Registration Dossier

Diss Factsheets

Administrative data

Description of key information

90-day repeated dose toxicity study by inhalation: NOAEC for male rats = 100 ppm

90-day repeated dose toxicity study by inhalation: NOAEC for female rats = 200 ppm

90-day repeated dose toxicity study by inhalation: NOAEC for female mice = 50 ppm

90-day repeated dose toxicity study by inhalation: LOAEC for male mice = 100 ppm

4-week repeated dose toxicity by oral route: NOAEC for male and female rats = 12 000 ppm (higher dose tested)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August - 29 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline required
Principles of method if other than guideline:
The purpose of this study was to assess the systemic toxic potential of the test item in a 21-day oral study (dietary administration) in Sprague-Dawley rats, and to aid in the selection of a suitable high dose for a subsequent OECD 421 screening study.
GLP compliance:
no
Remarks:
This study is a preliminary study. Therefore the GLP complinace is not required.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females nulliparous and non-pregnant
- Age at study initiation: male: 69-76 days and female: 83-90 days
- Weight at study initiation: male: 343 to 397g and female: 225 g to 285 g.
- Housing: Each sex was allocated separately . Four animal per cage
- Diet SDS VRF1 Certified powdered diet. ad libitum
- Water ad libitum: potable water
- Acclimation period: Six days before the beginning of the treatment

DETAILS OF FOOD AND WATER QUALITY:

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

Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
corn oil
Remarks:
test item to corn oil ratio 5:1
Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly. Stability of the formulations was confirmed for one day at ambient temperature and for eight days frozen for formulations between 1000 ppm and 15000 ppm
- Mixing appropriate amounts with (Type of food): SDS VRF1 Certified diet.
- Storage temperature of food: Deep-frozen (nominally -20°C) for 22 days.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
21 days
Frequency of treatment:
continuous
Dose / conc.:
3 000 ppm
Dose / conc.:
6 000 ppm
Dose / conc.:
12 000 ppm
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a recent GLP study conducted according to OECD guideline 423, the LD50 of the test item was found to be between 300 and 2000 mg/kg bw. Also, palatability issues were expected with this test item therefore the highest dietary concentration to be tested was decided to be 12000 ppm, corresponding to an anticipated test item intake around 800 mg/kg bw/day. Then a two-fold factor was applied to obtain the lower concentrations, i.e. 6000 and 3000 ppm
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cages were inspected daily for evidence of animal ill-health amongst the occupants.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on Days 1, 4, 8, 11, 15, 18 and 21 on each animal to monitor general health.

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

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

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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at week 3.
- How many animals: all animals
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile aids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb)

ESTROUS CYCLES:
- Daily smears were taken for 21 days, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the estrous cycle
Sacrifice and pathology:
SACRIFICE: Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY:
All animals were subject to a detailed necropsy. Only the thoracic and abdominal cavities were opened. The cranial cavity was not opened as there were no observations during the study to indicate a possible neurotoxic action. 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 fixative. The retained tissues were checked before disposal of the carcass.
The organs weighed and tissue samples fixed are detailed in table 1
Other examinations:
ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed individually and summed for presentation in the tables. Requisite organs were weighed for animals killed at the scheduled interval.
Statistics:
No statistical analysis of the data was performed on this study.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For males and females receiving 3000, 6000 and 12000 ppm, overall mean bodyweight gain was low when compared with Control (84, 77 and 78%, respectively for males and 74, 74 and 86%, respectively for females) after three weeks of treatment. This was due to an initial bodyweight loss over Days 1-2 in males and Days 1-3 in females at 6000 and 12000 ppm and Days 1-2 in females at 3000 ppm, and low overall bodyweight gain in males at 3000, 6000 and 12000 ppm over Days 1-7 of treatment and females at 6000 and 12000 ppm over Days 7 14 of treatment when compared with Control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption for males and females receiving the test item at 3000, 6000 and 12000 ppm was lower than that of Control on Day 1 of treatment, with a dose-dependent decrease. Food consumption remained slightly low in all treated males thereafter. Lower food consumption was also observed on Day 2 of treatment in females receiving 12000 ppm but food consumption was generally similar to that of the Controls and pre treatment intake in all treated groups thereafter.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Thymus mean weights were higher than that of the Control for males receiving 3000, 6000 and 12000 ppm; a dose response was apparent.
All other organ weights for males and all organ weights for females were unaffected by treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Remarks on result:
not determinable
Critical effects observed:
no
Conclusions:
The administration of the test item to Sprague Dawley rats by dietary administration for 21 days at dietary levels of 3000, 6000, or 12000 ppm was well tolerated. No animals died and the clinical condition of the animals was satisfactory. At 6000 and 12000 ppm in males and females and 3000 ppm in females, weight loss was observed over Days 1-2 or Days 1-3 which correlated with the dose-dependent initial low food consumption observed at all treatment levels in both sexes. Estrous cycles of the females and blood chemistry investigations in both sexes were unaffected by treatment. There were no findings attributable to treatment at macroscopic examination, but mean adjusted thymus weights were higher among males receiving 3000, 6000 or 12000 ppm.
It was therefore concluded that there were no findings that would preclude the use of 12000 ppm as the high dietary concentration to be tested on the main OECD 421 study.
Executive summary:

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

The overall mean achieved dosages were 196, 410 and 788 mg/kg bw/day in males and 201, 411 and 827 mg/kg bw/day in females receiving 3000, 6000 and 12000 ppm, respectively.

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

For males and females receiving 3000, 6000 and 12000 ppm, overall mean bodyweight gain was low when compared with Control (84, 77 and 78%, respectively for males and 74, 74 and 86%, respectively for females) after three weeks of treatment. This was due to an initial bodyweight loss over Days 1-2 in males and Days 1-3 in females at 6000 and 12000 ppm and days 1-2 in females at 3000 ppm, and low overall bodyweight gain in males at 3000, 6000 and 12000 ppm over Days 1-7 of treatment and females at 6000 and 12000 ppm over Days 7-14 of treatment when compared with Control.

The food consumption for males and females receiving alpha-pinene multiconstituent at 3000, 6000 and 12000 ppm was lower than that of Control on Day 1 of treatment, with a dose-dependent decrease. Food consumption remained slightly low in all treated males thereafter. Lower food consumption was also observed on Day 2 of treatment in females receiving 12000 ppm but food consumption was generally similar to that of the Controls and pre‑treatment intake in all treated groups thereafter.

Thymus mean weights were higher than that of the Control for males receiving 3000, 6000 and 12000 ppm; a dose response was apparent.

It was therefore concluded that there were no findings that would preclude the use of 12000 ppm as the high dietary concentration to be tested on the main OECD 421 study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 weeks in 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Whole body exposure, and fixation of cauda tissue at 65°C
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
no data on food consumption, no ophthalmological examination, some organ weights were not recorded (Adrenals, brain, heart, Ovaries, thyroids, Uterus). Animals weighed weekly and not twice weekly
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58).
Limit test:
no
Specific details on test material used for the study:
- lot 4KB705 supplied from Millennium Specialty Chemicals (Jacksonville, FL), purity 96%
- water content of 27 ppm (Karl Fischer titration)
- 69% (+)-α-pinene and 31% (–)-α-pinene (GC/FID)
- Identified impurities: camphene (1.77%) and β-pinene (1.73%) identified by GC/FID, and tricyclene (0.51%) identified by GC/MS.
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
Male and female B6C3F1/N mice were obtained from the NTP colony maintained at Taconic Farms, Inc. (Germantown, NY),
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Male and female B6C3F1/N mice were obtained from the NTP colony maintained at Taconic Farms, Inc. (Germantown, NY),
- Age at the study initiate: 5 to 6 weeks
- Animals were quarantined for 12 days before the study and after, assigned at random to treatment groups.
- Housing: Mice were housed individually in stainless steel, wire bottom cages (LabProducts, Inc., Seaford, DE); changed weekly.
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods); changed weekly.
Water: Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum

- Chamber environment:
Temperature: 72° ± 3° F
Relative humidity: 50% ± 15%
Room fluorescent light: 12 hours/day
Chamber air changes: 15 ± 2/hour
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber and room concentrations of α-pinene were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period.
Average concentration measured were 24.9 ± 1.1ppm for the 25 ppm group, 49.8 ± 0.8 ppm for the 50 ppm group, 99.6 ± 1.4 ppm for the 100 ppm group, 200 ± 4 ppm for the 200 ppm group, 401 ± 7 ppm for the 400 ppm group.
Duration of treatment / exposure:
6 hours plus T90 (10 minutes) per day, 5 days per week, for 14 weeks
Frequency of treatment:
Five times per week, weekdays only
Dose / conc.:
25 ppm
Remarks:
nominal conc.
Dose / conc.:
50 ppm
Remarks:
nominal conc.
Dose / conc.:
100 ppm
Remarks:
nominal conc.
Dose / conc.:
200 ppm
Remarks:
nominal conc.
Dose / conc.:
400 ppm
Remarks:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Before the studies began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease.
- Core study animals were weighed initially, and body weights and clinical findings were recorded on day 8, weekly thereafter, and at the end of the studies.
- Serologic analyses were performed on five male and five female sentinel mice at 1 week and at the end of the studies using the protocols of the NTP Sentinel Animal Program
- Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital plexus of clinical pathology rats on days 4 and 23 and from core study mice at the end of the studies for hematology analyses.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes for moribundity and death
- Time schedule: twice daily, at least 6 hours apart (before 10:00 AM and after 2:00 PM)

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical findings were recorded on day 7 (female rats) and day 8 (male rats), weekly thereafter, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially on Day 1, on days 7 (female rats) and days 8 (male rats), weekly thereafter, and at the end of the studies;

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, collected from the retroorbital plexuscof clinical pathology rats at the end of the study.
- Parameters examined: hematocrit; packed cell volume; hemoglobin; erythrocyte, reticulocyte, and platelet counts; Howell-Jolly bodies (mice); mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and differentials.

CLINICAL CHEMISTRY: Yes, collected from the retroorbital plexuscof clinical pathology rats at the end of the study.
- Parameters examined: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
METHOD OF KILL: Carbon dioxide asphyxiation
GROSS PATHOLOGY: : Yes
Heart, right kidney, liver, lung, spleen, right testis, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.

HISTOPATHOLOGY: Yes, complete histopathologic examinations were performed by the study laboratory pathologist on all chamber control and 400 ppm animals and 200 ppm female rats.
In addition to gross lesions and tissue masses, the following tissues were examined:
adrenal gland, bone, brain, clitoral gland, esophagus, eyes, Harderian gland, gallbladder, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder, and uterus. The liver of male rats and the kidney of rats were examined in the remaining groups.
Other examinations:
SPERM MOTILITY AND VAGINAL CYTOLOGY: At the end of the study, sperm samples were collected for sperm motility evaluations. Sperm heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. were evaluated. The left cauda, left epididymis, and left testis were weighed. The numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Sperm density was then determined microscopically with the aid of a hemacytometer
Vaginal samples were collected for up to 12 consecutive days prior to the end of the study for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Statistics:
Kaplan-Meier used for probability of survival. Statistical analyses used for possible dose-related effect on survival was Cox (Cox D.R. (1972) Regression models and life tables. J.R. Stat. Soc. B34: 187-220.) for testing two groups for equality; and Tarone’s (Tarone R.E. (1975) Tests for trend in life table analysis. Biometrika 62; 679-682) life table test for a dose-related trend.
Fisher exact test (Gart et al., 1979) was used to determine significance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
no mortality
Body weight and weight changes:
no effects observed
Description (incidence and severity):
(See table 1 for raw data)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Changes were observed but were either considered as secondary treatment-associated stress effects or not considered as toxicologically relevant. (See table 4)
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Changes were observed but no gross or microscopic lesions were associated with these organ weight findings. (See table 2)
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperplasia of the transitional epithelium of the urinary bladder, were observed from 100 ppm in males and females. However, there was no evidence of histopathological changes to the clitoris, ovaries, uterus, epididymis, preputial gland, seminal vesicles, and testes in any groups of animals. (See table 3)
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There were significantly decreased sperm count per mg cauda in males treated at 200 and 400 ppm and in cauda sperm counts in 100, 200, and 400 ppm groups (See table 5)
However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). Also, these decreases might be secondary to stress induced by hyperplasia of bladder epithelium at the 3 highest doses.
Details on results:
MORTALITY
All mice survived until the terminal sacrifice.

CLINICAL EXAMINATION
There were no treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was comparable for all test animals when compared to controls.

CLINICAL CHEMISTRY
No data available.

ORGAN WEIGHTS
Absolute liver weights were increased for both sexes at the 400 ppm and relative and absolute liver weights were increased for both sexes at 200 ppm and 400 ppm, and for female only at 100 ppm. The 400 ppm male group showed decreased absolute and relative thymus weight. The basolute kidney weights for 200 and 400 ppm males were significantly less than those of the control group. No gross or microscopic lesions were associated with these organ weight findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination of male and female mice exposed to atmospheres of ≥100 ppm of α-pinene revealed evidence of hyperplasia of the transitional epithelium of the urinary bladder. However, there was no evidence of histopathological changes to the clitoris, ovaries, uterus, epididymis, preputial gland, seminal vesicles, and testes any of the control or test groups of animals.

HEMATOLOGY:
At the end of the study, there were small but statistically significant decreases in erythrocyte counts in 200 and 400 ppm females and in the hemoglobin concentration and the hematocrit value in 400 ppm females compared to concurrent controls. Decreases in erythrocyte count and hematocrit value also occurred in 400 ppm males.
Leukocyte and lymphocyte counts were significantly decreased in 400 ppm males. The leukocyte changes likely represent a secondary treatment-associated stress effect. The exact mechanism for the mild decreases in the erythron are not known. Other significant changes in hematology parameters were not toxicologically relevant.

SPERM MOTILITY AND VAGINAL CYTOLOGY:
There were significantly decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males. There were no changes in the proportion of regularly cycling females, estrous cycle length, or percentage of time spent in the individual stages of the estrous cycle of female mice at any exposure concentration and there were no ovarian histopathologic findings.
Therefore, α-pinene exposure via inhalation exhibits the potential to be a reproductive toxicant in male mice, but not in female mice
Key result
Dose descriptor:
LOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: significantly decreased sperm count per mg cauda in males treated at 200 and 400 ppm and in cauda sperm counts in 100, 200, and 400 ppm groups
Dose descriptor:
NOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
male reproductive system
Organ:
cauda epididymis
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Mean of body weights and body weight gainsa:


 
















































































































 Concentration(ppm)Survivalb

Initial Body Weight (g) Final Body Weight (g)Change in Body Weight(g) Final Weight Relative to Controls (%)
Male010/1022.9 ± 0.237.1 ± 0.614.3 ± 0.6 
2510/1023.0 ± 0.336.9 ± 0.713.9 ± 0.899
5010/1022.7 ± 0.338.3 ± 0.915.6 ± 0.8103
10010/1022.5 ± 0.235.9 ± 0.713.4 ± 0.797
20010/1022.8 ± 0.335.5 ± 1.012.7 ± 0.996
40010/1022.8 ± 0.236.2 ± 0.513.5 ± 0.498
Female010/1019.5 ± 0.431.5 ± 0.612.0 ± 0.5 
2510/1019.6 ± 0.430.3 ± 0.610.8 ± 0.796
5010/1019.7 ± 0.332.7 ± 0.712.9 ± 0.7104
10010/1019.7 ± 0.431.5 ± 1.111.8 ± 0.9100
20010/1019.3 ± 0.330.7 ± 0.611.4 ± 0.697
40010/1019.4 ± 0.330.6 ± 0.511.2 ± 0.497

a  Weights and weight changes are given as mean ± standard error.


b Number of animals surviving at 14 weeks/number initially in group


 


Table 2: Mean of absolute and relative organ weight


 
































































































































 Chamber control25 ppm50 ppm100 ppm200 ppm400 ppm
Male 
n101010101010
Necropsy body wt37.1 ± 0.636.9 ± 0.738.3 ± 0.935.9 ± 0.735.5 ± 1.036.2 ± 0.5
R Kidney absolute0.330 ± 0.0060.318 ± 0.0090.336 ± 0.0100.309 ± 0.0080.295 ± 0.006*0.307 ± 0.007*
R kidney relative8.903 ± 0.1678.629 ± 0.2088.793 ± 0.2678.617 ± 0.2058.348 ± 0.1458.469 ± 0.155
Liver absolute1.617 ± 0.0221.589 ± 0.0281.702 ± 0.0401.637 ± 0.0241.660 ± 0.0431.957 ± 0.057**
Liver relative43.671 ± 0.88043.123 ± 0.45844.487 ± 0.80645.651 ± 0.67846.903 ± 0.750*54.009 ± 1.465**
Thymus absolute0.066 ± 0.0040.063 ± 0.0040.067 ± 0.0030.057 ± 0.0010.062 ± 0.0040.051 ± 0.003**
Thymus relative1.777 ± 0.0811.699 ± 0.0901.742 ± 0.0631.591 ± 0.0521.739 ± 0.1151.397 ± 0.081**
Female
n101010101010
Necropsy body wt31.5 ± 0.630.3 ± 0.632.7 ± 0.731.5 ± 1.130.7 ± 0.630.6 ± 0.5
Liver absolute1.466 ± 0.0411.475 ± 0.0531.442 ± 0.0361.548 ± 0.0531.587 ± 0.0371.730 ± 0.032**
Liver relative46.542 ± 0.98848.567 ± 1.23944.214 ± 0.88049.280 ± 0.672*51.728 ± 0.795**56.511 ± 0.705**

aOrgan weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).


* Significantly different (P≤0.05) from the chamber control group by Williams’ or Dunnett’s test


** Significantly different (P≤0.01) from the chamber control group by Williams’ or Dunnett’s test


 


Table 3: Incidence of Non neoplastic lesions of the urinary bladder


 























































SexeTreatment concentration (ppm) 0 (control group)2550100200400
MaleNumber Examined Microscopically101010101010
MaleTransitional Epithelium, Hyperplasiaa0007** (1.0)10** (2.0)b 10** (2.5)b 
FemaleNumber Examined Microscopically101010101010
 FemaleTransitional Epithelium, Hyperplasiaa0006** (1.0)10** (1.6)10** (2.2)b 

 a Number of animals with lesion


 b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked


 


Table 4: Hematology data


 
















































































































































































 Treatment concentration (ppm) 0 (control group) 25 50 100 200 400
Male      
Hematocrit (spun) (%)51.3 ± 0.350.5 ± 0.450.1 ± 0.351.1 ± 0.350.9 ± 0.449.8 ± 0.3*
Hemoglobin (g/dL)16.0 ± 0.116.0 ± 0.115.7 ± 0.116.0 ± 0.016.1 ± 0.115.7 ± 0.1
Erythrocytes (106/µL)10.51 ± 0.0610.47 ± 0.0610.23 ± 0.0910.52 ± 0.0410.55 ± 0.0810.10 ± 0.07**
Reticulocytes(103/µL)Nucleated erythrocytes/100 leukocytes223.7 ± 19.4 0.00 ± 0.00200.3 ± 14.9 0.00 ± 0.00193.9 ± 16.4 0.00 ± 0.00205.2 ± 13.0 0.00 ± 0.00214.3 ± 16.4 0.00 ± 0.00202.2 ± 15.9 0.00 ± 0.00
Mean cell volume (fL)49.3 ± 0.349.2 ± 0.249.6 ± 0.249.4 ± 0.249.6 ± 0.250.6 ± 0.2**
Mean cell hemoglobin (pg)Mean cell hemoglobin concentration (g/dL)15.3 ± 0.1 31.0 ± 0.215.2 ± 0.1 31.0 ± 0.115.4 ± 0.1 31.0 ± 0.115.2 ± 0.0 30.8 ± 0.215.2 ± 0.0 30.7 ± 0.115.6 ± 0.1* 30.8 ± 0.1
Leukocytes (103/µL)3.10 ± 0.402.94 ± 0.432.03 ± 0.262.47 ± 0.152.31 ± 0.261.87 ± 0.17*
Lymphocytes (103/µL)2.60 ± 0.342.41 ± 0.391.73 ± 0.232.03 ± 0.131.93 ± 0.221.48 ± 0.14*
 Female      
Hematocrit (spun) (%)49.6 ± 0.350.1 ± 0.449.4 ± 0.550.1 ± 0.348.9 ± 0.348.3 ± 0.3*
Hemoglobin (g/dL)15.8 ± 0.116.0 ± 0.115.7 ± 0.215.9 ± 0.115.5 ± 0.115.5 ± 0.1*
Erythrocytes (106/µL)10.21 ± 0.0510.26 ± 0.0610.10 ± 0.1110.14 ± 0.069.96 ± 0.09*9.85 ± 0.08**
Reticulocytes(103/µL)Nucleated erythrocytes/100 leukocytes269.5 ± 15.4 0.00 ± 0.00248.9 ± 14.9 0.00 ± 0.00251.9 ± 16.5 0.00 ± 0.00282.5 ± 18.3 0.00 ± 0.00240.1 ± 20.8 0.00 ± 0.00251.2 ± 15.3 0.00 ± 0.00
Mean cell volume (fL)49.3 ± 0.249.7 ± 0.249.5 ± 0.350.1 ± 0.2*49.7 ± 0.249.7 ± 0.2
Mean cell hemoglobin (pg)Mean cell hemoglobin concentration (g/dL)15.5 ± 0.1 31.5 ± 0.215.6 ± 0.1 31.4 ± 0.115.5 ± 0.1 31.4 ± 0.215.6 ± 0.1 31.1 ± 0.115.6 ± 0.1 31.4 ± 0.115.7 ± 0.1 31.6 ± 0.1
Leukocytes (103/µL)3.65 ± 0.353.10 ± 0.273.34 ± 0.322.80 ± 0.293.11 ± 0.323.16 ± 0.34
Lymphocytes (103/µL)3.09 ± 0.302.65 ± 0.232.78 ± 0.302.39 ± 0.252.60 ± 0.262.66 ± 0.27

* Significantly different (P≤0.05) from the chamber control group by Dunn’s or Shirley’s test


** P≤0.01


Data are presented as mean ± standard error. Statistical tests were performed on unrounded data.


 


Table 5 Mean of epididymal spermatozoal measurements


 











































 Chamber control100 ppm200 ppm400 ppm
n10101010
Epididymal spermatozoal measurements
Sperm motility (%)90.25 ± 0.3488.31 ± 0.8689.74 ± 0.8087.95 ± 1.08
Sperm (103/mg cauda epididymis)704.8 ± 64.9690.7 ± 55.9537.5 ± 27.0*445.8 ± 13.5**
Sperm (106/cauda epididymis)24.45 ± 0.9518.40 ± 0.41**16.48 ± 0.72**14.64 ± 0.25**

* Significantly different (P≤0.05) from the chamber control group by Shirley’s test


** P≤0.01

Conclusions:
The NOAEC for females is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated from 100 ppm.

The LOAEC for males is 100 ppm based on significantly decreased sperm count per mg cauda in males treated at 200 and 400 ppm and in cauda sperm counts in 100, 200, and 400 ppm groups.
However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). Also, these decreases might be secondary to stress induced by hyperplasia of bladder epithelium at the 3 highest doses. However, in a conservative approach, this effect was selected as the critical effect to calculate DNELs.

The study suffers from several limitations:
- animals were exposed whole body by inhalation which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming).
- the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples
Executive summary:

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5  days per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals.


Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.


Similar effects were observed in male and female mice from the same dose level (100 ppm): minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder.


Decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males were observed. However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). Also, these decreases might be secondary to strss induced by hyperplasia of bladder epithelium at the 3 highest doses. In addition, animals were exposed whole body by inhalation, which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming). However, in a conservative approach, this effect was selected as the critical effect to calculate DNELs.


The NOAEC for female mice is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm. The LOAEC for males is 100 ppm based on significantly decreased sperm count per mg cauda in males treated at 200 and 400 ppm and in cauda sperm counts in 100, 200, and 400 ppm groups.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 weeks in 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Whole body exposure, and fixation of cauda tissue at 65°C
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
no data on food consumption, no ophthalmological examination, some organ weights were not recorded(Adrenals, Brain, Ovaries, Thyroids, Uterus), animals weighed weekly and not twice weekly at the beginning.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58).
Limit test:
no
Specific details on test material used for the study:
- lot 4KB705 supplied from Millennium Specialty Chemicals (Jacksonville, FL), purity 96%
- water content of 27 ppm (Karl Fischer titration)
- 69% (+)-α-pinene and 31% (–)-α-pinene (GC/FID)
- Identified impurities: camphene (1.77%) and β-pinene (1.73%) identified by GC/FID, and tricyclene (0.51%) identified by GC/MS.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Male and female F344/N rats were obtained from the NTP colony maintained at Taconic Farms, Inc. (Germantown, NY),
- Age at the study initiate: 6 weeks
- Animals were quarantined for 13 days befor the study.
- Housing: Rats were housed individually in stainless steel, wire bottom cages (LabProducts, Inc., Seaford, DE); changed weekly.
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods); changed weekly.
Water: Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum

Chamber environment:
Temperature: 72° ± 3° F
Relative humidity: 50% ± 15%
Room fluorescent light: 12 hours/day
Chamber air changes: 15 ± 2/hour
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber and room concentrations of α-pinene were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period.
The average concentration measured were: 24.9 ± 1.1 ppm for the 25 ppm group, 49.8 ± 0.8 for the 50 ppm group, 99.6 ± 1.4ppm for the 100 ppm group, 200 ± 5 ppm for the 200 ppm group and 401 ± 6 ppm for the 400 ppm group.
Duration of treatment / exposure:
14 weeks; 6 hours plus T90 (10 minutes) per day .
Groups of 10 male and 10 female clinical pathology rats were exposed to the same concentrations for 23 days.
Frequency of treatment:
five times per week, weekdays only
Dose / conc.:
25 ppm
Remarks:
nominal conc.
Dose / conc.:
50 ppm
Remarks:
nominal conc.
Dose / conc.:
100 ppm
Remarks:
nominal conc.
Dose / conc.:
200 ppm
Remarks:
nominal conc.
Dose / conc.:
400 ppm
Remarks:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease.
- Core study animals were weighed initially, and body weights and clinical findings were recorded on day 7 (female rats), day 8 (male rats), weekly thereafter, and at the end of the studies.
- Serologic analyses were performed on five male and five female sentinel rats at 1 week and at the end of the studies using the protocols of the NTP Sentinel Animal Program
- Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital plexus of clinical pathology rats on days 4 and 23 and from core study rats at the end of the studies for hematology and clinical chemistry analyses.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes for moribundity and death
- Time schedule: twice daily, at least 6 hours apart (before 10:00 AM and after 2:00 PM)

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical findings were recorded on day 7 (female rats) and day 8 (male rats), weekly thereafter, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially on Day 1, on days 7 (female rats) and days 8 (male rats), weekly thereafter, and at the end of the studies;

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, collected from the retroorbital plexuscof clinical pathology rats at the end of the study.
- Parameters examined: hematocrit; packed cell volume; hemoglobin; erythrocyte, reticulocyte, and platelet counts; Howell-Jolly bodies (mice); mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and differentials.

CLINICAL CHEMISTRY: Yes, collected from the retroorbital plexuscof clinical pathology rats at the end of the study.
- Parameters examined: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
METHOD OF KILL: Carbon dioxide asphyxiation
GROSS PATHOLOGY: : Yes
Heart, right kidney, liver, lung, spleen, right testis, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.

HISTOPATHOLOGY: Yes, complete histopathologic examinations were performed by the study laboratory pathologist on all chamber control and 400 ppm animals and 200 ppm female rats.
In addition to gross lesions and tissue masses, the following tissues were examined:
adrenal gland, bone, brain, clitoral gland, esophagus, eyes, Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder, and uterus. The liver of male rats and the kidney of rats were examined in the remaining groups.
Other examinations:
SPERM MOTILITY AND VAGINAL CYTOLOGY: At the end of the study, sperm samples were collected for sperm motility evaluations. Sperm heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. were evaluated. The left cauda, left epididymis, and left testis were weighed. The numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Sperm density was then determined microscopically with the aid of a hemacytometer
Vaginal samples were collected for up to 12 consecutive days prior to the end of the study for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Statistics:
Kaplan-Meier used for probability of survival. Statistical analyses used for possible dose-related effect on survival was Cox (Cox D.R. (1972) Regression models and life tables. J.R. Stat. Soc. B34: 187-220.) for testing two groups for equality; and Tarone’s (Tarone R.E. (1975) Tests for trend in life table analysis. Biometrika 62; 679-682) life table test for a dose-related trend
Fisher exact test (Gart et al., 1979), was used to determine significance.
Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
In the high dose group (400 ppm), 6 females were found dead before the end of the study (See Table 1 for more information)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: the final mean body weights and mean body weight gains of exposed males were similar to those of the chamber controls.
Females: In the high dose group (400 ppm), the final mean body weights and the mean body weight gains of females exposed to 400 ppm were significantly less than those of the chamber controls.
(See Table 1 for more information)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
On day 4, mild decreases in leukocyte and lymphocyte counts were observed in 200 and 400 ppm male rats. However, these findings were not observed in females, they can be considered as mild but not severe (23% and 26% from control values for leukocytes and lymphocytes counts, respectively) and are not persistent (not found at Day 23 and Week 14). Also, no histopathological findings could be associated to these changes. Therefore, these treatment-related effects can be considered as not toxicologically significant.

In males, limited decreases of 4, 4 and 5% of controls were observed for haemoglobin, haematocrit and erythrocytes, respectively. (See table 5)
Historical data from Charles River, 1984 on 19-21-week-old Fischer 344 male rats show ranges ± 2 SD between 13.5-17.1 g/dL, 41-51 % and 6.52-8.74 106/µL for haemoglobin, haematocrit and erythrocytes, respectively. Hematocrit and haemoglobin values found in the alpha-pinene study are within these ranges. The erythrocytes values from all groups are above the historical control range but the coefficient of variation (SD/mean=0.56/7.62=7%), that can be considered as the normal biological variability, is higher than the 5% decrease observed in this parameter. Also, Chayne, 2006 report hematology ranges in Fischer 344/Cr rats of 13.4-17.2 g/dL, 46-52.5% and 6.68-9.15 106/µL for haemoglobin, haematocrit and erythrocytes, respectively. Like the previous historical control data, values found in the alpha-pinene study are within these ranges except for erythrocytes values of control and 50 ppm groups that are slightly above this range. This means that the erythrocytes values obtained in the control group of this study may be high compared to normal values and the decrease observed in this parameter may therefore be due to those study-specific high control values.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The remaining significant differences in clinical chemistry parameters were not considered to be toxicologically relevant. (See table 3 for details)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The organ weight changes (liver, heart, thymus, spleen and kidneys) in male and female rats were not accompanied by histopathologic lesions except for the male kidney. The weight changes in lymphoid tissues were not accompanied by clinical chemistry or histopathologic changes indicative of immunotoxicity and, therefore, were not considered toxicologically relevant. (See table 2 for more information)
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Examination of the male kidneys at all dose levels revealed lesions including granular casts and hyaline droplets. The severities of these lesions increased with increasing exposure concentration. (See table 4)
The presence of these nonneoplastic lesions in the kidney is suggestive of α2μ-globulin nephropathy, a renal syndrome that occurs in male but not female F334/N rats and that has been linked to the development of renal tubule neoplasms. This syndrome has been produced by structurally diverse chemicals and is thought to be secondary to toxicity caused by accumulation of hyaline droplets within the renal tubule epithelial cells.
Alpha 2μ-globulin is a protein that is synthesized in vivo in large quantities exclusively by male rats. The presence of this protein renders male rats uniquely sensitive to a chemically induced syndrome that is manifest acutely as the accumulation of α2μ-globulin in renal proximal tubule cells. Alpha-pinene (like d-limonene, the prototypical compound for this effect) induces α2μ-globulin nephropathy (and renal tumors) in male rats. The accumulation of alpha2µ-globulin in the form of hyaline droplets in the renal tubules, and then a chronic renal cellular protein overload lead to renal cell necrosis and compensatory cell proliferation.
Ref: Meek M.E. et al. (2003) A Framework for Human Relevance Analysis of Information on Carcinogenic Modes of Action, Critical Reviews in Toxicology, 33(6): 591 -653.

The histopathological examination done in this study showed that the effects observed in male kidneys correspond to this kind of nephropathy. In addition, they were not observed in female rats and in mice. Therefore, this effect is sexe- and species-specific and not relevant for humans.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Decreased sperm count in cauda epididymis in 200 and 400 ppm males vs control group (see table 6).
Details on results:
MORTALITY
In the high dose group (400 ppm), 6 females were found dead before the end of the study (4 on Day 36, 1 on Day 50 and 1 on Day 91). All other animals survived until terminal sacrifice.

CLINICAL EXAMINATION
No signs of toxicity (e.g., abnormal breathing or behavior) were noted during clinical observations

BODY WEIGHT AND WEIGHT GAIN
Males: the final mean body weights and mean body weight gains of exposed males were similar to those of the chamber controls.
Females: All surviving females at 400 ppm lost weight between week 12 and week 14. The final mean body weights of females exposed to 400 ppm were significantly less than those of the chamber controls (159g vs 194g in the control group) . The mean body weight gains of female exposed to 400 ppm were significantly less than those of the chamber controls (72g vs 105 g in the control group). .

CLINICAL CHEMISTRY
Males showed statistically significant reductions in sorbitol dehydrogenase activity at 400 ppm, alanine aminotransferase activity at levels ≥50 ppm, and alkaline phosphatase activity at levels ≥100 ppm. Females showed statistically significant reductions in alanine aminotransferase activity at levels ≥200 ppm, and alkaline phosphatase activity at the 400 ppm. There were significant decreases at lower levels of exposure for females but these changes were not dose-dependent. None of these changes in enzyme activity were related to organ weight changes or evidence of histopathology.

ORGAN WEIGHTS
Relative liver weights, were statistically increased in males at 100 ppm and greater and in all females treated groups. Absolute liver weights were statistically increased in males at 400 ppm and in females at 50, 100 and 200 ppm.
Absolute kidney weights were increased in males at 100 ppm and greater, and relative kidney weights were increased in males at 50 ppm and greater. In females, absolute liver weights were increased at levels of 50 and 200 ppm, relative were increased at level of 200 ppm and greater. There were no increases in either hepatic enzymes or any evidence of histopathological changes at any of these dose levels.
Females showed statistically significant decreases in absolute and relative thymus weights, and significant decreases in relative thymus weights at 200 ppm, and increased relative and absolute heart weight at the 100 ppm level.
With the exception of the male kidney, the organ weight changes in male and female rats were not accompanied by histopathologic lesions. Therefore, they are either considered as toxicologically not significant or only adaptative effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
Examination of the male kidneys at all dose levels revealed lesions including granular casts and hyaline droplets indicative of α2u-globulin nephropathy. and the severities of these lesions increased with increasing exposure concentration.
In females there was no evidence of histopathology in any organ at any dose level. Specifically, there was no evidence of histopathological changes to the clitoris, ovaries, uterus, epididymis, preputial gland, seminal vesicles, and testes for any of the control or test groups of animals.

HEMATOLOGY: On day 4, there were mild exposure-related significant decreases in the leukocyte counts paired with mild significant decreases in the lymphocyte counts in 200 ppm male rats (7.57 ppm vs 9.08ppm) and in 400 ppm male rats (6.95ppm vs 9.08ppm) vs controls. These decreases ameliorated by day 23. The leukocyte changes likely represent a secondary treatment-associated stress effect.
At week 14, there were mild significant decreases in erythrocyte counts, hemoglobin concentrations, and hematocrit values in males exposed to 100 ppm or greater.
The remaining significant differences in hematology parameters were not considered to be toxicologically relevant

SPERM MOTILITY AND VAGINAL CYTOLOGY: There were significantly decreased sperm count in cauda epididymis in 200 and 400 ppm males vs control group.
Females in the 400 ppm group displayed an apparent increase in cycle length and a slight increase in the percentage of the cycle spent in metestrus, relative to the chamber control group. However, the apparent increase in cycle length may be secondary to stress, as evidenced by lower body weight and mortality in the 400 ppm group. Alternatively, the apparent changes in the 400 ppm females may have been an artifact of having too few animals available to allow for meaningful interpretation.
α-pinene exposure by inhalation exhibits the potential to be a reproductive toxicant in male rats, but not in female rats.
Dose descriptor:
NOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
other: increased incidences of kidney lesions in male,
Dose descriptor:
NOAEC
Effect level:
200 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Critical effects observed:
not specified
Lowest effective dose / conc.:
25 ppm
System:
urinary
Organ:
kidney
Critical effects observed:
not specified
Lowest effective dose / conc.:
100 ppm
System:
male reproductive system
Organ:
cauda epididymis

Table 1 Survival and Body Weights ; body weight gains of Rats


 
















































































































 Concentration(ppm)Survivalb

Initial BodyWeight (g)Final BodyWeight (g)Change in BodyWeight (g)Final Weight Relative to Controls(%)
Male010/1098 ± 3335 ± 6238 ± 5 
2510/1098 ± 2329 ± 11231 ± 998
5010/1098 ± 2333 ± 6235 ± 599
10010/1098 ± 2334 ± 7236 ± 5100
20010/1096 ± 2330 ± 4234 ± 498
40010/1097 ± 2322 ± 6225 ± 796
Female010/1089 ± 2194 ± 3105 ± 3 
2510/1089 ± 2199 ± 4110 ± 4102
5010/1089 ± 2206 ± 4117 ± 4106
10010/1088 ± 2199 ± 3112 ± 2103
20010/1088 ± 2201 ± 3113 ± 2104
4004/10c89 ± 2159 ± 5**72 ± 5**82

** Significantly different (P≤0.01) from the chamber control group by Dunnett’s test


bNumber of animals surviving at 14 weeks/number initially in group


   Weeks of death: 6, 6, 6, 6, 8, 13


 


Table 2- Mean of absolute and relative organ weight


 






















































































































































































 Chamber control25 ppm50 ppm100 ppm200 ppm400 ppm
Male 
n101010101010
Necropsy body wt335 ± 6329 ± 11333 ± 6334 ± 7330 ± 4322 ± 6
R Kidney absolute1.025 ± 0.0191.012 ± 0.0371.061 ± 0.0261.137 ± 0.027**1.209 ± 0.020**1.286 ± 0.039**
R kidney relative3.058 ± 0.0383.073 ± 0.0373.186 ± 0.042*3.405 ± 0.036**3.660 ± 0.040**3.991 ± 0.056**
Liver absolute10.54 ± 0.2710.31 ± 0.4010.44 ± 0.3211.08 ± 0.3611.37 ± 0.2611.87 ± 0.45*
Liver relative31.402 ± 0.37531.270 ± 0.31731.298 ± 0.49033.152 ± 0.569*34.393 ± 0.531**36.807 ± 0.864**
Spleen absolute0.628 ± 0.0120.630 ± 0.0130.663 ± 0.0140.659 ± 0.0090.655 ± 0.0100.677 ± 0.023*
Spleen relative1.874 ± 0.0281.925 ± 0.0451.997 ± 0.0581.978 ± 0.0301.983 ± 0.0222.103 ± 0.057**
Female
n10101010104
Necropsy body wt194 ± 3199 ± 4206 ± 4199 ± 3201 ± 3159 ± 5**
Heart absolute0.584 ± 0.0100.612 ± 0.0120.618 ± 0.0100.629 ± 0.012*0.638 ± 0.011**0.530 ± 0.006*
Heart relative3.010 ± 0.0393.081 ± 0.0543.002 ± 0.0413.156 ± 0.034*3.175 ± 0.049*3.349 ± 0.084**
R Kidney absolute0.618 ± 0.0110.641 ± 0.0090.680 ± 0.013**0.659 ± 0.0150.679 ± 0.014**0.595 ± 0.021
R kidney relative3.185 ± 0.0403.230 ± 0.0623.301 ± 0.0413.307 ± 0.0583.376 ± 0.050*3.757 ± 0.138**
Liver absolute5.486 ± 0.1795.990 ± 0.1216.270 ± 0.115**6.269 ± 0.151**6.424 ± 0.144**4.840 ± 0.247
Liver relative28.216 ± 0.63730.152 ± 0.550**30.438 ± 0.319**31.459 ± 0.586**31.916 ± 0.317**30.470 ± 0.715**
Thymus absolute0.347 ± 0.0120.349 ± 0.0100.352 ± 0.0100.346 ± 0.0100.330 ± 0.0140.204 ± 0.010**
Thymus relative1.785 ± 0.0541.751 ± 0.0291.707 ± 0.0411.739 ± 0.0481.638 ± 0.058*1.286 ± 0.035**

* Significantly different (P≤0.05) from the chamber control group by Williams’ or Dunnett’s test


** P≤0.01


 


Table 3: Chemistry data


 
















































































































































































































































































































































































































































Male
Dose treatment (ppm)0 (control group)25 50100200400
Urea nitrogen (mg/dL)      
Day 47.5 ± 0.47.8 ± 0.47.5 ± 0.37.4 ± 0.37.0 ± 0.47.5 ± 0.4
Day 239.9 ± 0.58.9 ± 0.49.1 ± 0.29.5 ± 0.39.8 ± 0.411.4 ± 0.6
Week 1412.3 ± 0.313.7 ± 0.3*12.8 ± 0.313.3 ± 0.213.3 ± 0.313.6 ± 0.4*
Creatinine (mg/dL)      
Day 40.29 ± 0.010.26 ± 0.020.23 ± 0.02*0.25 ± 0.020.25 ± 0.020.24 ± 0.02
Day 230.30 ± 0.000.32 ± 0.010.32 ± 0.030.31 ± 0.010.36 ± 0.02**0.38 ± 0.01**
Week 140.37 ± 0.020.37 ± 0.020.37 ± 0.030.39 ± 0.020.39 ± 0.010.40 ± 0.03
Glucose (mg/dL) Day 4 137 ± 3 134 ± 1 133 ± 5 137 ± 3 139 ± 6 130 ± 2
Day 23145 ± 12126 ± 7134 ± 9127 ± 5117 ± 4116 ± 5
Week 14127 ± 2130 ± 3124 ± 2129 ± 3136 ± 6128 ± 3
Total protein (g/dL)      
Day 46.0 ± 0.06.0 ± 0.16.1 ± 0.16.0 ± 0.06.1 ± 0.16.1 ± 0.0
Day 236.5 ± 0.16.5 ± 0.16.5 ± 0.16.5 ± 0.16.8 ± 0.1**6.8 ± 0.1**
Week 147.4 ± 0.17.4 ± 0.17.5 ± 0.17.4 ± 0.17.5 ± 0.17.5 ± 0.0
Albumin (g/dL)      
Day 44.3 ± 0.04.3 ± 0.04.3 ± 0.04.3 ± 0.04.4 ± 0.04.4 ± 0.0
Day 234.6 ± 0.04.6 ± 0.04.5 ± 0.04.5 ± 0.14.7 ± 0.04.7 ± 0.1
Week 144.9 ± 0.14.9 ± 0.04.9 ± 0.14.8 ± 0.04.9 ± 0.04.9 ± 0.0
Globulin (g/dL)      
Day 41.7 ± 0.01.7 ± 0.01.8 ± 0.01.7 ± 0.01.8 ± 0.01.8 ± 0.0
Day 231.9 ± 0.02.0 ± 0.02.0 ± 0.02.0 ± 0.02.1 ± 0.0**2.1 ± 0.0**
Week 142.6 ± 0.02.5 ± 0.02.6 ± 0.02.5 ± 0.02.6 ± 0.02.6 ± 0.0
A/G ratioDay 4 2.5 ± 0.0 2.5 ± 0.0 2.5 ± 0.0 2.5 ± 0.0 2.5 ± 0.0 2.4 ± 0.0
Day 232.4 ± 0.02.4 ± 0.02.3 ± 0.12.3 ± 0.02.3 ± 0.02.2 ± 0.0**
Week 141.9 ± 0.01.9 ± 0.01.9 ± 0.01.9 ± 0.01.9 ± 0.02.0 ± 0.0
Alanine aminotransferase (IU/L)      
Day 457 ± 157 ± 155 ± 153 ± 155 ± 152 ± 1**
Day 2341 ± 141 ± 141 ± 139 ± 238 ± 135 ± 0**
Week 1485 ± 383 ± 370 ± 3**60 ± 2**56 ± 2**51 ± 2**
Alkaline phosphatase (IU/L)      
Day 4575 ± 7578 ± 10566 ± 10566 ± 11554 ± 7546 ± 11*
Day 23406 ± 6423 ± 11433 ± 9407 ± 11420 ± 8404 ± 12
Week 14223 ± 5227 ± 7211 ± 4200 ± 3**204 ± 4**199 ± 6**
Creatine kinase (IU/L)      
Day 4545 ± 121507 ± 42430 ± 52449 ± 56515 ± 54434 ± 44
Day 23404 ± 37390 ± 40409 ± 66393 ± 37354 ± 30413 ± 45
Week 14171 ± 8186 ± 18144 ± 14155 ± 13150 ± 14183 ± 15
Sorbitol dehydrogenase (IU/L)      
Day 413 ± 114 ± 013 ± 012 ± 0*14 ± 113 ± 0
Day 2314 ± 114 ± 116 ± 115 ± 118 ± 1**15 ± 1
Week 1424 ± 124 ± 122 ± 122 ± 121 ± 1*20 ± 1**
Bile acids (µmol/L)      
Day 44.7 ± 0.44.7 ± 0.55.6 ± 0.84.6 ± 0.47.2 ± 1.34.6 ± 0.7
Day 235.7 ± 0.93.3 ± 0.2**4.9 ± 0.6*3.6 ± 0.3**3.8 ± 0.3**3.6 ± 0.7**
Week 143.3 ± 0.13.5 ± 0.43.4 ± 0.33.2 ± 0.13.8 ± 0.63.0 ± 0.1


 


 





















































































































































































































































































































































































































































Female
Dose treatment (ppm)0 (control group)2550100200400
Urea nitrogen (mg/dL)      
Day 47.8 ± 0.48.5 ± 0.3 8.1 ± 0.38.3 ± 0.4 9.1 ± 0.3*8.6 ± 0.3 
Day 2311.5 ± 0.311.8 ± 0.411.5 ± 0.410.6 ± 0.410.8 ± 0.39.1 ± 0.4**
Week 1414.1 ± 0.414.4 ± 0.413.0 ± 0.513.6 ± 0.513.4 ± 0.511.3 ± 0.5*
Creatinine (mg/dL)      
Day 40.29 ± 0.010.28 ± 0.010.29 ± 0.020.26 ± 0.020.28 ± 0.010.26 ± 0.02
Day 230.31 ± 0.010.30 ± 0.000.28 ± 0.010.30 ± 0.000.30 ± 0.000.31 ± 0.01
Week 140.37 ± 0.020.35 ± 0.020.36 ± 0.020.38 ± 0.010.34 ± 0.020.35 ± 0.03
Glucose (mg/dL)      
Day 4138 ± 2135 ± 2136 ± 4136 ± 2139 ± 5130 ± 2
Day 23127 ± 3123 ± 6133 ± 5123 ± 3122 ± 3122 ± 5
Week 14141 ± 8131 ± 5123 ± 2133 ± 3131 ± 4114 ± 12
Total protein (g/dL)      
Day 45.9 ± 0.06.0 ± 0.16.1 ± 0.06.0 ± 0.06.1 ± 0.1*6.1 ± 0.0
Day 236.3 ± 0.16.4 ± 0.16.4 ± 0.16.5 ± 0.16.5 ± 0.16.6 ± 0.1
Week 147.5 ± 0.17.4 ± 0.17.5 ± 0.17.6 ± 0.17.5 ± 0.17.2 ± 0.1
Albumin (g/dL)      
Day 44.3 ± 0.04.4 ± 0.04.4 ± 0.04.4 ± 0.04.4 ± 0.04.4 ± 0.0
Day 234.5 ± 0.04.6 ± 0.04.6 ± 0.04.6 ± 0.14.6 ± 0.04.7 ± 0.1
Week 145.2 ± 0.15.2 ± 0.15.2 ± 0.15.3 ± 0.05.2 ± 0.05.0 ± 0.1
Globulin (g/dL)      
Day 41.6 ± 0.01.6 ± 0.01.6 ± 0.01.7 ± 0.01.7 ± 0.0*1.6 ± 0.0
Day 231.8 ± 0.01.8 ± 0.01.8 ± 0.01.9 ± 0.0*1.9 ± 0.0*2.0 ± 0.0**
Week 142.3 ± 0.12.2 ± 0.02.3 ± 0.02.3 ± 0.02.4 ± 0.02.2 ± 0.1
A/G ratio      
Day 42.8 ± 0.02.7 ± 0.12.7 ± 0.02.7 ± 0.12.6 ± 0.12.7 ± 0.0
Day 232.6 ± 0.02.5 ± 0.02.5 ± 0.02.4 ± 0.02.5 ± 0.02.4 ± 0.0**
Week 142.3 ± 0.12.4 ± 0.02.3 ± 0.02.3 ± 0.02.2 ± 0.02.4 ± 0.1
Alanine aminotransferase (IU/L)
Day 447 ± 149 ± 149 ± 146 ± 145 ± 144 ± 2
Day 2335 ± 136 ± 135 ± 136 ± 134 ± 131 ± 1
Week 1469 ± 465 ± 555 ± 3**56 ± 4*47 ± 2**49 ± 5**
Alkaline phosphatase (IU/L) 
Day 4 487 ± 8 493 ± 10475 ± 6 468 ± 7 454 ± 5**457 ± 8** 
Day 23305 ± 5311 ± 8304 ± 5302 ± 8289 ± 8289 ± 7
Week 14197 ± 6182 ± 4182 ± 8177 ± 8**181 ± 5*164 ± 13*
Creatine kinase (UI/L)      
Day 4364 ± 20b332 ± 27388 ± 29b443 ± 74460 ± 39b375 ± 48
Day 23299 ± 30305 ± 27292 ± 41369 ± 43338 ± 26250 ± 16
Week 14162 ± 16165 ± 38172 ± 22139 ± 14170 ± 22145 ± 26
Sorbitol dehydrogenase (IU/L      
Day 413 ± 113 ± 014 ± 012 ± 0*11 ± 1*12 ± 0*
Day 2314 ± 015 ± 115 ± 115 ± 114 ± 116 ± 0
Week 1421 ± 120 ± 118 ± 117 ± 117 ± 118 ± 1
Bile acids (µmol/L)      
Day 45.3 ± 0.55.0 ± 0.56.5 ± 1.15.8 ± 0.66.8 ± 1.34.9 ± 0.5
Day 234.0 ± 0.34.7 ± 0.45.4 ± 0.74.5 ± 0.43.9 ± 0.44.0 ± 0.7
Week 149.1 ± 2.34.9 ± 0.5**4.7 ± 0.4**4.3 ± 0.3**5.1 ± 1.1**16.9 ± 4.7*

 


Table 4 Incidences of Non neoplastic Lesions of the Kidney in Male Rats


 


 


















































 Chamber control25 ppm50 ppm100 ppm200 ppm400 ppm
Number Examined Microscopically 10 10 10 10 10 10
Casts, Granulara09** (1.0)b10** (1.2)10** (1.5)10** (2.5)10** (3.0)
Accumulation, Hyaline Droplet1 (2.0)10** (1.1)10** (1.8)10** (2.0)10** (2.7)10** (3.0)
Nephropathy9 (1.1)10 (1.6)10 (2.0)10 (2.0)10 (2.5)10 (3.0)

** Significantly different (P≤0.01) from the chamber control group by the Fisher exact test


a    Number of animals with lesion


bAverage severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked


 


Table 5 Hematology data


 


 















































































































































































































































































































































Treatment dose (ppm)02550100200400
Male
 Hematocrit (spun) (%)      
Day 445.6 ± 0.345.1 ± 0.646.5 ± 0.645.5 ± 0.444.8 ± 0.544.4 ± 0.4
Day 2347.9 ± 0.548.0 ± 0.647.4 ± 0.447.8 ± 0.447.7 ± 0.348.6 ± 0.5
Week 1449.5 ± 0.548.3 ± 0.449.0 ± 0.348.3 ± 0.7*47.6 ± 0.3**47.7 ± 0.4**
Packed cell volume (mL/dL)      
Day 444.6 ± 0.444.1 ± 0.645.0 ± 0.644.2 ± 0.443.1 ± 0.3*43.3 ± 0.4*
Day 2347.4 ± 0.447.1 ± 0.546.5 ± 0.547.2 ± 0.446.9 ± 0.448.1 ± 0.4
Week 1449.9 ± 0.548.9 ± 0.449.5 ± 0.348.3 ± 0.3*48.0 ± 0.3**47.8 ± 0.6**
Hemoglobin (g/dL)      
Day 413.5 ± 0.113.4 ± 0.213.7 ± 0.113.6 ± 0.213.3 ± 0.113.2 ± 0.1
Day 2314.9 ± 0.114.9 ± 0.114.6 ± 0.115.0 ± 0.114.8 ± 0.115.1 ± 0.1
Week 1415.7 ± 0.115.5 ± 0.115.5 ± 0.115.3 ± 0.1*15.0 ± 0.1**15.1 ± 0.2**
Erythrocytes (106/µL)      
Day 47.15 ± 0.097.12 ± 0.117.27 ± 0.077.26 ± 0.087.09 ± 0.077.06 ± 0.07
Day 238.06 ± 0.067.95 ± 0.077.84 ± 0.098.04 ± 0.087.92 ± 0.078.10 ± 0.06
Week 149.35 ± 0.079.09 ± 0.059.25 ± 0.068.94 ± 0.05**8.92 ± 0.08**8.86 ± 0.10**
Mean cell volume (fL)      
Day 462.3 ± 0.362.0 ± 0.461.8 ± 0.360.9 ± 0.560.9 ± 0.4*61.3 ± 0.4
Day 2358.8 ± 0.259.2 ± 0.459.4 ± 0.258.8 ± 0.459.3 ± 0.459.4 ± 0.3
Week 1453.4 ± 0.253.9 ± 0.453.5 ± 0.354.0 ± 0.453.9 ± 0.353.9 ± 0.3
Mean cell hemoglobin (pg)      
Day 418.8 ± 0.118.8 ± 0.118.9 ± 0.018.8 ± 0.118.8 ± 0.118.7 ± 0.0
Day 2318.5 ± 0.118.7 ± 0.118.7 ± 0.118.6 ± 0.118.6 ± 0.118.6 ± 0.1
Week 1416.9 ± 0.117.0 ± 0.116.8 ± 0.117.1 ± 0.116.9 ± 0.117.1 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 430.2 ± 0.130.4 ± 0.230.5 ± 0.130.9 ± 0.2*30.9 ± 0.1**30.5 ± 0.1
Day 2331.5 ± 0.231.7 ± 0.231.4 ± 0.231.7 ± 0.231.5 ± 0.131.4 ± 0.2
Week 1431.6 ± 0.1 31.6 ± 0.1 31.4 ± 0.1 31.5 ± 0.131.3 ± 0.131.7 ± 0.2
Leukocytes (103/µL)      
Day 49.08 ± 0.458.94 ± 0.329.75 ± 0.449.04 ± 0.727.57 ± 0.36*6.95 ± 0.25**
Day 236.29 ± 0.237.10 ± 0.257.44 ± 0.298.12 ± 0.42**7.52 ± 0.647.20 ± 0.31
Week 147.01 ± 0.366.99 ± 0.397.86 ± 0.257.34 ± 0.376.71 ± 0.406.69 ± 0.47
 Lymphocytes (103/µL)      
Day 47.99 ± 0.407.82 ± 0.308.39 ± 0.387.75 ± 0.616.44 ± 0.33**5.88 ± 0.24**
Day 235.25 ± 0.235.91 ± 0.256.38 ± 0.22d6.89 ± 0.40*6.10 ± 0.576.06 ± 0.27
Week 145.36 ± 0.355.31 ± 0.396.18 ± 0.255.66 ± 0.405.12 ± 0.414.93 ± 0.38

 















































































































































































































































































































































Treatment dose (ppm)02550100200400
female
 Hematocrit (spun) (%)      
Day 447.7 ± 0.447.0 ± 0.247.0 ± 0.247.5 ± 0.347.1 ± 0.6 46.2 ± 0.4
Day 2348.7 ± 0.449.2 ± 0.549.1 ± 0.449.2 ± 0.348.9 ± 0.549.7 ± 0.6
Week 1448.9 ± 0.447.2 ± 0.4*47.8 ± 0.248.3 ± 0.448.7 ± 0.450.9 ± 0.8
Packed cell volume (mL/dL)      
Day 446.7 ± 0.546.1 ± 0.346.0 ± 0.346.8 ± 0.446.1 ± 0.545.3 ± 0.5 
Day 2348.5 ± 0.449.0 ± 0.449.3 ± 0.349.2 ± 0.348.8 ± 0.350.0 ± 0.6*
Week 1449.1 ± 0.348.6 ± 0.348.7 ± 0.49.0 ± 0.549.7 ± 0.452.7 ± 0.4
Hemoglobin (g/dL)      
Day 414.3 ± 0.114.2 ± 0.114.2 ± 0.114.5 ± 0.114.3 ± 0.114.1 ± 0.1
Day 2315.3 ± 0.115.5 ± 0.115.5 ± 0.115.5 ± 0.115.3 ± 0.115.7 ± 0.1
Week 1415.7 ± 0.115.4 ± 0.115.5 ± 0.115.6 ± 0.115.8 ± 0.116.7 ± 0.1
Erythrocytes (106/µL)      
Day 47.64 ± 0.07 7.56 ± 0.057.56 ± 0.057.74 ± 0.07 7.67 ± 0.107.55 ± 0.08 
Day 238.13 ± 0.088.13 ± 0.088.20 ± 0.07 8.24 ± 0.05 8.13 ± 0.06 8.31 ± 0.09
Week 148.67 ± 0.068.53 ± 0.058.54 ± 0.068.62 ± 0.098.71 ± 0.069.23 ± 0.09
Mean cell volume (fL)      
Day 461.1 ± 0.360.9 ± 0.360.9 ± 0.260.5 ± 0.360.2 ± 0.560.0 ± 0.3*
Day 2359.6 ± 0.360.3 ± 0.360.1 ± 0.359.7 ± 0.360.1 ± 0.360.2 ± 0.2
Week 1456.6 ± 0.256.9 ± 0.157.0 ± 0.156.9 ± 0.157.0 ± 0.257.1 ± 0.3
Mean cell hemoglobin (pg)      
Day 418.7 ± 0.118.8 ± 0.118.8 ± 0.118.7 ± 0.118.7 ± 0.118.7 ± 0.1
Day 2318.8 ± 0.119.0 ± 0.118.9 ± 0.118.8 ± 0.118.8 ± 0.118.9 ± 0.1
Week 1418.1 ± 0.018.1 ± 0.118.2 ± 0.118.1 ± 0.118.1 ± 0.018.1 ± 0.0
Mean cell hemoglobin concentration (g/dL)
Day 430.6 ± 0.230.8 ± 0.130.8 ± 0.131.0 ± 0.231.1 ± 0.231.2 ± 0.2
Day 2331.6 ± 0.231.5 ± 0.131.4 ± 0.131.6 ± 0.131.4 ± 0.131.4 ± 0.1
Week 14 32.1 ± 0.131.8 ± 0.131.9 ± 0.131.9 ± 0.131.8 ± 0.131.7 ± 0.2
Leukocytes (103/µL)      
Day 410.52 ± 0.5210.89 ± 0.2610.25 ± 0.3411.26 ± 0.6110.39 ± 0.638.52 ± 0.68
Day 237.96 ± 0.368.01 ± 0.247.87 ± 0.438.04 ± 0.397.78 ± 0.566.84 ± 0.48
Week 145.86 ± 0.275.70 ± 0.246.05 ± 0.295.60 ± 0.295.22 ± 0.336.08 ± 0.58
 Lymphocytes (103/µL)      
Day 49.34 ± 0.529.58 ± 0.258.90 ± 0.309.76 ± 0.599.19 ± 0.51d7.34 ± 0.62
Day 236.79 ± 0.356.86 ± 0.206.83 ± 0.416.96 ± 0.386.62 ± 0.545.83 ± 0.42
Week 144.67 ± 0.244.44 ± 0.254.67 ± 0.234.36 ± 0.284.17 ± 0.294.93 ± 0.53

* Significantly different (P≤0.05) from the chamber control group by Dunn’s or Shirley’s test


** P≤0.01


 


Table 6: Mean of epididymal spermatozoal measurements


 











































 Chamber control100 ppm200 ppm400 ppm
n1010910
Epididymal spermatozoal measurements
Sperm motility (%)91.73 ± 1.2691.40 ± 0.9391.24 ± 0.8090.93 ± 0.89
Sperm (103/mg cauda epididymis)615.0 ± 34.3596.5 ± 31.8526.3 ± 19.0547.4 ± 14.0
Sperm (106/cauda epididymis)120.89 ± 6.79113.16 ± 3.1197.52 ± 3.51**98.40 ± 3.02**

 


** Significantly different (P≤0.01) from the chamber control group by Shirley’s test.

Conclusions:
The effects observed on kidneys of male rats are not relevant to humans as they are based on renal effects linked to alpha2µ-globulin accumulation. When considering effects other than those on kidneys in males, significant decreased sperm counts in cauda epididymis at 200 and 400 ppm were observed when compared to controls. Therefore the NOAEC idenitified for male rats is 100 ppm.
A NOAEC could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls.
The overall NOAEC relevant for humans is therefore 100 ppm.

However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.).

The study suffers from several limitations:
- animals were exposed whole body by inhalation which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming).
- the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples
Executive summary:

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 days per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals, including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.


A lower body weight gain than in controls was observed in the high dose group in female. In addition, 6 females died in the high dose group too. Female rats appeared to be more sensitive to α-pinene than male rats. A NOAEC could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls.


Apart from lesions including granular casts and hyaline droplets indicative of alpha2µ-globulin nephropathy observed in all treated group males, a lower body weight gain than in controls was observed in the high dose group in males. The effects observed on kidneys of male rats are not relevant to humans as they are based on renal effects linked to alpha2µ-globulin accumulation


When considering effects other than those on kidneys in males, significant decreased sperm counts in cauda epididymis at 200 and 400 ppm were observed when compared to controls. Therefore the NOAEC idenitified for male rats is 100 ppm.


However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.).


In addition animals were exposed whole body by inhalation, which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
566.5 mg/m³
Study duration:
subchronic
Species:
mouse
Quality of whole database:
Study conducted similarly to OECD guideline 413
System:
male reproductive system
Organ:
cauda epididymis

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In 2-week inhalation preliminary studies (rats and mice) animals were exposed to concentration of 800 and 1600 ppm.

 

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, male rats exposed to alpha-pinene showed effects on renal and reproductive systems. The effects on kidneys are not relevant to humans as they are based on sex- and species-specific renal effects linked to alpha2µ-globulin accumulation. A decrease in sperm count in cauda epididymis at 200 and 400 ppm was observed. Therefore the NOAEC identified for male rats is 100 ppm.

However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). In addition, animals were exposed whole body by inhalation, which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming).

A lower body weight gain and death of 6 out of 10 females in the high dose group (400 ppm) were observed. A NOAEC could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain.

 

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, mice were exposed to alpha-pinene. Minimal to moderate hyperplasia have been observed in the transitional epithelium of the urinary bladder from 100 ppm in both sexes.

Decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males were observed.

However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). Also, these decreases might be secondary to stress induced by hyperplasia of bladder epithelium at the 3 highest doses. In addition,animals were exposed whole body by inhalation, which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming). In a conservative approach, this effect was selected as the critical effect to calculate DNELs and a LOAEC was established for males at 100 ppm.

A NOAEC for female mice has been set at 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder.

In a repeated dose toxicity study conducted according to Guideline OECD 421, dietary administration of the test item to Sprague-Dawley rats at concentrations of 3000, 6000 or 12000 ppm for five weeks to reproductive phase males and for three weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but did elicit histopathological changes in the kidneys in the reproductive males, indicative of the species- and sex-specific alpha 2μ-globulin nephropathy.  

A NOAEC for male and female rats has been set at 12 000 ppm, the higher tested dose.

Justification for classification or non-classification

The CLP classification for Specific Target Organ Toxicity in Repeated Exposure is based on results found in rats. Therefore the results found in the 90-day repeated dose toxicity study by inhalation in mice are not taken into account. Two repeated dose toxicity studies are available in rats: one by oral route and one by inhalation route.The LOAEC in male rats reported in the study by inhalation is not relevant to humans as it is based on sex- and species-specific renal effects linked to alpha-2µ-globulin accumulation. When considering other toxicological effects, a lower body weight gain at 400 ppm and decreased sperm count in cauda epididymis from 200 ppm were observed, therefore a conservative NOAEC for males could be defined at 100 ppm. A NOAEC could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls.

Mortality was observed in females at 400 ppm, corresponding to 2.26 mg/L, which is above the limit concentration for classification (1 mg/L for vapours). The decreased sperm count in males is not relevant for STOT-RE classification and was not observed in the study by oral route (although exposure by oral route was much higher). Also, it was observed from 200 ppm, corresponding to 1.13 mg/L, which is above the limit concentration for classification (1 mg/L for vapours).

Therefore, alpha-pinene multiconstituent does not need to be classified for Specific Target Organ Toxicity in Repeated Exposure according to CLP Regulation (EC) n° 1272/2008.