Registration Dossier

Administrative data

Description of key information

The NOAEL for repeated dose toxicity of Terpenehydrocarbon alcohols 100 mg/kg bw, at maximum containing 17.45% Camphor.

This is based on read-across from Camphor, which was tested in Sage oil containing at least 7% Camphor, in an 8wk repeated dose toxicity study. The NOAEL in the Sage oil was 250 mg/kg bw, resulting a Camphor NOAEL of 17.5 mg/kg bw for 100% Camphor.

Other Terpenehydrocarbon alcohol constituents all have higher NOAELs. Terpineol multi is a representative of this group and did not show adverse repeated dose effects in a 90 -day inhalation study up to 2230 mg/m3 (OECD TG 413, Rel. 1).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across information.
Justification for type of information:
The read-across justification is presented in the Endpoint summary Repeated dose toxicity. The accompanying files are also attached there.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
103 other: mg/kg bw/d based on Camphor. Value corrected for highest concentration of Camphor present in the different Terpene hydrocarbon alcohols (17.45%).
Based on:
act. ingr.
Sex:
not specified
Basis for effect level:
clinical signs
Remarks on result:
other: read-across from Camphor
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
206 other: mg/kg bw/d based on Camphor. Value corrected for highest concentration of Camphor present in the different Terpene hydrocarbon alcohols (17.45%).
System:
other: convulsions
Organ:
not specified
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
412 other: mg/kg bw/d based on Camphor. Value corrected for highest concentration of Camphor present in the different Terpene hydrocarbon alcohols (17.45%).
System:
other: mortaliy
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The NOAEL is 103 mg/kg bw/d, based on read-across to Camphor. The NOAEL for Camhor was determined to be 18 mg/kg bw/d and is corrected for the highest percentage Camphor in the Terpene hydrocarbon alcohols (17.45%).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1959
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
This information is used for read-across to Terpene hydrocarbon alcohols
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 5 rats were exposed daily for 8 weeks by gavage.
GLP compliance:
not specified
Limit test:
no
Species:
other: white rat
Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
8 weeks
Frequency of treatment:
daily
Dose / conc.:
250 other: mg/kg bw Salvia oil containing 7-50% camphor
Dose / conc.:
500 other: mg/kg bw Salvia oil containing 7-50% camphor
Dose / conc.:
1 000 other: mg/kg bw Salvia oil containing 7-50% camphor
Dose / conc.:
1 250 other: mg/kg bw Salvia oil containing 7-50% camphor
No. of animals per sex per dose:
5
Control animals:
not specified
Sacrifice and pathology:
Clinical signs and mortality
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
a daily dose of 250 mg/kg bw Salvia oil was well tolerated. When the dose was increased to 500 mg/kg bw/d, some convulsing was observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
Upon increase to 1000 mg/kg bw/d, most animals died and all animals died when the level was increased to 1250 mg/kg bw/d.
Key result
Dose descriptor:
NOAEL
Effect level:
18 mg/kg bw/day (nominal)
Based on:
other: camphor
Remarks:
(7% present in Salvia oil).
Sex:
not specified
Basis for effect level:
clinical signs
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
36 other: mg/kg bw/d based on Camphor (7% present in Salvia oil).
System:
other: convulsions
Organ:
not specified
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
72 other: mg/kg bw/d based on Camphor (7% present in Salvia oil).
System:
other: mortality
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

The effect value above presented is based on a reported NOAEL of 250 mg/kg bw Salvia oil containing 7-50% Camphor. In calculation of the NOAEL for Camphor, the lower limit of 7% was used as a conservative approach.

Conclusions:
For the test substance Savia oil, the NOAEL was determined to be 250 mg/kg bw/d based on convulsions oberved at a test concentration of 500 mg/kg bw/d. Salvia oil containins 7-50% Camphor. In calculation of the NOAEL based on Camphor, the lower limit of 7% was used as a conservative approach.
Executive summary:

In an 8 -week toxicity study with groups of 5 white rats were given an oral daily dose of 250 mg/kg bw Salvia oil (sage oil) which was well tolerated. When the dose was increased to 500 mg/kg bw/d, some convulsing was observed. Further increase of the dose level resulted in mortality of most (1000 mg/kg bw/d), or all animals (1250 mg/kg bw/d). The observed No Observed Adverse Effect Level (NOAEL) is 250 mg/kg bw/d based on sage oil, corresponding to a NOAEL of 18 to 125 mg/kg bw/d, based on Camphor.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
103 mg/kg bw/day
Study duration:
subacute
Species:
rat
Organ:
not specified

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across information
Justification for type of information:
The read-across justification is presented in the Endpoint summary Repeated dose toxicity. The accompanying files are also attached there.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEC
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
read-across from Terpineol multi
Critical effects observed:
no
Conclusions:
In a 13-week repeated dose inhalation study no adverse toxic effects were observed. The NOAEC is determined to be ≥ 2230 mg/m3, based on the results of the source substance.
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS Limited, Huntingdon, UK
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals 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:
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
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; 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
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"
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
Dose descriptor:
NOAEC
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical 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:
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).

Endpoint conclusion
Study duration:
subchronic

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across information
Justification for type of information:
The read-across justification is presented in the Endpoint summary Repeated dose toxicity. The accompanying files are also attached there.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEC
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
read-across from Terpineol multi
Critical effects observed:
no
Conclusions:
In a 13-week repeated dose inhalation study no adverse toxic effects were observed. The NOAEC is determined to be ≥ 2230 mg/m3, based on the results of the source substance.
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS Limited, Huntingdon, UK
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals 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:
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
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; 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
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"
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
Dose descriptor:
NOAEC
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical 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:
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).

Additional information

Repeated dose toxicity for Terpene hydrocarbon alcohols is derived from Camphor because this substance has the lowests NOAEL of constituents. First the repeated dose toxicity study of Camphor is summarised and the Terpineol multi supporting repeated dose toxicity via inhalation. Thereafter the read across rationale is presented.

Repeated dose toxicity of Camphor (as tested in Sage oil, with a Camphor content of minimally 7%)

For Camphor, data is available from an 8-week toxicity study in white rats. Groups of 5 rats were given an oral daily dose of 250, 500, 1000 and 1250 mg/kg bw Salvia oil (sage oil). which was well At 250 mg/kg bw no adverse effects were seen. At 500 mg/kg bw some convulsing was observed. At 1000 mg/kg bw mortality was seen in most animals. At 1250 mg/kg bw all animals died.The NOAEL is 250 mg/kg bw. It is assumed that the toxicity comes from Camphor in this oil. The Camphor content in the oil is 7 to 50%. Assuming the lower Camphor content the NOAEL for 100% Camphor is 17.5 mg/kg bw (250 mg/kg bw Sage oil*0.07 fraction of Camphor).

Terpineol multi repeated dose inhalation toxicity

A repeated dose toxicity study with Terpineol multi is available which was performed according to OECD TG 413, and in compliance with GLP criteria. In this study, Terpineol multi 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 group could 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 groups particle 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).

The repeated dose toxicity of Terpene hydrocarbon alcohols using read across from Camphor (CAS# 76-22-2) and Terpineol multi (CAS# 8000-41-7).

 

Introduction and hypothesis for the analogue approach

Terpene hydrocarbon alcohols have the followingconstituent types of substances: Solely hydrocarbons-terpene type, Alcohol-type, Ketone-type and Ether-type all having a saturated or unsaturated cyclic hydrocarbon backbone.For this substance no repeated dose toxicity data are available. In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro, QSARs, grouping and read-across. For assessing the repeated dose toxicity of the Terpene hydrocarbon alcohols, the analogue approach is selected. The main constituent type is the Alcohol-type for which the repeated dose toxicity from Terpineol multi can be used for read across. There is one Ketone-type constituent, Camphor, which is more toxic, compared to other constituents and therefore the repeated dose from Camphor will be used for read across.

Hypothesis: Terpene hydrocarbon alcohols can have repeated dose toxicity similar to Camphor at maximum Camphor concentration of 17.5%. Other Terpene hydrocarbon alcohols have NOAELs similar to Terpineol multi.

Available information: The key information fromCamphoris derived from an EFSA evaluation on Camphor of 2008. In this evaluation the key information is where Camphor was tested in an 8-week toxicity study with rats in the form of Sage oil, which contained potentially 7 to 50% Camphor. Groups of 5 rats were given daily oral doses of 250, 500, 1000 and 1250 mg/kg bw Sage oil. At 250 mg/kg bw no adverse effects were seen. At 500 mg/kg bw some convulsing was observed. At 1000 mg/kg bw most animals died and at 1250 mg/kg bw 100% mortality was seen. The NOAEL for Sage oil in this study is 250 mg/kg bw. When converting this Sage oil NOAEL to 100% Camphor, the NOAEL for Camphor is 18 mg/kg bw using the 7% lowest concentration of Camphor in Sage oil.

Terpineol multiis tested via oral gavage in an OECDTG 422, which resulted in an repeated dose NOAEL of 750 mg/kg bw but also some fertility effects were seen and therefore further repeated dose toxicity and developmental toxicity was performed. Thereafter, Terpineol multi is tested via the inhalation-aerosol route according to OECD TG 413 (Rel. 1). In this study 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. No adverse effects were seen in any of the parameters and the NOAEC in this study is >=2230 mg/m3 (1250 mg/kg bw using 50 and 100% via inhalation and oral route, respectively).

Target chemical and source chemical(s)

Constituent types of the target substance and chemical structures of the source substances are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for repeated dose oral toxicity.

Purity / Impurities

Constituent types of the target substanceare covered by the presented constituent types, there are no other constituent that impacts the repeated dose toxicity.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue selection: For the Terpene hydrocarbon alcohols for repeated dose toxicity Camphor is selected because for this constituent repeated dose toxicity information showing the lower NOAEL compared to information from other constituent type e.g. Alcohol-type of which Terpineol multi is a representative. This information covers the repeated dose endpoint.

Structural similarities and differences: Terpene hydrocarbon alcohols contain a.o. the Ketone-type of which Camphor is the representative. Terpene hydrocarbon alcohols almost all have a similar hydrocarbon backbone with an alcohol as a functional group. The secondary alcohols in this group can be oxidised to a ketone but it is more likely that the ketone is reduced to a secondary alcohol to give handles for further processing. The Terpene hydrocarbon constituents other than Camphor can be well represented by Terpineol multi because this substance contains a variety of secondary and tertiary alcohols.

Toxico-kinetic:All Terpene hydrocarbon alcohols are absorbed somewhat similar via all routes based on the similarities in molecular weight and physico-chemical properties and Camphor and Terpineol multi are no exception.

Metabolism: Terpene hydrocarbon alcohols metabolites result in similar metabolites as those from Camphor and Terpineol Multi. The first metabolic step will result in primary, secondary or tertiary alcohols if these are not already constituents as such. Thereafter these metabolites will be further oxidised e.g. the primary alcohols can turn into acids and excreted as such or will be conjugated. The secondary and tertiary alcohols can be conjugated as such. They can also be conjugated with alpha-2 u globulin and as such be transported to the kidneys. This route is known to occur in male rats because the alpha-2u globulin sediments in the kidney and cause alpha-hydrocarbon nephropathy.

The key feature for initiation this binding to the alpha2u globulin is that the substance has a lipophilic hydrocarbon backbone and at least one hydrogen donor (alcohol) to bind to this proton and thus globulin. Therefore hydrocarbons as such (Limonene), secondary (Borneol) and tertiary alcohols, ketones that can be reduced to alcohols are susceptible to binding (Borghoff et al., 1991).

Toxico-dynamics: For Terpene hydrocarbons alcohols the Camphor constituent is the key toxicant, because repeated dose toxicity shows that the Camphor NOAEL, based on neurotoxicity, is much lower compared to Terpineol-multi. There other Terpene hydrocarbon alcohols constituents have toxicity similar to Terpineol multi. Terpineol multi showing no adverse effects up to the limit dose of 2230 mg/m3 or when converted to the oral route 1250 mg/kg bw.

Conversion of the NOAEL from Sage oil to Terpene hydrocarbon alcohols: Conversion is needed based on the Terpene hydrocarbon alcohol concentration of Camphor compared to the concentration of Camphor in Sage oil, which was the tested substance.

In Sage oil containing at least 7% Camphor, the NOAEL is 250 mg/kg bw. Assuming that Camphor is the key toxicant 100% Camphor NOAEL would be 18 mg/kg bw (250*0.07). Terpene hydrocarbon alcohols contain maximally 17.45% Camphor and therefore the NOAEL of the Terpene hydrocarbons using Camphor is 103 mg/kg bw (18/0.1745).

Other Terpene hydrocarbon alcohol constituents all have higher NOAELs. Terpineol multi is a representative of this group and did not show adverse repeated dose effects up to 750 mg/kg bw and in a follow up 90 -day inhalation study up to 2230 mg/m3 (OECD TG 413, Rel. 1).

Uncertainty of the prediction: There are no other uncertainties not already addressed above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix.

Conclusions on repeated dose toxicity for hazard and risk assessment

For Terpene hydrocarbon alcohols no repeated dose toxicity information is available but for some constituents such information is present, which can be used for read across. When using read across the result derived should be applicable for C&L and/or risk assessment, cover an exposure period duration comparable or longer than the corresponding method and be presented with adequate and reliable documentation.This documentation is presented in the current text. The key constituent for repeated dose toxicity is Camphor, in view of its toxicity seen inan 8-week toxicity study with Sage oil. The converted NOAEL 103 mg/kg bw (NOAEL in Sage oil is 250 mg/kg bw containing at least 7% Camphor converted to Terpene hydrocarbon alcohols containing maximally 17.45% Camphor. The oral and inhalation NOAELs of Terpineol multi, representing the other constituents are >=750 mg/kg bw and 2230 mg/m3 (or 1250 mg/kg bw when converted to the oral route using the same absorption percentages for both routes).

Final conclusion:For theTerpene hydrocarbon alcoholsthe NOAEL is 103 mg/kg bw.

 


Data matrix supporting the Terpene hydrocarbon alcohol repeated dose toxicity assessment by usingread acrossfrom Camphor and Terpineol multi.

Terpene hydrocarbon alcohols

Terpineol hydrocarbon alcohols

Camphor

Terpineol multi

 

Target

Source

Supporting source

Structure

Not applicable

 

(α-Terpineol)

(γ-Terpineol)

CAS

Not applicable

76-22-2

8000-41-7 (generic CAS no)

EC No.

945-149-0

200-945-0

232-268-1

Reach registration

2018

Registered

Registered

Molecular weight

136-154

 

 

Phys-chem properties

 

 

 

Appearance

Liquid

Solid

Liquid

Vp (Pa)

51.9

(10-52, exp)

87

(ECHA site)

6.48

(exp. α-Terpineol)

Log Kow

4.9

(3.3-5.5; IFF)

2.4

(ECHA site)

2.6

(α-Terpineol, IFF)

Identity, Constituent type (%)

100%

Mono constituent

Multi constituent

Solely hydrocarbons

 

 

 

Limonene type

0-15

 

 

Alcohol type

 

 

>80

Tertiary alcohols

40-90

 

 

Secondary alcohols

7-40

 

 

Ketone type

-

 

 

Camphor-Type

0-17%

> 80

 

Ether type

 

 

 

Cineol type

<10%-

 

 

Aromatic ether type

<10%

 

 

Human health

 

 

 

Repeated dose, oral, sub-acute: NOAEL mg/kg bw

103*

(Read across from Camphor)

18

(8 wk oral repeated dose toxicity)

->=750

(OECD TG 422)

Repeated dose, inhalation, sub-chronic NOAEC in mg/m3)

Read across for other than Camphor constituents

 

≥2230

(OECD TG 413)

* Recalculated based on a NOAEL ofSage oil containing at least 7% Camphor, which is 250 mg/kg bw. Assuming that Camphor is the key toxicant 100% Camphor NOAEL would be 18 mg/kg bw (250*0.07). Terpene hydrocarbon alcohols contain maximally 17.45% Camphor and therefore the NOAEL of the Terpene hydrocarbons using Camphor is 103 mg/kg bw (18/0.1745).

References:

Belsito et al. A toxicologic and dermatologic assessment of cyclic and non-cyclic terpene alcohols when used as fragrance ingredients. Food and Chemical Toxicology. 46 (2008a) S1-71.

 

Borghoff, S.J., Miller, A.B., Bowen, J.P. and Swenberg, J.A., 1991, Characteristics of chemical binding to alph2u-globulin in vitro-Evaluation structure-acitivity relationships, Toxicol. Appl.Pharmacol. , 107, 228-238.

 

Camphor in flavourings and other food ingredients with flavouring properties, 2008, Scientific opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC) on a request from the Commission on Camphor in flavourings and other food ingredients with flavouring properties. The EFSA Journal 729, 1-15,http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/729.pdf

 

 

Justification for classification or non-classification

Based on the absence of adverse effects >100 mg/kg bw in a reliable repeated dose toxicity study, the substance does not need to be classified for repeated dose toxicity by the oral route according to EU CLP (EC No. 1272/2008 and its amendments).