation day.

- Fasting period before blood sampling for clinical biochemistry: One night. Animals were fasted from the evening on the day before necropsy until the necropsy.

Examinations

Observations and examinations performed and frequency:
1- Clinical signs:
Daily observations were conducted from the first day of exposure to the day of necropsy.
Exposure day: Twice a day (before and after exposure).
Other days: Once a day.

2- Body weight:
Body weight was measured according to the schedule below. Measurements were conducted before exposure on the day of exposure.

Male: Day 1, Day 6, Day 8, Day 13, Day 15, Day 20, Day 22, Day 27, Day 29, Day 34, Day 36, Day 41, Day 43, Day 48, Day 50, Day 55, Day 57, Day 62, Day 64, Day 69, Day 71, Day 76, Day 78, Day 83, Day 85, Day 90, and Day 91.

Female: Day 1, Day 5, Day 8, Day 12, Day 15, Day 19, Day 22, Day 26, Day 29, Day 33, Day 36, Day 40, Day 43, Day 47, Day 50, Day 54, Day 57, Day 61, Day 64, Day 68, Day 71, Day 75, Day 78, Day 82, Day 85, Day 89, and Day 91.

On the day of the scheduled necropsy (Day 92), body weight was measured to calculate
relative organ weights (relative body weight ratio).

3- Food consumption:
Gross weight of food per cage was measured according to the following schedule:
Day 1, Day 8, Day 15, Day 22, Day 29, Day 36, Day 43, Day 50, Day 57, Day 64, Day 71, Day 78, Day 85, Day 87, and Day 91

Mean daily food consumption per animal housed in the same cage was calculated from the weight difference between each measurement day.

4- Ophthalmological examination:
Schedule:
Pre-exposure: Week -1
Exposure period: Week 13

5- Urinalysis:
Urinalysis was conducted in 6 animals from each group.
Schedule:
Week 13 (Day 85 to Day 86).
Parameters are summarized below.

6- Hematology:
Schedule:
The day of scheduled necropsy (Day 92).
Parameters are summarized below.

7- Blood chemistry:
Schedule:
The day of scheduled necropsy (Day 92).
Parameters are summarized below. (Thyroid hormones were not included).
Sacrifice and pathology:
* Pathological examination:
Examined organs and tissues:
The organs and tissues collected and examined are listed in the file attached in "Overall remarks, attachments".

* Necropsy:
All animals were subjected to necropsy on Day 92.
Animals were subjected to necropsy after euthanization by exsanguination from the abdominal aorta after blood collection.

* Organ weight:
The organs and tissues listed in the file attached in "Overall remarks, attachments". Animals were weighed using an electronic balance (AG204).
Bilateral organs were weighed simultaneously. The pituitary and thyroid/parathyroid were weighed after fixation with formalin. Moreover, relative organ weight (relative body weight ratio) was calculated from body weight measured on the day of necropsy.

* Histopathological examination.
Microscopic examination was conducted on specimens from all animals in the control and high exposure concentration groups, and organs and tissues with gross abnormalities from all animals, which were collected at the end of the exposure period.
Other examinations:
* Examination of bronchoalveolar lavage fluid (BALF): The day of scheduled necropsy (Day 92).


Deviation from the study protocol:
Lung fixation:
The study protocol indicated that the lung was to be perfused with the fixation solution at 25 cmH2O and kept for 15 minutes. The actual duration was 15 to 25 minutes. A duration of 15 minutes was sufficient for lung fixation; therefore, the description in the study protocol was judged to be incorrect. Since sufficient fixation was conducted, it was concluded that this did not affect the reliability of the study.

Volume of BALF retrieval solution:
The study protocol indicated that 3 mL of BALF retrieval solution was to be used for the first sampling and 2 mL for subsequent samplings. There was concern that the volume used in males was too small and may influence the accuracy of the study results; therefore, sampling in females was conducted using 5 mL of retrieval solution. Since similar conditions were established according to sex, it was concluded that these operations did not affect the reliability of the study.
Statistics:
The following statistical analyses were conducted on the obtained data. Safety Study System was used for statistical analyses.
Method:
Numerical data:
Initially, variance was assessed using Bartlett's test (significance level: 5%). When the
variance was homogeneous, multiple comparisons test was conducted using Dunnett's
method. When the variance was heterogeneous, multiple comparisons test was conducted
using the Steel test. Multiple comparisons tests were conducted using a two-tailed test with
significance levels of 1% and 5%.

Examined items:
The items subjected to statistical analyses were as follows:
- Body weight.
- Food consumption.
- Urinalysis (specific gravity, volume, and electrolytes).
- Hematology.
- Blood chemistry.
- Organ weight (absolute and relative organ weights).
- BALF examination results.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Loss of fur on the head was observed in 1 female each in the control and 0.25 mg/L groups,
loss of fur on the back was observed in a male in the 2 mg/L group, and swelling of the ear
was observed in 1 male each in the 0.25 and 1 mg/L groups. Since these findings were
external changes caused by the animals being held in a restraining tube during the nose-only
inhalation exposure, it was judged that these findings were not caused by exposure to the test
substance.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in any of the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and any of the
groups exposed to the test substance during the entire observation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and any of the
groups exposed to the test substance during the entire observation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Particulate opacity in the lens and corneal opacity were both observed to a slight degree, as
spontaneous changes during the exposure period. No changes were caused by test substance
exposure.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was statistically significantly higher reticulocyte ratio, white blood cell count, and lymphocyte count in males in the 0.25 mg/L group; and higher neutrophil count in males in the 2 mg/L group compared to the control group. In absence of a dose-relationship for the observations in the low dose group, these were considered not related to test item exposure. The change in neutrophil count in the 2 mg/L group was not accompanied by a change in white blood cell count and there was no statistically significant difference in the neutrophil ratio. No similar effect on neutrophils was observed in females. Therefore, the changes in hematology were judged to be without toxicological significance. No effects were observed in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significantly lower glucose levels in males in the 2 mg/L group (approximately 10% decrease compared to controls) and higher inorganic phosphorous levels in males in the 1 and 2 mg/L groups compared to the control group (appr. +9% and +11.6% compared to controls for 1 and 1 mg/L groups, resp.). No effects were observed in females.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the examination of fresh urine, there was a tendency toward high levels of occult blood
and red blood cells in urinary sediment in males in the 1 mg/L group. Since there were no
exposure concentration dependencies, the changes were considered incidental and to be
without toxicological significance.

In the examination of 16-hour accumulated urine, there were no statistically significant
differences between the control group and any of the groups exposed to the test substance.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significantly higher absolute values for the spleen in males in the 0.25 mg/L group compared to the control group. (approximately +25% compared to controls). The high average value was contributed to two individual rats in this dose group. Since there was no exposure concentration dependency, the change was judged to be incidental and without toxicological significance. No other effects were observed in male and female rats.

There were statistically significantly lower values for the submandibular glands weights compared to body weights in males in the 0.25 mg/L group (-11% compared to controls), higher values for the heart in males in the 1 mg/L group (+ 7.7% compared to controls), higher values for the liver in males in the 2 mg/L group (+6% compared to controls), and higher values for the kidney in males in the 1 and 2 mg/L groups (+7% and +8% for the males at 1 and 2 mg/L, respectively, compared to the control group). The changes in the submandibular glands in males in the 0.25 mg/L group and heart in males in the 1 mg/L group did not show exposure concentration dependency. Thus, the changes were judged to be incidental and without toxicological significance. All changes were considered to be limited (<10% change compared to controls) and not accompanied by histopathological effects. Therefore these effects were concluded not toxicologically relevant. No effect on organ weight to body weight ratio were observed in females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes attributable to the test substance.
There were unilateral swelling of the pinna in two animals, impression of the cerebrum in one animal, and loss of fur in 3 animals. These were sporadic changes without exposure concentration dependency; therefore, it was judged that these changes were not attributable to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes attributable to the test substance.
Swelling of the pinna observed at necropsy in two animals was auricular chondropathy. Since dilatation of the cerebral ventricle was observed at the impression of the cerebrum in one animal, it was judged to be hydrocephalus. Since the above findings are incidental and commonly observed changes in rats, it was judged that these findings were not attributable to the test substance. There were no histological abnormalities in the skin, although loss of fur was observed in 3 animals.

In the control and 2 mg/L groups, the following histological changes were observed: focal inflammatory cell infiltration in the myocardium of the heart, mineralization of the arterial wall and alveolar macrophage aggregation in the lung, focal inflammatory cell infiltration in the liver, lobular atrophy in the pancreas, basophilic tubule in the kidney, lymphocyte infiltration in the prostate, presence of ultimobranchial remnants in the thyroid gland, and retinal rosette/folding in the eye. The above findings are incidental changes in rats, and there was no significant difference in the number of animals with these findings between groups. In the 2 mg/L group, the following histological alterations were observed: ectopic pancreatic tissue at the serosa of the glandular stomach, focal hypertrophy in the tubules in the kidney, cyst in the pars distalis of the pituitary gland, and accessory adrenocortical tissue in the adrenal gland. The number of animals found with these findings was one and the findings are non-specific changes in rats. Therefore, the findings in the 2 mg/L group were judged not to be attributable to the test substance.
In the control group, the following histological changes were observed: proliferation of epithelial cells in the thymus, focal necrosis of hepatocytes in the liver, cyst in the medulla and infarct in the cortex in the kidney, luteal cyst in the ovary, cyst and aberrant craniopharyngeal tissue of the pars intermedia in the pituitary gland, and focal inflammatory cell infiltration in the Harderian gland. These findings are non-specific changes in rats.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Examination of BALF:
There were no statistically significant differences between the control group and any of the
groups exposed to the test substance.
Details on results:
In blood chemistry, low glucose levels in males in the 2 mg/L group and high inorganic phosphorous levels in males in the 1 mg/L and 2 mg/L groups were observed. Higher relative weights (relative to body weight) of the liver in males in the 2 mg/L group and the kidney in males in the 1 and 2 mg/L groups were observed. Concerning the low glucose levels, the measured value in the 2 mg/L group was 117.0 mg/dL while the background value from the test facility was 141.04 ± 24.65 mg/dL. Concerning the high inorganic phosphorous levels, the measured values in the 1 and 2 mg/L groups were 7.76 and 7.93 mg/dL, respectively while the background value from the test facility was 7.27 ± 0.54 mg/L. Since the changes in blood chemistry were within the range of mean ± 2 S.D. of the background values, it was judged that the changes did not have toxicological significance. Regarding the changes in relative weights (relative to body weight) of the organs, there were no toxicologically significant changes in the blood chemistry or findings in the histopathological examination related to the changes in the kidney or liver. Moreover, there were no toxicologically significant changes in the urinalysis that were related to the change in the kidney. Therefore, although it was not possible to eliminate the possibility that these changes were caused by test substance exposure, it was judged that the changes were not adverse effects.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
2 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No adverse effects observed up to and including the highest dose tested.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

See the "Appendix" attached file in "Overall remarks, attachments".

Applicant's summary and conclusion

Conclusions:
Based on the results of a 90-day inhalation study, performed according to OECD guideline 413 and GLP principles, the NOAEL of 3-methoxy-3-methylbutanol was concluded to be 2 mg/L, the highest concentration tested.
Executive summary:

The sub chronic inhalation toxicity of MMB was examined in rats. Crl:CD (SD) rats were exposed to the test substance by nose only inhalation exposure for 6 hours a day, 5 days a week, for 13 weeks at the target exposure concentrations of 0.25, 1, and 2 mg/L. Each study group was composed of 10 males and 10 females. The actual exposure concentrations were 0.27, 1.04, and 2.07 mg/L. There were no remarkable changes in testing conditions such as temperature and humidity to affect the study results. 


No abnormalities were observed in clinical signs. Furthermore, there were no changes attributable to the test substance exposure in body weight, food consumption, ophthalmological observation, hematology, blood chemistry, or examination of bronchoalveolar lavage fluid. No abnormal findings attributable to the test substance exposure were observed in necropsy or histopathological examination.


Based on the results of this study, it was concluded that the no observed adverse effect level (NOAEL) of MMB in a 90 day sub chronic inhalation toxicity study was was 2 mg/L in males and females.