Registration Dossier

Administrative data

Description of key information

In a Preliminary Toxicity Study, the systemic toxic potential of the registered substance was assessed in a 21-day oral study (dietary administration) in Sprague-Dawley rats to aid in the selection of a suitable high dose for a subsequent OECD 421 screening study. Based on the results of this study, it was concluded that the effects observed at the high dose of 12000 ppm do not preclude the use of this dose level as the high dose for the main OECD 421 study.

In a GLP and OECD 413 compliant 90-day repeated dose toxicity study by inhalation with the registered substance, 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.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August to 23 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
The purpose of this study was to assess the systemic toxic potential of the test item in a 21-day oral study (dietary administration) in Sprague-Dawley rats, and to aid in the selection of a suitable high dose for a subsequent OECD 421 screening study.
GLP compliance:
no
Remarks:
The study is not GLP compliant due to the preliminary nature of the study.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD) rat
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females nulliparous and non-pregnant
- Age at study initiation: male: 68-74 days and female: 82-88 days
- Weight at study initiation: male: 324 to 373g and female: 252 g to 288 g.
- Housing: Each sex was allocated separately
. Four animal per cage
- Diet: SDS VRF1 Certified powdered diet. ad libitum
- Water ad libitum: potable water
- Acclimation period: Five days before the beginning of the treatment (observations of the animals and their cages were recorded at least once per day).

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes : Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark

Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
corn oil
Details on oral exposure:
- DIET PREPARATION
- Mixing appropriate amounts with (Type of food): SDS VRF1 Certified with corn oil stabilizer at a ratio of test item to corn oil of 5 to 1.
- Rate of preparation of diet (frequency): Weekly.
Before the beginning of treatment, the suitability of the proposed mixing procedures were determined and specimen formulations analyzed at concentrations of 1000 to 15000 ppm to assess the stability and homogeneity of the test item in the diet matrix as part of the main OECD 421 study (Covance Study Number: MR86GX).
- Storage temperature of food: Deep-frozen (nominally -20°C)

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
21 days
Frequency of treatment:
Continuously
Dose / conc.:
3 000 ppm
Dose / conc.:
6 000 ppm
Dose / conc.:
12 000 ppm
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a recent OECD 421 GLP study (Envigo Study GR06DS) using the structurally similar substance Alpha-pinene multiconstituent, the dietary concentrations of 3000, 6000 and 12000 ppm were tested. The same concentration levels were used in this study in order to enable comparison of the toxic effects observed between the two substances and to assess the potential similarity of toxicity between them.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cages were inspected daily for evidence of animal ill-health amongst the occupants.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on Days -3, 1, 4, 8, 11, 15, 18 and 22 (before necropsy) on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each animal was recorded daily throughout the study from Day -3 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily throughout the study from Day -3.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples (0.7 mL) were collected after overnight withdrawal of food at week 3, for all animals.
- Method: Animals were held under light general anesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Creatine Phosphokinase (CK), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Bile aids (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) and Albumin/globulin ration (A/G ratio) from total protein concentration and albumin concentration.

ESTROUS CYCLES: Yes
- Daily smears were taken for 21 days, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the estrous cycle

Sacrifice and pathology:
SACRIFICE: Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY:
All animals were killed on Day 22 and 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.

The organs weighed and tissue samples fixed are detailed in table 1.
Other examinations:
ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed individually and summed for presentation in the tables. Requisite organs were weighed for animals killed at the scheduled interval.

FIXATION: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of the testes initially in modified Davidson’s fluid.
The tissues were retained pending any future requirement for processing and examination
Statistics:
No statistical analysis of the data was performed on this study.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related changes in clinical condition.
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following the start of treatment at 12000 ppm, minor mean body weight loss was recorded for males between Days 1 and 2 of the study, while for females a mean progressive weight loss of 8 grams was recorded over Days 1-3 of the study. For both sexes at 12000 ppm weight gain was then recorded between Days 2 and 3 for males and Days 3 and 4 for females. Despite a second period of minor weight loss recorded between Days 5-7 of the study in females, the bodyweight gain between Day 4 and Day 22 was higher than Control (13 g vs. 17 g for Control and for 12000 ppm dose level, respectively) and mean bodyweights were similar at the end of treatment. For males at this dose level, mean bodyweight was only slightly lower than Controls (-0.5%) at the end of treatment and bodyweight gain during Days 4-22 was only 9% lower than Control, mainly due to one animal.

Following the start of treatment at 6000 or 3000 ppm, mean bodyweight gains in males were unaffected by treatment but mean weight gains in females at 3000 or 6000 ppm were slightly lower than in Controls. However, bodyweight gain between Days 4-22 in females was similar to Control at 3000 ppm and lower at 6000 ppm (while higher than Control at 12000 ppm).
In males, bodyweight gain between Days 4-22 was only slightly lower than Control at 3000 ppm and was lower than Control at 6000 ppm, due to one out of 4 males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following the start of treatment at 12000 ppm, food consumption in males was slightly lower on Days 1-2, while intake in females was markedly low on Days 1-2 and 2-3 of treatment; thereafter, food consumption of both sexes improved but females showed a second period of low intake on Days 6-9 of the study. Food intake in females receiving 6000 ppm was slightly low on Days 1-2 and 2-3 of study.
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:
no effects observed
Description (incidence and severity):
Blood chemistry parameters were unaffected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Absolute and body weight adjusted liver weights were higher at the end of the treatment period in males and females that received 6000 or 12000 ppm and males that received 3000 ppm when compared with controls, although not clearly dose-related.
- Absolute and body weight adjusted thymus weights were higher in males at all dose levels when compared with controls but without dose relationship. Absolute and body weight adjusted prostate weights were lower in males that received 6000 or 12000 ppm when compared with controls but without dose dependency.
- Absolute and body weight adjusted spleen, uterus/cervix/oviduct and vagina weights were higher in all female treated groups when compared with controls. Absolute and body weight adjusted thymus weight was lower in females that received 12000 ppm when compared with controls.

All other inter-group differences were considered to be minor or lacked dose-relationship and were therefore attributed to normal variation, or were attributed to the differences in terminal body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings after three weeks that were attributable to treatment with (-)-alpha-pinene. All findings were considered incidental and were unrelated to treatment
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
ESTROUS CYCLES:
3 out of 4 females at 6000 ppm, and 4 out of 4 females in Control and at 3000 and 12000 ppm showed a normal estrous cycle of 4/5 days in length throughout the treatment period. 1 out of 4 animals at 6000 ppm had an irregular cycle. Therefore it is considered that there was no effect of treatment on estrous cycles.
Details on results:
ACHIEVED DOSE:
The overall mean achieved dosages were 188, 363 and 717 mg/kg bw/day in males and 185, 355 and 681 mg/kg bw/day in females receiving 3000, 6000 and 12000 ppm, respectively.
Remarks on result:
not determinable
Critical effects observed:
no
Conclusions:
The administration of the test item to Sprague Dawley rats by dietary administration for 21 days at dietary levels of 3000, 6000, or 12000 ppm was well tolerated.
No animals died and the clinical condition of the animals was satisfactory.
Following the start of treatment at 12000 ppm, minor mean body weight loss was recorded for males between Days 1-2 of study, while for females mean progressive weight loss of 8 grams was recorded over Days 1-3 of study. For both sexes at 12000 ppm weight gain was recorded between Days 2-3 for males and Days 3-4 for females. Despite a second period of minor weight loss recorded between Days 5-7 of the study in females, the bodyweight gain between Day 4 and Day 22 was higher than Control (13 g vs. 17 g for Control and 12000 ppm, respectively) and mean bodyweights were similar at the end of treatment. For males at this dose level, mean bodyweight was only slightly lower than Controls (-0.5%) at the end of treatment and bodyweight gain during Days 4-22 was only 9% lower than Control, mainly due to one animal. Following the start of treatment at 6000 or 3000 ppm, mean bodyweight gain in males was unaffected by treatment but mean weight gain in females at 3000 or 6000 ppm were lower than Controls. However, bodyweight gain between Days 4-22 in females was similar to Control at 3000 ppm and lower at 6000 ppm (while higher than Control at 12000 ppm).
Following the start of treatment at 12000 ppm, food consumption in males was slightly lower on Days 1-2 while intake in females was markedly low on Days 1-2 and 2-3 of treatment. Thereafter, food consumption of both sexes improved but females showed a second period of low intake on days 6-9 of study. Food intake in females receiving 6000 ppm was slightly lower on Days 1-2 and 2-3 of the study.
Estrous cycles of the females and blood chemistry investigations in both sexes were unaffected by treatment. There were no findings attributable to treatment at macroscopic examination, but liver, thymus (males only), spleen (females only), uterus/cervix/oviducts and vagina weights were higher than that of Control for animals that received 3000, 6000 or 12000 ppm, with the exception of liver weight in females receiving 3000 ppm. Thymus weight was considered lower in females that received 12000 ppm.

Based on the results of this study, it was concluded that the effects observed at the high dose of 12000 ppm do not preclude the use of this dose level as the high dose for the main OECD 421 study.
Executive summary:

The purpose of this study was to assess the systemic toxic potential of the test item in a 21-day oral study (dietary administration) in Sprague-Dawley rats, and to aid in the selection of a suitable high dose for a subsequent OECD 421 screening study. Three groups, each comprising four male and four female Crl:CD(SD) rats received the test item at dietary concentrations of 3000, 6000 and 12000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil. During the study, clinical condition, body weight, food consumption, visual water consumption, estrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken.

The overall mean achieved dosages were 188, 363 and 717 mg/kg bw/day in males and 185, 355 and 681 mg/kg bw/day in females receiving 3000, 6000 and 12000 ppm, respectively.

Administration for 21 days at dose levels up to and including 12000 ppm was well tolerated. There were no premature deaths and no test item-related changes in clinical condition, estrous cycles, blood chemistry and macropathology.

 

Following the start of treatment at 12000 ppm, minor mean body weight loss was recorded for males between Days 1-2 of study, while for females mean progressive weight loss of 8 grams was recorded over Days 1-3 of study. For both sexes at 12000 ppm weight gain was recorded between Days 2-3 for males and Days 3-4 for females. Despite a second period of minor weight loss recorded between Days 5-7 of the study in females, the bodyweight gain between Day 4 and Day 22 was higher than Control (13 g vs. 17 g for Control and 12000 ppm, respectively) and mean bodyweights were similar at the end of treatment. For males at this dose level, mean bodyweight was only slightly lower than Controls (-0.5%) at the end of treatment and bodyweight gain during Days 4-22 was only 9% lower than Control, mainly due to one animal. Following the start of treatment at 6000 or 3000 ppm, mean bodyweight gain in males was unaffected by treatment but mean weight gain in females at 3000 or 6000 ppm were lower than Controls. However, bodyweight gain between Days 4-22 in females was similar to Control at 3000 ppm and lower at 6000 ppm (while higher than Control at 12000 ppm).

Following the start of treatment at 12000 ppm, food consumption in males was slightly lower on Days 1-2 while intake in females was markedly low on Days 1-2 and 2-3 of treatment. Thereafter, food consumption of both sexes improved but females showed a second period of low intake on days 6-9 of study. Food intake in females receiving 6000 ppm was slightly lower on Days 1-2 and 2-3 of the study.

Liver, thymus (males only), spleen (females only), uterus/cervix/oviducts and vagina weights were higher than that of Control for animals that received 3000, 6000 or 12000 ppm, with the exception of liver weight in females receiving 3000 ppm. Thymus weight was considered lower in females that received 12000 ppm.

 

It was therefore concluded that the effects observed at the high dose of 12000 ppm do not preclude the use of this dose level as the high dose for the main OECD 421 study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Preliminary study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to other study
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
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
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
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
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
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.
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
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

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  

 

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
300 mg/m³
Study duration:
subchronic
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
GLP and OECD guideline compliant study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to other study
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
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
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
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
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
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.
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
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

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  

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
GLP and OECD guideline compliant study

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The purpose of this study was to assess the systemic toxic potential of the test item in a 21-day oral study (dietary administration) in Sprague-Dawley rats, and to aid in the selection of a suitable high dose for a subsequent OECD 421 screening study. Three groups, each comprising four male and four female Crl:CD(SD) rats received the test item at dietary concentrations of 3000, 6000 and 12000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil. During the study, clinical condition, body weight, food consumption, visual water consumption, estrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken.
The overall mean achieved dosages were 188, 363 and 717 mg/kg bw/day in males and 185, 355 and 681 mg/kg bw/day in females receiving 3000, 6000 and 12000 ppm, respectively.
Administration for 21 days at dose levels up to and including 12000 ppm was well tolerated. There were no premature deaths and no test item-related changes in clinical condition, estrous cycles, blood chemistry and macropathology.
 
Following the start of treatment at 12000 ppm, minor mean body weight loss was recorded for males between Days 1-2 of study, while for females mean progressive weight loss of 8 grams was recorded over Days 1-3 of study. For both sexes at 12000 ppm weight gain was recorded between Days 2-3 for males and Days 3-4 for females. Despite a second period of minor weight loss recorded between Days 5-7 of the study in females, the bodyweight gain between Day 4 and Day 22 was higher than Control (13 g vs. 17 g for Control and 12000 ppm, respectively) and mean bodyweights were similar at the end of treatment. For males at this dose level, mean bodyweight was only slightly lower than Controls (-0.5%) at the end of treatment and bodyweight gain during Days 4-22 was only 9% lower than Control, mainly due to one animal. Following the start of treatment at 6000 or 3000 ppm, mean bodyweight gain in males was unaffected by treatment but mean weight gain in females at 3000 or 6000 ppm were lower than Controls. However, bodyweight gain between Days 4-22 in females was similar to Control at 3000 ppm and lower at 6000 ppm (while higher than Control at 12000 ppm).
Following the start of treatment at 12000 ppm, food consumption in males was slightly lower on Days 1-2 while intake in females was markedly low on Days 1-2 and 2-3 of treatment. Thereafter, food consumption of both sexes improved but females showed a second period of low intake on days 6-9 of study. Food intake in females receiving 6000 ppm was slightly lower on Days 1-2 and 2-3 of the study.
Liver, thymus (males only), spleen (females only), uterus/cervix/oviducts and vagina weights were higher than that of Control for animals that received 3000, 6000 or 12000 ppm, with the exception of liver weight in females receiving 3000 ppm. Thymus weight was considered lower in females that received 12000 ppm.
 
It was therefore concluded that the effects observed at the high dose of 12000 ppm do not preclude the use of this dose level as the high dose for the main OECD 421 study.

 

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 clinical signs 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.

Justification for classification or non-classification

Harmonised classification:

The test material has no harmonised classification for repeated dose toxicity according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self-classification:

Based on the available information, no additional self-classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.