Rectified Hydrocarbons by-products Limonene enriched from synthetic process

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 November 2004 - 16 December 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

of which it is a range-finding study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 March 2005 - 1 July 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

no tested on preferred specie rat, no data on food consumption, no ophthalmological and clinical chemistry exams, some organ weights were not recorded (Adrenals,Brain,Ovaries,Thyroids,Uterus), animals weighed weekly and not twice weekly at the beginning.

GLP compliance:

yes

Remarks:

In compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 22.5-23 g (male) and 19.3-19-7 g (female)
- Fasting period before study: not specified
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); rotated weekly. Cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: Animals were quarantined for 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber and room concentrations of alpha 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 ± 4 ppm for the 200 ppm group and 401 ± 7 ppm for the 400 ppm group.

Duration of treatment / exposure:

14 weeks; 6 hours plus T90 (10 minutes) per day.

Frequency of treatment:

Five times per week, weekdays only

Dose / conc.:

0 ppm

Dose / conc.:

25 ppm

Remarks:

(0.14 mg/L)

Dose / conc.:

50 ppm

Remarks:

(0.28 mg/L)

Dose / conc.:

100 ppm

Remarks:

(0.56 mg/L)

Dose / conc.:

200 ppm

Remarks:

(1.13 mg/L)

Dose / conc.:

400 ppm

Remarks:

(2.26 mg/L)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: doses were selected taken into account the results obtained in a previous 2-week range finding study conducted for exposure (inhalation) concentrations of 0, 100, 200, 400, 800, and 1,600 ppm.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on day 8, weekly thereafter, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: initially, on day 8, weekly thereafter, and at the end of the studies.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Not specified
- How many animals: 10 per sex per dose
- Parameters examined: Hematocrit; packed cell volume; hemoglobin; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and differentials

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.
HISTOPATHOLOGY: Yes. Complete histopathologic examinations were performed by the study laboratory pathologist on all chamber control and 400 ppm animals. In addition, the kidney and urinary bladder of mice were examined in the remaining groups. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.

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 tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei were counted with 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:

Calculation and Analysis of Lesion Incidences: Fisher exact test (Gart et al., 1979) was used to determine significance.
Analysis of Continuous Variables:
Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972),
Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test).
Proportions of regular cycling females in each exposed group were compared to the control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus were constructed based on a Markov chain model proposed by Girard and Sager (1987).
For each exposure group, a transition probability matrix was estimated for transitions among the proestrus, estrus, metestrus, and diestrus stages, with provision for extended stays within each stage as well as for skipping estrus or diestrus within a cycle. Equality of transition matrices among exposure groups and between the control group and each exposed group was tested using chi-square statistics.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs.

Mortality:

no mortality observed

Description (incidence):

All mice survived until the terminal sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Final mean body weights and body weight gains were comparable for all test animals when compared to controls.

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):

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.

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):

The absolute liver 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 than those of the chamber controls. The absolute and relative thymus weights of 400 ppm males were significantly less than those of the chamber controls. The absolute kidney weights of 200 and 400 ppm males were significantly less than those of the chamber controls. These organ weight changes were not accompanied by histopathologic lesions.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of transitional epithelium hyperplasia of the urinary bladder in males and females exposed to 100 ppm or more, the severity of which increased with increasing exposure concentration.
Transitional epithelium hyperplasia in the urinary bladder can be either reparative (e.g., regenerative or reactive) or preneoplastic, but there are no histologic features that can be used to reliably distinguish between the two etiologies.
Specific histopathologic indicators of either type of hyperplasia in male or female mice were not evident; therefore, the neoplastic potential of the transitional epithelium hyperplasia of the urinary bladder is uncertain.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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.
Thus, the test item exposure by inhalation exhibits the potential to be a reproductive toxicant in male mice, but not in female mice.

Key result

Dose descriptor:

LOEL

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

Key result

Dose descriptor:

LOEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

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:

not specified

Table 1: Survival and Body Weights

	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: Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios

	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**

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

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

a Organ 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).

Table 3: 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)	223.7 ± 19.4	200.3 ± 14.9	193.9 ± 16.4	205.2 ± 13.0	214.3 ± 16.4	202.2 ± 15.9
Nucleated erythrocytes/100 leukocytes	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	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)	15.3 ± 0.1	15.2 ± 0.1	15.4 ± 0.1	15.2 ± 0.0	15.2 ± 0.0	15.6 ± 0.1*
Mean cell hemoglobin concentration (g/dL)	31.0 ± 0.2	31.0 ± 0.1	31.0 ± 0.1	30.8 ± 0.2	30.7 ± 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)	269.5 ± 15.4	248.9 ± 14.9	251.9 ± 16.5	282.5 ± 18.3	240.1 ± 20.8	251.2 ± 15.3
Nucleated erythrocytes/100 leukocytes	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	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)	15.5 ± 0.1	15.6 ± 0.1	15.5 ± 0.1	15.6 ± 0.1	15.6 ± 0.1	15.7 ± 0.1
Mean cell hemoglobin concentration (g/dL)	31.5 ± 0.2	31.4 ± 0.1	31.4 ± 0.2	31.1 ± 0.1	31.4 ± 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

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

Table 4: Incidence of Non neoplastic lesions of the urinary bladder

Sex	Treatment concentration (ppm)	0 (control group)	25	50	100	200	400
Male	Number Examined Microscopically	10	10	10	10	10	10
Male	Transitional Epithelium, Hyperplasia^a	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, Hyperplasia^a	0	0	0	6** (1.0)^b	10** (1.6)^b	10** (2.2)^b

** 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: Epididymal Spermatozoal Measurements for Male Mice

	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

a Data are presented as mean ± standard error.

 

Conclusions:

The LOEL for alpha pinene after 14 weeks of vapour inhalation exposure to mice was determined to be 100 ppm based on the effects on transitional epithelium hyperplasia of the urinary bladder for males and females and decrease in sperm per cauda epididymis for males, both observed in the 100 ppm treated group or greater.

Executive summary:

A 90-day repeated dose toxicity study (inhalation route) was performed on alpha pinene according to a similar method to OECD guideline 413 under GLP conditions. Groups of 10 male and 10 female mice were exposed to the test item by whole body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 ppm, 6 hours per day, 5 days per week for 14 weeks. During the study, clinical observations, bodyweight, organ weight, haematology, macropathology, histopathology and sperm motility and vaginal cytology investigations were undertaken.

All exposed mice survived to the end of the studies. Some small changes in organ weights as liver or kidney, were noted but without accompanying histopathologic lesions.

The major targets for test item toxicity were the urinary system and male reproductive system.

There was an increased incidence of transitional epithelium hyperplasia of the urinary bladder in males and females exposed to 100 ppm or more (LOEL=100 ppm), the severity of which increased with increasing exposure concentration. Transitional epithelium hyperplasia in the urinary bladder can be either reparative (e.g., regenerative or reactive) or preneoplastic, but there are no histologic features that can be used to reliably distinguish between the two etiologies. Specific histopathologic indicators of either type of hyperplasia in male or female mice were not evident; therefore, the neoplastic potential of the transitional epithelium hyperplasia of the urinary bladder is uncertain.

There were also significantly decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males (LOEL=100 ppm). There was an accompanying minor decrease in epididymal weights that did not reach significance. Therefore, the possibility that the change in absolute sperm per cauda was due to a decrease in epididymal weight cannot be excluded. A definitive conclusion by histopathologic investigations could not be conducted due to an artifact resulting from formalin fixation of the male reproductive tract tissues. Thus, further studies on reproductive function are warranted. However, in females there were no changes in the percentage of time spent in the individual stages of the estrous cycle or in estrous cycle length at any exposure concentration. Also, there were no ovarian histopathologic findings. Thus, no evidence of reproductive toxicity in females was found.

Based on these results, the LOEL for alpha pinene was determined to be 100 ppm.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2016

Materials and methods

Principles of method if other than guideline:

- Principle of test: Mice were exposed to alpha pinene via whole body inhalation 5 days per week for 17 days
- Short description of test conditions: see below
- Parameters analysed / observed: see below

GLP compliance:

yes

Remarks:

In compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Test material form:

liquid

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 23.1-23.9 g (male) and 19.8-20.6 g (female)
- Fasting period before study: not specified
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); changed weekly. Cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: Animals were quarantined for 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber and room concentrations of alpha 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: 99.8 ± 1.6 ppm for the 100 ppm group, 200 ± 1 for the 200 ppm group, 404 ± 4ppm for the 400 ppm group, 794 ± 37 ppm for the 800 ppm group and 1540 ± 129 ppm for the 1600 ppm group.

Duration of treatment / exposure:

17 days; 6 hours plus T90 (12 minutes) per day.

Frequency of treatment:

Five times per week, weekdays only

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

100 ppm

Remarks:

(0.56 mg/L)

Doses / concentrations 3

Dose / conc.:

200 ppm

Remarks:

(1.13 mg/L)

Doses / concentrations 4

Dose / conc.:

400 ppm

Remarks:

(2.26 mg/L)

Doses / concentrations 5

Dose / conc.:

800 ppm

Remarks:

(4.53 mg/L)

Doses / concentrations 6

Dose / conc.:

1 600 ppm

Remarks:

(9.06 mg/L)

No. of animals per sex per dose:

5

Control animals:

yes

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily on exposure days and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: initially, on days 6 and 13, and at the end of the studies.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.
HISTOPATHOLOGY: Yes. Histopathology was performed on 0, 400, 800, and 1,600 ppm rats. In addition to gross lesions and tissue masses, the lung and nose were examined. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.

Statistics:

Calculation and Analysis of Lesion Incidences: Fisher exact test (Gart et al., 1979) was used to determine significance.
Analysis of Continuous Variables:
Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Lethargy and abnormal breathing were observed in three 800 ppm males and two 1,600 ppm males, and lethargy was observed in one 1,600 ppm female. Ataxia was observed in two 800 ppm males, two 1,600 ppm males, and four 800 ppm females.

Mortality:

mortality observed, treatment-related

Description (incidence):

All 800 and 1,600 ppm males and females died early.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The final mean body weights and mean body weight gains of all surviving groups of exposed mice were similar to those of the chamber controls.

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:

not examined

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):

The absolute and relative liver weights of 400 ppm males and females and the relative liver weight of 200 ppm males were significantly greater (up to 20%) than those of the chamber controls. The absolute and relative kidney weights of 100 ppm females were significantly greater (18% and 15%, respectively) than those of the chamber controls as was the relative kidney weight of 400 ppm males (12%).

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the nose, there were significantly increased incidences of minimal olfactory epithelial degeneration in 800 and 1,600 ppm males (chamber controls, 0/5; 100 ppm, 0/0; 200 ppm, 0/0; 400 ppm, 0/5; 800 ppm, 5/5; 1,600 ppm, 4/5) and females (0/5, 0/0, 0/0, 0/5, 4/5, 5/5). Degeneration was characterized by slight disorganization of the normal olfactory architecture in the Level I and II nasal sections and the epithelial cells in the mid layers of the olfactory epithelium were often pyknotic. The mucosa often had an undulating appearance, and strands of proteinaceous debris and/or mucus were present in the nasal passages.

Histopathological findings: neoplastic:

not examined

Details on results:

Based on decreased survival and nonneoplastic lesions in the nose of 800 and 1,600 ppm males and females observed in this study, exposure concentrations of 0, 25, 50, 100, 200, and 400 ppm alpha pinene were selected for the 3-month study in mice.

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

800 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Survival and Body Weights

	Concentration (ppm)	Survivalb	Initial Body Weight (g)	Final Body Weight (g)	Change in Body Weight (g)	Final Weight Relative to Controls (%)
Male	0	5/5	23.7 ± 0.4	27.9 ± 0.7	4.1 ± 0.4
	100	5 /5	23.4 ± 0.7	26.7 ± 0.7	3.4 ± 0.3	96
	200	5/5	23.1 ± 0.8	26.6 ± 0.9	3.5 ± 0.4	95
	400	5/5	23.9 ± 0.5	27.0 ± 0.6	3.1 ± 0.2	97
	800	0/5 c	23.5 ± 05	-	-	-
	1600	0/5 d	23.5 ± 0.6	-	-	-
Female	0	5/5	20.2 ± 0.4	23.0 ± 0.4	2.8 ± 0.3
	100	5/5	20.6 ± 0.5	23.6 ± 0.5	3.0± 0.3	103
	200	5/5	20.2 ± 0.5	23.2 ± 0.7	3.0 ± 0.7	101
	400	5/5	19.9 ± 0.5	22.6 ± 0.5	2.7 ± 0.3	99
	800	0/5 e	19.8 ± 0.3	-	-	-
	1600	0/5 f	19.8 ± 0.2	-	-	-

a Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study.

b Number of animals surviving at day 18/number initially in group

c Days of death: 2, 2, 3, 4, 16

d Days of death: 1, 1, 1, 1, 2

e Day of deaths: 2

f Day of deaths: 1

Table 2: Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios

			Chamber Control	100 ppm	200 ppm	400 ppm
		n	5	5	5	5
Male		Necropsy body wt	27.9 ± 0.7	26.7 ± 0.7	26.6 ± 0.9	27.0 ± 0.6
	Heart	Absolute	0.136 ± 0.006	0.124 ± 0.004	0.126 ± 0.005	0.136 ± 0.008
		Relative	4.872 ± 0.140	4.640 ± 0.127	4.743 ± 0.182	5.028 ± 0.223
	R. Kidney	Absolute	0.234 ± 0.011	0.246 ± 0.009	0.236 ± 0.015	0.254 ± 0.012
		Relative	8.377 ± 0.226	9.194 ± 0.191	8.832 ± 0.297	9.401 ± 0.337*
	Liver	Absolute	1.436 ± 0.066	1.394 ± 0.044	1.476 ± 0.069	1.672 ± 0.056*
		Relative	51.415 ± 1.352	52.117 ± 0.648	55.355 ± 0.721*	61.935 ± 1.281**
	Lung	Absolute	0.184 ± 0.011	0.186 ± 0.007	0.200 ± 0.017	0.184 ± 0.006
		Relative	6.579 ± 0.260	6.953 ± 0.154	7.498 ± 0.510	6.814 ± 0.120
	R. Testis	Absolute	0.099 ± 0.002	0.095 ± 0.004	0.089 ± 0.010	0.094 ± 0.002
		Relative	3.539 ± 0.048	3.536 ± 0.109	3.301 ± 0.287	3.478 ± 0.074
	Thymus	Absolute	0.057 ± 0.007	0.048 ± 0.004	0.050 ± 0.003	0.049 ± 0.007
		Relative	2.030 ± 0.192	1.801 ± 0.131	1.882 ± 0.105	1.831 ± 0.256
Female		Necropsy body wt	23.0 ± 0.4	23.6 ± 0.5	23.2 ± 0.7	22.6 ± 0.5
	Heart	Absolute	0.118 ± 0.004	0.122 ± 0.004	0.120 ± 0.003	0.118 ± 0.002
		Relative	5.135 ± 0.090	5.163 ± 0.137	5.165 ± 0.034	5.221 ± 0.137
	R. Kidney	Absolute	0.166 ± 0.005	0.196 ± 0.007*	0.184 ± 0.008	0.180 ± 0.006
		Relative	7.225 ± 0.137	8.283 ± 0.132**	7.919 ± 0.273	7.952 ± 0.234
	Liver	Absolute	1.230 ± 0.029	1.300 ± 0.033	1.320 ± 0.065	1.426 ± 0.036*
		Relative	53.557 ± 0.672	54.996 ± 0.772	56.718 ± 1.676	63.001 ± 0.995**
	Lung	Absolute	0.182 ± 0.006	0.190 ± 0.005	0.188 ± 0.004	0.198 ± 0.015
		Relative	7.932 ± 0.261	8.033 ± 0.085	8.114 ± 0.273	8.756 ± 0.661
	Thymus	Absolute	0.074 ± 0.003	0.073 ± 0.005	0.068 ± 0.003	0.060 ± 0.004
		Relative	3.220 ± 0.122	3.059 ± 0.157	2.913 ± 0.123	2.654 ± 0.114*

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

** P≤0.01

a Organ 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).

No data were available for the 800 and 1,600 ppm groups due to 100% mortality.

Applicant's summary and conclusion

Conclusions:

In the 2-week study with alpha-pinene, the two highest exposure concentrations (800 and 1600 ppm) were overtly toxic to mice, resulting in clinical findings of toxicity and death. Based on this experiment a maximal dose of 400 ppm was administrated in the 90-day study.

Executive summary:

A 2-week repeated dose toxicity study (inhalation route) was performed on alpha pinene as a dose range-finding study prior to a 3 month main test. Groups of 5 male and 5 female mice were exposed to the test item by whole body inhalation at concentrations of 0, 100, 200, 400, 800 and 1600 ppm, 6 hours per day, 5 days per week for 17 days. During the study, clinical observations, bodyweight, organ weight, macropathology and histopathology investigations were undertaken. The two highest exposure concentrations (800 and 1,600 ppm) were overtly toxic to mice, resulting in clinical findings of toxicity (e.g., ataxia, tremors, abnormal breathing) and death. In the remaining exposed groups, there was evidence of a general increase in absolute and/or relative liver and kidney weights compared to chamber control mice of both sexes, but not considered to be life threatening. In the histopathologic exams only minimal olfactory epithelial degeneration of nasal tissue was found in male and female mice exposed to 800 and 1600 ppm. Therefore, 400 ppm was selected as the high concentration for the 3-month studies in mice.