Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints.)
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3650
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints.)
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints.)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.99%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 70 days
- Weight at study initiation: 317-445 grams (males) and 201-269 grams (females)
- Fasting period before study: No
- Housing:
During Non-Exposure Periods: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks except during cohabitation, gestation/lactation as described below.
Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in solid bottom caging with bedding and enrichment. Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly.
Cage Rack Positioning: Cage racks were not relocated within the animal room.
- Diet (e.g. ad libitum): ad libitum except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30%–70%
- Air changes (per hr): 10-13
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
Vehicle:
other: filtered and conditioned air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A stainless steel baffle below the chamber turret inside the chambers promoted uniform chamber distribution of the test atmosphere
Exposure #s 1 to 10, all exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L
Exposure #s 11 to 71, control animals were exposed to air in the same chamber while test substance exposures were performed in stainless steel and glass (NYU style) chamber with a nominal internal volume of 150 L
- Method of holding animals in test chamber: individually placed in stainless steel, wire-mesh modules and exposed whole-body inside the exposure chamber; During cohabitation animals were housed as mating pairs and exposed in the same manner.
- Source and rate of air: filtered and conditioned air and oxygen
- System of generating particulates/aerosols: test substance vapour and supplemental oxygen were metered into a 1-liter 3-neck mixing flask by mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without test substance.
- Temperature, humidity, pressure in air chamber: 21-25ºC, 45-67%, and 21% oxygen, no data on pressure
- Air flow rate: Exposure #s 1 to 10 - chamber flow 60-64 L/min; Exposure #s 11 to 20 - chamber flow 29-33 L/min
- Air change rate: 10-13 per hour
- Treatment of exhaust air: exhausted from the bottom of each chamber through MSA filters using vacuum pumps and discharged into the fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: vapour concentration was directly injected into an Agilent model 6890 gas chromatograph (GC)
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each exposure, the vapour concentration was determined by gas chromatography at least once an hour from the test chambers. The control chamber was sampled at least twice a day. Samples of volumes of chamber atmosphere were directly injected into an Agilent model 6890 gas chromatograph (GC) equipped with an automated gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 80ºC on a 30m x 0.53 mm (nominal diameter) RTX-502.2 fused silica glass column. The vapour concentration was determined from a standard curve derived from gas standards.
Duration of treatment / exposure:
1 month
Frequency of treatment:
6 hr/day, 5 day/wk
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2500, 10000, and 50000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.0 ± 0.0; 2500 ± 11; 10000 ± 74; and 51000 ± 650 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
5 rats/sex/dose were designated for the Subchronic
5 rats/sex/dose were designated for Subchronic with Recovery
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502000 ppm. Based on these results, exposure concentrations of 0, 2500, 10000, and 50000 ppm were selected for the current study.
- Post-exposure recovery period in satellite groups: 28 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals – At least once daily during quarantine and prior to study start, and twice daily thereafter; once daily on exposure days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretest (baseline), and at least weekly (except necropsy and FOBs) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine: All animals – at least once
Pretest: Weekly
Subchronic & Subchronic with Recovery: Twice weekly for the first 2 weeks, and then at least once weekly up to and including terminal sacrifice

FOOD CONSUMPTION: Yes; weekly
Calculation of Food Consumption: Weighed each feeder at the beginning and end of interval and subtracted final weight and amount of spillage from the feeder from initial weight

FOOD EFFICIENCY: Yes
Calculation of Mean Daily Food Efficiency: From food consumption and body weight data
Subchronic & Subchronic with Recovery: weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and before sacrifice
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males and females from the Subchronic Subset after 4 weeks of exposure; males and females from the Recovery Subset on approximately test day 48
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males and females from the Subchronic Subset after 4 weeks of exposure; males and females from the Recovery Subset on approximately test day 48
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in Table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of blood: males and females from the Subchronic Subset after 4 weeks of exposure; males and females from the Recovery Subset on approximately test day 48
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes (refer to Section 7.9.1, DI.K1.Neuro.D-20964 for additional details).
- Time schedule for examinations: during acclimation (baseline) and after approximately 3 weeks of exposure, FOB and MA assessments
- Dose groups that were examined: 5 animals/sex/group of the Subchronic with Recovery Subset
- Battery of functions tested: Abbreviated Functional Observational Battery (FOB), Motor Activity Evaluation
- open field arena: righting reflex, approach and touch, sharp auditory stimulus, tail pinch
- Chatillon® Digital Force gauge: forelimb grip strength, hindlimb grip strength
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 4)
HISTOPATHOLOGY: Yes (see Table 5)

SACRIFICE SCHEDULE:
Subchronic - Test Day 29
Subchronic with Recovery Subset - Test Day 57

Gross examination was performed on all adult rats. Final body and organ weights were recorded for all rats at the scheduled sacrifice.

Gross Pathology - All animals
Organ Weights - All animals
Histopathology - Control and high-dose groups (target organs and gross lesions in intermediate-dose groups)
Other examinations:
OTHER:
For additional details on the Micronucleus In Vivo test, refer to Section 7.6.2 Genetic Toxicity – In Vivo: DI.K1.MicroNuc.R.D-19695-1422.KD
For additional details regarding Reproductive/Developmental test refer to Section 7.8.1 (DI.K1.1Gen.RD/REPRO/DEV.R.D-19695-1422.KD) and 7.8.2 (DI.K1.DEV.RD/REPRO/DEV.R.D-19695-1422.KD)
Statistics:
Male and female data were evaluated separately. For each parameter analysed with a trend test the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected.(18) The level of significance selected was p < 0.05. (See Table 6.)
see Table 4 Statistical Methods below.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
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
Unscheduled deaths did not occur in the animals assigned to the Subchronic and Subchronic with Recovery Subsets. No test substance-related clinical sigs of toxicity were observed in males or females at any exposure concentration. Incidental clinical signs common to the age and strain of rat were occasionally observed during the exposure and/or recovery periods.

BODY WEIGHT AND WEIGHT GAIN
No adverse, test substance-related effects on body weight or weight gain were observed for males or females exposed to any concentration of the test substance during the exposure or recovery periods. Body weight of the 10000 ppm males was statistically significantly greater compared to the control group on test days 3 and 7. However, this statistical difference was considered to be spurious since a dose-response relationship was not evident. Weight gain for 2500 ppm females was statistically significantly lower compared to the control group during test days 7-14. However, this statistical difference was considered to be spurious since a dose-response relationship was not present.

FOOD CONSUMPTION & FOOD EFFICIENCY
No test substance-related effects or statistically significant differences in food consumption or food efficiency were observed for males or females exposed to any concentration of the test substance during the exposure of recovery periods.

OPHTHALMOSCOPIC EXAMINATION
No test substance-related ophthalmological observations occurred in males or females exposed to any concentration of the test substance.

HAEMATOLOGY
There were no exposure-related changes in any group mean haematology parameter in male or female rats at any concentration tested.

The following statistically significant difference was not considered to be related to exposure to the test substance:
• Absolute large unstained cells (ALUC) were minimally higher in female rats exposed to 50000 ppm (90% above the control). There were no other statistically significant differences in other white blood cell parameter in any subsets of male or female rats at any concentration tested. Therefore, this change was considered unrelated to exposure and non-adverse.
• Coagulation - There were no statistically significant or exposure-related changes in any group mean coagulation parameter in male or female rats at any concentration tested.

CLINICAL CHEMISTRY
There were no exposure-related changes in group mean clinical chemistry parameters in male or female rats at any concentration tested.

The following statistically significant difference was not considered to be related to exposure to the test substance:
• Blood urea nitrogen (BUN) was minimally lower in female rats exposed to 50000 ppm (18% below the control). However, there were no statistically significant differences in other kidney-related clinical pathology parameters and no exposure-related changes in renal histopathology in this group. In addition, there were no changes in BUN in any other male or female group, including the 50000 ppm females from the reproduction subset. Therefore the lower BUN in the 50000 ppm females from the subchronic subset was considered unrelated to exposure and non-adverse.

URINALYSIS
There were no statistically significant differences in group mean urinalyses parameters in male or female rats at any concentration tested.
• Plasma and Urine Fluoride - There were no statistically significant changes in plasma and urine fluoride parameters in male or female rats.

NEUROBEHAVIOUR (only done on Subchronic with Recovery Subset)
• Forelimb and Hindlimb Grip Strength - No test substance-related or statistically significant effects occurred on forelimb or hindlimb grip strength for males or females exposed to any concentration of the test substance.
• Open Field Observations - No test substance-related or statistically significant effects were observed for any behavioural parameter evaluated in males or females exposed to any concentration of the test substance. The incidences for all groups were similar to the values for the control groups.
A wound observed during the FOB at week 6 in one male in the 50000 ppm group was considered to be incidental.
• Motor Activity - No test substance-related or statistically significant effects were observed on motor activity for males or females exposed to any concentration of the test substance. At week 4, the mean duration and number of movements for 50000 ppm females during the 2nd – 6th intervals as well as for the total session were decreased (not statistically significant) compared to control. However, these decreases, although they showed a trend, were not considered to be adverse because the decreases were not observed in males, were not statistically significant, and were within historical control. Mean values for duration and number of movements for the 2500 and 10000 ppm males and females and 50000 ppm males were similar to their respective controls.

ORGAN WEIGHTS
There were no test substance-related organ weight effects at inhalation exposures up to 50000 ppm.
All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure.
• In males, two of the accessory sex organs (ASO) mean weight parameters were statistically (p < 0.05) decreased in the 2500 ppm exposure group, as compared to the respective control values. However, since there was no dose response, no microscopic correlate, and no similar finding in the Subchronic with Recovery Subset or Reproduction Subset, the statistical finding was interpreted to be spurious.
• In females, the mean absolute adrenal weight and mean absolute kidneys weight were both statistically decreased in the 2500 ppm group, as compared to the respective control values. These were also considered spurious since there was no dose response, no microscopic correlate, and no similar finding in the Subchronic Subset males, Subchronic with Recovery Subset, or Reproduction Subset.

In the Subchronic with Recovery Subset, there were no test substance-related organ weight effects.
All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure.
• In males, both the mean relative (% body weight) heart weight and lung weight were statistically decreased in the 10000 ppm exposure group compared to the respective control values. These were both considered spurious since there was no dose response, no microscopic correlate, and no similar finding in the main study, recovery females, or reproduction study subsets. A statistically significant decrease in the 50000 ppm mean absolute lung weight was also interpreted as spurious because there was no microscopic correlate and no similar finding in the Subchronic Subset, Subchronic with Recovery females, or Reproduction Subsets.
• In females, the mean relative (% brain weight) lung weight was increased in the 50000 ppm group, as compared to the respective control values. This was also considered spurious since there was no microscopic correlate and no similar finding in the Subchronic, Subchronic with Recovery males, or Reproduction Subsets.

GROSS PATHOLOGY
At the terminal sacrifice, there were no test substance-related gross observations in either sex. All gross observations were consistent with normal background lesions in rats of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance-related microscopic findings in male or female rats in the 50000 ppm inhalation exposure group. All microscopic findings in this study were consistent with normal background lesions in rats of this age and strain.

For additional details on the Micronucleus In Vivo test, refer to Section 7.6.2 Genetic Toxicity – In Vivo: DI.K1.MicroNuc.R.D-19695-1422.KD
For additional details regarding Reproductive/Developmental test, refer to Section 7.8.1 (DI.K1.1Gen.RD/REPRO/DEV.R.D-19695-1422.KD) and 7.8.2 (DI.K1.DEV.RD/REPRO/DEV.R.D-19695-1422.KD)
For additional details on the Neurological Assessment, refer to Section 7.9.1 Neurotoxicity: DI.K1.Neuro.R.D-19695-1422.KD

Effect levels

Dose descriptor:
NOAEC
Effect level:
50 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the absence of effects in all endpoints at the highest concentration tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEC = 50000 ppm

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The purpose of this study was to determine the potential subchronic toxicity from repeated inhalation exposure of male and female rats to 0, 2500, 10000, or 50000 ppm of the test substance including a neurobehavioural evaluation.

Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were exposed to the test substance 6 hours per day, 5 days per week over a one-month period . Concentrations of test substance were generated by metering the test substance into the inhalation chambers and diluting it with conditioned, filtered air, supplemented with oxygen to achieve an oxygen concentration of 20% in the high level chamber. An air control group was also evaluated using a similar generation method. Vapour concentrations of the test substance were measured by gas chromatography (GC). Temperature, humidity, and airflow were also recorded periodically during each exposure day. 

Body weights, clinical signs, and food consumption were recorded throughout the study. On test day 29, blood and urine samples were collected from 5 males and 5 females for measurement of haematology, clinical chemistry, and urinalysis parameters. Blood and urine samples for measurement of haematology, clinical chemistry, and urinalysis parameters were also collected on test day 29 from the 5 male and 5 female rats per group assigned to the Recovery group and again near the end of the recovery period on test day 57. 

An abbreviated neurobehavioural evaluation was conducted on the 5 males and 5 females prior to test substance administration in order to obtain baseline measurements. This neurobehavioural evaluation was conducted again following approximately 4 weeks of exposure for the animals with Recovery Subset. An approximate 28-day recovery period was initiated after the end of the exposure period for rats with Recovery Subset during which time assessments of body weight, food consumption, and clinical signs of toxicity continued. Following collection of blood samples from the rats assigned, the rats were euthanized and selected organs were weighed, and selected tissues were evaluated microscopically. 

The mean concentrations (± standard error of the mean) were 2500 ± 11, 10000 ± 74, and 51000 ± 647 ppm in chambers targeted at 2500, 10000, and 50000 ppm, respectively. No test substance-related or adverse effects were observed on survival, clinical signs, ophthalmological observations, body weight, weight gain, food consumption, and food efficiency. There were no adverse, test substance-related effects on micronucleus endpoints, neurobehavioural parameters, or clinical pathology parameters. There were no test substance-related effects on organ weight parameters. There were no test substance-related effects on gross or microscopic observations.

Under the conditions of the study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity in males and females was 50000 ppm, the highest concentration tested, based on the absence of effects in all endpoints.