Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

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:
comparable to guideline study with acceptable restrictions
Remarks:
Whole body exposure, and fixation of cauda tissue at 65°C
Cross-reference
Reason / purpose:
read-across: supporting information

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, 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- batch 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:
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 and environmental conditions:
After a 12-day quarantine period, animals are assigned at random to treatment groups.
Age at the study initiate: 5 to 6 weeks
Mice 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.
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
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 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

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.
HEMATOLOGY: 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:
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

Effect levels

open allclose all
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

Target system / organ toxicity

open allclose all
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

Any other information on results incl. tables

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

 

Concentration

(ppm)

Survivalb


Initial Body Weight (g)

 

Final Body Weight (g)

Change in Body Weight(g)

 

Final Weight Relative to Controls (%)

 

Male

 

0

10/10

22.9 ± 0.2

37.1 ± 0.6

14.3 ± 0.6

 

25

10/10

23.0 ± 0.3

36.9 ± 0.7

13.9 ± 0.8

99

50

10/10

22.7 ± 0.3

38.3 ± 0.9

15.6 ± 0.8

103

100

10/10

22.5 ± 0.2

35.9 ± 0.7

13.4 ± 0.7

97

200

10/10

22.8 ± 0.3

35.5 ± 1.0

12.7 ± 0.9

96

400

10/10

22.8 ± 0.2

36.2 ± 0.5

13.5 ± 0.4

98

Female

0

10/10

19.5 ± 0.4

31.5 ± 0.6

12.0 ± 0.5

 

25

10/10

19.6 ± 0.4

30.3 ± 0.6

10.8 ± 0.7

96

50

10/10

19.7 ± 0.3

32.7 ± 0.7

12.9 ± 0.7

104

100

10/10

19.7 ± 0.4

31.5 ± 1.1

11.8 ± 0.9

100

200

10/10

19.3 ± 0.3

30.7 ± 0.6

11.4 ± 0.6

97

400

10/10

19.4 ± 0.3

30.6 ± 0.5

11.2 ± 0.4

97

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 control

25 ppm

50 ppm

100 ppm

200 ppm

400 ppm

Male

 

n

10

10

10

10

10

10

Necropsy body wt

37.1 ± 0.6

36.9 ± 0.7

38.3 ± 0.9

35.9 ± 0.7

35.5 ± 1.0

36.2 ± 0.5

R Kidney absolute

0.330 ± 0.006

0.318 ± 0.009

0.336 ± 0.010

0.309 ± 0.008

0.295 ± 0.006*

0.307 ± 0.007*

R kidney relative

8.903 ± 0.167

8.629 ± 0.208

8.793 ± 0.267

8.617 ± 0.205

8.348 ± 0.145

8.469 ± 0.155

Liver absolute

1.617 ± 0.022

1.589 ± 0.028

1.702 ± 0.040

1.637 ± 0.024

1.660 ± 0.043

1.957 ± 0.057**

Liver relative

43.671 ± 0.880

43.123 ± 0.458

44.487 ± 0.806

45.651 ± 0.678

46.903 ± 0.750*

54.009 ± 1.465**

Thymus absolute

0.066 ± 0.004

0.063 ± 0.004

0.067 ± 0.003

0.057 ± 0.001

0.062 ± 0.004

0.051 ± 0.003**

Thymus relative

1.777 ± 0.081

1.699 ± 0.090

1.742 ± 0.063

1.591 ± 0.052

1.739 ± 0.115

1.397 ± 0.081**

Female

n

10

10

10

10

10

10

Necropsy body wt

31.5 ± 0.6

30.3 ± 0.6

32.7 ± 0.7

31.5 ± 1.1

30.7 ± 0.6

30.6 ± 0.5

Liver absolute

1.466 ± 0.041

1.475 ± 0.053

1.442 ± 0.036

1.548 ± 0.053

1.587 ± 0.037

1.730 ± 0.032**

Liver relative

46.542 ± 0.988

48.567 ± 1.239

44.214 ± 0.880

49.280 ± 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

Sexe

Treatment concentration (ppm)

 0 (control group)

25

50

100

200

400

Male

Number Examined Microscopically

10

10

10

10

10

10

Male

Transitional Epithelium, Hyperplasiaa

0

0

0

7** (1.0)b

 

10** (2.0)b

 

10** (2.5)b

 

Female

Number Examined Microscopically

10

10

10

10

10

10

 Female

Transitional Epithelium, Hyperplasiaa

0

0

0

6** (1.0)b

 

10** (1.6)b

 

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 ± 0.3

50.5 ± 0.4

50.1 ± 0.3

51.1 ± 0.3

50.9 ± 0.4

49.8 ± 0.3*

Hemoglobin (g/dL)

16.0 ± 0.1

16.0 ± 0.1

15.7 ± 0.1

16.0 ± 0.0

16.1 ± 0.1

15.7 ± 0.1

Erythrocytes (106/µL)

10.51 ± 0.06

10.47 ± 0.06

10.23 ± 0.09

10.52 ± 0.04

10.55 ± 0.08

10.10 ± 0.07**

Reticulocytes(103/µL)Nucleated erythrocytes

/100 leukocytes

223.7 ± 19.4

 

0.00 ± 0.00

200.3 ± 14.9

 

0.00 ± 0.00

193.9 ± 16.4

 

0.00 ± 0.00

205.2 ± 13.0

 

0.00 ± 0.00

214.3 ± 16.4

 

0.00 ± 0.00

202.2 ± 15.9

 

0.00 ± 0.00

Mean cell volume (fL)

49.3 ± 0.3

49.2 ± 0.2

49.6 ± 0.2

49.4 ± 0.2

49.6 ± 0.2

50.6 ± 0.2**

Mean cell hemoglobin (pg)

Mean cell hemoglobin concentration (g/dL)

15.3 ± 0.1

 

31.0 ± 0.2

15.2 ± 0.1

 

31.0 ± 0.1

15.4 ± 0.1

 

31.0 ± 0.1

15.2 ± 0.0

 

30.8 ± 0.2

15.2 ± 0.0

 

30.7 ± 0.1

15.6 ± 0.1*

 

30.8 ± 0.1

Leukocytes (103/µL)

3.10 ± 0.40

2.94 ± 0.43

2.03 ± 0.26

2.47 ± 0.15

2.31 ± 0.26

1.87 ± 0.17*

Lymphocytes (103/µL)

2.60 ± 0.34

2.41 ± 0.39

1.73 ± 0.23

2.03 ± 0.13

1.93 ± 0.22

1.48 ± 0.14*

 

Female

 

 

 

 

 

 

Hematocrit (spun) (%)

49.6 ± 0.3

50.1 ± 0.4

49.4 ± 0.5

50.1 ± 0.3

48.9 ± 0.3

48.3 ± 0.3*

Hemoglobin (g/dL)

15.8 ± 0.1

16.0 ± 0.1

15.7 ± 0.2

15.9 ± 0.1

15.5 ± 0.1

15.5 ± 0.1*

Erythrocytes (106/µL)

10.21 ± 0.05

10.26 ± 0.06

10.10 ± 0.11

10.14 ± 0.06

9.96 ± 0.09*

9.85 ± 0.08**

Reticulocytes(103/µL)Nucleated erythrocytes

/100 leukocytes

269.5 ± 15.4

 

0.00 ± 0.00

248.9 ± 14.9

 

0.00 ± 0.00

251.9 ± 16.5

 

0.00 ± 0.00

282.5 ± 18.3

 

0.00 ± 0.00

240.1 ± 20.8

 

0.00 ± 0.00

251.2 ± 15.3

 

0.00 ± 0.00

Mean cell volume (fL)

49.3 ± 0.2

49.7 ± 0.2

49.5 ± 0.3

50.1 ± 0.2*

49.7 ± 0.2

49.7 ± 0.2

Mean cell hemoglobin (pg)

Mean cell hemoglobin concentration (g/dL)

15.5 ± 0.1

 

31.5 ± 0.2

15.6 ± 0.1

 

31.4 ± 0.1

15.5 ± 0.1

 

31.4 ± 0.2

15.6 ± 0.1

 

31.1 ± 0.1

15.6 ± 0.1

 

31.4 ± 0.1

15.7 ± 0.1

 

31.6 ± 0.1

Leukocytes (103/µL)

3.65 ± 0.35

3.10 ± 0.27

3.34 ± 0.32

2.80 ± 0.29

3.11 ± 0.32

3.16 ± 0.34

Lymphocytes (103/µL)

3.09 ± 0.30

2.65 ± 0.23

2.78 ± 0.30

2.39 ± 0.25

2.60 ± 0.26

2.66 ± 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 control

100 ppm

200 ppm

400 ppm

n

10

10

10

10

Epididymal spermatozoal measurements

Sperm motility (%)

90.25 ± 0.34

88.31 ± 0.86

89.74 ± 0.80

87.95 ± 1.08

Sperm (103/mg cauda epididymis)

704.8 ± 64.9

690.7 ± 55.9

537.5 ± 27.0*

445.8 ± 13.5**

Sperm (106/cauda epididymis)

24.45 ± 0.95

18.40 ± 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

Applicant's summary and conclusion

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 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.

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 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). 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.