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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:
22 March 2011 to 04 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted 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:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report: MTDID 20422
- Substance type: Pure active substance
- Physical state: Liquid
- Analytical purity: 99.99%
- Purity test date: 02/16/2011
- Expiration date of the lot/batch: 02/16/2013
- Storage condition of test material: At room temperature in the dark.

Test animals

Species:
rat
Strain:
other: Sprague Dawley Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: Approx. 39 days old
- Weight at study initiation:
- Fasting period before study: None
- Housing: Individually in suspended cages.
- Diet (e.g. ad libitum): Certified Rodent LabDiet 5002 (meal) (PMI Nutrition International, LLC) provided ad libitum.
- Water (e.g. ad libitum): Reverse osmosis treated water provided ad libitum.
- Acclimation period: 19 days previous to treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3-22
- Humidity (%): 42.8-52.8
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):12/12
IN-LIFE DATES: From: 22 March 2011 To: 20 May 2011

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: Air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 L stainless steel and glass whole-body inhalation exposure chamber. One chamber for each group
- Method of holding animals in test chamber: Animals were placed into exposure caging and placed into the chamber in the cage.
- Source and rate of air: HEPA and charcoal-filtered, temperature and humidity-controleed source.
- Method of conditioning air:
- System of generating particulates/aerosols: Vapor was generated by releasing test substance vapor from the orignial cylinder, heated to approximately 35 degrees Celsius.
- Temperature: 21.3-22 degrees Celsius.
-Humidity: 42.8-52.8% humidity
- Air flow rate:
- Air change rate: At least 12 to 15 air changes per hour.
- Treatment of exhaust air: HEPA and charcoal filter.
TEST ATMOSPHERE
- Brief description of analytical method used: Determined using a gas chromatogram. Samples collected from animal-breathing zone. Concentration in parts per million was calculated.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations were analyzed at 35-minute intervals using a gas chromatogram. Samples were collected from the approximate animal-breathing zone.
Duration of treatment / exposure:
6 hours a day
Frequency of treatment:
5 days a week, for 4 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 3000 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on results from a previous actue inhalation study conducted at 10,000 and 20,000 ppm.
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 2 week recovery period.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to exposure, during exposure (midpoint), and 0 to 1 hour following exposure.
BODY WEIGHT: Yes
- Time schedule for examinations: Recorded approximately weekly throughout the study starting during the pretest period.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded approximately weekly.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At primary necropsy (study day 26).
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 10 animals/sex/main study group and < or equal to 5 animals/sex in the control and 3000 ppm groups.
- Parameters checked in table appendix K were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At primary necropsy (study day 26).
- Animals fasted: Yes
- How many animals:10 animals/sex/main study group and < or equal to 5 animals/sex in the control and 3000 ppm groups.
- Parameters checked in table appendix K were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: At primary necropsy
- Metabolism cages used for collection of urine:No data
- Animals fasted: Yes
- Parameters checked in appendix K were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the initiation of exposure and during study week 3 (following 20 exposures.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity /
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, Appendix K
HISTOPATHOLOGY: Yes, Appendix K

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortality occured. Yellow and/or red material on various body surfaces were noted in the 500, 1000, and 3000 ppm main study and satellite groups.
BODY WEIGHT AND WEIGHT GAIN: No treatment related effects.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No effects.
HAEMATOLOGY: No effects.
CLINICAL CHEMISTRY: Lower globulin and glucose levels with corresponding higher albumin/globulin (A/G) ratios noted in the 500, 1000, and 3000 ppm group males and females.
URINALYSIS: No effects.
NEUROBEHAVIOUR: No effects.
ORGAN WEIGHTS: Higher lung weights were noted in the 3000 ppm main study group at the primary necropsy and correlated with microscopic alteration of increased alveolar macrophages, increased BALT, and perivascular mononuclear cell infiltrates at 500 ppm and greater. Laryngeal epithelial degeneration at 3000 ppm and cecal hyperplasia at 500 ppm or greater were also noted. Lung and cecal alterations were partially recovered on study day 40, and the laryngeal alteration was fully recovered.
GROSS PATHOLOGY: No effects.

Effect levels

Dose descriptor:
NOAEC
Effect level:
ca. 3 000 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Tthe No-Observed-Adverse-Effect Concentration (NOAEC) was 3000 ppm of the test article in male and female rats exposed by whole-body inhalation for 6 hours/day, 5 days/week, for 4 weeks based on the reversibility of the test article-related effects.
Executive summary:

The inhalation (vapor) toxicity potential of the test article (clear and colorless liquid, purity 99.98%) was evaluated in male and female Sprague-Dawley rats via whole-body inhalation for 6 hours/day, 5 days/week, for 4 weeks (20 total exposures). This study was performed in compliance with EPA GLP 40 CFR Part 792 (1989), OECD GLP C97 186 (1997), and Japanese MAFF GLP No. 3850 (1984). The study design was based on OECD Guideline 412 (2009). Main phase rats (10/sex/group) were exposed to filtered air (control) or filtered air containing 500 ppm, 1000 ppm, or 3000 ppm of the test article (equivalent doses were 5.44 mg/L, 10.88 mg/L, and 32.64 mg/L respectively), for 6 hour exposures, 5 days per week for 4 weeks (20 total exposures). Recovery rats (5/sex/group) were exposed to filtered air or 3000 ppm of the test article in the same manner to be followed for a 2 week recovery period after the exposure period. Satellite rats (5/sex/group) were exposed at 500 ppm, 1000 ppm, or 3000 ppm of the test article in the same manner as main phase rats. In all study animals, detailed physical examinations were preformed weekly, clinical observations were recorded daily, and body weights and food consumption were recorded weekly. A functional observational battery and motor activity tests were performed in all animals except the satellite rats at weeks 3 and 5 of the study. Main phase animals were necropsied on Day 26 and selected tissues were examined microscopically. In the satellite animals, necropsies were performed on Day 26 of the study and livers were collected for a peroxisome proliferation assay (PPAR). Necropsies were performed on the recovery group rats following the two week recovery period and selected tissues were examined microscopically. All animals survived. Test substance-related observations of yellow and/or red material on the body surface were noted in the 500, 1000, and 3000 ppm main study and satellite groups of animals. Test substance-related effects on serum chemistry parameters included lower globulin and glucose levels with corresponding higher albumin/globulin rations noted in 500, 1000, and 3000 ppm group male and female rats. These changes were considered non-adverse due to the small magnitude of the change being within historical parameters. Lung weights were higher in the 3000 ppm main study group at primary necropsy and correlated with increased alveolar macrophages, increased bronchial/bronchiolar associated lymphoid tissue (BALT), and perivascular mononuclear cell infiltrates at greater than or equal to 500 ppm. Laryngeal epithelial degeneration at 3000 ppm and cecal hyperplasia at greater than or equal to 500 ppm were observed. The lung and cecal alterations were found to be partially recovered on study day 40, and the laryngeal alteration was fully recovered. There also appeared to be a small sex difference in the rate of B-oxidation, but it did not appear to significantly induce peroxisomal proliferation. There were no toxicologically relevant changes in body weights, food consumption, FOB, hematology, coagulation or urinalysis parameters. In the female, the ovaries, uterus, cervix, and vagina were microscopically evaluated for changes in the estrous cycle, and a high incidence of diestrus was observed in the 3000 ppm group. All these tissues appeared normal, and consequently, the incidence of diestrus was considered normal. All other organs (weights and macroscopic observations) evaluated were considered to be not affected by the test substance. Based on the results of the study, the No-Observed-Adverse-Effect Concentration (NOAEC) was 3000 ppm of the test article in male and female rats exposed by whole-body inhalation for 6 hours/day, 5 days/week, for 4 weeks based on the reversibility of the test article-related effects.