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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:
18 March to 28 August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 17 October 2019 to 15 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
Non-GLP 2-week dose range finding inhalation study designed to select the appropriate dose levels for the upcoming 90-day inhalation study.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: males: 335 to 390 g; females: 195 to 265 g
- Housing: 3 rats/cage/sex in polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: Teklad 2014C diet, ad libitum (except at scheduled necropsy and during dosing)
- Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (except during dosing)
- Acclimation period: at least 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 ºC
- Humidity: 40-70 %
- Photoperiod: 12 h light / 12 h dark
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
The results indicated that no test item impacted on the stainless-steel substrates with all the sample being collected in the solvent trap. The achieved distribution meant that a MMAD value could not be calculated. A larger volume sample was collected; however, this did not offer any improvement in calculating a MMAD value. Therefore, no PSD samples were collected during study.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole body exposure chamber (total chamber volume = 137.5L).
Stainless steel and acrylic polymer construction comprising a base unit, cuboidal housing section and a semi-pyramidal top section incorporating a central inlet and an inverted cone to aid atmosphere distribution. The dimensions of the chamber give an internal volume of approximately 120 L. A large glass elutriator was incorporated into the exposure system which connected to the top of the semi-pyramidal section via aerosol ducting. The dimensions of the elutriator give an internal volume of approximately 10.5 L.
- Method of holding animals in test chamber: Individually in stainless steel mesh compartments within restraint caging positioned within the cuboidal section of the exposure system.
- Source and rate of air: from in-house compressed air system
– Breathing quality: Generator flow:14 - 49L/minute; Supplementary flow: 15 -35 L/minute
- System of generating particulates/aerosols: a stainless steel concentric jet atomiser, designed to produce and maintain an atmosphere containing a high proportion of respirable droplets. The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump.
- Method of particle size determination: by cascade impaction (Marple Cascade Impactor)
- Treatment of exhaust air: Drawn by in-house vacuum system and filtered locally; Extract flow: 30 - 50L/minute
- Temperature: Some of observed chamber temperatures for all groups were slightly abovethe recommended range (19 – 25°C) for inhalation exposure of rats. The chamber temperatures were similar for each group on each day of the study.

TEST ATMOSPHERE
- Brief description of analytical method used: test article concentration was analysed by chemical analysis (GC method of analysis) in solvent trap samples.
- Samples taken from breathing zone: yes, aerosol samples were collected with Dreschel head and solvent trap (bubbler) at 1 sample from Group 1(control)/day (taken at approximately 60 minutes during exposure) and 3 samples from Group 1 (test), 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test article concentration was analysed by chemical analysis (GC method of analysis: test samples were extracted with acetonitrile then injected by split injection onto GC column (ZB-50) with flame ionisation detection)
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
0/0.2, 0.6, 1.2 and 2.4 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
3
Control animals:
other: air only
Details on study design:
Rationale for Exposure Level Selection :
The target exposure levels used in this study (0/0.2, 0.6, 1.2 and 2.4 mg/L) were selected in conjunction with the Sponsor.
In a 2-week preliminary inhalation study conducted with the related substance alpha-pinene multiconstituent in F344/N rats, 100% mortality was observed from 800 ppm. The dose level of 400 ppm, corresponding to 2.4 mg/L, was used as the high dose for the subsequent 90-day repeated dose study.
In a 13-week inhalation study conducted with the related substance alpha-pinene multiconstituent in F344/N rats, at 400 ppm, 60% of female rats died with no cause of death established (from Week 6) and lower body weight gain was noted.
Therefore the same exposure level, 2.4 mg/L, and a lower exposure level, 1.2 mg/L, were initially used as a first step in order to determine that the level of toxicity was similar.

Exposure levels of 1.2 and 2.4 mg/L were considered too high and caused 4/6 decedents at 2.4 mg/L and 1/6 decedents at 1.2 mg/L following the second daily exposure. No dosing beyond exposure 2 was conducted in Group 2 and 3 or in the associated control animals. To make effective use of the animals assigned to the study, the first 2 control animals/sex were subsequently exposed to a concentration level of 0.2 mg/L for 2 weeks.

Group 1 animals tolerated an exposure concentration of 0.2 mg/L well. Based on this, Group 4 animals were exposed at a target concentration of 0.6 mg/L.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: yes
Time schedule: detailed observations were recorded daily, on exposure days, at the following times in relation to dose administration (pre-exposure observation); as each animal is returned to its home cage; as late as possible in the working day
In addition observations were made in the threatment period, on days without exposures, at the following times during the day: early in the working day (equivalent to pre-exposure observation); as late as possible in the working day.
A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: yes
Time schedule for examinations: The weight of each animal was recorded twice weekly before treatment commenced, daily throughout the study and before necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded twice weekly before the commencement of treatment and daily during Weeks 1 and 2 of the study.

WATER CONSUMPTION: no

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: no

CLINICAL CHEMISTRY: no

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: no
Sacrifice and pathology:
A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.

Schedule: animals were killed following 2 weeks of treatment.
Method of kill: overdose of intraperitoneal pentobarbitone sodium followed by exsanguination.
Sequence: to allow satisfactory inter-group comparison.

Organ weights
Brain, heart, kidneys, liver, lungs and spleen were weighed. For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for study animals killed at scheduled intervals. Organ weights were presented both as absolute and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

HISTOPATHOLOGY
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of testes (in modified Davidson’s fluid) and eyes (Davidson’s fluid).
Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with haematoxylin and eosin.
Tissues examined:
- All specified organs in table 7.5.2/1 were examined in all animals of control and high dose groups.
- Nasal turbinates, larynx, trachea, lungs, tracheobronchial lymph nodes and abnormalities were only examined in animals killed or dying prematurely and in all terminal animals of Groups 1 (air control) and 4.
Other examinations:
None
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Following the second exposure, all females and one male exposed to 2.75 mg/L and one female exposed to 1.61 mg/L were sacrificed due to clinical signs. Signs included combinations of decreased activity, breathing irregularities, muscle reaction (convulsion, tremor, spasms, repetitive movements), gait abnormalities (elevating/swaying/flattened, uncoordinated) and flattened posture noted on Day 1 and/or Day 2.

In animals exposed to 0.187 or 0.621 mg/L there were no test item related clinical signs following exposure or at the weekly physical examination.
Mortality:
mortality observed, treatment-related
Description (incidence):
Following the second exposure, all females and one male exposed to 2.75 mg/L and one female exposed to 1.61 mg/L were sacrificed due to clinical signs.
There were no pathological findings attributable to the early termination of any decedent animal and the major factor contributing to death in all decedents was considered to be poor clinical condition.

Exposures were terminated for the remaining two males at 2.75 mg/L and all other animals at 1.61 mg/kg/day, and these animals were retained off dose for the remaining 2 weeks of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no marked effects on body weight change.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was slightly lower than pre-exposure values in animals exposed to 1.61 or 2.75 mg/L on Day 1.
There were no test item related effects on food consumption in animals exposed to 0.187 or 0.621 mg/L.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no marked effects on the organ weights examined.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 2 weeks of exposure revealed the following changes in the teeth: pale lower incisors were observed in female No. 22 exposed to 0.187 mg/L. This finding was also observed in some decedent animals.

Pathological findings were as follows: At 1.61 mg/L, 26F had pale lower incisors macroscopically and no microscopic findings. At 2.75 mg/L, 9M had firm seminal vesicles noted macroscopically and no microscopic findings, 27F had no macroscopic or microscopic findings, 28F had pale lower incisors noted macroscopically and a microscopic finding of slight, focal bronchioalveolar hyperplasia in the lungs and 29F had pale lower incisors noted macroscopically and a microscopic finding of minimal, focal, mixed alveolar inflammatory cell infiltrate in the lungs. There were no pathological findings attributable to the early termination of any decedent animal and the major factor contributing to death in all decedents was considered to be poor clinical condition. The incidence and distribution of all findings were considered to be unrelated to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The microscopic examination performed after 2 weeks of exposure revealed no exposure related findings. The incidence and distribution of all microscopic findings were considered unrelated to the test item. There was no histopathological correlate for the pale incisors observed macroscopically in decedent animals (female No. 22 exposed to 0.187 mg/L was not examined microscopically. Incidental findings were observed in all females exposed to 0.621 mg/L which had a finding of minimal to slight inflammatory cell infiltrate in the larynx. This was due to a foreign body (likely food material) reaction in the ventral pouch of the larynx, a relatively common background finding in toxicity studies, and was therefore considered unrelated to the test item.
Histopathological findings: neoplastic:
no effects observed
Details on results:
See "Any other information on results incl.tables".
Remarks on result:
not determinable
Critical effects observed:
not specified

Atmosphere analysis and estimation of achieved dose

Table 7.5.2/2: Summary data of (-)-alpha-pinene aerosol concentrations (mean of daily means)

Groupa Atmosphere Concentration (mg/L)
Target Achieved
1 0 -
1 0.2 0.187
2 1.2 1.61
3 2.4 2.75
4 0.6 0.621

Group 2 and Group 3 exposure levels were considered too high after causing decedents; therefore, no further exposures were conducted after Exposure 1.2 (Week. Day). To make effective use of animals assigned to study, the initial cohort of Group 1 animals were subsequently used for exposures with the test item at a target concentration of 0.2 mg/L.

The mean achieved atmosphere concentrations were 94, 134, 115 and 104% of target for Groups 1 (exposed to test item), 2, 3 and 4, respectively.

 

Table 7.5.2/3: Body weight - Group mean values (g) - Males


Dose GroupConcentration (mg/L)

Control

1

0/0.187

(-)-alpha-pinene
2 - 3 - 4
1.61 - 2.75 - 0.621
Group/Sex Day Day Change
P1 P4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1-3 1-14

Air exposure

1M

Mean 307 329 351 355 361 363 365 370 376 380 413 419 420 428 435 442 443 - 81
SD 9.8 11.5 14.0 12.9 18.2 17.5 22.2 23.1 24.4 28.5 - - - - - - - - -
N 3 3 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 - 1

Test exposure

1M

Mean     365 367 368 376 375 383 384 389 387 395 399 401 405 406 419 -  
SD     17.1 13.5 16.0 16.7 15.1 15.0 13.1 17.0 19.2 18.3 14.2 13.1 18.6 18.0 18.5 -  
N     2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 -  
4M Mean 298 325 353 358 365 372 380 384 392 395 401 407 407 416 422 426 426 - 73
SD 5.8 6.7 8.4 8.7 6.4 9.2 11.4 13.5 9.6 9.9 13.4 10.2 14.9 12.0 15.7 17.1 15.1 - 10.2
N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 - 3
% of 1M                     - 90
2M Mean 320 335 362 366 367 376 377 388 392 399 406 410 413 418 421 428 427 5 66
SD 21.9 21.1 10.1 8.6 9.9 7.8 9.5 5.7 7.5 6.4 9.1 8.5 7.6 6.5 6.6 3.4 6.0 0.3 12.4
N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
% of 1M                     - 82
3M Mean 323 341 372 375 384 394 401  408  413  417  431  431  443  446  455  459  463 1  76
SD 15.5 14.3 20.5 20.5 18.4 20.9 23.1  23.5  23.6  22.8  23.8  17.2  29.0  28.5  27.9  32.0  30.5 7.9  21.5
N 3 3 3 3 2 2 2  2 2  2  2  2  2  2  2  2 2  2
% of 1M                     -  94

Group 1 animals 1 and 2 not exposed to air Days 3-7. Group 2 and 3 animals not exposed after Day 2.

 

Table 7.5.2/4: Body weight - Group mean values (g) – Females


Dose GroupConcentration (mg/L)

Control

1

0/0.187

(-)-alpha-pinene
2 - 3 - 4
1.61 - 2.75 - 0.621
Group/Sex Day Day Change
P1 P4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1-3 1-14

Air exposure

1F

Mean 195 207 214 215 212 219 221 226 227 232 206 207 206 210 212 219 217 - 24
SD 19.0 17.6 20.8 23.2 29.2 25.0 24.1 27.1 31.2 31.6 - - - - - - - - -
N 3 3 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 - 1

Test exposure

1F

Mean     247 251 246 248 260 260 259 257 264 272 278 265 277 280 273 -  
SD     25.3 23.3 17.7 30.5 29.9 21.4 19.1 30.8 28.0 26.4 23.5 27.5 23.9 28.6 29.1 -  
N     2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 -  
4F Mean 198 206 217 216 221 224 223 228 232 233 232 231 234 234 232 236 240 - 19
SD 5.1 8.3 6.0 3.8 5.6 5.3 7.4 5.2 5.1 5.0 3.5 5.6 2.9 6.8 5.2 6.1 3.6 - 8.9
N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 - 3
% of 1F                     - 81
2F Mean 198 203 214 219 222 230 230 231 237 240 241 242 244 245 249 245 251 4 27
SD 12.1 10.7 10.5 14.8 14.1 15.6 20.0 26.2 23.5 21.9 19.9 27.3 24.4 23.5 21.9 30.1 27.9 3.1 19.1
N 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
% of 1F                     - 115
3F Mean 186 194 206 206                              
SD 5.0 4.0 1.0 3.1                              
N 3 3 3 3                              
% of 1F                        

Group 1 animals 21 and 22 not exposed to air Days 3-7. Group 2 and 3 animals not exposed after Day 2

Table 7.5.2/5: Histopathology - group distribution of findings

 

Tissue/Organ and Findings Group/Sex Number of animals affected
1M 2M 3M 4M 1F 2F 3F 4F
No. of animals 1 3 2 3 1 2 0 3
Kidneys No. examined 0 0 0 1 0 0 0 3
Dilatation, Pelvic Moderate 0 0 0 1 0 0 0 0
Total 0 0 0 1 0 0 0 0
Accumulation, Hyaline Droplets Moderate 0 0 0 1 0 0 0 0
Total 0 0 0 1 0 0 0 0
Larynx No. examined 1 0 0 3 1 0 0 3
Infiltrate, Inflammatory Cell Minimal 0 0 0 0 0 0 0 2
Slight 0 0 0 0 0 0 0 1
Total 0 0 0 0 0 0 0 3
Lungs and Bronchi No. examined 1 0 0 3 1 0 0 3
Infiltrate, Inflammatory Cell

Minimal 1 0 0 1 1 0 0 0
Total 1 0 0 1 1 0 0 0
Alveolar Macrophage Aggregation Minimal 1 0 0 1 0 0 0 1
Total 1 0 0 1 0 0 0 1
Lymph Node, Tracheobronchial No. examined 1 0 0 3 1 0 0 3
Nose/Turbinates No. examined 1 0 0 3 1 0 0 3
Infiltrate, Inflammatory Cell Minimal 0 0 0 1 0 0 0 0
Total 0 0 0 1 0 0 0 0
Trachea No. examined 1 0 0 3 1 0 0 3
Tracheal Bifurcation No. examined 1 0 0 3 1 0 0 2

 

Conclusions:
(-)- alpha-pinene administration to Sprague Dawley rats by whole body inhalation exposure for 2 weeks at achieved exposure concentrations (mean of daily means) of 0.187 and 0.621 mg/L was well-tolerated, but at 1.61 and 2.75 mg/L, it was not tolerated and exposures were terminated after 2 days. Pale incisors noted macroscopically in some females given the test item were of uncertain toxicological significance. There were no test item related effects in animals exposed to 0.187 and 0.621mg/L.
It was concluded that a high target exposure level between 0.6 and 1.2 mg/L would be suitable for use in a subsequent 13-week study.
Executive summary:

In a 2-week dose range finding inhalation study, (-)-alpha-pinene was administered in the form of aerosol to Sprague Dawley rats (3 rats/sex/ group) by whole body inhalation exposure at nominal concentration levels of 0/0.2, 0.6, 1.2 and 2.4 mg/L for two weeks. Group 1 animals received the control, air (for 2 days)/test item (7 days after the start of the air exposure) or air alone, and Groups 2, 3 and 4 received the test item by whole body inhalation for 2 weeks (6 hours daily exposure for 5 days each week) for Group 4 and only 2 days for Groups 2 and 3. During the study, clinical condition, body weight, food consumption, organ weight, macropathology and histopathology investigations were undertaken.

 

The achieved exposure concentrations (mean of daily means) were 0.187 and 0.621 mg/L for 0.2 and 0.6 mg/L targeted, and at 1.61 and 2.75 mg/L for 2 consecutive days for 1.2 and 2.4 mg/L targeted. The mean achieved atmosphere concentrations were 94, 134, 115 and 104% of target for Groups 1, 2, 3 and 4, respectively.

 

Following the second exposure, all females and one male exposed to 2.75 mg/L and one female exposed to 1.61 mg/L were sacrificed due to clinical signs included decreased activity, breathing irregularities, tremor/spasms/repetitive movements, gait abnormalities (elevating/swaying/flattened, uncoordinated) and flattened posture. Microscopic changes were limited to pale incisors in most females and firm seminal vesicles in the male, but there were no pathological findings attributable to the early termination of any decedent animal. There were no test item related effects in animals exposed to 0.187 or 0.621 mg/L on clinical observations, body weight, food consumption or organ weights. One female exposed to 0.187 mg/L also had pale incisors noted macroscopically; no histopathology was conducted on this animal.

 

Exposure at 1.61 and 2.75 mg/L was not tolerated and exposures were terminated after 2 days. Pale incisors noted macroscopically in some females given the test item were of uncertain toxicological significance. There were no test item related effects in animals exposed to 0.187 and 0.621mg/L. It was concluded that a high target exposure level between 0.6 and 1.2 mg/L would be suitable for use in a subsequent 13-week study.

Data source

Reference
Reference Type:
other: Draft report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 1000037958
Purity : 94.4%
Name of test material (as cited in study report): (-)-alpha-pinene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 04 May 2020

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TO BE CHECKED/COMPLETED IF NEEDED (from range-finding study)

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: males: 335 to 390 g; females: 195 to 265 g
- Housing: 3 rats/cage/sex in polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: Teklad 2014C diet, ad libitum (except at scheduled necropsy and during dosing)
- Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (except during dosing)
- Acclimation period: at least 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 ºC
- Humidity: 40-70 %
- Photoperiod: 12 h light / 12 h dark

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
TO BE CHECKED/COMPLETED IF NEEDED (comes from range-finding study)
During preliminary characterization trials particle size distribution samples were collected from the exposure system using a Marple Cascade Impactor and submitted for chemical analysis. The results indicated that no test item impacted on the stainless-steel substrates with all the sample being collected in the solvent trap. The achieved distribution meant that a MMAD value could not be calculated. A larger volume sample was collected; however, this did not offer any improvement in calculating a MMAD value. Therefore, no PSD samples were collected during study.
Details on inhalation exposure:
TO BE CHECKED/COMPLETED IF NEEDED (comes from range-finding study)

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole body exposure chamber (total chamber volume = 137.5L).
Stainless steel and acrylic polymer construction comprising a base unit, cuboidal housing section and a semi-pyramidal top section incorporating a central inlet and an inverted cone to aid atmosphere distribution. The dimensions of the chamber give an internal volume of approximately 120 L. A large glass elutriator was incorporated into the exposure system which connected to the top of the semi-pyramidal section via aerosol ducting. The dimensions of the elutriator give an internal volume of approximately 10.5 L.
- Method of holding animals in test chamber: Individually in stainless steel mesh compartments within restraint caging positioned within the cuboidal section of the exposure system.
- Aerosol Generation: A stainless-steel concentric jet atomizer designed to produce and maintain an atmosphere containing a high proportion of respirable droplets. The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump.
- Inlet Airflow:
From in-house compressed air system – breathing quality
Generator flow:14 - 49L/minute
Supplementary flow: 15 -35 L/minute
- Extract Airflow:
Drawn by in-house vacuum system; filtered locally; Extract flow: 30-50 L/minute
- Airflow Monitoring:
High quality tapered tube flowmeters – calibrated daily
In-line flowmeters monitored continuously
- System containment: systems housed in separate ventilated cabinets
- Temperature in air chamber: Measured using an electronic thermometer probe placed in the breathing zone of the animals via a port on the exposure chamber. Chamber air temperature was monitored continuously and recorded at 30-minute intervals.
- Air flow rate: 2.0 L/minute
- Method of particle size determination: determined by cascade impaction.

Samples collected as follows:
During preliminary characterization trials particle size distribution samples were collected from the exposure system using a Marple Cascade Impactor and submitted for chemical analysis. A larger volume sample was collected; however, this did not offer any improvement in calculating a MMAD value. Therefore, no PSD samples were collected during study.

ATMOSPHERE ANALYSIS
Aerosol samples collected as follows:
Collection media: Dreschel head and solvent trap (bubbler)
Sample solvent: Acetonitrile
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: 1 sample from Group 1(control)/day (taken at approximately 60 minutes during exposure), 3 samples from Group1 (test), 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
Sample location: Port on exposure chamber
Sample analysis: Chemical.
Sample disposition: Bubblers sent to DFA for chemical analysis daily

During preliminary characterization trials an assessment was made of the percentage breakthrough of test item through the sample collection media; this was achieved by setting up two bubblers in series and collecting a sample of test atmosphere. The acceptable breakthrough limit to the second solvent trap is ≤ 10%. During preliminary characterization trials the amount of breakthrough observed was below the 10% threshold, therefore only one bubbler used to collect chamber atmosphere samples.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test article concentration was analysed by chemical analysis (GC method of analysis: test samples were extracted with acetonitrile then injected by split injection onto GC column (ZB-50) with flame ionisation detection).
Duration of treatment / exposure:
13-week exposure period following by a 4-week recovery period.
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0.15, 0.3 and 0.9 mg/L
Basis: nominal conc.
No. of animals per sex per dose:
10 sex/dose for the main study
5 sex/dose for the recovery phase
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: test concentrations were selected on the basis of the results of a preliminary toxicity study by inhalation administration to rats for 2 Weeks (Study Code: TW76LH), where (-)-alpha-pinene was administered in the form of aerosol to Sprague Dawley rats (3 rats/sex/ group) by whole body inhalation exposure at nominal concentration levels of 0/0.2, 0.6, 1.2 and 2.4 mg/L for two weeks. Group 1 animals received the control, air (for 2 days)/test item (7 days after the start of the air exposure) or air alone, and Groups 2, 3 and 4 received the test item by whole body inhalation for 2 weeks (6 hours daily exposure for 5 days each week) for Group 4 and only 2 days for Groups 2 and 3. During the study, clinical condition, body weight, food consumption, organ weight, macropathology and histopathology investigations were undertaken.
Exposure at 1.61 and 2.75 mg/L was not tolerated and terminated after 2 days. Pale incisors noted macroscopically in some females given the test item were of uncertain toxicological significance. There were no test item related effects in animals exposed to 0.187 and 0.621mg/L. It was concluded a high target exposure level between 0.6 and 1.2 mg/L would be suitable for use in a subsequent 13-week study.
- Rationale for animal assignment: randomly allocated on arrival; using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Post-exposure recovery period in satellite groups: each dose groups (5/sex/dose).
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
TO BE CHECKED/COMPLETED IF NEEDED

CAGE SIDE OBSERVATIONS: yes
Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: yes
Time schedule: detailed observations were recorded daily, on exposure days, at the following times in relation to dose administration (pre-exposure observation); as each animal is returned to its home cage; as late as possible in the working day
In addition observations were made in the threatment period, on days without exposures, at the following times during the day: early in the working day (equivalent to pre-exposure observation); as late as possible in the working day.
A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: yes
Time schedule for examinations: The weight of each animal was recorded twice weekly before treatment commenced, daily throughout the study and before necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded twice weekly before the commencement of treatment and daily during Weeks 1 and 2 of the study.

WATER CONSUMPTION: no

OPHTHALMOSCOPIC EXAMINATION: yes
Time schedule for examinations
- Pretreatment: all animals (Main, Recovery and spares)
- Week 13: all Main and Recovery animals of Groups 1 and 4
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide, ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: yes
Time schedule for collection of blood: during Week 13, all animals (Main and recovery); during Week 4 (recovery), all recovery animals.
Anaesthetic used for blood collection: yes;
Animals were held under light general anaesthesia induced by isoflurane.
Animals fasted: yes;
Blood samples were collected after overnight withdrawal of food and prior to dosing.
Parameters checked:
Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) & Large unstained cells (LUC), Platelet count (Plt), Morphology: Anisocytosis, Microcytosis, Macrocytosis, Hypochromasia & Hyperchromasia
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. In the presence of platelet clumping a manual count of the differential white blood cell parameters was performed.

Using citrate as anticoagulant - Prothrombin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: yes
Time schedule for collection of blood: Week 13, all animals (Main and recovery).
Anaesthetic used for blood collection: yes;
Animals were held under light general anaesthesia induced by isoflurane.
Animals fasted: yes;
Blood samples were collected after overnight withdrawal of food and prior to dosing.
Parameters checked: blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and albumin concentration was analysed.

URINALYSIS: yes
Time schedule for collection of urine: Week 13

BRONCHO-ALVEOLAR LAVAGE EXAMINATION: yes, at termination study (main and recovery)

NEUROBEHAVIOURAL EXAMINATION: no
Sacrifice and pathology:
TO BE CHECKED/COMPLETED IF NEEDED
Necropsy: animals were killed by overdose of intraperitoneal pentobarbitone sodium followed by exsanguination.
Schedule:
Main study animals were killed following 13 weeks of treatment.
Recovery animals were killed following 13 weeks of treatment and 4 weeks of recovery.

All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals (Table 7.5.2/1).

HISTOPATHOLOGY: yes
Fixation: tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below.
Testes were preserved in modified Davidson’s fluid.
Eyes were preserved in Davidson’s fluid.

Histology:
Processing: tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required (Table 7.5.2/1).
Full List: main study and recovery animals of Groups 1 and 4 were killed at a scheduled interval.
Nasal turbinates and nasal pharynx: main study animals of Groups 2 and 3, and Recovery Phase animals of Groups 1 and 4 were killed at a scheduled interval.
Routine staining: sections were stained with haematoxylin and eosin.

Light microscopy:
Tissues preserved for examination were examined as follows.
Main study
- All animals of Groups 1 and 4: all specified tissues are in Table 7.5.2/1.
- All animals of Groups 2 and 3 and recovery animals: abnormalities only.
The following tissues, which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study animals of Groups 2 and 3 and recovery animals of Groups 1 and 4: nasal turbinates and nasal pharynx.
Other examinations:
Haematology, bone marrow: bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods.
Fixation: smears were air dried and subsequently fixed in methanol.
Analysis: no examinations were performed, however, the smears were retained for possible future examination.
Statistics:
See section "Any other information on materials and methods incl. tables

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See above for details.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- There were two decedents in females exposed to 0.9 mg/L. Female 132 was sacrificed on Day 3 (Week 1) due to general poor clinical condition including tremor, piloerection, flattened posture, thin build and was abnormally cold to touch. At necropsy, pale areas were noted on the right caudal lobe of the lungs and there was bilateral distention of the periovarian sac of the ovaries. Female 137 was sacrificed on Day 9 (Week 2) due to general poor clinical condition including three consecutive convulsions (1-30 seconds) within 5 minutes. At necropsy, there were no macroscopic findings noted.
Clinical signs following exposure at 0.9 mg/L were noted on isolated occasions on return to cage after the end of exposure, with more females affected than males, and included flattened posture, elevated gait, hunched posture and, in females only, tremor. In males, these signs were noted on one or two occasions with the number of animals with elevated gait, flattened posture or hunched posture being nine, three and three respectively. In females, tremor, elevated gait, flattened posture and hunched posture were seen in the majority of females on between one and 11 occasions for tremor and elevated gait and up to six occasions for hunched or flattened posture. Unsteadiness was noted in two females on one or two occasions. These signs usually resolved by the end of the day examination and were not present prior to exposure the following day. A comparison of days the signs are seen and individual daily exposure concentrations will be made to ascertain if the signs are linked to days when exposure was higher, as it seems that a target aerosol concentration of 0.9 mg/L is close to that where adverse signs are noted.
- Clinical signs following exposure at 0.3 mg/L included tremor in one male, hunched posture in two males and four females and elevated gait in five males and seven females, seen on one or two occasions.
- Clinical signs following exposure at 0.15 mg/L included flattened gait on a single occasion in one male and one female, hunched posture on one occasion in two females and elevated gait on 1 or 2 occasions in three females.
- Clinical signs of red staining around the eyes and on head were noted on return to cage after exposure in a small number of animals from all groups (including control), and are considered likely to be associated with the route and duration of exposure and not test item-related.
- There were no test item-related signs noted during the detailed weekly physical examination.
- All other exposure phase animals and all recovery phase animals survived to their scheduled sacrifice.

BODY WEIGHT AND WEIGHT GAIN
- There were no changes in group mean body weight gain that were considered to be test item-related.

FOOD CONSUMPTION
- There was a slight and transient lower group mean food consumption in females exposed to 0.9 mg/L over the first four days of exposure in both Weeks 1 and 2 when compared to the control. Thereafter, group mean food consumption in these females was essentially similar to the controls such that overall consumption over the 13 weeks of exposure was similar to that of the control group.
- There were no test item-related effects on food consumption in males exposed to 0.9 mg/L or in males and females exposed to 0.15 or 0.3 mg/L.

OPHTHALMOSCOPIC EXAMINATION
There were no test item related ophthalmic changes in Week 13.

HAEMATOLOGY
- There were no test item related effects on the haematological parameters evaluated in Week 13. Occasional differences from control were inconsistent between the sexes, lacked dose relationship, or were considered to be due to high intra group variation and were therefore considered not to be test item related.

CLINICAL CHEMISTRY
- There were no test item related effects on the blood chemistry parameters evaluated in Week 13. Occasional differences from control were inconsistent between the sexes, lacked dose relationship, or were considered to be due to high intra group variation and were therefore considered not to be test item related.

URINALYSIS FINDINGS
- There were no test item related effects on the urinalysis parameters evaluated in Week 13. Occasional differences from control were inconsistent between the sexes, lacked dose relationship, or were considered to be due to high intra group variation and were therefore considered not to be test item related.

BRONCHO-ALVEOLAR LAVAGE EXAMINATIONS
- In the bronchoalveolar lavage supernatant samples, although there was some intergroup variability and some high values that will be evaluated when the histopathological examinations are completed., group mean lactate dehydrogenase and protein levels were lower than control in females exposed to 0.9 mg/L (0.6X Control).
- The total and differential cell count data is awaited.

ORGAN WEIGHTS
- Group mean body weight adjusted liver weights were statistically significantly higher than the control in males and females exposed to 0.9 mg/L (116% and 107% of controls respectively). Group mean body weight adjusted kidney weights was higher than the control in males exposed to 0.9 mg/L (121% of control).
- There were no other changes in organ weights considered to be test item-related. Occasional differences from control were inconsistent between the sexes, lacked dose relationship, or were considered to be due to high intra group variation and were therefore considered not to be test item related. Organ weight changes will be reviewed once the pathological examinations are completed.
- At the recovery sacrifice, no (-)-alpha-pinene-related statistically significant mean body weight adjusted organ weight differences from controls were noted.

GROSS PATHOLOGY
- In animals killed after 13 weeks of treatment, it was observed irregular kidney surface was seen in two males exposed to 0.9 mg/L. Pale areas in the lungs and bronchi were seen above the combined control incidence in animals exposed to 0.9 mg/L.
- All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age; therefore, they were not considered related to (-)-alpha-pinene.
- No (-)-alpha-pinene-related macroscopic findings were noted at recovery sacrifice. All macroscopic findings were examined microscopically and were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age; therefore, they were considered not related to previous exposure to (-)-alpha-pinene.

HISTOPATHOLOGY: NON-NEOPLASTIC
- In the kidneys of exposure phase animals, a minimal to moderate accumulation of hyaline droplets in the cortical tubular epithelium and minimal to moderate basophilia of the cortical tubular epithelium was seen in all males exposed to 0.9 mg/L. Minimal to moderate tubular granular casts in the outer medulla was seen in the majority of males exposed to 0.9 mg/L and minimal cortical tubular degeneration was also seen in two males of this exposure group.
- In females, minimal basophilia of the cortical tubular epithelium was seen in one control female and minimal cortical tubular degeneration was seen in one female exposed to 0.3 mg/L. In consequence, these test item-related findings were considered confined to males and accounted for the irregular renal surface seen at necropsy in two males that were exposed to 0.9 mg/L and for the higher than control mean body weight adjusted kidney weights for males exposed to 0.9 mg/L. The increase in hyaline droplets in the kidneys of males is consistent with the accumulation of alpha-2µ-globulin, a common finding in untreated male rats (Khan KNM et al., 2002). Hyaline droplet accumulation is both sex and species specific (Frazier et al., 2012) and is generally not considered to be significant in man. However, the association of hyaline droplets with basophilic tubules and granular casts, known as alpha-2µ-globulin nephropathy, is considered to be adverse in the animals affected.

- In the lungs and bronchi of exposure phase animals, minimal foamy alveolar macrophages were seen at a higher incidence in males exposed to 0.9 mg/L than in control males and minimal alveolar eosinophilic crystals with associated inflammatory cell infiltrate (generally mixed cell) was seen at a clearly higher incidence in males of this exposure group compared with male controls. Foamy alveolar macrophages, without any associated inflammatory cell infiltrate (neutrophilic or lymphocytic) or damage to the adjacent alveolar walls, may be a response induced by inhaled items ascribable to either phagocytosis of poorly soluble drug particles or to pharmacology (Lewis et al., 2013). The foamy macrophages generally accounted for the pale areas seen at necropsy in control and exposed animals. Although alveolar eosinophilic crystals, with or without associated inflammatory cell infiltrate, may be seen as a background finding in rodent lungs (Renne et al., 2009), as the incidence of both foamy alveolar macrophages and alveolar eosinophilic crystals were clearly higher in males exposed to 0.9 mg/L than in controls these finding were considered to have an uncertain relationship to exposure in this sex.

- In the tongue, minimal or slight myofiber degeneration/regeneration/necrosis and minimal or slight hemorrhage were seen in control animals and males and female exposed to 0.9 mg/L. These findings were considered directly related to the blood sampling procedure from the sublingual vein.

- In the eyes, minimal to moderate bilateral retinal atrophy was seen at similar incidences in control males and females and males and females exposed to 0.9 mg/L. In consequence, this finding was considered incidental and not related to exposure to (-)-alpha-pinene.

- All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague Dawley rats of this age; therefore, they were considered not test item-related.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
0.3 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.9 mg/L air (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

ATMOSPHERE ANALYSIS AND ESTIMATION OF ACHIEVED DOSE

Table 7.5.2/1: Summary data

Group Target concentration (mg/L) Achieved concentration (mg/L)
1 - -
2 0.15 0.140
3 0.3 0.297
4 0.9 0.809

The achieved aerosol concentrations were 93, 99 and 90% of the target concentration for Groups 2, 3 and 4, respectively. Settings for Group 4 were reduced in Week 2 and as they were then running low, were again increased from Week 6.

 

ORGAN WEIGHTS

 

Table 7.5.2/2: Test Item-Related Effects in Organ Weight Parameters –Terminal Sacrifice 

Sex  (-)-alpha-pinene 
Males  Females 
Target exposure level (mg/L)  0.15  0.3  0.9  0.15  0.3  0.9 

Kidneys 

Absolute Weight (g) 

3.097  112  112  122  1.830  103  106  103 
Body Weight Adjusted (g)  3.123  108  114  121**  1.829  102  106  105 

Liver 

Absolute Weight (g) 

16.612  111  105  118  10.889  106  104  105 
Body Weight Adjusted (g)  16.740  108  106  116**  10.879  105  104  107* 
*/** = p≤0.05/p≤0.01 statistically significant difference (absolute or body weight adjusted) compared with respective control mean value. Note: Values for absolute weight and body weight adjusted organ weights for exposed groups expressed as percentage of control mean value (Percent control is 101%, (NOT 1%, 98%, not -2%). 

MACROSCOPIC FINDINGS

Table 7.5.2/3: Incidence of Test Item-Related Macroscopic Findings – Terminal Sacrifice 

Sex  (-)-alpha-pinene 
Males  Females 
Target exposure level (mg/L)  0.15  0.3  0.9  0.15  0.3  0.9 
Kidneys                 
Number Examined  10  10  10  10  10  10  10 
  Irregular surface 
Lungs and bronchi                 
Number Examined  10  10  10  10  10  10  10 
  Pale area(s) 

MICROSCOPIC FINDINGS

Table 7.5.2/4: Incidence and Severity of Test Item-Related Microscopic Findings –Terminal Sacrifice 

Sex  (-)-alpha-pinene 
Males  Females 
Target exposure level (mg/L)  0.15  0.3  0.9  0.15  0.3  0.9 
Kidneys                 
Number Examined  10  10  10 
Accumulation, Hyaline Droplets, Epithelial, Tubular                 
Minimal  
Slight 
Moderate 
Basophilia, Epithelial, Tubular                 
Minimal  
Slight 
Moderate 
Casts, granular, Tubular                 
Minimal  
Slight 
Moderate 
Degeneration, Tubular                 
Minimal  

 

Table 7.5.2/5: Incidence and Severity of Microscopic Findings of Uncertain Relationship to Exposure– Terminal Sacrifice 

Sex  (-)-alpha-pinene 
Males  Females 
Target exposure level (mg/L)  0.15  0.3  0.9  0.15  0.3  0.9 
Lungs and Bronchi                 
Number Examined  10  10  10 
  Alveolar Macrophages, Foamy                 
Minimal  
Slight 
  Eosinophilic Crystals with Associated Inflammatory Cell Infiltrate, Alveoli                 
Minimal  

 

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.3 mg/L, based on two females decedents (No. 132 and 137) at the concentration level of 0.9 mg/L due to general poor clinical condition.
Executive summary:

In a repeated dose toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, (-)-alpha-pinene was administered by inhalation-aerosol to groups of Sprague Dawley rats (10 rats/sex/group) by whole-body inhalation exposure at target exposure levels of 0.15, 0.3 and 0.9 mg/L for 6 hours per day, 5 days per week for 13 weeks. Control animals received air only. Recovery animals were similarly treated for 13 weeks followed by a 4 week off dose period. During the study, clinical condition, body weight, food consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry, organ weight, broncho-alveolar lavage examinations, macropathology and histopathology investigations were undertaken.

 

The achieved aerosol concentrations were 93, 99 and 90% of the target concentration for Groups 2, 3 and 4, respectively. Settings for Group 4 were reduced in Week 2 and as they were then running low, were again increased from Week 6.

 

There were no treatment related deaths or effects on food consumption, blood chemistry, ophthalmoscopy, urinalysis, organ weights or broncho-alveolar lavage examinations.

 

There were two unscheduled female deaths during the exposure phase of the study in the group exposed to 0.9 mg/L. Following microscopic examination, no histopathological cause for either death was established. 

In the kidneys of exposure phase animals, test item-related accumulation of hyaline droplets in the cortical tubular epithelium and basophilia of the cortical tubular epithelium were seen in all males exposed to 0.9 mg/L. Tubular granular casts in the outer medulla were seen in the majority of males exposed to 0.9 mg/L and cortical tubular degeneration was also seen in two males of this exposure group. These findings accounted for the irregular renal surface seen at necropsy in two males that were exposed to 0.9 mg/L and for the higher than control mean body weight adjusted kidney weights for males exposed to 0.9 mg/L. These test item-related findings were considered confined to males. 

In the lungs and bronchi of exposure phase animals, findings of an uncertain relationship to the test item were seen in males. Foamy alveolar macrophages were seen at a higher incidence in males exposed to 0.9 mg/L than in control males and alveolar eosinophilic crystals with associated inflammatory cell infiltrate (generally mixed cell) were seen at a clearly higher incidence in males exposed to 0.9 mg/L than in male controls. The foamy macrophages generally accounted for the pale areas seen at necropsy. 

Therefore, the No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.3 mg/L, based on the two females decedents at the concentration level of 0.9 mg/L due to general poor clinical condition.