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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
read-across source

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: National Toxicology Program. Toxicology and Carcinogenesis study
Deviations:
not specified
Principles of method if other than guideline:
NTP study, fully valid for assessment
GLP compliance:
yes
Remarks:
According to Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)

Test material

Constituent 1
Reference substance name:
Alpha-pinene
IUPAC Name:
Alpha-pinene
Constituent 2
Chemical structure
Reference substance name:
Pin-2(3)-ene
EC Number:
201-291-9
EC Name:
Pin-2(3)-ene
Cas Number:
80-56-8
Molecular formula:
C10H16
IUPAC Name:
2,6,6-trimethylbicyclo[3.1.1]hept-2-ene
Constituent 3
Reference substance name:
Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-
IUPAC Name:
Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-
Test material form:
liquid
Details on test material:
Purity/Composition: >=97%
Specific details on test material used for the study:
CASRN: 80-56-8
Formula: C10-H16
Synonyms/Common Names: Acintene A, Cyclic Dexadiene, 2-Pinene, 2,6,6-Trimethylbicyclo(3.1.1)-2-hept-2-ene
Appereance: a colorless oily liquid with a strong piney odor
Source: Millennium Specialty Chemicals (Jacksonville, FL)
Batch: 4KB705

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species: B6C3F1 mice
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- On receipt, animals were approximately 4 weeks old.
- Acclimatation: 12 (male and female mice) days
- Study start age: 5 to 6 weeks old on the first day of the studies
- Housing: All mice were housed individually.
- Water: Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); changed weekly


ENVIRONMENTAL CONDITIONS
- Temperature: 72° ± 3° F
- Relative humidity: 50% ± 15%
- Room fluorescent light: 12 hours/day
- Chamber air changes: 15 ± 2/hour
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods); changed weekly

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
not specified
Details on inhalation exposure:
groups of 10 male and 10 female rats and mice were exposed to α-pinene by whole body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 14 weeks.
Duration of treatment / exposure:
6 hours plus T90 (10 minutes) per day, 5 days per week, for 14 weeks
Frequency of treatment:
6 hours plus T90 (10 minutes) per day, 5 days per week, for 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
25 ppm
Remarks:
Calculated to correspond to a daily intake of 36 mg/kg bw per day.
Dose / conc.:
50 ppm
Remarks:
Calculated to correspond to a daily intake of 72 mg/kg bw per day.
Dose / conc.:
100 ppm
Remarks:
Calculated to correspond to a daily intake of 144 mg/kg bw per day.
Dose / conc.:
200 ppm
Remarks:
Calculated to correspond to a daily intake of 288 mg/kg bw per day.
Dose / conc.:
400 ppm
Remarks:
Calculated to correspond to a daily intake of 576 mg/kg bw per day.
No. of animals per sex per dose:
Treatment: 10 female and 10 male- 5 test groups
Controls: 10 female and 10 male- 1 test group
Animals for clinical lab studies: 10 female and 10 male- 5 test groups
Control animals for clinical lab studies: 10 female and 10 male- 1 test group
Control animals:
yes, concurrent no treatment
Details on study design:
Animals were acclimataded for a period of 10- to 14-days, then they were assigned at random treatment groups. The study included five treatment groups each one corresponded to a different concentration of the test material plus the control group. Each group included 10 animals per sex.
The animals were treated with alpha pinene at different concentrationn by inhalation route. The control group received untreated water or feed or vehicle alone. Animals were exposed five times per week, weekdays only until the day prior to necropsy.

Examinations

Observations and examinations performed and frequency:
Animals were observed twice daily; the core study animals were weighed initially, on day 8 (male and female mice), weekly thereafter, and at the end of the studies.
The clinical findings were recorded on day 8 (male and female mice), weekly thereafter, and at the end of the studies.

Sacrifice and pathology:
Necropsies were performed on all core study animals.
Organs weighed: heart, right kidney, liver, lung, spleen, right testis, and thymus.
Treatment-related lesions (target organs) were identified and these organs plus gross lesions were examined to a no-effect level: (respiratory system)

Histopathology: performed on 0 and 400 ppm core study mice
Gross lesions and tissue masses, and tissues examinated:
adrenal gland, bone, brain, clitoral gland, esophagus, eyes, gallbladder (mice), 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, kidney of rats and mice, and
urinary bladder of mice were examined in the remaining groups.
Other examinations:
Blood: it was collected from the retroorbital plexus of clinical pathology rats on days 4 and 23 and from core study animals at the end of the studies for hematology and clinical chemistry (rats only).
Hematology: 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: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts.

Sperm samples: collected from male animals in the 0, 100, 200, and 400 ppm groups.
Examinations: sperm motility
Parameters: sperm heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed.
Vaginal samples: collected for up to 12 consecutive days prior to the end of the studies from females exposed to 0, 100, 200, or 400 ppm.
Examinations: vaginal cytology evaluations, percentage of time spent in the various estrous cycle stages and estrous cycle length.
Statistics:
The Fisher exact test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
None
Mortality:
no mortality observed
Description (incidence):
None
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The final mean body weights and body weight gains of exposed males and females were similar to those of controls
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The exact mechanism for the mild decreases in the erythron are not known. Other significant changes in hematology parameters were not considered toxicologically relevant.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
LIVER: The absolute weights of 400 ppm males and females and the relative liver weights of 200 and 400 ppm males and 100, 200, and 400 ppm females were significantly greater (up to 24%) than those of the chamber controls.
THYMUS: The absolute and relative weights of 400 ppm males were significantly less than those of the chamber controls.
KIDNEY: The absolute weights of 200 and 400 ppm males were significantly less than those of the chamber controls (11% and 7%, respectively).
These organ weight changes were not accompanied by histopathologic lesions.

URINARY BLADDER: significantly increased incidences of transitional epithelium hyperplasia in males and females exposed to 100 ppm or greater. The incidences and the severities of this lesion increased in an exposure concentration-related manner. This lesion was characterized by a relatively uniform increase
in mucosal thickness with an increase in the size of the transitional epithelial cells, and the number of epithelial cell layers from two or three in controls to four or more layers in affected mice.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The organ weight changes were not accompanied by histopathologic lesions.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
The organ weight changes were not accompanied by histopathologic lesions.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
MALE:: There were significantly decreased numbers of sperm per mg cauda in 200 and 400 ppm males (24% and 37%, respectively) and cauda sperm in 100, 200, and 400 ppm males (25%, 33%, and 40%, respectively).
FEMALE: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.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased sperm per cauda and increased incidences of transitional epithelium hyperplasia of the urinary bladder in male mice
Key result
Dose descriptor:
NOAEL
Effect level:
50 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: increased incidences of transitional epithelium hyperplasia of the urinary bladder in female mice

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
other: Reproductive organs and Urinary system
Organ:
bladder
cauda epididymis
Treatment related:
yes
Relevant for humans:
no

Any other information on results incl. tables

The full report is available: https://ntp.niehs.nih.gov/results/pubs/shortterm/reports/abstracts/tox081/index.html

Applicant's summary and conclusion

Conclusions:
Based on the result of the study, 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.
Executive summary:

A NTP (National Toxicology Program) 90 -Day study was conducted on alpha-pinene. Female and male B6C3F1 MICE were treated with alpha-pinene at concentration of 0, 25, 50, 100, 200, or 400 ppm for 6h/days per week via inhalation route for 14 weeks. Animals were observed twice per day and weighed once per week.

Histopathologic evaluation were performed on all early death animals regardless of dose group, all control animals, all animals, and all animals in the highest treatment group. Treatment-related lesions (target organs) were identified and these organs plus gross lesions were examined to a no-effect level.

All mice survived until the study was terminated. Body weights gain were comparable for all test animals when compared to controls.

At 400 ppm, an increse of absolute liver weights was observed in both sexes. At 200 and 400 ppm, an increase of absolute liver weights were recorded for both sexes. The 400 ppm male group showed decreased absolute and relative thymus weight. No gross or microscopic lesions were associated with these organ weight findings.

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. No changes have been reported 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.

In the urinary bladder, there were significantly increased incidences of transitional epithelium hyperplasia in males and females exposed to 100 ppm or greater. This was found dose-response related.

Based on the result of the study, 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.

The full report is available: https://ntp.niehs.nih.gov/results/pubs/shortterm/reports/abstracts/tox081/index.html