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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January to 21 July 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:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
Adopted 25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
p-mentha-1(7),2-diene
EC Number:
209-081-9
EC Name:
p-mentha-1(7),2-diene
Cas Number:
555-10-2
Molecular formula:
C10H16
IUPAC Name:
3-isopropyl-6-methylenecyclohexene
Constituent 2
Chemical structure
Reference substance name:
(R)-p-mentha-1,8-diene
EC Number:
227-813-5
EC Name:
(R)-p-mentha-1,8-diene
Cas Number:
5989-27-5
Molecular formula:
C10H16
IUPAC Name:
(4R)-isopropenyl-1-methylcyclohexene
Constituent 3
Chemical structure
Reference substance name:
(S)-p-mentha-1,8-diene
EC Number:
227-815-6
EC Name:
(S)-p-mentha-1,8-diene
Cas Number:
5989-54-8
Molecular formula:
C10H16
IUPAC Name:
(4S)-isopropenyl-1-methylcyclohexene
impurity 1
Chemical structure
Reference substance name:
p-cymene
EC Number:
202-796-7
EC Name:
p-cymene
Cas Number:
99-87-6
Molecular formula:
C10H14
IUPAC Name:
1-isopropyl-4-methylbenzene
impurity 2
Chemical structure
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 3
Chemical structure
Reference substance name:
p-mentha-1,3-diene
EC Number:
202-795-1
EC Name:
p-mentha-1,3-diene
Cas Number:
99-86-5
Molecular formula:
C10H16
IUPAC Name:
1-isopropyl-4-methylcyclohexa-1,3-diene
impurity 4
Chemical structure
Reference substance name:
(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
Cas Number:
20296-50-8
Molecular formula:
C10H16
IUPAC Name:
(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
impurity 5
Chemical structure
Reference substance name:
(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
EC Number:
207-856-6
EC Name:
(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
Cas Number:
498-15-7
Molecular formula:
C10H16
IUPAC Name:
(1S,6R)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
impurity 6
Chemical structure
Reference substance name:
7-methyl-3-methyleneocta-1,6-diene
EC Number:
204-622-5
EC Name:
7-methyl-3-methyleneocta-1,6-diene
Cas Number:
123-35-3
Molecular formula:
C10H16
IUPAC Name:
7-methyl-3-methyleneocta-1,6-diene
impurity 7
Chemical structure
Reference substance name:
(5S)-isopropyl-2-methylcyclohexa-1,3-diene
Cas Number:
2243-33-6
Molecular formula:
C10H16
IUPAC Name:
(5S)-isopropyl-2-methylcyclohexa-1,3-diene
impurity 8
Chemical structure
Reference substance name:
(R)-5-isopropyl-2-methylcyclohexa-1,3-diene
EC Number:
224-167-6
EC Name:
(R)-5-isopropyl-2-methylcyclohexa-1,3-diene
Cas Number:
4221-98-1
Molecular formula:
C10H16
IUPAC Name:
(5R)-isopropyl-2-methylcyclohexa-1,3-diene
impurity 9
Chemical structure
Reference substance name:
p-mentha-1,4-diene
EC Number:
202-794-6
EC Name:
p-mentha-1,4-diene
Cas Number:
99-85-4
Molecular formula:
C10H16
IUPAC Name:
1-isopropyl-4-methylcyclohexa-1,4-diene
impurity 10
Chemical structure
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.: F290519
Purity: 74.8% (sum of the three main constituents)
Name of test material (as cited in study report): Reaction mass of beta-phellandrene and d-limonene and l-limonene
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 28 May 2020

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
RATIONALE FOR ANIMAL MODEL:
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 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.
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: males: 273 to 355 g; females: 191 to 242 g
- Housing: 3 or 4 rats/cage/sex in polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals and wood based bedding which was changed at appropriate intervals each week.
- 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).
- Environmental enrichment: Aspen gnawing material and plastic shelter provided to each cage throughout the study and replaced when necessary.
- Acclimation period: 12 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 ºC
- Humidity: 40-70 %
- Photoperiod: 12 h light / 12 h dark
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
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. To obtain
particle size measurements during the study an Aerodynamic Particle Size Spectrometer was
used.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through nose-only chamber.
Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber.
- Method of holding animals in test chamber: With a plastic nose-only restraint tube.
- Source and rate of air: from in-house compressed air system
– Breathing quality: Generator flow: 29 L/minute (Groups 1-4) and Supplementary flow: 20 L/minute (Groups 1, 3 and 4) and 50 L/minute (Group 2).
- System of generating particulates/aerosols: a stainless steel concentric jet atomiser, designed to produce and maintain an atmosphere containing a high proportion of respirable droplets. The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump.
- Method of particle size determination: by cascade impaction (Marple Cascade Impactor)
- Treatment of exhaust air: Drawn by in-house vacuum system and filtered locally; Extract flow: 50 L/minute (Groups 1, 3 and 4) and 80 L/minute (Group 2).
- Temperature: The observed chamber temperatures for all groups remained within the recommended range (19 – 25°C) for inhalation exposure of rats. The chamber temperatures were similar for each group on each day of the study.


TEST ATMOSPHERE
- Brief description of analytical method used: test article concentration was analysed by chemical analysis (GC method of analysis) in solvent trap samples.
- Samples taken from breathing zone: yes, aerosol samples were collected with Dreschel head and solvent trap (bubbler) at 1 sample from Group 1/day (taken at approximately 60 minutes during
exposure) and 3 samples from Group 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test article concentration was analysed by chemical analysis (GC method of analysis: test samples were extracted with ethyl acetate 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 / concentrationsopen allclose all
Dose / conc.:
0 mg/L air (nominal)
Remarks:
Group 1 (Air control)
Dose / conc.:
0.1 mg/L air (nominal)
Remarks:
Group 2 (test item)
Dose / conc.:
0.25 mg/L air (nominal)
Remarks:
Group 3 (test item)
Dose / conc.:
0.75 mg/L air (nominal)
Remarks:
Group 4 (test item)
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:
RATIONALE FOR EXPOSURE CONCENTRATION SELECTION
The target exposure concentrations used in this study (0, 0.1, 0.25 and 0.75 mg/L) were selected in conjunction with the Sponsor based on results of a 2-week study in the rat (Covance Study Number XG42WX) in which exposure at target concentrations of 1 mg/L and above was associated with body weight loss/reduced gain over the study, especially in females and particularly during the 5 days exposure each week. Target exposure concentration of 1.5 mg/L was also associated with adverse clinical signs such as hunched posture, decreased activity, partially closed eyelids and piloerection.

ANIMAL ASSIGNEMENT
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.

ANIMAL REPLACEMENT
Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch. Replacement before treatment commenced: one ill-health female.

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 tube restraint. 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.

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 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 main study 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 0.5 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: Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute and percentage reticulocyte count (Retic/%), Mean cell hemoglobin (MCH), Mean cell hemoglobin 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) and 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 study 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), 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.

URINALYSIS:
Time schedule for collection of urine: Week 13, all main study 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(R)500 instrument:
• Glucose (Gluc)
• Blood pigments (UBld)
Using a Roche Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)

BRONCHO-ALVEOLAR LAVAGE EXAMINATION (BAL):
Time schedule: at termination, All main study and recovery animals
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 (Sysmex UK Ltd). The sample was vortexed for approximately 5 seconds and analyzed. A 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 the testes in modified Davidson’s fluid and the eyes in Davidson’s fluid.

Histology:
Processing: For all main study animals of Groups 1 and 4 killed at a scheduled interval, 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. For all main study animals of Groups 2 and 3 and all recovery phase animals killed at a scheduled interval, the abnormalities only were retained.
Routine staining: sections were stained with haematoxylin and eosin.
See Table 7.5.2/1 for detailed terminal investigations.

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 all recovery animals: abnormalities only.
Other examinations:
Haematology, bone marrow: bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods and from animals killed prematurely during the study.
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):
Test item related clinical signs following exposure comprised elevated gait in a small number of males and females exposed to 0.234 mg/L and males exposed to 0.765 mg/L on one or a few occasions and all females exposed to 0.765 mg/L on one or generally more occasions. In addition, abnormal gait of flattened, swaying or splayed hind limbs was seen in a small number of females exposed to 0.765 mg/L on a few occasions. These signs were noted on return to cage and occasionally persisting to the end of working day check but were not present the following morning.
Clinical signs associated with the exposure procedure and tube restraint included wet fur and red staining of the head/muzzle/nose/eyes, on occasion, for animals of all groups (including control) and are considered to be associated with the method and duration of restraint and not test item related.
Red staining on the coat was noted at the weekly examination in some males and females exposed to 0.234 and 0.765 mg/L and some females exposed to 0.108 mg/L, with the highest number of animals affected being exposed to 0.765 mg/L.
These clinical signs were considered transient, not seen after all exposures and are thus considered not adverse.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Over Days 1 to 92, group mean body weight gain was statistically significantly lower than the concurrent controls for males and females exposed to 0.765 mg/L (65% and 40% of Control, respectively) and females exposed to 0.234 mg/L (71% of Control). Those lower bodyweight gains led to final bodyweights -14.5% and -11% lower than Control in males and females exposed to 0.765 mg/L, respectively, without attaining statistical significance.
Body weight gain over Days 1 to 92 was essentially similar to the controls in males exposed to 0.234 mg/L and both sexes exposed to 0.108 mg/L.
Following 4 weeks of recovery, group mean body weight gains were statistically significantly higher than the concurrent controls in males and females previously exposed to 0.765 mg/L and 0.234 mg/L. Group mean body weight gain was also higher, without attaining statistical significance, in females previously exposed to 0.108 mg/L. This led to final mean bodyweights close to Control, showing full recovery of any previous body weight effect, except for males exposed to 0.765 mg/L where a -10% lower mean bodyweight than Control was still observed at the end of the recovery period.

There were no test item related effects on group mean body weight gain of males previously exposed to 0.234 mg/L or males and females previously exposed to 0.108 mg/L; any intergroup differences were considered of no toxicological significance as there were no test item-related body weight changes noted during the 13 week exposure period.
The lower body weight gain in animals exposed to 0.234 or 0.765 mg/L was associated with, at 0.765 mg/L, slightly lower food consumption and as there were no further changes throughout the study and recovery was seen, is considered not adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption over Days 1-92 was slightly lower than the concurrent control in both males and females exposed to 0.765 mg/L (86% and 89% of Control respectively); full recovery was seen over recovery Weeks 1 to 4.
There were no test item-related effects on food consumption in animals exposed to 0.108 or 0.234 mg/L over Days 1 to 92 or in the recovery period.
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 effects on the ophthalmoscopic examination in Week 13.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related effects.
All differences from control were inconsistent between the sexes, lacked dose relationship, were considered to be of low magnitude or 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.
All differences from control were inconsistent between the sexes, lacked dose relationship, or were considered to be of low magnitude or 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.
All differences from control were inconsistent between the sexes, lacked dose relationship, or were considered to be of low magnitude or 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, non-treatment-related
Description (incidence and severity):
A slight non-statistically significant intergroup difference in absolute testes/epididymal weights was observed between animals in the control group and those exposed to 0.765 mg/L. This was mainly due to one specific animal. No similar changes were seen at the end of the recovery period.

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; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item related macroscopic findings were observed at the terminal or recovery sacrifice.

Small testes and small epididymides were observed in one male exposed to 0.234 mg/L and one male exposed to 0.765 mg/L. This finding correlated with testicular tubular degeneration/atrophy seen on microscopic observation. This finding has already been observed in the Historical Control Data and is therefore considered as part of the common background.

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 considered not test-item related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No test-item related microscopic findings occurred in terminal and recovery sacrifice animals.
Testicular tubular degeneration/atrophy was seen in three males exposed to 0.765 mg/L and one male exposed to 0.234 mg/L. In males exposed to 0.765 mg/L, the severity ranged from minimal to marked, and in one male exposed to 0.234 mg/L, the severity was slight.
Testicular atrophy was also seen in one control male at minimal severity. This change is often seen in controls of this strain on similar study types with variable incidences. The incidence (30%) of this finding in animals exposed to 0.765 mg/L is slightly above the upper limit of the Covance historical control range (0.0% - 10%).
Due to small number of animals affected and absence of findings in the recovery animals, this minor intergroup difference is considered to be incidental to treatment with the test item. It is suggested that all studies in which rats are nose-only exposed for longer than 4 hours demonstrate across-group testicular atrophy, independent of the method of acclimation, the strain of rat used, or the exposure conditions employed (Rothenberg et al., 1999). The higher incidence of testicular degeneration/atrophy was ascribed to restraint stress associated with treatment period (Greaves, 2012), due to either heat or immobilisation (Gopinath and Mowat, 2014).

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 this animal age and strain therefore, they were considered not test-item related.
Especially, 3/10 males at the high dose group were identified with minimal tubular focal basophilia (in the absence of kidney weight changes). This change is predominantly minimal unilateral foci of basophilia, it occurred in the absence of any other changes in the kidneys (accumulation of eosinophilic inclusions in the cortical tubules, granular casts etc), and the increased incidence is within Historical Control Data of the laboratory, therefore it is considered unlikely that this isolated finding would be mediated by alpha-2-microglubulin but that it is more probably reflecting background biological variation.
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.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
0.765 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects including abnormal but transient signs on occasion and lower body weight gain and/or food consumption which showed full recovery at 0.234 or 0.765 mg/L, and no test item-related pathological findings indicative of systemic toxicity.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

ATMOSPHERE ANALYSIS AND ESTIMATION OF ACHIEVED DOSE


Table 7.5.2/2: Summary data of reaction mass of beta-phellandrene and d-limonene and l-limonene concentrations (mean of daily means)

























GroupAchieved concentration (mg/L)Particle size DP (µm)
20.1080.68-0.74
30.2340.66-0.71
40.7650.66-0.70

The achieved aerosol concentrations were 108, 94 and 102% of the target concentrations for Groups 2, 3 and 4, respectively. The particle diameters for Groups 2, 3 and 4 indicate the generated aerosols were respirable to the rat.


 


See table of results available in the field "attached backgroung material" for individual, group mean and pathology report data and tables.

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.765 mg/L, based on no adverse effects or test item-related pathological findings observed at the highest concentration level tested.
Executive summary:

In a repeated dose toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, Reaction mass of beta-phellandrene and d-limonene and l-limonene was administered by inhalation-aerosol to groups of Sprague Dawley rats (10 rats/sex/group) by snout-only inhalation exposure at target exposure levels of 0.10, 0.25 and 0.75 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 108, 94 and 102% of the target concentrations for Groups 2, 3 and 4, respectively, for an achieved exposure concentrations (mean of daily means) of 0.108, 0.234 or 0.765 mg/L.

The particle diameters for Groups 2, 3 and 4 indicate the generated aerosols were respirable to the rat.


Test item related clinical signs following exposure, and occasionally persisting to the end of working day, included elevated gait in a small number of males and females exposed to 0.234 mg/L and males exposed to 0.765 mg/L on one or a few occasions and in all females exposed to 0.765 mg/L on one or generally more occasions. Abnormal gait including flattened, swaying or splayed hind limbs was seen in a small number of females exposed to 0.765 mg/L on a few occasions. Red staining on the coat was noted at the weekly examination in some males and females exposed to 0.234 and 0.765 mg/L and some females exposed to 0.108 mg/L, with the highest number of animals affected being exposed to 0.765 mg/L.


Lower body weight gain over the 13 weeks of exposure in males and females exposed to 0.765 mg/L and females exposed to 0.234 mg/L and slightly lower food consumption over the same period in animals exposed to 0.765 mg/L was seen. Full recovery was seen by the end of the recovery phase.

There were no test item-related effects on ophthalmoscopic changes, or on haematology, blood chemistry or urinalysis parameters investigated in Week 13 or in broncho-alveolar lavage responses at termination.


There were no test-item related effects on organ weights or the incidence of macroscopic or microscopic pathology findings. The incidence of testicular tubular degeneration/atrophy correlating with lower testes weight, in one male exposed to 0.765 mg/L was considered of uncertain relationship to treatment with the test item and, due to the small numbers affected (within historical control data for the macroscopic finding) and the absence of findings in the recovery animals, not considered adverse.  


3/10 males at the high dose group were identified with minimal tubular focal basophilia (in the absence of kidney weight changes). This change is predominantly minimal unilateral foci of basophilia, it occurred in the absence of any other changes in the kidneys (accumulation of eosinophilic inclusions in the cortical tubules, granular casts etc), and the increased incidence is within Historical Control Data of the laboratory, therefore it is considered unlikely that this isolated finding would be mediated by alpha-2-microglubulin but that it is more probably reflecting background biological variation.


Therefore, exposure at 0.234 or 0.765 mg/L was associated with non adverse effects including abnormal but transient signs on occasion and lower body weight gain and/or food consumption which showed full recovery. 


There were no test item-related pathological findings indicative of systemic or local toxicity, although a low incidence of testicular tubular degeneration/atrophy was not considered adverse.


In conclusion, the No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.765 mg/L, based on no adverse effects or test item-related pathological findings observed at the highest concentration level tested.