Terpene hydrocarbon alcohols, terpene processing by-products

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 February - 01 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read-across to Terpene hydrocarbon alcohols

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February - 01 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read-across to Terpene hydrocarbon alcohols

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo CRS Limited, Huntingdon, UK

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 60-67 days
- Weight at study initiation: 264-338 g (males); 173-219 g (females)
- Housing: 5 rats/cage/sex in polycarbonate cages with a stainless steel mesh lid.
- Diet: Harlan Teklad 2014C pelleted diet, ad libitum (except overnight before blood sampling for haematology or blood chemistry and during exposure)
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 ºC
- Humidity: 40-70 %
- Air supply: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h light / 12 h dark

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

other: snout-only

Mass median aerodynamic diameter (MMAD):

<= 1.6 µm

Remarks on MMAD:

MMAD / GSD: MMAD: <0.52, 0.7 and 1.6 µm for achieved concentrations of 0.202, 0.572 and 2.23 mg/L, respectively.
GSD: 2.99 and 1.75 for achieved concentrations of 0.572 and 2.23 mg/L, respectively.

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through snout only chamber; Aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre chamber; Groups 3 and 4 exposure chamber contained a chamber liner to reduce the internal volume.
- Method of holding animals in test chamber: during exposure, the rats were held in restraining tubes with their snouts protruding from the ends of the tubes into the exposure chambers.
- Training for dosing: the animals were acclimated to the method of restraint, over a 5 day period immediately preceding the first test substance exposure
- Aerosol Generation: a stainless steel concentric jet atomiser (manufactured in house by Inhalation Engineering Services), designed to produce and maintain an atmosphere containing a high proportion of respirable droplets. The test substance was supplied to the generator, via a Polyethylene feed line, from a plastic syringe driven at a constant rate by a syringe pump (Harvard).
- Inlet Airflow: From in-house compressed air system – breathing quality; Generator flow: 9 L/minute per system
- Extract Airflow: Drawn by in-house vacuum system; filtered locally; Flow: 25 L/minute per system
- Airflow Monitoring: High quality tapered tube flowmeters – calibrated daily; In-line flowmeters monitored continuously

- Temperature in air chamber: Measured using an electronic thermometer and recorded at 60 minute intervals during exposure. The chamber temperatures were similar for each group on each day of the study. The observed temperatures for all groups remained within the acceptable range for inhalation exposure of rats.
- Air flow rate: 2.0 L/minute

- Method of particle size determination: determined by cascade impaction. Samples collected as follows:
-- Impactor type: Marple 298
-- Collection media: stainless steel substrates and glass fibre (GF10) final stage filter
-- Sample flow: 2.0 L/minute
-- Sample volume: measured by Apex Pro pump
-- Sample frequency: minimum of 1 sample/week/group
-- Sample location: animal exposure port
-- Sample analysis: gravimetric

ATMOSPHERE ANALYSIS
Aerosol samples collected as follows:
- Filter type: glass fibre filter (GF10) held in open faced filter holder
- Sample flow: 2.0 L/minute
- Sample volume: measured by Apex Pro pump
- Sample frequency: minimum of 3 sample/group/day (test groups); 1 sample/day (Group 1)
- Sample location: animal exposure port
- Sample analysis: gravimetric

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total amount of test material on the collection substrates were determined by gravimetric analysis and test article concentration by chemical analysis (GC method of analysis)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

0.2 mg/L air

Remarks:

target concentration; corresponding to 0.202 mg/L (achieved concentration)

Dose / conc.:

0.6 mg/L air

Remarks:

target concentration; corresponding to 0.572 mg/L (achieved concentration)

Dose / conc.:

2 mg/L air

Remarks:

target concentration; corresponding to 2.23 mg/L (achieved concentration)

No. of animals per sex per dose:

10

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: OAD0029), where terpineol multiconstituent was administered to Crl:CD(SD) rats for six hours per day, for five days, using a snout-only exposure system at achieved exposure levels of 0.194, 0.637 and 2.15 mg/L. The only treatment related findings observed in life consisted of reduced body weight gain for all males exposed to terpineol multiconstituent and for females exposed to 2.15 mg/L. Histopathological changes related to treatment were evident for both sexes at an exposure level of 2.15 mg/L. These consisted of minimal to slight hyperplasia/inflammation of the nasal epithelium. The No Observed Adverse Effect Level (NOAEL) for the 2 week study was 2.15 mg/L. Consequently, the same exposure levels were used for this 13 week study. An exposure level of 2.15 mg/L was expected to be tolerated for 13 weeks, with minor body weight effects described above. The lowest exposure level of 0.2 mg/L is a tenth of the highest level and is expected to provide no adverse effects. The intermediate exposure level of 0.6 mg/L is the geometric mean of the high and low exposure target levels and provided information on the dose relationship of any effects seen.

- 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: control and high dose groups (10/sex/dose)

Positive control:

No

Parental animals: Observations and examinations:

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.
During the acclimatisation and recovery periods, 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 at the following times in relation to dose administration:
- Pre-exposure observation and during exposure. However, observation is severely restricted due to tube restraint.
- As each animal is returned to its home cage,
- As late as possible in the working day.
In addition, observations were made in the treatment period, on days without exposures and 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.
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 from Week 1 to Week 4, weekly from Week 5 to Week 13 and before necropsy.

FOOD CONSUMPTION: yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment and for each week throughout 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: 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, Prothrombin time, Activated partial thromboplastin time, Blood film (prepared for all samples) - Romanowsky stain

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: 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: no

NEUROBEHAVIOURAL EXAMINATION: no

Postmortem examinations (parental animals):

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.
- 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.

Statistics:

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

Reproductive indices:

Weight, histology and pathology on ovaries, uturus with cervix, testes and epididymides.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Salivation and chin rubbing were observed from Day 23 onwards in all animals exposed to 2.23 mg/L, in several animals exposed to 0.572 mg/L and one female exposed to 0.202 mg/L. These signs were observed on consecutive days for the majority of animals.
- Unsteady gait was observed between Day 30 and 64 of treatment in 7/20 males and 14/20 females exposed to 2.23 mg/L and in 1/10 females exposed to 0.572 mg/L. Partially closed or closed eyelids were observed between Day 26 and 60 of treatment in 2/20 males and 7/20 females exposed to 2.23 mg/L and in 1/10 females exposed to 0.572 mg/L. Elevated posture was observed between Day 30 and 33 in 8/20 males and 7/20 females exposed to 2.23 mg/L. With the exception of a few isolated incidences, the aforementioned signs resolved prior to the final check at the end of the working day. These observations were intermittent for all effected animals, usually presenting on non-consecutive days.
- Signs associated with the method of restraint included wet fur and red/brown staining of the head, muzzle and nose which were observed in some animals from all groups on return to the home cage.
- There were no test article related clinical signs during the recovery period.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males exposed to 2.23 mg/L showed a group mean body weight loss of 6 g between Day 1 and 4, compared with a group mean gain of 5 g in Control males. Body weight changes for females exposed to 2.23 mg/L showed a group mean body weight loss of 2 g over the same period, compared with a group mean gain of 1 g in Control females.
- Over the 13 weeks of exposures, group mean body weight gains for males exposed to 0.202, 0.572 and 2.23 mg/L were lower than controls (0.87X, 0.87X and 0.81X respectively), with changes for males exposed to 2.23 mg/L achieving statistical significance. For females exposed to 0.572 and 2.23 mg/L, group mean body weights were statistically significantly lower than controls (0.81X and 0.84X respectively). Females exposed to 0.202 mg/L had a similar weight gain to controls.
- Over the 4 week recovery period, body weight gains for males and females previously exposed to 2.23 mg/L showed complete recovery to control values.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The group mean reticulocyte percentage and the absolute reticulocyte count were lower than control values for males exposed to 0.572 (0.90 X and 0.92 X respectively) and males exposed to 2.23 mg/L (0.82 X and 0.82 X respectively), with males exposed to 2.23 mg/L achieving statistical significance. A similar effect was seen for females exposed to 2.23 mg/L (0.92 X and 0.87 X control values for reticulocyte percentage and absolute reticulocyte count, respectively), but this did not attain statistical significance. During Recovery Week 4, values for both sexes previously exposed to 2.23 mg/L were similar to controls. Other changes from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra-group variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects. Differences from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra-group variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals killed after 13 weeks of treatment:
- Treatment related findings: Changes related to treatment with terpineol multiconstituent were seen in the nasal turbinates and nasal pharynx.
- Nasal turbinates: hyperplasia of the mucous cells (minimal to slight severity) was present in almost all males treated with terpineol multiconstituent, the majority of females receiving 0.572 mg/L and 2.23 mg/L and several females receiving 0.202 mg/L. The respiratory epithelium associated with the ventral nasal septum in the anterior portion of the nasal cavity was predominantly affected. Minimal inflammation was present in the respiratory epithelium of two animals exposed to 2.23 mg/L and was typified by the presence of a predominantly neutrophilic cellular infiltrate. Minimal degeneration of the olfactory and/or respiratory epithelium lining the anterior portion of the dorsal meatus was present, predominantly in females treated with 2.23 mg/L terpineol multiconstituent.
- Nasal pharynx: hyperplasia of the mucous cells was present in the respiratory epithelium lining the nasal pharynx of predominantly females treated with 0.572 mg/L or 2.23 mg/L of terpineol multiconstituent.
- Incidental findings: all other changes were considered to be incidental and consistent with the common background lesions seen in Sprague-Dawley rats at these laboratories.

Animals killed after 4 weeks of recovery:
- Treatment related findings: changes related to treatment with terpineol multiconstituent were seen in the nasal turbinates following the 4 week recovery period.
- Nasal turbinates: hyperplasia of the mucous cells (minimal to slight severity) was present in the majority of males and females treated with 2.23 mg/L and in one control male. The respiratory epithelium associated with the ventral nasal septum in the anterior portion of the nasal cavity was predominantly affected. Minimal degeneration of the olfactory epithelium lining the anterior portion of the dorsal meatus was present in one male and two females treated with 2.23 mg/L terpineol multiconstituent.
- Incidental findings: All other changes were considered to be incidental and consistent with the common background lesions seen in Sprague-Dawley rats at these laboratories.

Histopathological findings: neoplastic:

no effects observed

No treatment related effects were seen in the ovaries, uturus with cervix, testes and epididymides with respect to histological and pathological findings and the organ weight of these organs.

Dose descriptor:

NOAEC

Remarks:

General toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEC

Remarks:

Fertility

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Remarks on result:

not measured/tested

Reproductive effects observed:

no

DISCUSSION

Minimal to slight hyperplasia of the mucous cells was observed primarily in the respiratory epithelial lining of the ventral nasal septum in the anterior nasal cavity. This finding is consistent with that previously identified in the preliminary 2 week study for this compound (Envigo Study Number OAD0029). The nasal turbinate mucous cell hyperplasia was present at all exposure levels and did not exhibit a clear dose response in terms of incidence or severity in males, although there were slightly higher incidences in females at 0.572 mg/L or 2.23 mg/L compared with females exposed to 0.202 mg/L. The changes in the respiratory epithelium of the nasal turbinates were generally not associated with an inflammatory cell infiltrate or cellular degeneration; such changes were observed in a minority of animals and were of minimal severity. Following a 4 week recovery period, similar incidences of mucous cell hyperplasia in the respiratory epithelium were observed in animals exposed to 2.23 mg/L compared to those at the end of the main study phase, although most were of minimal severity. Therefore only partial recovery of mucous cell hyperplasia in the respiratory epithelium of the nasal turbinates was seen after 4 weeks of recovery.

Mucous cell hyperplasia is considered to be an adaptive change in the epithelium in response to chronic irritation (Renne et al 2009). This may correlate with clinical signs of salivation and chin rubbing that were observed on consecutive days (from Day 23 onwards) and displayed a dose related response in terms of the number of animals affected. Given that the histopathology changes were of minimal or slight severity and were generally not associated with inflammatory or degenerative changes in the respiratory epithelium, particularly at lower exposure levels, it would be expected that these changes would reverse following a suitable recovery period. Therefore, mucous cell hyperplasia in the nasal turbinates is also not considered adverse.

In the nasal pharynx, minimal hyperplasia of the mucous cells was also seen in the respiratory epithelium in a small number of animals exposed to 0.572 mg/L or 2.23 mg/L. The changes in the nasal pharynx were also not associated with an inflammatory cell infiltrate or degenerative changes. Examination of recovery phase animals showed no changes in the nasal pharynx respiratory epithelium, suggesting complete recovery after 4 weeks which is therefore not considered adverse.

Minimal and typically focal regions of cellular degeneration were present in the olfactory epithelium of the anterior dorsal meatus of the nasal cavity in several animals exposed to 2.23 mg/L. This was typified by loss of organisation of the cell layers and increased intercellular spacing, but was not associated with inflammatory cell infiltrates. Degeneration of the olfactory epithelium may follow injury to the olfactory sensory neurons or supporting sustentacular cells induced by exposure to chronic irritants (Harkema et al 2006). The olfactory epithelium retains the ability to regenerate although this is dependent on the severity of the damage induced and it can take a period of weeks to months to resume a normal histological appearance (Harkema et al 2006). The degenerative changes observed in this study were very minimal and were restricted to a small portion of the olfactory epithelium in the nasal turbinates. Additionally, they were only observed in animals exposed to 2.23 mg/L (2/10 males and 4/10 females). This was still apparent in a small number of animals exposed to 2.23 mg/L following a 4 week recovery period (1/10 male and 2/10 females), however, a longer recovery period may allow complete recovery to be reached. Therefore, due to the minimal severity, limited number of animals affected and the capacity of the tissue to regenerate, these changes are not considered adverse.

Clinical signs of unsteady gait and partially closed or closed eyelids were observed on nonconsecutive days between Day 30 and 64 of treatment for animals exposed to 2.23 mg/L and for a single female exposed to 0.572 mg/L. These signs were more prevalent in females compared with males. Elevated posture was also observed for a few days at the start of this period in animals exposed to 2.23 mg/L. These signs generally resolved by the end of the day and usually presented on non-consecutive days. With the absence of any associated histopathology findings, these signs are not considered adverse at the degree observed.

Reduced body weight gain was evident over the 13 week exposure period for all males exposed to terpineol multiconstituent and females exposed to 0.572 and 2.23 mg/L although clearly related to treatment, this did not exhibit a clear dose response.

Reticulocyte counts were lower than control values for males exposed to 0.572 and both sexes exposed to 2.23 mg/L. As these values were similar to control values during recovery week 4, and in the absence of any associated histopathology findings, it is not considered adverse at the degree observed.

ATMOSPHERE ANALYSIS AND ESTIMATION OF ACHIEVED DOSE

Table 7.5.2/2: Summary data

Group	Achieved concentration (mg/L)	Particle size
		MMAD (µm)	GSD
1	-	-	-
2	0.202	<0.52	-
3	0.572	0.7	2.99
4	2.23	1.6	1.75

MMAD: Mass median aerodynamic diameter

GSD: Geometric standard deviation

The achieved levels were 101, 95 and 112% of the target concentrations for Groups 2, 3 and 4 respectively.

The MMAD values showed a general increase with increasing aerosol concentration. The general trend to larger MMAD with increasing aerosol concentration is normal and is attributed to increased particle interaction and subsequent aggregation.

The MMAD for Group 4 was within the ideal range of 1 to 3 µm indicating that the terpineol multiconstituent aerosol was respirable to the rats.

The Group 3 particle size distribution values show a bi-modal distribution across the impactor stages with an average of 49% of the captured droplet having an MMAD below 0.52 µm.

The MMAD for Group 2 was not calculable as virtually all the measurable test material was captured on the final filter stage. The value presented is based on the cut point of the penultimate stage of the impactor (Stage 8) which has a cut point of 0.52 µm. This value is below the ideal range of 1 to 3 µm. The concern with how respirable particles with an MMAD below 1 µm are that they may not impact on airway surfaces and be exhaled, thus reducing exposure. In this study, due to the delivered aerosol being a liquid, it is likely that inhaled droplets would impact on airway surfaces and not be exhaled.

HISTOPATHOLOGY

Table 7.5.2/3: Summary of treatment related findings in the nasal cavity for animals killed after 13 weeks of treatment 

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Achieved Exposure Level (mg/L)	0	0.202	0.572	2.23	0	0.202	0.572	2.23
Hyperplasia, Mucous Cell, Respiratory Epithelium
Minimal	0	5	5	5	0	3	6	5
Slight	0	4	4	5	0	1	2	3
Total	0	9	9	10	0	4	8	8
Degeneration, Respiratory Epithelium
Minimal	0	0	0	0	0	0	0	2
Total	0	0	0	0	0	0	0	2
Degeneration, Olfactory Epithelium
Minimal	0	0	0	2	0	0	0	4
Total	0	0	0	2	0	0	0	4
Inflammation, Respiratory Epithelium
Minimal	0	0	0	1	0	0	0	1
Total	0	0	0	1	0	0	0	1
Number of tissues examined	10	10	10	10	10	10	10	10

Table 7.5.2/4: Summary of treatment related findings in the nasal pharynx for animals killed after 13 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Achieved Exposure Level (mg/L)	0	0.202	0.572	2.23	0	0.202	0.572	2.23
Hyperplasia, Mucous Cell
Minimal	0	0	1	1	0	0	2	4
Total	0	0	1	1	0	0	2	4
Number of tissues examined	10	10	10	10	10	10	10	10

 

Table 7.5.2/5: Summary of treatment-related changes in the nasal turbinates for animals killed after 4 weeks of recovery

Group/sex	1M	4M	1F	4F
Achieved Exposure Level (mg/L)	0	2.23	0	2.23
Hyperplasia, Mucous Cell, Respiratory Epithelium
Minimal	1	5	0	7
Slight	0	4	0	2
Total	1	9	0	9
Degeneration, Olfactory Epithelium
Minimal	0	1	0	2
Total	0	1	0	2
Number of tissues examined	10	10	10	10

Conclusions:

No adverse effects were seen on the organs related to reproduction. Therefore, the No Observed Adverse Effect Concentration (NOAEC) for fertility was considered to be ≥ 2230 mg/m3 (2.23 mg/L).

Executive summary:

In a repeated dose toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, terpineol multiconstituent was administered by inhalation-aerosol to groups of Crl:CD(SD) rats (10 rats/sex/ group) by snout-only inhalation exposure at target exposure levels of 0.2, 0.6 and 2 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. Control and high dose recovery groups were included (10/sex/group). During the study, clinical condition, body weight, food consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry,organ weight, macropathology and histopathology investigations were undertaken.

The achieved levels were 101, 95 and 112% of the target concentrations of 0.2, 0.6 and 2 mg/L, respectively (achieved concentrations 0.202, 0.572 and 2.23 mg/L).MMAD: <0.52, 0.7 and 1.6 µm for achieved concentrations of 0.202, 0.572 and 2.23 mg/L, respectively.GSD: 2.99 and 1.75 for achieved concentrations of 0.572 and 2.23 mg/L, respectively.MMAD showed a general increase with increasing aerosol concentration. The MMAD for the low dose groupcould not be calculated, as virtually all the measurable test material was captured on the final filter stage, and the value presented is based on the cut point of the penultimate impactor stage. The mid dose groupsparticle size distribution values showed a bi-modal distribution with an average of 49% of the captured droplet having a MMAD below 0.52 µm. The MMAD value for the high dose group was within the ideal range (1 to 3 µm), indicating that the aerosol was respirable to the rats. The MMADs for the low and mid dose groups were below the ideal range of 1 to 3 µm. However, since the delivered aerosol was a liquid, it is likely that those inhaled droplets with an aerodynamic diameter below 1 µm would still have impacted on airway surfaces and not been exhaled.

There were no treatment related deaths or effects on food consumption, blood chemistry, ophthalmoscopy, organ weights or macropathology findings.Group mean body weight gains were lower than control for males exposed to 0.202 mg/L and for both sexes exposed to 0.572 and 2.23 mg/L. In both sexes, no relationship between exposure concentration and body weight gain was observed but the decrease in mean body weight gain was statistically significant for males exposed to 2.23 mg/L. Body weights showed full recovery for animals previously exposed to 2.23 mg/L.Clinical pathology measurements following 13 weeks of exposure revealed statistically significantly lower group mean reticulocyte percentages and absolute counts for males exposed to 0.572 or 2.23 mg/L, compared to control (as low as 0.82X control). A similar effect was observed for females exposed to 2.23 mg/L (as low as 0.87X control) but this did not attain statistical significance. During Recovery Week 4, values for both sexes previously exposed to 2.23 mg/L were similar to controls.Histopathological changes related to treatment were observed in the nasal turbinates for the majority of animals given the test substance and nasal pharynx for a limited number of animals given 0.572 or 2.23 mg/L.In the nasal turbinates, minimal to slight hyperplasia of the mucous cells in the respiratory epithelium was present at all exposure levels and did not exhibit a clear dose response in terms of incidence or severity in males, although there were slightly higher incidences in females at 0.572 mg/L or 2.23 mg/L compared with females exposed to 0.202 mg/L. After 4 weeks of recovery, partial recovery was evident, in terms of the severity observed. After 13 weeks of exposures, these changes were generally not associated with an inflammatory cell infiltrate or cellular degeneration; such changes were observed in a minority of animals and were of minimal severity. After 4 weeks of recovery, degeneration and inflammation of the respiratory epithelium showed completed recovery. However, degeneration of the olfactory epithelium showed only partial recovery in terms of the incidence observed. In the nasal pharynx, minimal hyperplasia of the mucous cells was also observed but at a lower incident and severity and was only evident for animals exposed to 0.572 or 2.23 mg/L. Complete recovery from this effect was observed following the 4 week recovery period. The aforementioned changes were typically associated with chronic exposure to an irritant material and are not considered adverse at the incidence and severity seen.

No effects were observed on the organ weight of the reproductive organs. In addition, gross macroscopy and histopathology of the reproductive organs did not show any changes due to treatment. The No Observed Adverse Effect Concentration (NOAEC) for general toxicity and fertility is considered to be ≥2.23 mg/L (2230 mg/m3).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo CRS Limited, Huntingdon, UK

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Terpineol multi: 8000-41-7

IUPAC Name:

Terpineol multi: 8000-41-7

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Terpineol; CAS 8000-41-7
- Physical state: Clear, colourless, lightly viscous liquid
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 60-67 days
- Weight at study initiation: 264-338 g (males); 173-219 g (females)
- Housing: 5 rats/cage/sex in polycarbonate cages with a stainless steel mesh lid.
- Diet: Harlan Teklad 2014C pelleted diet, ad libitum (except overnight before blood sampling for haematology or blood chemistry and during exposure)
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 ºC
- Humidity: 40-70 %
- Air supply: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h light / 12 h dark

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

other: snout-only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

<= 1.6 µm

Remarks on MMAD:

MMAD / GSD: MMAD: <0.52, 0.7 and 1.6 µm for achieved concentrations of 0.202, 0.572 and 2.23 mg/L, respectively.
GSD: 2.99 and 1.75 for achieved concentrations of 0.572 and 2.23 mg/L, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through snout only chamber; Aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre chamber; Groups 3 and 4 exposure chamber contained a chamber liner to reduce the internal volume.
- Method of holding animals in test chamber: during exposure, the rats were held in restraining tubes with their snouts protruding from the ends of the tubes into the exposure chambers.
- Training for dosing: the animals were acclimated to the method of restraint, over a 5 day period immediately preceding the first test substance exposure
- Aerosol Generation: a stainless steel concentric jet atomiser (manufactured in house by Inhalation Engineering Services), designed to produce and maintain an atmosphere containing a high proportion of respirable droplets. The test substance was supplied to the generator, via a Polyethylene feed line, from a plastic syringe driven at a constant rate by a syringe pump (Harvard).
- Inlet Airflow: From in-house compressed air system – breathing quality; Generator flow: 9 L/minute per system
- Extract Airflow: Drawn by in-house vacuum system; filtered locally; Flow: 25 L/minute per system
- Airflow Monitoring: High quality tapered tube flowmeters – calibrated daily; In-line flowmeters monitored continuously

- Temperature in air chamber: Measured using an electronic thermometer and recorded at 60 minute intervals during exposure. The chamber temperatures were similar for each group on each day of the study. The observed temperatures for all groups remained within the acceptable range for inhalation exposure of rats.
- Air flow rate: 2.0 L/minute

- Method of particle size determination: determined by cascade impaction. Samples collected as follows:
-- Impactor type: Marple 298
-- Collection media: stainless steel substrates and glass fibre (GF10) final stage filter
-- Sample flow: 2.0 L/minute
-- Sample volume: measured by Apex Pro pump
-- Sample frequency: minimum of 1 sample/week/group
-- Sample location: animal exposure port
-- Sample analysis: gravimetric

ATMOSPHERE ANALYSIS
Aerosol samples collected as follows:
- Filter type: glass fibre filter (GF10) held in open faced filter holder
- Sample flow: 2.0 L/minute
- Sample volume: measured by Apex Pro pump
- Sample frequency: minimum of 3 sample/group/day (test groups); 1 sample/day (Group 1)
- Sample location: animal exposure port
- Sample analysis: gravimetric

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total amount of test material on the collection substrates were determined by gravimetric analysis and test article concentration by chemical analysis (GC method of analysis)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.2 mg/L air

Remarks:

target concentration; corresponding to 0.202 mg/L (achieved concentration)

Doses / concentrations 2

Dose / conc.:

0.6 mg/L air

Remarks:

target concentration; corresponding to 0.572 mg/L (achieved concentration)

Doses / concentrations 3

Dose / conc.:

2 mg/L air

Remarks:

target concentration; correspdonding to 2.23 mg/L (achieved concentration)

No. of animals per sex per dose:

10

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: OAD0029), where terpineol multiconstituent was administered to Crl:CD(SD) rats for six hours per day, for five days, using a snout-only exposure system at achieved exposure levels of 0.194, 0.637 and 2.15 mg/L. The only treatment related findings observed in life consisted of reduced body weight gain for all males exposed to terpineol multiconstituent and for females exposed to 2.15 mg/L. Histopathological changes related to treatment were evident for both sexes at an exposure level of 2.15 mg/L. These consisted of minimal to slight hyperplasia/inflammation of the nasal epithelium. The No Observed Adverse Effect Level (NOAEL) for the 2 week study was 2.15 mg/L.
Consequently, the same exposure levels were used for this 13 week study. An exposure level of 2.15 mg/L was expected to be tolerated for 13 weeks, with minor body weight effects described above. The lowest exposure level of 0.2 mg/L is a tenth of the highest level and is expected to provide no adverse effects. The intermediate exposure level of 0.6 mg/L is the geometric mean of the high and low exposure target levels and provided information on the dose relationship of any effects seen.
- 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: control and high dose groups (10/sex/dose)

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
During the acclimatisation and recovery periods, 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 at the following times in relation to dose administration:
- Pre-exposure observation and during exposure. However, observation is severely restricted due to tube restraint.
- As each animal is returned to its home cage,
- As late as possible in the working day.
In addition, observations were made in the treatment period, on days without exposures and 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.
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 from Week 1 to Week 4, weekly from Week 5 to Week 13 and before necropsy.

FOOD CONSUMPTION: yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment and for each week throughout 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: 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, Prothrombin time, Activated partial thromboplastin time, Blood film (prepared for all samples) - Romanowsky stain

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: 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: no

NEUROBEHAVIOURAL EXAMINATION: no

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: 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.
- 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.

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):

- Salivation and chin rubbing were observed from Day 23 onwards in all animals exposed to 2.23 mg/L, in several animals exposed to 0.572 mg/L and one female exposed to 0.202 mg/L. These signs were observed on consecutive days for the majority of animals.
- Unsteady gait was observed between Day 30 and 64 of treatment in 7/20 males and 14/20 females exposed to 2.23 mg/L and in 1/10 females exposed to 0.572 mg/L. Partially closed or closed eyelids were observed between Day 26 and 60 of treatment in 2/20 males and 7/20 females exposed to 2.23 mg/L and in 1/10 females exposed to 0.572 mg/L. Elevated posture was observed between Day 30 and 33 in 8/20 males and 7/20 females exposed to 2.23 mg/L. With the exception of a few isolated incidences, the aforementioned signs resolved prior to the final check at the end of the working day. These observations were intermittent for all effected animals, usually presenting on non-consecutive days.
- Signs associated with the method of restraint included wet fur and red/brown staining of the head, muzzle and nose which were observed in some animals from all groups on return to the home cage.
- There were no test article related clinical signs during the recovery period.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males exposed to 2.23 mg/L showed a group mean body weight loss of 6 g between Day 1 and 4, compared with a group mean gain of 5 g in Control males. Body weight changes for females exposed to 2.23 mg/L showed a group mean body weight loss of 2 g over the same period, compared with a group mean gain of 1 g in Control females.
- Over the 13 weeks of exposures, group mean body weight gains for males exposed to 0.202, 0.572 and 2.23 mg/L were lower than controls (0.87X, 0.87X and 0.81X respectively), with changes for males exposed to 2.23 mg/L achieving statistical significance. For females exposed to 0.572 and 2.23 mg/L, group mean body weights were statistically significantly lower than controls (0.81X and 0.84X respectively). Females exposed to 0.202 mg/L had a similar weight gain to controls.
- Over the 4 week recovery period, body weight gains for males and females previously exposed to 2.23 mg/L showed complete recovery to control values.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The group mean reticulocyte percentage and the absolute reticulocyte count were lower than control values for males exposed to 0.572 (0.90 X and 0.92 X respectively) and males exposed to 2.23 mg/L (0.82 X and 0.82 X respectively), with males exposed to 2.23 mg/L achieving statistical significance. A similar effect was seen for females exposed to 2.23 mg/L (0.92 X and 0.87 X control values for reticulocyte percentage and absolute reticulocyte count, respectively), but this did not attain statistical significance. During Recovery Week 4, values for both sexes previously exposed to 2.23 mg/L were similar to controls. Other changes from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra-group variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects. Differences from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra-group variation.

Urinalysis findings:

not examined

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):

There were no treatment related effects. Differences from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra-group variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Animals killed after 13 weeks of treatment: the macroscopic examination performed after 13 weeks of treatment revealed no test substance related lesions. The incidence and distribution of all findings were consistent with the common background changes seen in Sprague-Dawley rats at these laboratories.
- Animals killed after 4 weeks of recovery: the macroscopic examination performed after 4 weeks of recovery revealed no test substance related lesions. The incidence and distribution of all findings were consistent with the common background changes seen in Sprague-Dawley rats at these laboratories.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals killed after 13 weeks of treatment:
- Treatment related findings: Changes related to treatment with terpineol multiconstituent were seen in the nasal turbinates and nasal pharynx.
- Nasal turbinates: hyperplasia of the mucous cells (minimal to slight severity) was present in almost all males treated with terpineol multiconstituent, the majority of females receiving 0.572 mg/L and 2.23 mg/L and several females receiving 0.202 mg/L. The respiratory epithelium associated with the ventral nasal septum in the anterior portion of the nasal cavity was predominantly affected. Minimal inflammation was present in the respiratory epithelium of two animals exposed to 2.23 mg/L and was typified by the presence of a predominantly neutrophilic cellular infiltrate. Minimal degeneration of the olfactory and/or respiratory epithelium lining the anterior portion of the dorsal meatus was present, predominantly in females treated with 2.23 mg/L terpineol multiconstituent.
- Nasal pharynx: hyperplasia of the mucous cells was present in the respiratory epithelium lining the nasal pharynx of predominantly females treated with 0.572 mg/L or 2.23 mg/L of terpineol multiconstituent.
- Incidental findings: all other changes were considered to be incidental and consistent with the common background lesions seen in Sprague-Dawley rats at these laboratories.

Animals killed after 4 weeks of recovery:
- Treatment related findings: changes related to treatment with terpineol multiconstituent were seen in the nasal turbinates following the 4 week recovery period.
- Nasal turbinates: hyperplasia of the mucous cells (minimal to slight severity) was present in the majority of males and females treated with 2.23 mg/L and in one control male. The respiratory epithelium associated with the ventral nasal septum in the anterior portion of the nasal cavity was predominantly affected. Minimal degeneration of the olfactory epithelium lining the anterior portion of the dorsal meatus was present in one male and two females treated with 2.23 mg/L terpineol multiconstituent.
- Incidental findings: All other changes were considered to be incidental and consistent with the common background lesions seen in Sprague-Dawley rats at these laboratories.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEC

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

DISCUSSION

Minimal to slight hyperplasia of the mucous cells was observed primarily in the respiratory epithelial lining of the ventral nasal septum in the anterior nasal cavity. This finding is consistent with that previously identified in the preliminary 2 week study for this compound (Envigo Study Number OAD0029). The nasal turbinate mucous cell hyperplasia was present at all exposure levels and did not exhibit a clear dose response in terms of incidence or severity in males, although there were slightly higher incidences in females at 0.572 mg/L or 2.23 mg/L compared with females exposed to 0.202 mg/L. The changes in the respiratory epithelium of the nasal turbinates were generally not associated with an inflammatory cell infiltrate or cellular degeneration; such changes were observed in a minority of animals and were of minimal severity. Following a 4 week recovery period, similar incidences of mucous cell hyperplasia in the respiratory epithelium were observed in animals exposed to 2.23 mg/L compared to those at the end of the main study phase, although most were of minimal severity. Therefore only partial recovery of mucous cell hyperplasia in the respiratory epithelium of the nasal turbinates was seen after 4 weeks of recovery.

Mucous cell hyperplasia is considered to be an adaptive change in the epithelium in response to chronic irritation (Renne et al 2009). This may correlate with clinical signs of salivation and chin rubbing that were observed on consecutive days (from Day 23 onwards) and displayed a dose related response in terms of the number of animals affected. Given that the histopathology changes were of minimal or slight severity and were generally not associated with inflammatory or degenerative changes in the respiratory epithelium, particularly at lower exposure levels, it would be expected that these changes would reverse following a suitable recovery period. Therefore, mucous cell hyperplasia in the nasal turbinates is also not considered adverse.

In the nasal pharynx, minimal hyperplasia of the mucous cells was also seen in the respiratory epithelium in a small number of animals exposed to 0.572 mg/L or 2.23 mg/L. The changes in the nasal pharynx were also not associated with an inflammatory cell infiltrate or degenerative changes. Examination of recovery phase animals showed no changes in the nasal pharynx respiratory epithelium, suggesting complete recovery after 4 weeks which is therefore not considered adverse.

Minimal and typically focal regions of cellular degeneration were present in the olfactory epithelium of the anterior dorsal meatus of the nasal cavity in several animals exposed to 2.23 mg/L. This was typified by loss of organisation of the cell layers and increased intercellular spacing, but was not associated with inflammatory cell infiltrates. Degeneration of the olfactory epithelium may follow injury to the olfactory sensory neurons or supporting sustentacular cells induced by exposure to chronic irritants (Harkema et al 2006). The olfactory epithelium retains the ability to regenerate although this is dependent on the severity of the damage induced and it can take a period of weeks to months to resume a normal histological appearance (Harkema et al 2006). The degenerative changes observed in this study were very minimal and were restricted to a small portion of the olfactory epithelium in the nasal turbinates. Additionally, they were only observed in animals exposed to 2.23 mg/L (2/10 males and 4/10 females). This was still apparent in a small number of animals exposed to 2.23 mg/L following a 4 week recovery period (1/10 male and 2/10 females), however, a longer recovery period may allow complete recovery to be reached. Therefore, due to the minimal severity, limited number of animals affected and the capacity of the tissue to regenerate, these changes are not considered adverse.

Clinical signs of unsteady gait and partially closed or closed eyelids were observed on nonconsecutive days between Day 30 and 64 of treatment for animals exposed to 2.23 mg/L and for a single female exposed to 0.572 mg/L. These signs were more prevalent in females compared with males. Elevated posture was also observed for a few days at the start of this period in animals exposed to 2.23 mg/L. These signs generally resolved by the end of the day and usually presented on non-consecutive days. With the absence of any associated histopathology findings, these signs are not considered adverse at the degree observed.

Reduced body weight gain was evident over the 13 week exposure period for all males exposed to terpineol multiconstituent and females exposed to 0.572 and 2.23 mg/L although clearly related to treatment, this did not exhibit a clear dose response.

Reticulocyte counts were lower than control values for males exposed to 0.572 and both sexes exposed to 2.23 mg/L. As these values were similar to control values during recovery week 4, and in the absence of any associated histopathology findings, it is not considered adverse at the degree observed.

ATMOSPHERE ANALYSIS AND ESTIMATION OF ACHIEVED DOSE

Table 7.5.2/2: Summary data

Group	Achieved concentration (mg/L)	Particle size
		MMAD (µm)	GSD
1	-	-	-
2	0.202	<0.52	-
3	0.572	0.7	2.99
4	2.23	1.6	1.75

MMAD: Mass median aerodynamic diameter

GSD: Geometric standard deviation

 

The achieved levels were 101, 95 and 112% of the target concentrations for Groups 2, 3 and 4 respectively.

The MMAD values showed a general increase with increasing aerosol concentration. The general trend to larger MMAD with increasing aerosol concentration is normal and is attributed to increased particle interaction and subsequent aggregation.

The MMAD for Group 4 was within the ideal range of 1 to 3 µm indicating that the terpineol multiconstituent aerosol was respirable to the rats.

The Group 3 particle size distribution values show a bi-modal distribution across the impactor stages with an average of 49% of the captured droplet having an MMAD below 0.52 µm.

The MMAD for Group 2 was not calculable as virtually all the measurable test material was captured on the final filter stage. The value presented is based on the cut point of the penultimate stage of the impactor (Stage 8) which has a cut point of 0.52 µm. This value is below the ideal range of 1 to 3 µm. The concern with how respirable particles with an MMAD below 1 µm are that they may not impact on airway surfaces and be exhaled, thus reducing exposure. In this study, due to the delivered aerosol being a liquid, it is likely that inhaled droplets would impact on airway surfaces and not be exhaled.

 

HISTOPATHOLOGY

Table 7.5.2/3: Summary of treatment related findings in the nasal cavity for animals killed after 13 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Achieved Exposure Level (mg/L)	0	0.202	0.572	2.23	0	0.202	0.572	2.23
Hyperplasia, Mucous Cell, Respiratory Epithelium
Minimal	0	5	5	5	0	3	6	5
Slight	0	4	4	5	0	1	2	3
Total	0	9	9	10	0	4	8	8
Degeneration, Respiratory Epithelium
Minimal	0	0	0	0	0	0	0	2
Total	0	0	0	0	0	0	0	2
Degeneration, Olfactory Epithelium
Minimal	0	0	0	2	0	0	0	4
Total	0	0	0	2	0	0	0	4
Inflammation, Respiratory Epithelium
Minimal	0	0	0	1	0	0	0	1
Total	0	0	0	1	0	0	0	1
Number of tissues examined	10	10	10	10	10	10	10	10

Table 7.5.2/4: Summary of treatment related findings in the nasal pharynx for animals killed after 13 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Achieved Exposure Level (mg/L)	0	0.202	0.572	2.23	0	0.202	0.572	2.23
Hyperplasia, Mucous Cell
Minimal	0	0	1	1	0	0	2	4
Total	0	0	1	1	0	0	2	4
Number of tissues examined	10	10	10	10	10	10	10	10

 

Table 7.5.2/5: Summary of treatment-related changes in the nasal turbinates for animals killed after 4 weeks of recovery

Group/sex	1M	4M	1F	4F
Achieved Exposure Level (mg/L)	0	2.23	0	2.23
Hyperplasia, Mucous Cell, Respiratory Epithelium
Minimal	1	5	0	7
Slight	0	4	0	2
Total	1	9	0	9
Degeneration, Olfactory Epithelium
Minimal	0	1	0	2
Total	0	1	0	2
Number of tissues examined	10	10	10	10

Applicant's summary and conclusion

Conclusions:

In a 13-week repeated dose inhalation study no adverse toxic effects were observed. The NOAEC is determined to be ≥ 2230 mg/m3.

Executive summary:

In a repeated dose toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, terpineol multiconstituent was administered by inhalation-aerosol to groups of Crl:CD(SD) rats (10 rats/sex/ group) by snout-only inhalation exposure at target exposure levels of 0.2, 0.6 and 2 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. Control and high dose recovery groups were included (10/sex/group). During the study, clinical condition, body weight, food consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry,organ weight, macropathology and histopathology investigations were undertaken.The achieved levels were 101, 95 and 112% of the target concentrations of 0.2, 0.6 and 2 mg/L, respectively (achieved concentrations 0.202, 0.572 and 2.23 mg/L). MMAD: <0.52, 0.7 and 1.6 µm for achieved concentrations of 0.202, 0.572 and 2.23 mg/L, respectively.GSD: 2.99 and 1.75 for achieved concentrations of 0.572 and 2.23 mg/L, respectively.MMAD showed a general increase with increasing aerosol concentration. The MMAD for the low dose groupcould not be calculated, as virtually all the measurable test material was captured on the final filter stage, and the value presented is based on the cut point of the penultimate impactor stage. The mid dose groupsparticle size distribution values showed a bi-modal distribution with an average of 49% of the captured droplet having a MMAD below 0.52 µm. The MMAD value for the high dose group was within the ideal range (1 to 3 µm), indicating that the aerosol was respirable to the rats. The MMADs for the low and mid dose groups were below the ideal range of 1 to 3 µm. However, since the delivered aerosol was a liquid, it is likely that those inhaled droplets with an aerodynamic diameter below 1 µm would still have impacted on airway surfaces and not been exhaled.There were no treatment related deaths or effects on food consumption, blood chemistry, ophthalmoscopy, organ weights or macropathology findings.Group mean body weight gains were lower than control for males exposed to 0.202 mg/L and for both sexes exposed to 0.572 and 2.23 mg/L. In both sexes, no relationship between exposure concentration and body weight gain was observed but the decrease in mean body weight gain was statistically significant for males exposed to 2.23 mg/L. Body weights showed full recovery for animals previously exposed to 2.23 mg/L. Clinical pathology measurements following 13 weeks of exposure revealed statistically significantly lower group mean reticulocyte percentages and absolute counts for males exposed to 0.572 or 2.23 mg/L, compared to control (as low as 0.82X control). A similar effect was observed for females exposed to 2.23 mg/L (as low as 0.87X control) but this did not attain statistical significance. During Recovery Week 4, values for both sexes previously exposed to 2.23 mg/L were similar to controls. Histopathological changes related to treatment were observed in the nasal turbinates for the majority of animals given the test substance and nasal pharynx for a limited number of animals given 0.572 or 2.23 mg/L. In the nasal turbinates, minimal to slight hyperplasia of the mucous cells in the respiratory epithelium was present at all exposure levels and did not exhibit a clear dose response in terms of incidence or severity in males, although there were slightly higher incidences in females at 0.572 mg/L or 2.23 mg/L compared with females exposed to 0.202 mg/L. After 4 weeks of recovery, partial recovery was evident, in terms of the severity observed. After 13 weeks of exposures, these changes were generally not associated with an inflammatory cell infiltrate or cellular degeneration; such changes were observed in a minority of animals and were of minimal severity. After 4 weeks of recovery, degeneration and inflammation of the respiratory epithelium showed completed recovery. However, degeneration of the olfactory epithelium showed only partial recovery in terms of the incidence observed. In the nasal pharynx, minimal hyperplasia of the mucous cells was also observed but at a lower incident and severity and was only evident for animals exposed to 0.572 or 2.23 mg/L. Complete recovery from this effect was observed following the 4 week recovery period. The aforementioned changes were typically associated with chronic exposure to an irritant material and are not considered adverse at the incidence and severity seen. Therefore, the No Observed Adverse Effect Concentration (NOAEC) was considered to be ≥2.23 mg/L (2230 mg/m3).