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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:
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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
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.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
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 day befor the study
- Rats were housed individually
- Feed was available ad libitum except during exposure periods; water was 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

Administration / exposure

Route of administration:
inhalation
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
Doses / concentrationsopen allclose all
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

Examinations

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: No data

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 of the test, after 7 days and at weekly intervals thereafter

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: : Yes
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.
HEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes
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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
Key result
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
Key result
Dose descriptor:
NOAEC
Effect level:
200 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality

Target system / organ toxicity

open allclose all
Critical effects observed:
not specified
Lowest effective dose / conc.:
25 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Critical effects observed:
not specified
Lowest effective dose / conc.:
100 ppm
System:
male reproductive system
Organ:
cauda epididymis
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

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(%)

 

Male

 

0

10/10

98 ± 3

335 ± 6

238 ± 5

 

25

10/10

98 ± 2

329 ± 11

231 ± 9

98

50

10/10

98 ± 2

333 ± 6

235 ± 5

99

100

10/10

98 ± 2

334 ± 7

236 ± 5

100

200

10/10

96 ± 2

330 ± 4

234 ± 4

98

400

10/10

97 ± 2

322 ± 6

225 ± 7

96

Female

0

10/10

89 ± 2

194 ± 3

105 ± 3

 

25

10/10

89 ± 2

199 ± 4

110 ± 4

102

50

10/10

89 ± 2

206 ± 4

117 ± 4

106

100

10/10

88 ± 2

199 ± 3

112 ± 2

103

200

10/10

88 ± 2

201 ± 3

113 ± 2

104

400

4/10c

89 ± 2

159 ± 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

c    Weeks of death: 6, 6, 6, 6, 8, 13

Table 2- Mean of absolute and relative organ weight

 

Chamber control

25 ppm

50 ppm

100 ppm

200 ppm

400 ppm

Male

 

n

10

10

10

10

10

10

Necropsy body wt

335 ± 6

329 ± 11

333 ± 6

334 ± 7

330 ± 4

322 ± 6

R Kidney absolute

1.025 ± 0.019

1.012 ± 0.037

1.061 ± 0.026

1.137 ± 0.027**

1.209 ± 0.020**

1.286 ± 0.039**

R kidney relative

3.058 ± 0.038

3.073 ± 0.037

3.186 ± 0.042*

3.405 ± 0.036**

3.660 ± 0.040**

3.991 ± 0.056**

Liver absolute

10.54 ± 0.27

10.31 ± 0.40

10.44 ± 0.32

11.08 ± 0.36

11.37 ± 0.26

11.87 ± 0.45*

Liver relative

31.402 ± 0.375

31.270 ± 0.317

31.298 ± 0.490

33.152 ± 0.569*

34.393 ± 0.531**

36.807 ± 0.864**

Spleen absolute

0.628 ± 0.012

0.630 ± 0.013

0.663 ± 0.014

0.659 ± 0.009

0.655 ± 0.010

0.677 ± 0.023*

Spleen relative

1.874 ± 0.028

1.925 ± 0.045

1.997 ± 0.058

1.978 ± 0.030

1.983 ± 0.022

2.103 ± 0.057**

Female

n

10

10

10

10

10

4

Necropsy body wt

194 ± 3

199 ± 4

206 ± 4

199 ± 3

201 ± 3

159 ± 5**

Heart absolute

0.584 ± 0.010

0.612 ± 0.012

0.618 ± 0.010

0.629 ± 0.012*

0.638 ± 0.011**

0.530 ± 0.006*

Heart relative

3.010 ± 0.039

3.081 ± 0.054

3.002 ± 0.041

3.156 ± 0.034*

3.175 ± 0.049*

3.349 ± 0.084**

R Kidney absolute

0.618 ± 0.011

0.641 ± 0.009

0.680 ± 0.013**

0.659 ± 0.015

0.679 ± 0.014**

0.595 ± 0.021

R kidney relative

3.185 ± 0.040

3.230 ± 0.062

3.301 ± 0.041

3.307 ± 0.058

3.376 ± 0.050*

3.757 ± 0.138**

Liver absolute

5.486 ± 0.179

5.990 ± 0.121

6.270 ± 0.115**

6.269 ± 0.151**

6.424 ± 0.144**

4.840 ± 0.247

Liver relative

28.216 ± 0.637

30.152 ± 0.550**

30.438 ± 0.319**

31.459 ± 0.586**

31.916 ± 0.317**

30.470 ± 0.715**

Thymus absolute

0.347 ± 0.012

0.349 ± 0.010

0.352 ± 0.010

0.346 ± 0.010

0.330 ± 0.014

0.204 ± 0.010**

Thymus relative

1.785 ± 0.054

1.751 ± 0.029

1.707 ± 0.041

1.739 ± 0.048

1.638 ± 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

 

50

100

200

400

Urea nitrogen (mg/dL)

 

 

 

 

 

 

Day 4

7.5 ± 0.4

7.8 ± 0.4

7.5 ± 0.3

7.4 ± 0.3

7.0 ± 0.4

7.5 ± 0.4

Day 23

9.9 ± 0.5

8.9 ± 0.4

9.1 ± 0.2

9.5 ± 0.3

9.8 ± 0.4

11.4 ± 0.6

Week 14

12.3 ± 0.3

13.7 ± 0.3*

12.8 ± 0.3

13.3 ± 0.2

13.3 ± 0.3

13.6 ± 0.4*

Creatinine (mg/dL)

 

 

 

 

 

 

Day 4

0.29 ± 0.01

0.26 ± 0.02

0.23 ± 0.02*

0.25 ± 0.02

0.25 ± 0.02

0.24 ± 0.02

Day 23

0.30 ± 0.00

0.32 ± 0.01

0.32 ± 0.03

0.31 ± 0.01

0.36 ± 0.02**

0.38 ± 0.01**

Week 14

0.37 ± 0.02

0.37 ± 0.02

0.37 ± 0.03

0.39 ± 0.02

0.39 ± 0.01

0.40 ± 0.03

Glucose (mg/dL) Day 4

 

137 ± 3

 

134 ± 1

 

133 ± 5

 

137 ± 3

 

139 ± 6

 

130 ± 2

Day 23

145 ± 12

126 ± 7

134 ± 9

127 ± 5

117 ± 4

116 ± 5

Week 14

127 ± 2

130 ± 3

124 ± 2

129 ± 3

136 ± 6

128 ± 3

Total protein (g/dL)

 

 

 

 

 

 

Day 4

6.0 ± 0.0

6.0 ± 0.1

6.1 ± 0.1

6.0 ± 0.0

6.1 ± 0.1

6.1 ± 0.0

Day 23

6.5 ± 0.1

6.5 ± 0.1

6.5 ± 0.1

6.5 ± 0.1

6.8 ± 0.1**

6.8 ± 0.1**

Week 14

7.4 ± 0.1

7.4 ± 0.1

7.5 ± 0.1

7.4 ± 0.1

7.5 ± 0.1

7.5 ± 0.0

Albumin (g/dL)

 

 

 

 

 

 

Day 4

4.3 ± 0.0

4.3 ± 0.0

4.3 ± 0.0

4.3 ± 0.0

4.4 ± 0.0

4.4 ± 0.0

Day 23

4.6 ± 0.0

4.6 ± 0.0

4.5 ± 0.0

4.5 ± 0.1

4.7 ± 0.0

4.7 ± 0.1

Week 14

4.9 ± 0.1

4.9 ± 0.0

4.9 ± 0.1

4.8 ± 0.0

4.9 ± 0.0

4.9 ± 0.0

Globulin (g/dL)

 

 

 

 

 

 

Day 4

1.7 ± 0.0

1.7 ± 0.0

1.8 ± 0.0

1.7 ± 0.0

1.8 ± 0.0

1.8 ± 0.0

Day 23

1.9 ± 0.0

2.0 ± 0.0

2.0 ± 0.0

2.0 ± 0.0

2.1 ± 0.0**

2.1 ± 0.0**

Week 14

2.6 ± 0.0

2.5 ± 0.0

2.6 ± 0.0

2.5 ± 0.0

2.6 ± 0.0

2.6 ± 0.0

A/G ratio

Day 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 23

2.4 ± 0.0

2.4 ± 0.0

2.3 ± 0.1

2.3 ± 0.0

2.3 ± 0.0

2.2 ± 0.0**

Week 14

1.9 ± 0.0

1.9 ± 0.0

1.9 ± 0.0

1.9 ± 0.0

1.9 ± 0.0

2.0 ± 0.0

Alanine aminotransferase (IU/L)

 

 

 

 

 

 

Day 4

57 ± 1

57 ± 1

55 ± 1

53 ± 1

55 ± 1

52 ± 1**

Day 23

41 ± 1

41 ± 1

41 ± 1

39 ± 2

38 ± 1

35 ± 0**

Week 14

85 ± 3

83 ± 3

70 ± 3**

60 ± 2**

56 ± 2**

51 ± 2**

Alkaline phosphatase (IU/L)

 

 

 

 

 

 

Day 4

575 ± 7

578 ± 10

566 ± 10

566 ± 11

554 ± 7

546 ± 11*

Day 23

406 ± 6

423 ± 11

433 ± 9

407 ± 11

420 ± 8

404 ± 12

Week 14

223 ± 5

227 ± 7

211 ± 4

200 ± 3**

204 ± 4**

199 ± 6**

Creatine kinase (IU/L)

 

 

 

 

 

 

Day 4

545 ± 121

507 ± 42

430 ± 52

449 ± 56

515 ± 54

434 ± 44

Day 23

404 ± 37

390 ± 40

409 ± 66

393 ± 37

354 ± 30

413 ± 45

Week 14

171 ± 8

186 ± 18

144 ± 14

155 ± 13

150 ± 14

183 ± 15

Sorbitol dehydrogenase (IU/L)

 

 

 

 

 

 

Day 4

13 ± 1

14 ± 0

13 ± 0

12 ± 0*

14 ± 1

13 ± 0

Day 23

14 ± 1

14 ± 1

16 ± 1

15 ± 1

18 ± 1**

15 ± 1

Week 14

24 ± 1

24 ± 1

22 ± 1

22 ± 1

21 ± 1*

20 ± 1**

Bile acids (µmol/L)

 

 

 

 

 

 

Day 4

4.7 ± 0.4

4.7 ± 0.5

5.6 ± 0.8

4.6 ± 0.4

7.2 ± 1.3

4.6 ± 0.7

Day 23

5.7 ± 0.9

3.3 ± 0.2**

4.9 ± 0.6*

3.6 ± 0.3**

3.8 ± 0.3**

3.6 ± 0.7**

Week 14

3.3 ± 0.1

3.5 ± 0.4

3.4 ± 0.3

3.2 ± 0.1

3.8 ± 0.6

3.0 ± 0.1


 

Female

Dose treatment (ppm)

0 (control group)

25

50

100

200

400

Urea nitrogen (mg/dL)

 

 

 

 

 

 

Day 4

7.8 ± 0.4

8.5 ± 0.3

 

8.1 ± 0.3

8.3 ± 0.4

 

9.1 ± 0.3*

8.6 ± 0.3

 

Day 23

11.5 ± 0.3

11.8 ± 0.4

11.5 ± 0.4

10.6 ± 0.4

10.8 ± 0.3

9.1 ± 0.4**

Week 14

14.1 ± 0.4

14.4 ± 0.4

13.0 ± 0.5

13.6 ± 0.5

13.4 ± 0.5

11.3 ± 0.5*

Creatinine (mg/dL)

 

 

 

 

 

 

Day 4

0.29 ± 0.01

0.28 ± 0.01

0.29 ± 0.02

0.26 ± 0.02

0.28 ± 0.01

0.26 ± 0.02

Day 23

0.31 ± 0.01

0.30 ± 0.00

0.28 ± 0.01

0.30 ± 0.00

0.30 ± 0.00

0.31 ± 0.01

Week 14

0.37 ± 0.02

0.35 ± 0.02

0.36 ± 0.02

0.38 ± 0.01

0.34 ± 0.02

0.35 ± 0.03

Glucose (mg/dL)

 

 

 

 

 

 

Day 4

138 ± 2

135 ± 2

136 ± 4

136 ± 2

139 ± 5

130 ± 2

Day 23

127 ± 3

123 ± 6

133 ± 5

123 ± 3

122 ± 3

122 ± 5

Week 14

141 ± 8

131 ± 5

123 ± 2

133 ± 3

131 ± 4

114 ± 12

Total protein (g/dL)

 

 

 

 

 

 

Day 4

5.9 ± 0.0

6.0 ± 0.1

6.1 ± 0.0

6.0 ± 0.0

6.1 ± 0.1*

6.1 ± 0.0

Day 23

6.3 ± 0.1

6.4 ± 0.1

6.4 ± 0.1

6.5 ± 0.1

6.5 ± 0.1

6.6 ± 0.1

Week 14

7.5 ± 0.1

7.4 ± 0.1

7.5 ± 0.1

7.6 ± 0.1

7.5 ± 0.1

7.2 ± 0.1

Albumin (g/dL)

 

 

 

 

 

 

Day 4

4.3 ± 0.0

4.4 ± 0.0

4.4 ± 0.0

4.4 ± 0.0

4.4 ± 0.0

4.4 ± 0.0

Day 23

4.5 ± 0.0

4.6 ± 0.0

4.6 ± 0.0

4.6 ± 0.1

4.6 ± 0.0

4.7 ± 0.1

Week 14

5.2 ± 0.1

5.2 ± 0.1

5.2 ± 0.1

5.3 ± 0.0

5.2 ± 0.0

5.0 ± 0.1

Globulin (g/dL)

 

 

 

 

 

 

Day 4

1.6 ± 0.0

1.6 ± 0.0

1.6 ± 0.0

1.7 ± 0.0

1.7 ± 0.0*

1.6 ± 0.0

Day 23

1.8 ± 0.0

1.8 ± 0.0

1.8 ± 0.0

1.9 ± 0.0*

1.9 ± 0.0*

2.0 ± 0.0**

Week 14

2.3 ± 0.1

2.2 ± 0.0

2.3 ± 0.0

2.3 ± 0.0

2.4 ± 0.0

2.2 ± 0.1

A/G ratio

 

 

 

 

 

 

Day 4

2.8 ± 0.0

2.7 ± 0.1

2.7 ± 0.0

2.7 ± 0.1

2.6 ± 0.1

2.7 ± 0.0

Day 23

2.6 ± 0.0

2.5 ± 0.0

2.5 ± 0.0

2.4 ± 0.0

2.5 ± 0.0

2.4 ± 0.0**

Week 14

2.3 ± 0.1

2.4 ± 0.0

2.3 ± 0.0

2.3 ± 0.0

2.2 ± 0.0

2.4 ± 0.1

Alanine aminotransferase (IU/L)

Day 4

47 ± 1

49 ± 1

49 ± 1

46 ± 1

45 ± 1

44 ± 2

Day 23

35 ± 1

36 ± 1

35 ± 1

36 ± 1

34 ± 1

31 ± 1

Week 14

69 ± 4

65 ± 5

55 ± 3**

56 ± 4*

47 ± 2**

49 ± 5**

Alkaline phosphatase (IU/L)

 

Day 4

 

487 ± 8

 

493 ± 10

475 ± 6

 

468 ± 7

 

454 ± 5**

457 ± 8**

 

Day 23

305 ± 5

311 ± 8

304 ± 5

302 ± 8

289 ± 8

289 ± 7

Week 14

197 ± 6

182 ± 4

182 ± 8

177 ± 8**

181 ± 5*

164 ± 13*

Creatine kinase (UI/L)

 

 

 

 

 

 

Day 4

364 ± 20b

332 ± 27

388 ± 29b

443 ± 74

460 ± 39b

375 ± 48

Day 23

299 ± 30

305 ± 27

292 ± 41

369 ± 43

338 ± 26

250 ± 16

Week 14

162 ± 16

165 ± 38

172 ± 22

139 ± 14

170 ± 22

145 ± 26

Sorbitol dehydrogenase (IU/L

 

 

 

 

 

 

Day 4

13 ± 1

13 ± 0

14 ± 0

12 ± 0*

11 ± 1*

12 ± 0*

Day 23

14 ± 0

15 ± 1

15 ± 1

15 ± 1

14 ± 1

16 ± 0

Week 14

21 ± 1

20 ± 1

18 ± 1

17 ± 1

17 ± 1

18 ± 1

Bile acids (µmol/L)

 

 

 

 

 

 

Day 4

5.3 ± 0.5

5.0 ± 0.5

6.5 ± 1.1

5.8 ± 0.6

6.8 ± 1.3

4.9 ± 0.5

Day 23

4.0 ± 0.3

4.7 ± 0.4

5.4 ± 0.7

4.5 ± 0.4

3.9 ± 0.4

4.0 ± 0.7

Week 14

9.1 ± 2.3

4.9 ± 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 control

25 ppm

50 ppm

100 ppm

200 ppm

400 ppm

Number Examined Microscopically

 

10

 

10

 

10

 

10

 

10

 

10

Casts, Granulara

0

9** (1.0)b

10** (1.2)

10** (1.5)

10** (2.5)

10** (3.0)

Accumulation, Hyaline Droplet

1 (2.0)

10** (1.1)

10** (1.8)

10** (2.0)

10** (2.7)

10** (3.0)

Nephropathy

9 (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

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

Table 5 Hematology data

Treatment dose (ppm)

0

25

50

100

200

400

 

 

 

 

 

 

 

Male

 

Hematocrit (spun) (%)

 

 

 

 

 

 

Day 4

45.6 ± 0.3

45.1 ± 0.6

46.5 ± 0.6

45.5 ± 0.4

44.8 ± 0.5

44.4 ± 0.4

Day 23

47.9 ± 0.5

48.0 ± 0.6

47.4 ± 0.4

47.8 ± 0.4

47.7 ± 0.3

48.6 ± 0.5

Week 14

49.5 ± 0.5

48.3 ± 0.4

49.0 ± 0.3

48.3 ± 0.7*

47.6 ± 0.3**

47.7 ± 0.4**

Packed cell volume (mL/dL)

 

 

 

 

 

 

Day 4

44.6 ± 0.4

44.1 ± 0.6

45.0 ± 0.6

44.2 ± 0.4

43.1 ± 0.3*

43.3 ± 0.4*

Day 23

47.4 ± 0.4

47.1 ± 0.5

46.5 ± 0.5

47.2 ± 0.4

46.9 ± 0.4

48.1 ± 0.4

Week 14

49.9 ± 0.5

48.9 ± 0.4

49.5 ± 0.3

48.3 ± 0.3*

48.0 ± 0.3**

47.8 ± 0.6**

Hemoglobin (g/dL)

 

 

 

 

 

 

Day 4

13.5 ± 0.1

13.4 ± 0.2

13.7 ± 0.1

13.6 ± 0.2

13.3 ± 0.1

13.2 ± 0.1

Day 23

14.9 ± 0.1

14.9 ± 0.1

14.6 ± 0.1

15.0 ± 0.1

14.8 ± 0.1

15.1 ± 0.1

Week 14

15.7 ± 0.1

15.5 ± 0.1

15.5 ± 0.1

15.3 ± 0.1*

15.0 ± 0.1**

15.1 ± 0.2**

Erythrocytes (106/µL)

 

 

 

 

 

 

Day 4

7.15 ± 0.09

7.12 ± 0.11

7.27 ± 0.07

7.26 ± 0.08

7.09 ± 0.07

7.06 ± 0.07

Day 23

8.06 ± 0.06

7.95 ± 0.07

7.84 ± 0.09

8.04 ± 0.08

7.92 ± 0.07

8.10 ± 0.06

Week 14

9.35 ± 0.07

9.09 ± 0.05

9.25 ± 0.06

8.94 ± 0.05**

8.92 ± 0.08**

8.86 ± 0.10**

Mean cell volume (fL)

 

 

 

 

 

 

Day 4

62.3 ± 0.3

62.0 ± 0.4

61.8 ± 0.3

60.9 ± 0.5

60.9 ± 0.4*

61.3 ± 0.4

Day 23

58.8 ± 0.2

59.2 ± 0.4

59.4 ± 0.2

58.8 ± 0.4

59.3 ± 0.4

59.4 ± 0.3

Week 14

53.4 ± 0.2

53.9 ± 0.4

53.5 ± 0.3

54.0 ± 0.4

53.9 ± 0.3

53.9 ± 0.3

Mean cell hemoglobin (pg)

 

 

 

 

 

 

Day 4

18.8 ± 0.1

18.8 ± 0.1

18.9 ± 0.0

18.8 ± 0.1

18.8 ± 0.1

18.7 ± 0.0

Day 23

18.5 ± 0.1

18.7 ± 0.1

18.7 ± 0.1

18.6 ± 0.1

18.6 ± 0.1

18.6 ± 0.1

Week 14

16.9 ± 0.1

17.0 ± 0.1

16.8 ± 0.1

17.1 ± 0.1

16.9 ± 0.1

17.1 ± 0.1

Mean cell hemoglobin concentration (g/dL)

Day 4

30.2 ± 0.1

30.4 ± 0.2

30.5 ± 0.1

30.9 ± 0.2*

30.9 ± 0.1**

30.5 ± 0.1

Day 23

31.5 ± 0.2

31.7 ± 0.2

31.4 ± 0.2

31.7 ± 0.2

31.5 ± 0.1

31.4 ± 0.2

Week 14

31.6 ± 0.1 

31.6 ± 0.1 

31.4 ± 0.1 

31.5 ± 0.1

31.3 ± 0.1

31.7 ± 0.2

Leukocytes (103/µL)

 

 

 

 

 

 

Day 4

9.08 ± 0.45

8.94 ± 0.32

9.75 ± 0.44

9.04 ± 0.72

7.57 ± 0.36*

6.95 ± 0.25**

Day 23

6.29 ± 0.23

7.10 ± 0.25

7.44 ± 0.29

8.12 ± 0.42**

7.52 ± 0.64

7.20 ± 0.31

Week 14

7.01 ± 0.36

6.99 ± 0.39

7.86 ± 0.25

7.34 ± 0.37

6.71 ± 0.40

6.69 ± 0.47

 Lymphocytes (103/µL)

 

 

 

 

 

 

Day 4

7.99 ± 0.40

7.82 ± 0.30

8.39 ± 0.38

7.75 ± 0.61

6.44 ± 0.33**

5.88 ± 0.24**

Day 23

5.25 ± 0.23

5.91 ± 0.25

6.38 ± 0.22d

6.89 ± 0.40*

6.10 ± 0.57

6.06 ± 0.27

Week 14

5.36 ± 0.35

5.31 ± 0.39

6.18 ± 0.25

5.66 ± 0.40

5.12 ± 0.41

4.93 ± 0.38

Treatment dose (ppm)

0

25

50

100

200

400

 

 

 

 

 

 

 

female

 

Hematocrit (spun) (%)

 

 

 

 

 

 

Day 4

47.7 ± 0.4

47.0 ± 0.2

47.0 ± 0.2

47.5 ± 0.3

47.1 ± 0.6 

46.2 ± 0.4

Day 23

48.7 ± 0.4

49.2 ± 0.5

49.1 ± 0.4

49.2 ± 0.3

48.9 ± 0.5

49.7 ± 0.6

Week 14

48.9 ± 0.4

47.2 ± 0.4*

47.8 ± 0.2

48.3 ± 0.4

48.7 ± 0.4

50.9 ± 0.8

Packed cell volume (mL/dL)

 

 

 

 

 

 

Day 4

46.7 ± 0.5

46.1 ± 0.3

46.0 ± 0.3

46.8 ± 0.4

46.1 ± 0.5

45.3 ± 0.5

 

Day 23

48.5 ± 0.4

49.0 ± 0.4

49.3 ± 0.3

49.2 ± 0.3

48.8 ± 0.3

50.0 ± 0.6*

Week 14

49.1 ± 0.3

48.6 ± 0.3

48.7 ± 0.

49.0 ± 0.5

49.7 ± 0.4

52.7 ± 0.4

Hemoglobin (g/dL)

 

 

 

 

 

 

Day 4

14.3 ± 0.1

14.2 ± 0.1

14.2 ± 0.1

14.5 ± 0.1

14.3 ± 0.1

14.1 ± 0.1

Day 23

15.3 ± 0.1

15.5 ± 0.1

15.5 ± 0.1

15.5 ± 0.1

15.3 ± 0.1

15.7 ± 0.1

Week 14

15.7 ± 0.1

15.4 ± 0.1

15.5 ± 0.1

15.6 ± 0.1

15.8 ± 0.1

16.7 ± 0.1

Erythrocytes (106/µL)

 

 

 

 

 

 

Day 4

7.64 ± 0.07

 

7.56 ± 0.05

7.56 ± 0.05

7.74 ± 0.07

 

7.67 ± 0.10

7.55 ± 0.08

 

Day 23

8.13 ± 0.08

8.13 ± 0.08

8.20 ± 0.07

 

8.24 ± 0.05

 

8.13 ± 0.06

 

8.31 ± 0.09

Week 14

8.67 ± 0.06

8.53 ± 0.05

8.54 ± 0.06

8.62 ± 0.09

8.71 ± 0.06

9.23 ± 0.09

Mean cell volume (fL)

 

 

 

 

 

 

Day 4

61.1 ± 0.3

60.9 ± 0.3

60.9 ± 0.2

60.5 ± 0.3

60.2 ± 0.5

60.0 ± 0.3*

Day 23

59.6 ± 0.3

60.3 ± 0.3

60.1 ± 0.3

59.7 ± 0.3

60.1 ± 0.3

60.2 ± 0.2

Week 14

56.6 ± 0.2

56.9 ± 0.1

57.0 ± 0.1

56.9 ± 0.1

57.0 ± 0.2

57.1 ± 0.3

Mean cell hemoglobin (pg)

 

 

 

 

 

 

Day 4

18.7 ± 0.1

18.8 ± 0.1

18.8 ± 0.1

18.7 ± 0.1

18.7 ± 0.1

18.7 ± 0.1

Day 23

18.8 ± 0.1

19.0 ± 0.1

18.9 ± 0.1

18.8 ± 0.1

18.8 ± 0.1

18.9 ± 0.1

Week 14

18.1 ± 0.0

18.1 ± 0.1

18.2 ± 0.1

18.1 ± 0.1

18.1 ± 0.0

18.1 ± 0.0

Mean cell hemoglobin concentration (g/dL)

Day 4

30.6 ± 0.2

30.8 ± 0.1

30.8 ± 0.1

31.0 ± 0.2

31.1 ± 0.2

31.2 ± 0.2

Day 23

31.6 ± 0.2

31.5 ± 0.1

31.4 ± 0.1

31.6 ± 0.1

31.4 ± 0.1

31.4 ± 0.1

Week 14

 

32.1 ± 0.1

31.8 ± 0.1

31.9 ± 0.1

31.9 ± 0.1

31.8 ± 0.1

31.7 ± 0.2

Leukocytes (103/µL)

 

 

 

 

 

 

Day 4

10.52 ± 0.52

10.89 ± 0.26

10.25 ± 0.34

11.26 ± 0.61

10.39 ± 0.63

8.52 ± 0.68

Day 23

7.96 ± 0.36

8.01 ± 0.24

7.87 ± 0.43

8.04 ± 0.39

7.78 ± 0.56

6.84 ± 0.48

Week 14

5.86 ± 0.27

5.70 ± 0.24

6.05 ± 0.29

5.60 ± 0.29

5.22 ± 0.33

6.08 ± 0.58

 Lymphocytes (103/µL)

 

 

 

 

 

 

Day 4

9.34 ± 0.52

9.58 ± 0.25

8.90 ± 0.30

9.76 ± 0.59

9.19 ± 0.51d

7.34 ± 0.62

Day 23

6.79 ± 0.35

6.86 ± 0.20

6.83 ± 0.41

6.96 ± 0.38

6.62 ± 0.54

5.83 ± 0.42

Week 14

4.67 ± 0.24

4.44 ± 0.25

4.67 ± 0.23

4.36 ± 0.28

4.17 ± 0.29

4.93 ± 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 control

100 ppm

200 ppm

400 ppm

n

10

10

9

10

Epididymal spermatozoal measurements

Sperm motility (%)

91.73 ± 1.26

91.40 ± 0.93

91.24 ± 0.80

90.93 ± 0.89

Sperm (103/mg cauda epididymis)

615.0 ± 34.3

596.5 ± 31.8

526.3 ± 19.0

547.4 ± 14.0

Sperm (106/cauda epididymis)

120.89 ± 6.79

113.16 ± 3.11

97.52 ± 3.51**

98.40 ± 3.02**

 

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

Applicant's summary and conclusion

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 weekdays 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 alpha 2µ-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).