(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March to 28 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

other: Draft report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(-)-pin-2(3)-ene

EC Number:

232-077-3

EC Name:

(-)-pin-2(3)-ene

Cas Number:

7785-26-4

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

impurity 1

Reference substance name:

(+)-pin-2(3)-ene

EC Number:

232-087-8

EC Name:

(+)-pin-2(3)-ene

Cas Number:

7785-70-8

Molecular formula:

C10H16

IUPAC Name:

(1R,5R)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

impurity 2

Reference substance name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-337-8

EC Name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-04-7

Molecular formula:

C10H16

IUPAC Name:

(1S,4R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 3

Reference substance name:

(-)-pin-2(10)-ene

EC Number:

242-060-2

EC Name:

(-)-pin-2(10)-ene

Cas Number:

18172-67-3

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

impurity 4

Reference substance name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-336-2

EC Name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-03-6

Molecular formula:

C10H16

IUPAC Name:

(1R,4S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 5

Reference substance name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

EC Number:

208-083-7

EC Name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

Cas Number:

508-32-7

Molecular formula:

C10H16

IUPAC Name:

1,7,7-trimethyltricyclo[2.2.1.0~2,6~]heptane

impurity 6

Reference substance name:

(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Cas Number:

19902-08-0

Molecular formula:

C10H16

IUPAC Name:

(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Test material form:

liquid

Details on test material:

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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited (Germany)
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: males: 335 to 402 g; females: 201 to 252 g
- Housing: three or four/cage/sex in polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: Teklad 2014C diet, ad libitum (removed overnight before blood sampling for hematology or blood chemistry or during exposure)
- Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (except during exposures)
- Acclimation period: 13 days before commencement of treatment

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

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

Analytical report awaited (not yet available)

Details on inhalation exposure:

Analytical report awaited (not yet available)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test article concentration was analysed by chemical analysis (GC method of analysis: test samples were extracted with acetonitrile then injected by split injection onto GC column (ZB-50) with flame ionisation detection).

Duration of treatment / exposure:

13-week exposure period following by a 4-week recovery period.

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.15 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

0.3 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

0.9 mg/L air (nominal)

No. of animals per sex per dose:

10 sex/dose for the main study
5 sex/dose for the recovery phase

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: test concentrations were selected on the basis of the results of a preliminary toxicity study by inhalation administration to rats for 2 Weeks (Study Code: TW76LH), where (-)-alpha-pinene was administered in the form of aerosol to Sprague Dawley rats (3 rats/sex/ group) by whole body inhalation exposure at nominal concentration levels of 0/0.2, 0.6, 1.2 and 2.4 mg/L for two weeks. Group 1 animals received the control, air (for 2 days)/test item (7 days after the start of the air exposure) or air alone, and Groups 2, 3 and 4 received the test item by whole body inhalation for 2 weeks (6 hours daily exposure for 5 days each week) for Group 4 and only 2 days for Groups 2 and 3. During the study, clinical condition, body weight, food consumption, organ weight, macropathology and histopathology investigations were undertaken.
Exposure at 1.61 and 2.75 mg/L was not tolerated and terminated after 2 days. Pale incisors noted macroscopically in some females given the test item were of uncertain toxicological significance. There were no test item related effects in animals exposed to 0.187 and 0.621mg/L. It was concluded a high target exposure level between 0.6 and 1.2 mg/L would be suitable for use in a subsequent 13-week study.
- Rationale for animal assignment: randomly allocated on arrival; using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Post-exposure recovery period in satellite groups: each dose groups (5/sex/dose).

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
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 acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Exposure:
Detailed observations were recorded, in the treatment period, on exposure days, at the following times in relation to exposure administration:
• Pre-exposure observation
• As each animal was returned to its home cage
• As late as possible in the working day
Observation during exposure was severely restricted due to the construction of the exposure chamber.
In addition observations were made in the treatment period, on days without exposures, at the following times during the day:
• Early in the working day (equivalent to pre-exposure observations)
• As late as possible in the working day

Clinical Signs:
A detailed weekly physical examination was performed on each animal to monitor general health.

Mortality:
A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases.


BODY WEIGHT:
The weight of each animal was recorded twice weekly during Weeks -1 to 4 and recovery Week 1, weekly during Weeks 5 to 13 and recovery Weeks 2 to 4 throughout the study and on the day of necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded twice weekly during Weeks -1 to 4 and recovery Week 1, weekly during Weeks 5 to 13 and recovery Weeks 2 to 4.

OPHTHALMOSCOPIC EXAMINATION:
Time schedule for examinations
- Pretreatment: all animals (Main, Recovery and spares)
- Week 13: all Main 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:
Time schedule for collection of blood: during Week 13, all animals (Main and recovery);
Blood samples were collected after overnight withdrawal of food and prior to dosing.
Sampling was performed on the morning after overnight collection of urine. Animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
Animals were held under light general anesthesia induced by isoflurane.

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 and percentage 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)

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.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY:
Time schedule for collection of blood: Week 13, all main animals
Blood samples were collected after overnight withdrawal of food and prior to dosing
Sampling was performed on the morning after overnight collection of urine. Animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
Animals were held under light general anesthesia induced by isoflurane. 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 Cobas 6000 Analyzer in respect of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), 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.

URINALYSIS:
Time schedule for collection of urine: Week 13, all main animals
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight.
The individual samples were examined for the following characteristics:
Using manual methods:
• Appearance (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek500 instrument:
• Glucose (Gluc)
• Blood pigments (UBld)
Using a Roche Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)

BRONCHO-ALVEOLAR LAVAGE EXAMINATION:
Time schedule: at termination (main and recovery)
The right lung was used for bronchoalveolar lavage sampling and the left lung was processed for histology and light microscopy.
A total and differential cell count of the BAL cells was performed using the XT-2000iV. total and differential cell count (neutrophils, eosinophils, mononuclear cells (included monocytes and macrophages) and lymphocytes) were reported as number of cells per animal and the differential cell count also as a percentage of the total cell count.
The BALF supernatants were analyzed on the Cobas 6000, on the day of collection, for the following characteristics:
Lactate dehydrogenase (LDH)
Total protein (Total Prot)

Sacrifice and pathology:

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:
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.
- 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, kidneys, lungs and bronchi.

Routine staining: Sections were stained with hematoxylin and eosin.
Special staining: α2μ globulin staining in kidneys was performed in representative animals as treatment-related changes were seen in kidneys

Other examinations:

Haematology, bone marrow: bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods.
Fixation: smears were air dried and subsequently fixed in methanol.
Analysis: no examinations were performed, however, the smears were retained for possible future examination.

Statistics:

See section "Any other information on materials and methods incl. tables

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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. There appeared to be a general trend that the days on which chamber aerosol levels were higher, the more frequent the signs were observed.
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.

Mortality:

mortality observed, treatment-related

Description (incidence):

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 muscle tremor, piloerection, flattened posture, thin build and was abnormally cold to touch. At necropsy, there was bilateral distention of the periovarian sac of the ovaries and pale areas were noted on the right caudal lobe of the lungs. At microscopic examination, bilateral periovarian (bursal) cysts accounted for the distention of the periovarian sacs. As only the left lung was processed for microscopy (the right lung being used for bronchoalveolar lavage sampling) the pale areas noted on the right caudal lobe could not be examined. No microscopic findings related to exposure to (-)-alpha-pinene were seen. Therefore, the major factor contributory to the welfare sacrifice of this animal was the poor clinical condition.

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. At microscopy, no findings were seen in the central nervous system to account for the convulsions. No microscopic findings related to exposure to (-)-alpha-pinene were seen, although slight alveolar eosinophilic crystals with associated inflammatory cell infiltrate were present that was considered a finding of uncertain relationship to exposure in terminal sacrifice animals. Therefore, the major factor contributory to the welfare sacrifice of this animal was the poor clinical condition.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

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 and the 4 weeks of recovery 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.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

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

Haematological findings:

no effects observed

Description (incidence and severity):

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 biochemistry findings:

no effects observed

Description (incidence and severity):

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.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

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.

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):

Mean body weight adjusted kidney weights were statistically significantly higher in males exposed to 0.9 mg/L (121% of control) when compared with controls.
Mean body weight adjusted liver weights were statistically significantly higher in males and females exposed to 0.9 mg/L (116% and 107% of controls, respectively). Following microscopic examination, no findings were seen to account for these statistically significant higher than control mean body weight adjusted liver weights in males and females exposed to 0.9 mg/L (See Table 7.5.2/2).
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in body weight adjusted but not both; lack of a relationship to exposure or correlative findings; and/or the magnitude was considered small.
At the recovery sacrifice, no (-)-alpha-pinene-related statistically significant mean body weight adjusted organ weight differences from controls were noted.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the kidneys of exposure phase animals, an irregular surface was seen in two males exposed to 0.9 mg/L. In the lungs and bronchi, pale areas were seen above the combined control incidence in animals that were exposed to 0.9 mg/L (Table 7.5.2/3).

All other macroscopic findings for exposure phase animals 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 (-)-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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidneys of exposure phase males, 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 (-)-alpha-pinene and demonstrated a relationship to concentration in terms of severity (See Table 7.5.2/4). Minimal to moderate tubular granular casts in the outer medulla was seen in the majority of males exposed to 0.3 or 0.9 mg/L and a few males exposed to 0.15 mg/L and minimal or slight cortical tubular degeneration was also seen in two males of each test exposure group. In females, minimal basophilia of the cortical tubular epithelium was seen in one control female and one female exposed to 0.3 mg/L and minimal cortical tubular degeneration was seen in another 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 kidneys of recovery phase males (See Table 7.5.2/5), a minimal accumulation of hyaline droplets in the cortical tubular epithelium was seen in one male of each group previously exposed to (-)-alpha-pinene. Minimal to moderate basophilia of the cortical tubular epithelium was seen in all males previously exposed to (-)-alpha-pinene and at the minimal severity in two control males and minimal or slight tubular granular casts in the outer medulla was seen in the minority of males previously exposed to 0.15, 0.3 or 0.9 mg/L. Minimal cortical scars, which were considered the result of previous tubular degeneration, were seen in one male and three males previously exposed to 0.3 or 0.9 mg/L, respectively. Therefore, accumulation of hyaline droplets and tubular granular casts both exhibited partial recovery in terms of severity and relative incidence, however, basophilia of the cortical tubular epithelium did not demonstrate recovery. In females, none of these findings were seen in previously exposed animals or in controls.


Findings of uncertain relationship to exposure:
In the lungs and bronchi of exposure phase animals (See Table 7.5.2/6), minimal or slight foamy alveolar macrophages were seen at a higher incidence in males exposed to 0.3 or 0.9 mg/L than in control males and minimal alveolar eosinophilic crystals with associated inflammatory cell infiltrate (generally mixed cell) were seen at a clearly higher incidence in males exposed to 0.15 or 0.9 mg/L compared with male controls and therefore, did not exhibit a clear concentration relationship. In females, both of these minimal findings were seen at similar incidences in control and exposed animals. 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 findings were considered to have an uncertain relationship to exposure in this sex.
In the lungs and bronchi of recovery phase animals (See Table 7.5.2/7), minimal or slight foamy alveolar macrophages were seen at, or lower than, the control incidence in males and females previously exposed to (-)-alpha-pinene and minimal alveolar eosinophilic crystals with associated inflammatory cell infiltrate (mixed cell) were seen in one male previously exposed to 0.15 mg/L. In consequence, these two findings were considered to be within the background level in these previously exposed male and female animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Bronchoalveolar Lavage: there were no test item-related changes in group mean lactate dehydrogenase and protein levels, or differential white cell counts in the right lung after 13 weeks of treatment or 4 weeks of recovery. Data, although variable, showed no exposure concentration-related trends and there were no pathological correlates in the lungs.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

0.3 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.9 mg/L air (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

ATMOSPHERE ANALYSIS AND ESTIMATION OF ACHIEVED DOSE

To be completed when available

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	0.15	0.3	0.9	0	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	0.15	0.3	0.9	0	0.15	0.3	0.9
Kidneys
Number Examined	10	10	10	10	10	10	10	8
Irregular surface	0	0	0	2	0	0	0	0
Lungs and bronchi
Number Examined	10	10	10	10	10	10	10	8
Pale area(s)	2	0	1	3	0	2	2	3

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	0.15	0.3	0.9	0	0.15	0.3	0.9
Kidneys
Number Examined	10	10	10	10	10	10	10	8
Accumulation, Hyaline Droplets, Epithelial, Tubular
Minimal	0	6	4	1	0	0	0	0
Slight	0	4	6	8	0	0	0	0
Moderate	0	0	0	1	0	0	0	0
Basophilia, Epithelial, Tubular
Minimal	0	7	3	3	1	0	1	0
Slight	0	2	5	4	0	0	0	0
Moderate	0	1	2	3	0	0	0	0
Casts, Granular, Tubular
Minimal	0	2	4	2	0	0	0	0
Slight	0	2	3	3	0	0	0	0
Moderate	0	0	0	1	0	0	0	0
Degeneration, Tubular
Minimal	0	2	1	2	0	0	1	0
Slight	0	0	1	0	0	0	0	0

 

Table 7.5.2/5: Incidence and Severity of Test Item-Related Microscopic Findings – Recovery

Sex	(-)-alpha-pinene
	Males				Females
Target exposure level (mg/L)	0	0.15	0.3	0.9	0	0.15	0.3	0.9
Kidneys
Number Examined	5	5	5	5	5	5	5	5
Accumulation, Hyaline Droplets, Epithelial, Tubular
Minimal	0	1	1	1	0	0	0	0
Basophilia, Epithelial, Tubular
Minimal	2	3	2	3	0	0	0	0
Slight	0	2	2	1	0	0	0	0
Moderate	0	0	1	1	0	0	0	0
Casts, Granular, Tubular
Minimal	0	1	0	1	0	0	0	0
Slight	0	0	2	1	0	0	0	0
Scars, Cortical
Minimal	0	0	1	3	0	0	0	0

Table 7.5.2/6: Incidence and Severity of Microscopic Findings of Uncertain Relationship to Exposure– Terminal Sacrifice

Sex	(-)-alpha-pinene
	Males				Females
Target exposure level (mg/L)	0	0.15	0.3	0.9	0	0.15	0.3	0.9
Lungs and Bronchi
Number Examined	10	10	10	10	10	10	10	8
Alveolar Macrophages, Foamy
Minimal	1	1	4	5	2	2	3	1
Slight	1	0	1	0	0	0	0	0
Eosinophilic Crystals with Associated Inflammatory Cell Infiltrate, Alveoli
Minimal	1	4	2	4	1	1	1	2

 

Table 7.5.2/7: Incidence and Severity of Microscopic Findings of Uncertain Relationship to Exposure– Recovery

Sex	(-)-alpha-pinene
	Males				Females
Target exposure level (mg/L)	0	0.15	0.3	0.9	0	0.15	0.3	0.9
Lungs and Bronchi
Number Examined	5	5	5	5	5	5	5	5
Alveolar Macrophages, Foamy
Minimal	1	1	1	1	2	2	1	1
Slight	1	0	0	0	0	0	0	0
Eosinophilic Crystals with Associated Inflammatory Cell Infiltrate, Alveoli
Minimal	0	1	0	0	0	0	0	0

 

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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

 

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 males, test item-related accumulation of hyaline droplets in the cortical tubular epithelium and basophilia of the cortical tubular epithelium was seen in all males exposed to (-)-alpha-pinene and demonstrated a relationship to concentration in terms of severity. Immunohistochemical (IHC) staining for alpha‑2µ-globulin in the kidneys of representative exposure phase animals confirmed the presence of this protein in the epithelial hyaline droplets. Tubular granular casts in the outer medulla was seen in the majority of males exposed to 0.3 or 0.9 mg/L and a few males exposed to 0.15 mg/L and cortical tubular degeneration was also seen in two males of each test exposure group. 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. These findings accounted for the irregular renal surface seen at necropsy in two males 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 confined to males.

In the kidneys of recovery phase males, accumulation of hyaline droplets in the cortical tubular epithelium was seen in one male of each group previously exposed to (‑)‑alpha‑pinene. Basophilia of the cortical tubular epithelium was seen in all males previously exposed to (-)-alpha-pinene and in two control males, and tubular granular casts in the outer medulla was seen in a few males previously exposed to 0.15, 0.3 or 0.9 mg/L. Cortical scars, which were considered the result of previous tubular degeneration, were seen in one male and three males previously exposed to 0.3 or 0.9 mg/L, respectively. Therefore, accumulation of hyaline droplets and tubular granular casts both exhibited partial recovery in terms of severity and relative incidence, however, basophilia of the cortical tubular epithelium did not demonstrate recovery.

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. 

In the lungs and bronchi of recovery phase animals, foamy alveolar macrophages were seen at, or lower than, the control incidence in males and females previously exposed to (‑)‑alpha‑pinene and alveolar eosinophilic crystals with associated inflammatory cell infiltrate (mixed cell) were seen in one male previously exposed to 0.15 mg/L. In consequence, these two findings were considered to be within the background level in these previously exposed male and female animals, and thus the foamy alveolar macrophages was considered to show full recovery.

Clinical signs after exposure noted at 0.9 mg/L tended to be more noticeable on days when the chamber aerosol concentrations were higher than target, and resulted in two deaths in female animals that were considered test item related. These signs were thus considered adverse. A few signs noted at 0.15 or 0.3 mg/L were considered due to the incidence and frequency to be not adverse.

The slight, transient, lower food consumption in females in Week 1 and 2 was not accompanied by any change in body weight and as such the food consumption change is considered not adverse.

There was no pathological correlates seen in the liver to account for the slightly higher weight in animals exposed to 0.9 mg/L; hence this change was considered not adverse and of no toxicological significance.

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.