Reaction mass of Terpenes and Terpenoids, turpentine-oil, limonene fraction, 1-methyl-4-(1-methylethenyl)cyclohexene and turpentine-oil beta-pinene fraction terpenes, dimers and Terpenes and Terpenoids, turpentine-oil, limonene fraction, 1-methyl-4-(1-methylethenyl)cyclohexene and turpentine-oil beta-pinene fraction terpenes, trimers

Endpoint summary

Description of key information

Two study results are available from two US National Toxicology Program (NTP) studies conducted on alpha pinene in which female and male F344/N rats and B6C3F1 mice were exposed to concentrations ranging from 0 to 400 ppm by inhalation. Alpha pinene is structurally related to Terpenes and terpenoids, turpentine oil, alpha pinene fraction oligomers, however its less complex structure and lower formula weight suggest it is should be more readily available than the oligomer. Hence results from these two sub-chronic inhalation studies will be used to predict the potential systemic toxicity of Terpenes and terpenoids, turpentine oil, alpha pinene fraction oligomers (read-across using the analogue approach).

In the first study (NTP, 2005c), female and male F344/N rats were exposed to 0, 25, 50, 100, 200, or 400 ppm alpha-pinene by inhalation for 6h/days for 14 weeks. In the first study (NTP, 2005), female and male F344/N rats were exposed to 0, 25, 50, 100, 200, or 400 ppm alpha-pinene by inhalation for 6h/days for 14 weeks. All males rats survived to scheduled study termination, however body weight gain was decreased when compared to controls. Absolute and relative liver weights were statistically increased in males exposed to >200 ppm with relative and absolute kidney weights increased >100 ppm. Males also showed statistically significant decreases in sorbitol dehydrogenase activity at 400 ppm, alanine aminotransferase activity at levels ≥50 ppm, and alkaline phosphatase activity at levels ≥100 ppm. None of these changes in enzyme activity were related to organ weight changes or evidence of histopathology. Examination of the male kidneys revealed lesions at all dose levels including granular casts and hyaline droplets indicative of a2u-globulin nephropathy. Based on these observations, the NOEL for male rats was 25 ppm (based on a2u-globulin related changes in kidney), with a NOAEL of 200 ppm, based on increased liver weights (in the absence of associated changes in histopathology) recorded at 400 ppm. In females, six animals from the 400 ppm group were found dead during the study and three animals from this same high exposure group displayed mild tremors. Relative and absolute liver weights were increased in females at exposure levels of ≥50 ppm, but there were no increases in either hepatic enzymes or any evidence of histopathological changes at any of these dose levels. Absolute and relative thymus weights were significantly decreased, and relative lung weight increased, in high dose level females. Alanine aminotransferase activity was decreased at ≥200 ppm, and alkaline phosphatase activity decreased at the 400 ppm, in females. None of these changes in enzyme activity were related to organ weight changes or evidence of histopathology (no evidence of histopathology in any organ at any dose level).

According to the result of the study, the LOEL was =25 ppm based on increased

incidences of kidney lesions in male rats and increased relative liver weights in female rats without accompanying histopathologic changes.

In the second study (NTP, 2006) female and male B6C3F1 mice were exposed to alpha-pinene via inhalation at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6h/days per week for 14 weeks. All mice survived until scheduled study termination, and body weight gain were comparable for all test and control animals. At 400 ppm, an increase 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. Histopathological examination of male and female mice exposed to ≥100 ppm of α-pinene revealed evidence of hyperplasia of the transitional epithelium of the urinary bladder. Histopathological examination confirmed that there were no changes in clitoris, ovaries, uterus, epididymis, preputial gland, seminal vesicles, and testes compared to the control groups. Based on the result of the study, the LOEL is 100 ppm for males and females, based on the presence of transitional cell hyperplasia of the urinary bladder.

This information is available from the NTP website: https://ntp.niehs.nih.gov/results/pubs/shortterm/reports/abstracts/tox081/index.html

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

read-across source

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)

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

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

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

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.

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

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.

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

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

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

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

Reference 2

Endpoint:

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

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)

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

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: F344/N rats
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- On receipt, the rats were approximately 4 weeks old.
- Acclimatation: 12 (Male) or 13 (Female)
- Study start age: 5 to 6 weeks old on the first day of the studies
- Housing: 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

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Duration of treatment / exposure:

Groups of 10 male and 10 female rats were exposed to α-pinene via 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

Frequency of treatment:

Groups of 10 male and 10 female rats were exposed to α-pinene via 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

Dose / conc.:

0 ppm

Dose / conc.:

25 ppm

Remarks:

Correspond to a daily intake of 36, mg/kg bw per day.

Dose / conc.:

50 ppm

Remarks:

Correspond to a daily intake of 72 mg/kg bw per day.

Dose / conc.:

100 ppm

Remarks:

Correspond to a daily intake of 144 mg/kg bw per day.

Dose / conc.:

200 ppm

Remarks:

Correspond to a daily intake of 288 mg/kg bw per day

Dose / conc.:

400 ppm

Remarks:

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

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.

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.


Treatment-related lesions (target organs) were identified and these organs plus gross lesions were examined to a no-effect level: (respiratory system)

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

Clinical signs:

no effects observed

Description (incidence and severity):

None

Mortality:

no mortality observed

Description (incidence):

Six 400 ppm female rats died during the study with no specific cause of death identified through gross examination or histopathologic analysis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

FEMALE: The final mean body weights and mean body weight gains of female rats exposed to 400 ppm were significantly less than those of the chamber controls.
MALE: The final mean body weights and mean body weight gains of exposed male rats were similar to those of the chamber controls.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 and 400 ppm male rats compared to concurrent controls. At day 23 the values came back to normal.

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 leukocyte changes likely represent a secondary treatment-associated stress effect. The exact mechanisms for the mild decreases in the erythron are not known.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Alanine aminotransferase activities were significantly decreased in males and females exposed to 50 ppm or greater at week 14. Significantly decreased alanine aminotransferase activities were also seen in 400 ppm male rats on days 4 and 23. Significant decreases in alkaline phosphatase activities were observed in 400 ppm males and 200 and 400 ppm females on day 4 and in males and females exposed to 100 ppm or greater at week 14.

The reason for the decreases in these enzyme activities is not known but may be related to alterations in liver metabolism or enzyme inhibition. The remaining significant differences in hematology and clinical chemistry parameters were not considered to be toxicologically relevant.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

LIVER:
- males: absolute liver weight of the 400 ppm group and the relative liver weights of groups exposed to 100 ppm or greater were significantly greater (up to 17%) than those of the chamber controls.
- female: the absolute liver weights of the 50, 100, and 200 ppm groups and the relative liver weights of all exposed groups were significantly greater (up to 17%) than those of the chamber controls.

HEART: The absolute heart weights of 100 and 200 ppm females and the relative heart weights of females exposed to 100 ppm or greater were significantly greater (up to 11%) than those of the chamber controls.

KIDNEY:
- male: The absolute kidney weights of male rats exposed to 100 ppm or greater and the relative kidney weights of males exposed to 50 ppm or greater were significantly greater (absolute: 11% to 25%; relative: 4% to 31%) than those of the chamber controls.
- female: the absolute kidney weights of the 50 and 200 ppm groups and the relative kidney weights of the 200 and 400 ppm groups were significantly greater (up to 18%) than those of the chamber controls.

THYMUS:
- female: The absolute and relative thymus weights of 400 ppm females and the relative thymus weight of 200 ppm females were significantly less than those of the chamber controls.
- male: The absolute and relative spleen weights of 400 ppm males were significantly greater than those of the chamber control group.

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. With the exception of the male kidney, the organ weight changes in male and female rats were not accompanied by histopathologic lesions

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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. With the exception of the male kidney, the organ weight changes in male and female rats were not accompanied by histopathologic lesions

KIDNEY: Renal tubule lesions including granular casts, hyaline droplets, and nephropathy were observed in male rats. In all male groups exposed to α-pinene, there were significantly increased incidences of granular casts and hyaline droplet accumulation and the severities of these lesions increased with increasing exposure concentration. Granular casts were not observed in the chamber controls and in exposed groups the mean severity ranged from minimal in males exposed to 25 ppm to moderate in males exposed to 400 ppm.
Hyaline droplet accumulation occurred in the cytoplasm of the renal proximal convoluted tubule epithelial cells, and the severity ranged from minimal in males exposed to 25 ppm to moderate in males exposed to 400 ppm. In the chamber controls, the droplets occurred as individual, uniformly fine, round eosinophilic globules in the epithelial cells of clusters of tubules.
With the exception of one chamber control rat, nephropathy occurred in all males, with the mean severity increasing from minimal in chamber control males to moderate in males exposed to 400 ppm. There were no exposure-related microscopic findings in females, including those that died before the end of the study

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

CAUDA SPERM: Significantly decreased numbers of cauda sperm in 200 and 400 ppm males with 19% lower sperm per cauda in the 200 and 400 ppm groups compared to the chamber controls.
CYCLE: in the 400 ppm female 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.
In addition, consideration of the complete cycle using a Markov analysis indicated that these exposed females did not spend significantly more time in any of the estrous stages than did the chamber control group.
The minor changes in cycle length observed only in the exposure concentration group exhibiting overt toxicity, combined with a lack of ovarian histopathology findings, did not provide sufficient evidence for female reproductive toxicity potential under the conditions of the study.

https://ntp.niehs.nih.gov/ntp/htdocs/st_rpts/tox081_508.pdf

Key result

Dose descriptor:

LOEL

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: liver, kidney

Remarks on result:

other:

Remarks:

increased incidences of kidney lesions in male rats [lowest-observed-effect level (LOEL)=25 ppm], increased relative liver weights in female rats (LOEL=25 ppm) without accompanying histopathologic changes

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: cauda sperm

Critical effects observed:

yes

Lowest effective dose / conc.:

25 ppm

System:

other: hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Conclusions:

Under the conditions of the 3-month inhalation studies, the major findings were observed in the liver, urinary system (kidney of rats), and cauda epididymal sperm. The LOEL was set at 25 ppm due to the kidney lesions in male rats and increased relative liver weights in female rats (without accompanying histopathologic changes), decreased sperm per cauda (NOAEL 100 ppm).
 

Executive summary:

A NTP (National Toxicology Program) 90 -Day study was conducted on alpha-pinene. Female and male F344/N rats were exposed to alpha-pinene by inhalation route at concentration of 0, 25, 50, 100, 200, or 400 ppm for 6h/days for 14 weeks. Animals were observed twice per day and weighed once per week.

Six of the 10 female rats exposed to 400 ppm α-pinene died before the end of the study with no specific reason.

In males and females the absolute liver weight and the relative liver weights of groups exposed to 100 ppm or greater were significantly greater compared to the control groups.

Increased liver weight is a common finding in toxicity studies and can be associated with induction of liver metabolizing enzymes. α-Pinene has been shown to increase both phase I and phase II metabolizing enzymes in vitro and in vivo.

The absolute kidney weights of male and female rats and the relative kidney weights were also significantly greater compared to the control group.

Renal tubule lesions including granular casts, hyaline droplets, and nephropathy were observed in male rats exposed to α-pinene and hyaline droplet accumulation occurred in the cytoplasm of the renal proximal convoluted tubule epithelial cells. The severity ranged from minimal in males exposed to 25 ppm to moderate in males exposed to 400 ppm. Nephropathy occurred in all males, with the mean severity increasing from minimal in chamber control males to moderate in males exposed to 400 ppm.

The presence of these non neoplastic 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.

 

There were significantly decreased numbers of cauda sperm in 200 and 400 ppm males with 19% lower sperm per cauda in the 200 and 400 ppm groups compared to the chamber controls.

 

Under the conditions of the 3-month inhalation studies, the major findings were observed in the liver, urinary system (kidney of rats), and cauda epididymal sperm. The LOEL was set at 25 ppm due to the kidney lesions in male rats and increased relative liver weights in female rats (without accompanying histopathologic changes), decreased sperm per cauda (NOAEL 100 ppm).

 

Additional information

The substanceTerpenes and terpenoids, turpentine-oil, alpha-pinene fractionis a reactant which undergoes a Lewis acid catalysed reaction which yieldsTerpenes and terpenoids, turpentine-oil, alpha-pinene fraction, dimers and trimers.

The reaction products are dimeric and trimeric species of reactants covalently coupled twice or three times. In other words, these species are structurally similar, i.e. they contain the same repeating reactant. It however has to be kept in mind that most of Physical and Chemical properties are altered significantly as a result of this increase in molecular weight going from the reactant to dimeric and trimeric species. For example, the vapor pressure is very significantly reduced. Overall, the change in physical properties between reactant and dimeric and trimeric species, results in a substance with significantly reduced hazardous properties.

Although it is proposed to read across for repeated dose toxicity endpoint (inhalation) fromTerpenes and terpenoids, turpentine-oil, alpha-pinene fraction it is believed that this represents a very worst case forTerpenes and terpenoids, turpentine-oil, alpha-pinene fraction, dimers and trimers

Justification for classification or non-classification

Not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 orUN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).