Registration Dossier

Administrative data

Description of key information

Weight of evidence: Repeated dose toxicity studies are available on the components d-limonene, camphene and cineole by oral route, and on component alpha pinene by inhalation route. The absence of any d-limonene-induced renal lesions in the study with dogs provides evidence that hydrocarbon induced nephropathy in the male rat is species- and sex-specific. Therefore, the male rat response to d-limonene,

alpha pinene, camphene or cineole may not be appropriate for assessing the potential risk of a similar nephrotoxic response in any other species, including humans. According to CLP annex I 3.9.2.8.1. (e), substance-induced species-specific mechanisms of toxicity, i.e. demonstrated with reasonable certainty to be not relevant for human health, shall not justify classification.

The key study was then selected to be the 180-d toxicity study by oral route in dogs (Webb, 1990) for DNEL derivation since mammalian exposure is more relevant than rodent exposure regarding human

health effect assessment. The key value has been then selected to be the LOAEL at 1000 mg/kg bw/d. When administered orally by gavage for at least 6 months, d-limonene induces some toxicological effects at 1000 mg/kg bw/day. At this dose level, following 90 days of exposure, d-limonene induces decreased bodyweight gain and clinical signs in mice. 180 days of exposure to d-limonene at this dose level decreased bodyweight gain and increased relative and absolute kidney weights of dogs (with protein casts in the renal tubules of females).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The study does not meet the main criteria of the specific testing guideline (duration, number of tested animals, haematology, clinical biochemistry, gross necropsy, etc.)
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the effect of camphene on serum lipids and apolipoproteins (apoproteins) was investigated in male Wistar rats in order to obtain more information on the physiological role of essential oils. Camphene was mixed with the powdered commercial rat ration at the level of 1%. A control group received the commercial rat ration alone. Three or four rats were used. The rats were fasted overnight prior to blood sampling from the abdominal aorta. Serum lipids and apoproteins were determined.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kyudo Co., Kumamoto
- Weight at study initiation: 248 ± 4.1 g
- Housing: The animals were kept in an air conditioned room
- Diet (e.g. ad libitum): ad libitum; NMF, Oriental Yeast Co, Tokyo.
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Camphene was mixed with the powdered commercial rat ration.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
14 Days
Frequency of treatment:
Daily
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Basis: 1 % of camphene mixed in diet
No. of animals per sex per dose:
3-4 male rats per group
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at beginning and at the end of study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of study.
- Animals fasted: Yes. The rats were fasted overnight prior to blood sampling.
- How many animals: 3-4
- Parameters examined: Serum lipids (cholesterol and triacylglycerol) and apoproteins (Apo A-I)

OTHER: RELATIVE LIVER WEIGHT
- Time schedule for collection of blood: At the end of study.
Sacrifice and pathology:
GROSS PATHOLOGY: No

HISTOPATHOLOGY: No
Statistics:
Student's t test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences were observed in body weight gain between treated and control animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant differences were observed in food consumption between treated and control animals.
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:
no effects observed
Description (incidence and severity):
No significant differences in cholesterol, triacylglycerol and Apolipoprotein Apo A-I were observed between treated and control animals.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Liver weight per 100 g of body weight tended to increase by the supplementary essential oils. No significant increase was recorded for the test item.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Critical effects observed:
no

After 14 days, the NOAEL was equal or greater than 500 mg/kg bw/day for male rats.

Conclusions:
After 14 days of oral exposure, the NOAEL of camphene was equal or greater than 500 mg/kg bw/day for male rats.

Executive summary:

The effect of camphene on serum lipids and apolipoproteins (apoproteins) was investigated in male Wistar rats in order to obtain more information on the physiological role of essential oils. Camphene was mixed with the powdered commercial rat ration at the level of 1% (ca. 500 mg/kg bw/day). It was daily administered for 14 days. A control group received the commercial rat ration without test substance. Three or four rats were used. The rats were fasted overnight prior to blood sampling from the abdominal aorta. Serum lipids and apoproteins were determined. No significant differences were observed in body weight gain and/or food consumption between treated and control animals. Liver weight per 100 g of body weight tended to increase by the supplementary essential oils but not significantly. No significant differences in cholesterol, triacylglycerol and Apolipoprotein Apo A-I were observed between treated and control animals. Thus, the NOAEL of camphene was equal or greater than 500 mg/kg bw/day for male rats.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations.
Principles of method if other than guideline:
- Principle of test: Subacute toxicity study (1 or 4 weeks). Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
5 or 26 days (5 days/week)
Frequency of treatment:
5 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five males
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
- All animals were weighed daily and feed consumption was noted weekly.
- On Days 6 and 27, approximately 24 hours after the 5th and 20th doses, respectively, animals from the appropriate groups were weighed, anaesthetized by ethyl ether inhalation, and killed by exsanguinations from the posterior vena cava. The liver and kidneys were removed and weighed.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.

HISTOPATHOLOGY: Yes

-Transverse sections were fixed and preserved in 10% neutral buffered formalin and stained with haematoxylin/eosin. Additional kidney sections were treated with Mallory’s Heidenhain stain.
-Tissues from right kidney of three control and three d-limonene-treated (150 mg/kg bw/day) animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation.
Clinical signs:
no effects observed
Description (incidence and severity):
daily in-life observation did not reveal any grossly evident indication of dose-related toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight gains values for treatment groups were, at both time points, similar to those of the vehicle control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption values for treatment groups were, at both time points, similar to those of the vehicle control groups.
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 specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Examination of animals at necropsy did not reveal any grossly evident indication of dose-related toxicity.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-related hyaline droplet formation associated with renal accumulation of α2μ-globulin was observed in all rats killed on Day 6.
Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male
Basis for effect level:
other: nephrotoxicity and accumulation of hyaline droplets (male rat specific)
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Key result
Critical effects observed:
not specified
Conclusions:
As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study., but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Executive summary:

In a subacute toxicity study, d-limonene was administered through gavage to groups of 5 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 75, 150 and 300 mg/kg bw/day for 1 or 4 weeks (5 days/week). Animals were weighed daily and feed consumption was recorded weekly. On Days 6 and 27, approximately 24 hours after the 5th and 20th doses, respectively, animals from the appropriate groups were subjected to gross necropsy during which weights of liver and kidneys were recorded and transverse sections were processed for histological examination. Tissues from right kidney of animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation. Neither daily in-life observation nor examination of animals at necropsy revealed any grossly evident indication of dose-related toxicity. Weight gains and feed consumption values for treatment groups were similar to those of the vehicle control groups. Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27. Dose-related hyaline droplet formation associated with renal accumulation of α2µ-globulin was observed in all rats killed on Day 6. Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27. As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Test method and results not sufficiently detailed. Study designed for the assessment of kidney effects of d-limonene
Principles of method if other than guideline:
- Principle of test: 91-day subchronic toxicity study: d-limonene (0, 150, 300, 600, 1200 and 2400 mg/kg bw/day) was administered orally (gavage) to rats for 91 days and kidney sections of the surviving male rats were processed for histological examination.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
91 days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Dose / conc.:
1 200 mg/kg bw/day (nominal)
Dose / conc.:
2 400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- At 0 and 1200 mg/kg bw/day: 10 males and 5 females
- At 150-600 mg/kg bw/day: 10 males
- At 2400 mg/kg bw/day: 5 males and 1 female
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
- Mortality and body-weight data, in-life clinical observations and renal histopathological data were derived from the Prechronic Test Phase Review and Addendum supplied by the NCI.
Sacrifice and pathology:
- Histopathology: Kidney sections (5 µm) of male rats were processed, stained with haematoxylin and eosin and scanned for lesion count, hyaline droplets and other more subtle alterations.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute mean bodyweight gain at 600, 1200 and 2400 mg/kg bw/day decreased by 12, 19 and 46%, respectively, relative to controls.
Food consumption and compound intake (if feeding study):
not specified
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 specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-related increase in the severity of chronic nephrosis and granular cast formation were observed in male rats at concentrations ranging from 150 to 1200 mg/kg bw/day. At 2400 mg/kg bw/day, the severity of chronic nephrosis was similar to that at 150 mg/kg bw/day and formation of granular cast was observed in 1/10 male.
- Chronic nephrosis was characterised by cytoplasmic basophilia of PCT epithelial cells, tubular hyperplasia or atrophy, fibrosis of Bowman’s capsule and an interstitial fibrolymphocytic response.
- Chronic nephrosis was much more severe in the kidneys of all the male rats treated with d-limonene than in the controls.
- No lesions were observed in kidneys of female rats.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Basis for effect level:
other: chronic nephrosis and granular cast formation were observed at all dose levels
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Key result
Critical effects observed:
not specified
Conclusions:
As chronic nephrosis and granular casts formation were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Executive summary:

In a subchronic toxicity study, d-limonene was administered through gavage to groups of male and female rats at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 91 days. Surviving animals from the appropriate groups were subjected to gross necropsy and transverse sections of right and left kidneys were processed for histological examination. Absolute mean bodyweight gain at 600, 1200 and 2400 mg/kg bw/day decreased by 12, 19 and 46%, respectively, relative to controls. Dose-related increase in the severity of chronic nephrosis and granular cast formation were observed in male rats at concentrations ranging from 150 to 1200 mg/kg bw/day. At 2400 mg/kg bw/day, the severity of chronic nephrosis was similar to that at 150 mg/kg bw/day and formation of granular cast was observed in 1/10 male. Treatment-related lesions were not observed in kidneys of female rats. As chronic nephrosis and granular casts formation were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 1979 - October 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 407 but with deviations: study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 24.4-26.2 g; females: 19.6-21.9 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-76 °F
- Humidity (%): 80-90%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
12 doses over 16 days
Frequency of treatment:
Once per day; 5 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
413 mg/kg bw/day (nominal)
Dose / conc.:
825 mg/kg bw/day (nominal)
Dose / conc.:
1 650 mg/kg bw/day (nominal)
Dose / conc.:
3 300 mg/kg bw/day (nominal)
Dose / conc.:
6 600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Random
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on six mice from survivors of highest dose groups
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- No compound-related clinical signs were observed in mice that received 1650 mg/kg bw/day and lived to the end of the studies.
Mortality:
mortality observed, treatment-related
Description (incidence):
- All but one of 20 mice that received 3300 or 6600 mg/kg bw/day died within 3 days.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Vehicle control mice gained little or no weight.
- Slight bodyweight loss was observed in all treated groups, but without any clear dose-response relationship.
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:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- No treatment-related histopathologic lesions were observed.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality at 3300 and 6600 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
3 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
not specified

Table 1: Survival and mean body weights of mice in the 16-day gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (grams)

Final weight relative to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

 5/5

25.4 ± 0.4

26.0 ± 0.9

0.6 ± 0.9

-

413

 5/5

25.2 ± 0.5

23.0 ± 0.9

 -2.2 ± 0.8

88.5

825

 5/5

24.6 ± 0.2

24.2 ± 0.7

-0.4 ± 0.6

93.1

1650

(d) 4/5

25.6 ± 0.6

25.3 ± 1.3

-0.5 ± 1.7

97.3

3300

(e) 1/5

24.8 ± 0.4

19

-5

73.1

6600

(f) 0/5

24.6 ± 0.2

(g)

(g)

(g)

Female

0

 5/5

20.2 ± 0.2

21.8 ± 1.1

 1.6 ± 1.1

-

413

 5/5

21.4 ± 0.5

20.8 ± 0.5

 -0.6 ± 0.5

95.4

825

 5/5

20.0 ± 0.3

19.8 ± 0.6

 -0.2 ± 0.4

90.8

1650

 (h) 4/5

21.2 ± 0.4

22.3 ± 0.6

1.0 ± 1.0

102.3

3300

 (i) 0/5

20.4 ± 0.4

(g)

(g)

(g)

6600

 (j) 0/5

19.8 ± 0.2

(g)

(g)

(g)

(a) Number surviving/number initially in group

(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d) Day of death: 2

(e) Day of death: 2, 2, 3, 3

(f) Day of death: all 1

(g) No data are reported due to the 100% mortality in this group.

(h) Death due to gavage error

(i) Day of death: 1, 2, 2, 2, 2

(j) Day of death: 1, 1, 1, 2, 2

Conclusions:
Under the test conditions, the NOAEL in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.
Executive summary:

In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene was administered through gavage to groups of five B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on six mice from survivors of highest dose groups. All but one of 20 mice that received 3300 or 6600 mg/kg bw/day died within 3 days. Vehicle control mice gained little or no weight. Slight and not treatment- related bodyweight loss was observed in all treated groups. No compound-related clinical signs were observed in mice that received 1650 mg/kg bw/day and lived to the end of the studies. No treatment-related histopathologic lesions were observed. Under the test conditions, the NOAEL in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 1979 - October 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 407 but with deviations: study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 109-117 g; females: 97-103 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 12 or 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-76 °F
- Humidity (%): 80-90%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
12 doses over 16 days
Frequency of treatment:
Once per day; 5 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
413 mg/kg bw/day (nominal)
Dose / conc.:
825 mg/kg bw/day (nominal)
Dose / conc.:
1 650 mg/kg bw/day (nominal)
Dose / conc.:
3 300 mg/kg bw/day (nominal)
Dose / conc.:
6 600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Random
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on seven rats from survivors of highest dose groups
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower.
Mortality:
mortality observed, treatment-related
Description (incidence):
- All rats in 6600 mg/kg bw/day group and 5/5 males and 3/5 females in 3300 mg/kg bw/day group died within the first 2 days.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls.
- Final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle 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:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
825 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: mortality at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 1650 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mortalities at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 3300 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
3 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
body weight and weight gain
Key result
Critical effects observed:
not specified

Table 1: Survival and mean body weights of rats in the 16-day gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (grams)

Final weight relative
to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

  5/5

115 ± 2

173 ± 3

58 ± 3

-

413

  5/5

113 ± 2

171 ± 4

58 ± 3

99

825

  5/5

113 ± 3

173 ± 4

60 ± 5

100

1650

  5/5

113 ± 3

156 ± 4

43 ± 3

90

3300

 (d) 0/5

114 ± 2

(e)

(e)

(e)

6600

 (f) 0/5

111 ± 2

(e)

(e)

(e)

Female

0

  5/5

98 ± 1

123 ± 1

25 ± 1

-

413

  5/5

101 ± 2

(g) 139 ± 2

38 ± 3

113

825

  5/5

100 ± 2

131 ± 3

31 ± 4

107

1650

  5/5

101 ± 2

127 ± 1

27 ± 2

103

3300

 (f) 2/5

102 ± 2

113 ± 8

10 ± 5

92

6600

 (f) 0/5

103 ± 4

(e)

(e)

(e)

(a) Number surviving/number initially in the group

(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d)Day of death: 1, 1, 1, 1, 2

(e) No data are reported due to the 100% mortality in this group.

(f) Day of death all 1

(g) One final body weight not recorded; weight change based on remaining four animals.

Conclusions:
Under the test conditions, the NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.
Executive summary:

In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene was administered through gavage to groups of 5 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on seven rats from survivors of highest dose groups. All rats that received 6600 mg/kg bw/day and 5/5 males and 3/5 females that received 3300 mg/kg bw/day d-limonene died within the first 2 days. The final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls. The final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls. No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower. No treatment-related lesions were observed. Under the test conditions, the NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 1980 - April 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: Males: 23.8-29.5 g; females: 20.2-21.5 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Apparatus: Periodic analysis for d-limonene in dose preparations was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/min, and a flame ionization detector.
- Sampling frequency: Once
- Results: 101-109% of the target concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once per day; 5 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gallbladder, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea and urinary bladder.
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Rough hair coats and decreased activity were observed at 1000 and 2000 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
- 1/10 male and 2/10 females died at 2000 mg/kg bw/day
- 1/10 female died at 500 mg/kg bw/day
- Several animals in other groups died as a result of gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females.
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 specified
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:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality at 2000 mg/kg bw/day; decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity) at 1000 and 2000 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Key result
Critical effects observed:
not specified

Table 1. Survival and mean body weights of mice in the 13-week gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (grams)

Final weight relative to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

10/10

 26.6 ± 1.0 

 37.1 ± 1.0 

 +10.5 ± 1.3 

-

125

10/10

 28.8 ± 0.7 

 37.9 ± 1.1 

 +9.1 ± 0.7 

102.2

250

(d) 9/10

 26.5 ± 0.8 

 33.9 ± 0.8 

 +7.6 ± 0.8 

91.4

500

(d) 7/10

 24.7 ± 0.9 

 34.4 ± 0.9 

 +9.7± 1.1 

92.7

1000

(d) 9/10

 28.2 ± 0.9 

 33.3 ± 0.8 

 +5.1 ± 1.1 

89.8

2000

(e) 9/10

 27.7±0.7 

 33.0 ± 0.8 

 +5.6 ± 0.8 

88.9

Female

0

10/10

 21.3 ± 0.2 

 24.7±0.5 

 +3.4 ±0.4 

-

125

(d) 9/10

 20.6 ± 0.3 

 25.9± 0.5 

 +5.2 ± 0.4 

104.9

250

10/10

 20.7 ± 0.3 

 25.4 ± 0.6 

 +4.7 ± 0.4 

102.8

500

(f) 9/10

 20.9 ± 0.2 

 24.9 ±0.5 

 +4.1 ± 0.4 

100.8

1000

10/10

 20.4 ±0.2 

 24.1 ± 0.7 

 +3.7 ±0.7 

97.6

2000

(g) 8/10

 21.0 ± 0.3 

 24.1 ± 0.4 

 +3.4 ± 0.3 

97.6

(a) Number surviving/number initially in group

(b) Initial group mean body weight f standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d) Death due to gavage error

(e) Week of death: 1

(f) Week of death: 5

(g) Week of death: 3,4

Conclusions:
Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
Executive summary:

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the studies. Several animals in other groups died as a result of gavage error. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day. Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 1980 - April 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 136-153 g; females: 101-120 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 or 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Apparatus: Periodic analysis for d-limonene in dose preparations was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/min, and a flame ionization detector.
- Sampling frequency: Once
- Results: 101-109% of the target concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once per day; 5 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
1 200 mg/kg bw/day (actual dose received)
Dose / conc.:
2 400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals and all female rats in the 1200 mg/kg bw/day group. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Kidneys examined for all male rats.
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
- 5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls.
- Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle 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 specified
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:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups.
- Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium.
- Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Sex:
male
Basis for effect level:
other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
Remarks on result:
other: no NOAEL identified
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
Key result
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
not specified

Table 1. Survival and mean body weights of rats in the 13-week gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (g)

Final weight relative to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

10/10

 144 ± 2 

 333 ± 6 

 +189 ± 5 

-

150

10/10

 145 ± 3 

 332 ± 4 

 +187 ± 3

100

300

10/10

 149 ±2 

 330 ± 3 

 +181 ± 4

99

600

10/10

 148 ± 2 

 314± 5 

 +166 ± 5

94

1200

10/10

 139 ± 3 

 292 ± 5 

 +153 ± 6

88

2400

(d) 5/10

 150 ±3 

 255 ± 10 

 +103 ± 10 

77

Female

0

10/10

118 ± 2

185 ± 2

+67± 4

-

150

10/10

115 ± 1

186± 2

+71± 2

101

300

10/10

105 ± 4

181 ± 2

+76± 4

98

600

10/10

114 ± 1

184± 2

+70± 1

99

1200

10/10

116 ± 2

182 ± 3

+66± 3

98

2400

(d) 1/10

113 ± 1

164

 + 56

89

(a) Number surviving/number initially in group

(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d) Week of death: all 1

 

Table 2. Severity of kidney lesions in male rats in the 13-week gavage study of d-limonene (a)

 

Lesion 

Dose (mg/kg)

 Vehicle Control 

 150 

 300 

 600 

1200

2400

 Regeneration

 (b) 0.8 

 2.4 

 2.5 

 2.5 

 3.7 

0.9

Granular casts

 0 

 1.6 

 2.4 

 2.7 

 3.5 

 0.3 

(a) Severity grades: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

(b) Average severity grade for all rats in the group

Conclusions:
Under the test conditions, the NOAEL for male and female rats was considered to be 600 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
Executive summary:

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.

Under the test conditions, the NOAEL for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats had primarily been identified in this study.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 January 1991 - 02 May 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD guideline 407 and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Box reason SPF breeding
- Age at study initiation: Approximately 6 weeks
- Housing: In air-conditioned spaces, in Makrolon cages (type 4) on granulate softwood, in groups of 5 animals.
- Diet (e.g. ad libitum): Rat diet Altromin 1324 (Altromin GmbH, Lage / Lippe), ad libitum, except the time in which the animals were in metabolism cages
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum, except the time in which the animals were in metabolism cages
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 10.01.1991 To: 07.02.1991
Route of administration:
oral: gavage
Vehicle:
other: Sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared daily, immediately before the administration. The test material was homogeneously dispersed using a magnetic stirrer.
Volume administered: 5 mL/kg bw

VEHICLE
Oleum Sesami, Ph. Eur III, Fa Mainland, Pharmazeutische Fabrik GmbH, Ffm.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 Days
Frequency of treatment:
Daily
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Remarks:
(1.25 % w/v)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
(5 % w/v)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
(20 % w/v)
No. of animals per sex per dose:
5 male and 5 female rats per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Randomization: Animals in cages randomisation = 946/90 and 947/90
Cages in the rack randomisation = 949/90
Observations and examinations performed and frequency:
In all experimental groups, the behavior and general health of animals were observed twice daily during the experiment, once a day on weekends and holidays. Every week neurological disturbances, clouding of the ocular media, adverse effects on oral mucosa and tooth development disorders were studied.
The body weight was determined at the beginning of the experiment and twice a week.
Food consumption was determined twice a week and w ater consumption, once a week.
At the end of the experiment, the bllod count was investigated without prior food deprivation in all male and female animals. The blood was taken from the retro-orbital venous plexus. The following hematological parameters were determined: erythrocyte count, hemoglobin, hematocrit, MCV, MCH, MCHC, WBC, platelet count, differential count, reticulocyte count, Heinz'sche inner body, clotting time.
The urine tests were performed on all male and female animals and took place overnight from day 26 to 27 of the experimental period. The following parameters were determined: Appearance, color, pH, hemoglobin, protein, glucose, ketone body, bilirubin, urobilinogen, density and sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
At the end of the study, animals were macroscopically examined. Alterations in organs were recorded, organs were weighed and their relative weights calculated. Histological preparations from the main organs were examined for microscopic changes. Chemical analysis were performed. The following parameters were analyzed: sodium, potassium, anorg. Phosphorus, uric acid, total and direct bilirubin, creatinine, serum glucose, urea nitrogen (BUN), calcium, chloride, AST (GOT), ALT (GPT), alkaline phosphatase (AP), gamma-glutamyl transferase (GGT), total protein, albumin.
Statistics:
Body weight, hematological and clinical tests, and organ weights (absolute and relative) were statistically compared with the control group.
The analysis were carried out using a program package for evaluation of toxicological tests, according to the Standard Operating Procedure (Department of Pharmaceutical Research).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The highest dose group (1000 mg/kg bw) showed an increased salivation. Behaviour and general health from other treated groups were not significantly different from control group.
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight was not affected by the administration of the test substance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not affected by the administration of the test substance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The water consumption was not affected by the administration of the test substance.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological tests revealed no evidence of compound-related toxicity.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In male animals from the highest dose group, clinical chemistry tests revealed an increase in urea nitrogen levels and a decrease in phosphorus levels.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urine was normal and showed no evidence of compound-related toxicity.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behaviour and general health from other treated groups were not significantly different from control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In male and female animals from the highest dose group, absolute and relative liver weights were increased.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examinations showed spotted kidneys in two male animals from the lowest dose group (62.5 mg / kg bw). In 3 mid-dose male animals and in all males from the highest dose group, colourless kidneys were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals from the highest dose group showed an increased vacuolization of the hepatocytes. Female animals from dose groups of 62.5 and 250 mg / kg bw did not show toxic effects. In all dose groups of male rats deposits of the test substance in the epithelium of the proximal renal tubules associated with necrosis of single cells have been observed. These effects seem to be specific for male rats and contingent upon alpha-2 microglobinemia. The renal toxic effects found in all dose levels groups in male rats are interpreted as uniquely specific for male rats, and as having no relevance for other animal species and humans.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
NOEL
Effect level:
< 62.5 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: renal toxic effects
Critical effects observed:
not specified
For female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day).
Conclusions:
For female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day).
Executive summary:

Camphene was daily administered to SPF Wistar rats (male and female) for 28 days at doses of 0, 62.5, 250 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. In all experimental groups, behaviour and general health were daily examined. The body weight and food consumption were determined twice a week, water consumption was determined once a week. Haematological and clinical tests, and urinalysis were also carried out. At the end of the study, animals were macroscopically examined. Alterations in organs were determined, organs were weighed and their relative weights calculated. Histologicalpreparations from the main organs were examined for microscopic changes. Body weight, haematological and clinical tests, and organ weights (absolute and relative) were statistically compared with the control group.

The highest dose group (1000 mg/kg bw/day) showed an increased salivation. Behaviour and general health from other treated groups were not significantly different from control group. The body weight and food- and water-consumption were not affected by the administration of the test substance.

Haematological tests revealed no evidence of compound-related toxicity. In male animals from the highest dose group, clinical chemistry tests revealed an increase in urea nitrogen levels and a decrease in phosphorus levels. The urine was normal and showed no evidence of compound-related toxicity.
In male and female animals from the highest dose group, absolute and relative liver weights were increased.

Macroscopic examinations showed spotted kidneys in two male animals from the lowest dose group (62.5 mg / kg bw/day). In 3 mid-dose male animals and in all males from the highest dose group, colourless kidneys were observed. Animals from the highest dose group showed an increased vacuolization of the hepatocytes. Female animals from dose groups of 62.5 and 250 mg / kg bw/day did not show toxic effects. In all dose groups of male rats deposits of the test substance in the epithelium of the proximal renal tubules associated with necrosis of single cells have been observed. These effects seem to be specific for male rats and contingent upon alpha-2 microglobinemia. The renal toxic effects found in all dose levels groups in male rats are interpreted as uniquely specific for male rats, and as having no relevance for other animal species and humans.

Based on the results of this study, for female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Well-conducted study but reported in Japanese language
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: A combination of rules from OECD Guidelines 409 and 452 were followed (by anticipation as thoses guidelines did not exist when the study was conducted).
- Short description of test conditions: d-limonene was administered by gavage to dogs for 6 months. This duration was chosen as usual when developing new medicinal products (d-limonene was explored in this study as a possible gallstone solubiliser).
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
other: Japanese beagle
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Details on oral exposure:
no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 months
Frequency of treatment:
once a day
Dose / conc.:
0.4 other: mL/kg-bw/day
Remarks:
Equivalent to 340 mg/kg-bw/day
Dose / conc.:
1.2 other: mL/kg-bw/day
Remarks:
Equivalent to 1000 mg/kg-bw/day
Dose / conc.:
3.6 other: mL/kg-bw/day
Remarks:
Equivalent to 3000 mg/kg-bw/day
No. of animals per sex per dose:
3/sex/dose
Control animals:
yes
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: yes

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Frequent vomiting and nausea were caused, which appeared to depend on the dose used.
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All males in the high dose group, all females in the intermediate group and 2/3 females in the high dose group lost weight over the 6 month-study. Not dose-related in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Not affected by treatment except in the intermediate dose group females. Not dose-related.
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
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Total cholesterol and glucose decrease in blood in the high dose group. The initial cholesterol level was recovered at the end of the 6-month treatment period in both sexes (when the level had increased in other control and treated groups).
Urinalysis findings:
effects observed, non-treatment-related
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 relative to body weight kidney and liver weights were slightly higher in the high dose group males than in other groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 1.2 and 3.6 mL/kg bw/day, protein casts were observed in the renal tubule of most animals. No remarkable treatment-related change was observed in other organs.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased body weight and protein casts observed in the renal tubule at 3000 mg/lg bw/d.
Key result
Dose descriptor:
LOAEL
Effect level:
3 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased body weight and protein casts observed in the renal tubule at 3000 mg/lg bw/d.
Key result
Dose descriptor:
NOAEL
Effect level:
340 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased body weight and protein casts observed in the renal tubule at 1000 mg/lg bw/d.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased body weight and protein casts observed in the renal tubule at 1000 mg/lg bw/d.
Key result
Critical effects observed:
not specified

Table 1: Protein casts observed in the renal tubule

  Male Female
  Control 0.4 1.2 3.6 Control 0.4 1.2 3.6
No. animals examined 3 3 3 3 3 2 3 3
Protein casts 1 1 2 3 1 2 3 3
Conclusions:
The NOAEL in this study is 1.2 mL/kg bw/day (equivalent to 1000 mg/kg bw/day) in males and 0.4 mL/kg bw/day (equivalent to 340 mg/kg bw/day) in females on the basis of decreased body weight and protein casts observed in the renal tubule at the next dose level. Food consumption was also decreased in the intermediate dose females.

Executive summary:

Three dogs per sex and per dose group were administered d-limonene by gavage once per day for 6 months at the dose level of 0, 0.4, 1.2 or 3.6 mL/kg bw/day. All 6 animals (males and females) from the high dose group except one female and all females in the intermediate dose group lost weight between the first and the last day of study. Food consumption only decreased in the intermediate dose group females. Urinalysis and hematology were not affected by treatment. The glucose and total cholesterol levels in blood decreased in the high dose group males and females when compared to the pre-treatment levels; the total cholesterol level recovered the pre-test level by the end of the 6 -month treatment period. The relative to body weight kidney and liver weights were slightly higher in the high dose group males than in other groups. A dose-related increased incidence of protein casts were observed in the renal tubule: all males in the high dose group and all females in the intermediate and high dose groups showed this effect.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study designed to evaluate effects of substance on kidneys of rats: dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed
Principles of method if other than guideline:
- Principle of test: Study designed to evaluate effects of substance on kidneys of rats: dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
Additional information: D-limonene used for preliminary acute study:
Radiolabelled d-limonene [9-14C]
- Source: Wizard Laboratories (Davies, CA, USA)
- Radiochemical purity (if radiolabelling): 99% (GC)
- Specific activity (if radiolabelling): 8.7 mCi/mmol
Species:
rat
Strain:
Fischer 344
Sex:
male
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- At 0, 10 and 75 mg/kg bw/day: 5 or 10 males
- At 2, 5 and 30 mg/kg bw/day: 10 males
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Preliminary acute toxicity study:
- D-limonene (200 mg/kg bw; 200 µCi/kg bw in corn oil) was administered to a group of male and female Fischer 344 rats by oral gavage. After 24 hours, all animals were sacrificed and kidney tissues were processed for histological examinations and 2D-gel electrophoresis.

Subchronic toxicity study:
- Rats were observed daily during the experimental period for signs of toxicity.
- Body weights were recorded before each daily administration of d-limonene and at the time of sacrifice.
- Feed consumption values were recorded weekly throughout the study and were used to determine weekly body-weight gain and feed efficiency values.
- Interim necropsies were conducted on 5 animals/group on Days 8 and 15 (group 4), and Days 8, 15, 22 and 29 (groups 1 and 6). All remaining rats (10/group) were sacrificed at the end of the 91-day study. Liver and kidneys were isolated and weighed. All tissues were collected and fixed in 10% neutral-buffered formalin for routine histological processing and light microscopic evaluation of sections stained with H&E. An additional kidney section was also stained with Mallory's Heidenhain stain and examined for hyaline droplets under light microscopy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The cumulative body-weight gain for treated males did not differ significantly from those of the control males.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption for treated males did not differ significantly from those of the control males.
Food efficiency:
no effects observed
Description (incidence and severity):
Feed efficiency for treated males did not differ significantly from those of the control males.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg/kg bw/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Incidence and type of gross pathological lesions observed at necropsy for treated males did not differ significantly from those of the control males.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at 10 mg/kg bw/day.
Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: chronic nephrosis and a dose-related trend in the increased relative weights of the kidney and liver were observed at 30 and 75 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: chronic nephrosis and a dose-related trend in the increased relative weights of the kidney and liver were observed at 30 and 75 mg/kg bw/day
Key result
Critical effects observed:
not specified

Preliminary acute toxicity study:

- An increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2µ-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [14C]d-limonene.

 

Conclusions:
Under the test conditions, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, based on observation of chronic nephrosis. Mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Executive summary:

In a subchronic toxicity study, d-limonene was administered through gavage to groups of 5 or 10 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 2, 5, 10, 30 and 75 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed and weighed daily, and feed consumption was recorded weekly. Rats from selected dose groups received interim necropsies from Days 8-29, while all groups were necropsied at the end of the study. In the preliminary acute toxicity study, d-limonene (200 mg/kg bw; 200 µCi/kg bw in corn oil) was administered to a group of male and female Fischer 344 rats by oral gavage. After 24 hours, an increase in the incidence and severity of hyaline droplets containing alpha-2µ-globulin was observed in the kidneys of males only. In the main study, incidence and type of gross pathological lesions observed at necropsy, the cumulative body-weight gain, feed consumption and feed efficiency for treated males did not differ significantly from those of the control males. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg/kg bw/day. Histological examination of kidney tissue confirmed induction of chronic nephrosis characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose.

Under the test conditions, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, but Mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD Guideline 409 with deviations: age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
yes
Remarks:
age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
GLP compliance:
not specified
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-15 months
- Housing: Housed in runs with outdoor access
- Diet (e.g. ad libitum): Meals provided for only 1 hour
- Water (e.g. ad libitum): Ad libitum
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was directly dosed by gavage in doses of 0.12 and 1.2 ml/kg body weight/day.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
180 days
Frequency of treatment:
Each daily dose was divided into two equal amounts, administered at approximately 9.30 am and 2.30 pm.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
(1.2 ml tap-water/kg bw/day)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
(0.12 ml test item/kg bw/day)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
(1.2 ml test item/kg bw/day)
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Highest daily dose was determined in a pilot 28-day study conducted in 5 male and 5 female beagles. Kidney weights for the d-limonene-treated animals were unaffected, but absolute and relative liver weights were slightly increased. Based on these findings, the 1.2 mL/kg bw/day treatment was chosen.
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for at least 1 hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: At study initiation, weekly during the study and at the time of sacrifice

FOOD CONSUMPTION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters examined: White cell count, red blood cell count, haemoglobin, haematocrit, platelet count, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Animals fasted: No data
- Parameters examined: Blood urea nitrogen (BUN), BUN/creatinine, creatinine, aspartate amino transferase, alanine amino transferase, phosphorus, glucose, albumin, total protein, globulins, albumin/globulin, alkaline phosphatase, cholesterol, triglycerides, sodium, potassium, calcium and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: 24-hour urine samples were collected at approximately 2 week pre-study and again at 6 months.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Colour and appearance, specific gravity, occult blood, protein, pH, glucose, ketones, bilirubin and urobilinogen and urinary sediment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; each animal anaesthetized with pentobarbital, killed by exsanguination and observed for gross post-mortem examinations
HISTOPATHOLOGY: Yes; samples of the following tissues were collected and fixed in 10% buffered formalin for routine histological processing, and light microscopical evaluation of haematoxylin and eosin stained sections: lungs, bronchial lymph node, heart, thoracic aorta, tongue, oesophagus, trachea, thyroid, parathyroid, submandibular lymph node, mesenteric lymph node, stomach, parotid salivary gland, palatine tonsil, liver, gall bladder, duodenum, jejunum, ileum, colon, rectum, urinary bladder, kidneys, testicles with epididymis, prostate, ovaries, uterus, vagina, cervix, adrenals, thymus, psoas muscle, spleen, pancreas, bone/marrow, skin, brain, spinal cord, sciatic nerve, pituitary gland, and eyes. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation. Kidney weights were determined immediately on removal from the animal. Absolute organ weights were calculated as percentages of the body weights (relative weights).
Other examinations:
None
Statistics:
- Statistically significant differences (P < 0.05, two-sided risk level) were established using least significant difference criteria, provided that Bartlett's test of homogeneity of variance was nonsignificant.
- Dose-response data were also analysed by linear regression (Dixon and Massey, 1969).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Excretion of soft faeces; dose-related occasional mild discomfort during defaecation (presumably because of perianal contact with unabsorbed d-limonene as it passed with the faeces); sporadic episodes of emesis and diarrhoea
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs.
Urinalysis findings:
no effects observed
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):
Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased absolute and relative female kidney weight and relative male kidney weight at 1000 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
not specified

Table 1. Organ and bodyweight data for dogs after 6 months daily administration of d-limonene

 

 

 Dose (mg/kg bw/day) 

 

 Control 

 100 

 1000

Male

 Final body weight (kg) 

 11.381 ± 0.828 

 10.889 ± 0.595 

 11.008 ± 0.688 

 Kidney weight (g) 

 57.09 ± 6.02 

 64.56 ± 6.60 

 73.33 ± 8.91 

 Kidney/body weight (%) 

 0.498 ± 0.023 

 0.588 ± 0.035 

 0.661 ± 0.61*

Female

 Final body weight (kg) 

 9.158 ± 0.789 

 9.513 ± 0.315 

 9.176 ± 0.823 

 Kidney weight (g) 

 42.18 ± 3.48 

 45.61 ± 2.29 

 55.36 ±2.58* 

 Kidney/body weight (%) 

 0.461 ± 0.006 

 0.479 ± 0.016 

 0.614 ± 0.032* 

* Statistical significance at P < 0.05,

Values are means ± SEM for 5 dogs.

Conclusions:
Under the test conditions, the NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.

Executive summary:

In a 6-month subchronic toxicity study performed similarly to OECD Guideline 409, d-limonene was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (0, 100 or 1000 mg/kg bw/day) in two divided doses for 180 days. Animals were observed daily and weighed at study initiation, weekly during the study and at the time of sacrifice. Feed consumptions were determined throughout the study and blood samples were obtained at 2 week pre-study (baseline) and then 1, 3 and 6 months during the study. At termination all animals were subjected to gross necropsy during which weights of kidneys were recorded and several tissues were processed for microscopical evaluation of haematoxylin and eosin stained sections. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation.

 

Feed consumption and body weight were unaffected by treatment. Clinical signs of toxicity noted were excretion of soft faeces, dose-related occasional mild discomfort during defaecation, sporadic episodes of emesis and diarrhoea. No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation or any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene.

 

Under the test conditions, the NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test method according to OECD guideline 407 with some modifications.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
No information reported
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Department of Physiology and Pharmacology of Federal University of Pernambuco (UFPE, Pernambuco, Brazil)
- Diet (e.g. ad libitum): industrialized dry food (Presence®, Purina, Brazil), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55-65 %
- Photoperiod: 12 hrs dark / 12 hrs light



Route of administration:
oral: gavage
Vehicle:
other: Tween-80 aqueous solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
1,8-cineole (CIN) was emulsified in a 1% Tween-80 aqueous solution before administration to the animals.
Volume administered: 10 mL/kg bw

VEHICLE
Tween-80 (CAS No: 9005-65-6) was obtained from Sigma-Aldrich® (St. Louis, MO, USA).

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
50 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
100 mg/kg dose was chosen according to previous results which showed significant gastric mucosa protection in the antiulcerogenic tests (Caldas et al., 2015). The others doses (500 and 1000 mg/kg) were extrapolated 5- and 10-fold the amount of the effective dose.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, the animals were observed for signs of toxicity, such as piloerection, diarrhea, changes in locomotor activity or mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 12 h
- Anaesthetic used for blood collection: Yes, thiopental (35 mg/kg, intraperitoneal route)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: red blood cell (RBC) count, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), white blood cell (WBC) count, platelets count, mean platelet volume (MPV) and differential leukocyte count (lymphocytes, monocytes and granulocytes).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 12 h
- Anaesthetic used for blood collection: Yes, thiopental (35 mg/kg, intraperitoneal route)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: glucose, blood urea nitrogen (BUN), creatinine, uric acid, sodium, potassium, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglycerides, gamma glutamyltranspeptidase (GGT), alkaline phosphatase (ALP); total, direct and indirect bilirrubin.



- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After blood collection, all the animals were euthanized in a CO2 chamber (by inhalation) for necropsy and evaluation of vital organs. For macroscopic analysis of organs were randomly selected 10 animals (5 males and 5 females) in each group.
The organs including heart, lung, liver, kidneys, adrenal glands, spleen, stomach, intestine, pancreas, brain and reproductive organs testicles and prostate (male) or uterus and ovaries (female) were carefully removed and weighed individually. Organ weights were expressed in absolute and relative terms (g and g/100 g of body weight, respectively).

HISTOPATHOLOGY: Yes
The remaining animals in each group (10 animals, 5 males and 5 females) were perfused with saline (to remove blood), and then, the organs previously described were removed and fixed "in totum" in 10% buffered formalin for 48h at room temperature. After fixing, each sample was washed with water and immersed in 70% ethyl alcohol for 3 to 4 days, then were embedded in paraffin. Paraffin sections of 5 μm were obtained and stained with hematoxylin/eosin (HE). Histological analyses of organs were made using an automatic microscopy system MICRO DIP® (Kacil Inc.).

Statistics:
Values were expressed as mean ± standard error of mean (S.E.M.) and the differences are analyzed by variance analysis (ANOVA) followed by Dunnett’s test or Student’s T test for unpaired samples. The level of significance for rejection of the null hypothesis was set at 5% (p < 0.05). Statistical analyses were performed using GraphPad Prism 5.0®.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No signs of toxicity (such as piloerection or alteration in locomotor activity) were recorded during the 50 consecutive days of treatment by oral route at doses of 100, 500 or 1000 mg/kg. However, animals treated at doses of 500 and 1000 mg/kg presented diarrhea during the first week of treatment although this ceased from the second week onwards.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Although it was observed a decrease in body weight gain in animals treated with test susbstance at doses of 500 and 1000 mg/kg during the first week, this reduction did not affect the total weight gain of animals, indicating that this response may be associated with the occurrence of diarrhea in rats treated during this period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Changes were observed in the consumption of food during all the treatment in animals of both sexes treated with all doses of test substance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Changes were observed in the consumption of water during all the treatment in animals of both sexes treated with all doses of test substance.

Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
For male rats, there was a significant increase of 6.93% in MCV (1000 mg/kg) and of 43.54 and 38.98% in the platelet count (500 and 1000 mg/kg, respectively) and a decrease of 6.74 and 6.67 % in MCHC (500 and 1000 mg/kg) and MPV of 10.40, 10.60 and 15.73% (100, 500 and 1000 mg/kg, respectively), when compared to the control group. In the female groups, no statistically significant clinical findings were recorded for any of the parameters examined.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No significant differences were observed in the serum levels of glucose, creatinine, uric acid, sodium, potassium, AST, ALT, total cholesterol, triglycerides, gamma glutamyltranspeptidase (GGT), total bilirubin, or direct and indirect bilirubin. However, a decrease was observed in the level of alkaline phosphatase (30.00%) in male rats treated with 100 mg/kg and an increase of 29.27% and 25.86% in the level of urea (BUN) in the female groups treated with 500 and 1000 mg/kg, respectively, in relation to the control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a decrease in absolute weight of the lungs (15.10%) and spleen (20.25%) in males rats at doses of 500 and 1000 mg/kg, respectively, as well as an increase in absolute (32.55%) and relative (39.33%) weight of the liver in females at a dose of 1000 mg/kg compared to control group.

Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic analysis of the target organs of the animals treated with 1,8-cineole did not show significant changes in the color or texture when compared with the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination of the organs showed the presence of eosinophilic and lymphocytic infiltrate in the lungs of males and females and into the uterus of females at all doses and lymphocytic infiltrate in the liver of males treated with all doses and females (500 and 1000 mg/kg). It was also observed increase of the glomerular space in the kidneys, weak for males (1000 mg/kg), and moderate for females (500 and 1000 mg/kg), which was more evident in females. The others organs of male and female animals in the treated and control groups exhibited no changes, with histological findings all within normal limits.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: water consumption and mean platelet volume and alkaline phosphatase
Key result
Critical effects observed:
no

Table 1: Effect of 1,8-cineole (CIN) on hematological parameters in male and female Wistar rats treated orally for 50 days

Parameters

Males

Females

control

100 mg/kg

500 mg/kg

1000 mg/kg

control

100 mg/kg

500 mg/kg

1000 mg/kg

RBC(106/μL)

5.31±0.20

5.79±0.31

6.33±0.49

6.16±0.39

5.63±0.33

5.14±0.13

5.61±0.32

5.89±0.42

Hb(g/dL)

11.30±0.41

12.29±0.48

12.76±0.99

13.06±0.67

12.39±0.59

11.88±0.22

12.39±0.46

13.20±0.78

Ht(%)

27.95±1.10

31.72±1.80

34.04±2.90

34.69±2.16

31.78±2.16

28.91±0.77

31.57±1.72

33.62±2.54

MCV(fL)

52.50±1.07

54.80±0.20

53.71±0.77

56.14±0.40*

56.00±0.68

56.33±0.33

56.11±0.58

56.90±0.56

MCH(pg)

21.36±0.57

21.37±0.35

20.24±0.79

21.26±0.32

22.12±0.37

23.13±0.28

22.26±0.40

22.56±0.35

MCHC(g/dL)

40.49±0.37

39.12±0.74

37.76±1.25*

37.77±0.49*

39.42±0.75

41.13±0.46

39.50±0.69

39.69±0.76

RDW(%)

13.81±0.09

13.67±0.27

14.57±0.24

14.56±0.23

13.84±0.22

13.77±0.10

14.04±0.21

13.73±0.11

WBC(103/μL)

12.99±0.74

11.06±1.06

11.11±1.72

12.69±0.65

9.93±0.65

8.73±0.96

12.28±1.05

8.81±0.49

Platelets(103/μL)

479.50±30.50

490.40±28.68

688.30±28.13*

666.40±38.02*

540.00±30.42

472.60±18.24

609.00±43.58

623.10±29.29

MPV(fL)

7.50±0.27

6.72±0.08*

6.70±0.17*

6.32±0.06*

6.32±0.13

6.35±0.07

6.56±0.26

6.22±0.06

Lymphocytes(%)

57.32±1.23

47.43±6.10

61.66±3.49

59.56±1.57

67.23±2.16

65.70±1.53

63.91±1.66

63.26±1.77

Monocytes(%)

11.98±0.40

15.53±1.54

15.40±3.07

13.51±0.78

10.59±0.73

10.73±0.49

11.70±0.83

11.96±1.12

Granulocytes(%)

30.70±0.96

37.04±4.62

22.94±1.83

26.93±1.30

22.18±1.67

23.57±1.16

24.39±1.00

24.78±0.90

RBC: red blood cell (RBC) count, Hb: hemoglobin, Ht: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: red cell distribution width, WBC: white blood cell, MPV: mean platelet volume. Values represent the mean ± S.E.M (n = 10/group). *Statistically different from control group (ANOVA followed by Dunnett's test, p < 0.05).

Table 2: Histopathological changes observed in male and female Wistar rats treated with 1,8-cineole (CIN) for 50 consecutive days

Organs

Males

Females

control

100 mg/kg

500 mg/kg

1000 mg/kg

control

100 mg/kg

500 mg/kg

1000 mg/kg

Heart

N

N

N

N

N

N

N

N

Lung

N

ELI (+)

ELI (+)

ELI (+)

N

ELI (+)

ELI (++)

ELI (+)

Liver

N

LI (+)

LI (+)

LI (+)

N

N

LI (+)

LI (+)

Kidney

N

N

N

IGS (+)

N

N

IGS (+)

IGS (++)

Adrenal

N

N

N

N

N

N

N

N

Spleen

N

N

N

N

N

N

N

N

Stomach

N

N

N

N

N

N

N

N

Intestine

N

N

N

N

N

N

N

N

Pancreas

N

N

N

N

N

N

N

N

Brain

N

N

N

N

N

N

N

N

Testicle

N

N

N

N

-

-

-

-

Prostate

N

N

N

N

-

-

-

-

Uterus

-

-

-

-

N

ELI (++)

ELI (++)

ELI (+)

Ovary

-

-

-

-

N

N

N

N

Histopathology changes: ELI (eosinophilic and lymphocytic infiltrate), LI (lymphocytic infiltrate) and IGS (increase of the glomerular space). Scores: (N) within normal range, (+) weak, (++) moderate or (+++) intense.

Conclusions:
In a short term repeated dose toxicity study, oral routed performed with 1,8-cineole using Wistar rats for a exposure time of 50 days, occasional alterations in rats of both sexes were observed, as evidenced in hematological and biochemical parameters and histopathological analysis. However, these changes showed low clinically relevant, since they occurred in a non-generalized manner between animals of the treated groups. Furthermore, a NOAEL was not established since effects were found at the lowest dose tested (100 mg/kg bw/day).




Executive summary:

1,8-cineole was daily administered to Wistar rats (male and female) for 50 days at doses of 0, 100, 500 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. In all experimental groups, animals were observed for signs of toxicity, such as piloerection, diarrhea, changes in locomotor activity or mortality. Body weight, food and water consumption were determined once a week. Haematological and clinical tests were also carried out. At the end of the study, animals were macroscopically examined. Alterations in organs were determined, organs were weighed and their relative weights calculated. Histological preparations from the main organs were examined for microscopic changes. Body weight, haematological and clinical tests, and organ weights (absolute and relative) were statistically compared with the control group.

No deaths or significant clinical signs of toxicity were reported during the study. Although it was observed a decrease in body weight gain in animals at doses of 500 and 1000 mg/kg during the first week, this reduction did not affect the total weight gain of animals, indicating that this response may be associated with the occurrence of diarrhea in rats treated during this period. Furthermore, oscillations in the consumption of water and food observed did not prevent an increase of the body weight from the second week of treatment in animals of both sexes with all doses.

Haematological tests revealed an increase in MCV and platelet count and a decrease in MCHC and MPV in male animals treated with 500 and 1000 mg/kg. Biochemical parameters showed a decreased level of alkaline phosphatase in male rats treated with 100 mg/kg and a slight increase in the level of urea in the group of females treated with 500 and 1000 mg/kg in relation to the control group. However, these values lie within the physiological limits described for the species (Harkness and Wagner, 2010).

There was a decrease in the absolute mass of the spleen and lungs in males (500 and 1000 mg/kg, respectively) and an increase in the absolute and relative mass of the liver in the females (1000 mg/kg). Although no change in the color or texture of organs has been found in rats of both sexes, the microscopic analysis showed eosinophilic and/or lymphocytic infiltrate in the lungs and liver of animals of both sexes and uterus of females treated with all doses. Moreover, an increase of the glomerular space in the kidneys of males females treated with highest doses was observed.

In conclusion, occasional alterations in rats of both sexes were observed, as evidenced in hematological and biochemical parameters and histopathological analysis. However, these changes showed low clinically relevant, since they occurred in a non-generalized manner between animals of the treated groups.

Since the lower dose (100 mg/kg) induced reduction of water consumption and mean platelet volume and alkaline phosphatase, it was not possible to establish the NOAEL.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Remarks:
(The experiment was carried out following the Regulations of Animal Experimentation of College of Veterinary Medicine, Sichuan Agricultural University, which is based on the Guidelines of the International Committee on Laboratory Animals)
Limit test:
no
Species:
mouse
Strain:
other: Kunming (a closed strain coming from Kunming, Yun nan province, P.R. China)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Chengdu Dossy Experimental Animals Co. Ltd. (License No. SCXK (Sichuan) 2008-24)
- Weight at study initiation: 18-22 g
- Housing: no data
- Diet (e.g. ad libitum): ad libitum, Laboratory animals-mice and rats formula feeds
- Water (e.g. ad libitum): ad libitum
- Acclimation period: The test animals were quarantined for 13 days before experiment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22ºC
- Humidity (%): 40-70%
- phothoperiod: 12 h light/dark cycle


Route of administration:
oral: gavage
Vehicle:
other: 2% Tween 80
Details on oral exposure:
VEHICLE
- Amount of vehicle (if gavage): 10 ml total dose/kg/day
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
30 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group I
Dose / conc.:
21.38 mg/kg bw/day (nominal)
Remarks:
Group II
Dose / conc.:
64.15 mg/kg bw/day (nominal)
Remarks:
Group III
Dose / conc.:
192.45 mg/kg bw/day (nominal)
Remarks:
Group IV
No. of animals per sex per dose:
10 animals per dose either sex
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, animal behavior and mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed on day 31. After fasted overnight, all the mice were euthanized. The organs/tissues were carefully examined macroscopically and gross lesions were recorded. The relative weight (organ to body weight ratios) of liver, spleen and double kidneys was immediately calculated.

HISTOPATHOLOGY: Yes
Heart, liver, spleen, lungs, kidney, testis and ovary of treatment groups and control group were excised and then fixed in 10% neutral buffered formalin. Following dehydration and embedding, they were sectioned at 5 μm, stained with Hematoxylin and Eosin (H and E) and examined microscopically.

Ultrastructural observation:

The organs were excised and prefixed in 2.5% glutaraldehyde solution, diced into 1 mm3, followed by three 15 min rinses with 0.1 M phosphate buffer (pH 7.4). Post-fixation was in cold 1% aqueous osmium tetroxide for 1 h. After rinsing with phosphate buffer again, the specimens were dehydrated in a graded ethanol series of 50~100% and then embedded in Epon 812. Ultra-thin sections were sliced with glass knives on a LKB-V ultramicrotome (Nova, Sweden), and stained with uranyl acetate and lead citrate. The sections were examined under a Hitachi H-600 transmission electron microscope.
Statistics:
The statistical significance of differences between means was calculated using One-way Analysis of Variance (ANOVA) followed by Dunnett’s test for multiple comparisons with the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
The animals in all groups were in good condition.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weights of the mice was increasing weekly in 1,8-cineole treated groups and the control group, and there was no significant difference among all the groups (P<0.05).
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 specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant differences in organ weights and relative organ weights between the 1,8-cineole treated groups and the control group after administration for 30 days.

Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 30 days of treatment the obvious pathological changes appeared in the liver and kidney of mice. There were no distinct histopathological changes in the other organs examined in this test.
Liver: Central venous congestion of liver lobule and granular degeneration of hepatocytes were appeared in Group II and III, while in Group IV, more serious pathological changes involving central venous congestion, granular degeneration, vacuolar degeneration and hepatic necrosis appeared.
Kidney: Normal appearances of kidney were appeared in Group I and II, in which glomerulus and renal tubule structure was clearly conserved. Group III and Group IV showed pathological changes: capillary of glomerulus and interstitial angiectasis hyperemia, renal tubular epithelial cells swelling, granular degeneration and partially separating from basement membrane; amount of eosinophilic protein exudation existing in tubular lumen.

Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Ultrastructural observation:
Electron microscope has revealed specific ultrastructural changes in hepatic cells and renal tubular epithelial cells under experimental conditions.
Hepatic cells: After 30 days of administrated with 1,8-cineole (192.45 mg/kg), the endoplasmic reticulum was distorted and fractured. The ribosomal fell off from the rough endoplasmic reticulum membrane and scattered into the cytoplasm. The mitochondria were found to be swollen and the cristae within the mitochondria were disorganized.

Kidney cells: After 30 days of administrated with 1,8-cineole (192.45 mg/kg), some proximal convoluted tubule epithelial cells was interrupted and escape from the cytoplasm to the lumen of the tubule. The usually elongated mitochondria in the basilar portion of the distal convoluted tubule epithelial cells became more spherical, with marked disorganization and swelling of the cristae. However, the basement membrane of mitochondria was intact. No significant alteration was observed in the glomeruli.
Key result
Dose descriptor:
NOAEL
Effect level:
64.15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
192.45 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
192.45 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver

Table 1: The weight and relative organ weight of liver, spleen and kidney in the subacute toxicity study

Dose

Liver

Spleen

Kidney

mg/kg

g

%

g

%

g

%

Group I: 0

0.95±0.03

4.15±0.37

0.09±0.02

0.44±0.14

0.25±0.03

1.16±0.04

Group II: 21.38

0.95±0.07

4.59±0.33

0.08±0.01

0.41±0.02

0.25±0.06

1.18±0.12

Group III: 64.15

1.06±0.11

4.55±0.52

0.08±0.02

0.37±0.11

0.28±0.09

1.22±0.36

Group IV: 192.45

0.95±0.08

4.39±0.26

0.08±0.01

0.39±0.04

0.28±0.05

1.29±0.22

Values are mean±S.D.; n=20. Values are absolute wet weight of organ (g) and relative organ weight (%, per body weight). No significant difference from the control group at P<0.05 (using one-way ANOVA followed by Dunnett’s test for multiple comparisons with the control group).

Conclusions:
The NOAEL of 1,8-cineole after an oral exposure of 30 days was 64.15 mg/kg bw/day in mice.

Executive summary:

A short term repeated dose toxicity study (oral route) was performed on 1,8-cineole according to OECD guideline 407. The substance was emulsified in 2% Tween 80 and administered daily to mice by oral gavage in doses of 0 (control, only 2% Tween 80), 21.38, 64.15 and 192.45mg/kg bw/day during 30 days.Animal behavior, body weight and mortality were recordedduring the study. After being sacrificed on day 31,organs/tissues were examined macroscopically and gross lesions were recorded. Relative weight of liver, spleen and double kidneys was calculated.Heart, liver, spleen, lungs, kidney, testis and ovary of treatment groups and control group were examined microscopically. Also, ultrastructural observation by electron microscopy was performed on those organ cells with effects. There were no significant differences in body weight and relative organ weight between the control group and treatment groups. The histopathological examinations showed that granular degeneration and vacuolar degeneration appeared in liver and kidney tissue after administration of high dose (192.45 mg/kg bw/day). Under electron microscopy, a series of ultrastructural changes were observed. At the subcellular level the influence of test substance was found on the mitochondria, endoplasmic reticulum and other membrane type structure of liver and kidney. Based on these results, the NOAEL of the test substance was determined to be 64.15 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
dog
Quality of whole database:
A weight of evidence approach has been applied. Several experimental studies are available with a Klimisch score of 2.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
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 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, 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

 

** 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.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 March 2005 - 29 June 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
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.
GLP compliance:
yes
Remarks:
In compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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: 96-98 g (male) and 88-89 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 (male rats) or 13 (female rats) 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 ± 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
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 7 (female), day 8 (male), weekly thereafter, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: initially, on day 7 (female), day 8 (male), 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: on days 4 and 23 for clinical pathology rats and at the end of the studies for core study animals
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Not specified
- How many animals: 10 per sex per dose (core study animals) and 10 per sex per dose (clinical pathology rats)
- 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: Yes
- Time schedule for collection of blood: on days 4 and 23 for clinical pathology rats and at the end of the studies for core study animals
- Animals fasted: Not specified
- How many animals: 10 per sex per dose (core study animals) and 10 per sex per dose (clinical pathology rats)
- Parameters examined: Urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts

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 and 200 ppm female rats. In addition, the kidney in the remaining groups and the liver in the remaining groups of male rats 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.
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):
No signs of toxicity (e.g., abnormal breathing or behavior) were noted during clinical observations.
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 with no specific cause of death identified through gross examination or histopathologic analysis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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. All surviving females at 400 ppm lost weight between week 12 and week 14.
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, there were mild exposure-related significant decreases in the leukocyte counts paired with mild significant decreases in the lymphocyte counts in 200 and 400 ppm male rats compared to concurrent controls. 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.
Also, no histopathological findings could be associated to these changes. Therefore, these treatment-related effects can be considered as not toxicologically significant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Alanine aminotransferase activities were significantly decreased in males and females exposed to 50 ppm or greater at week 14. Significantly decreased alanine aminotransferase activities were also seen in 400 ppm male rats on days 4 and 23. Significant decreases in alkaline phosphatase activities were observed in 400 ppm males and 200 and 400 ppm females on day 4 and in males and females exposed to 100 ppm or greater at week 14. The reason for the decreases in these enzyme activities is not known but may be related to alterations in liver metabolism or enzyme inhibition.
The remaining significant differences in clinical chemistry parameters were not considered to be toxicologically relevant.
None of these changes in enzyme activity were related to organ weight changes or evidence of histopathology.
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):
Relative liver weights were significantly greater compared to controls in males at 100 ppm and greater and in all females treated groups. Absolute liver weights were significantly greater compared to controls in males at 400 ppm and in females at 50, 100 and 200 ppm.
The absolute heart weights of 100 and 200 ppm females and the relative heart weights of females exposed to 100 ppm or greater were significantly greater compared to controls.
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 kidney weights were increased at levels of 50 and 200 ppm, relative were increased at level of 200 ppm and greater.
The absolute and relative thymus weights of 400 ppm females and the relative thymus weight of 200 ppm females were significantly less compared to controls. The absolute and relative spleen weights of 400 ppm males were significantly greater compared to controls.
The weight changes in lymphoid tissues were not accompanied by clinical chemistry or histopathologic changes indicative of immunotoxicity and, therefore, were not considered toxicologically relevant. With the exception of the male kidney, the organ weight changes in male and female rats were not accompanied by histopathologic lesions.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Renal tubule lesions including granular casts, hyaline droplets, and nephropathy were observed in male rats. In all male groups exposed to test item, there were significantly increased incidences of granular casts and hyaline droplet accumulation and the severities of these lesions increased with increasing exposure concentration.
Granular casts were not observed in the controls and in exposed groups the mean severity ranged from minimal in males exposed to 25 ppm to moderate in males exposed to 400 ppm.
Hyaline droplet accumulation occurred in the cytoplasm of the renal proximal convoluted tubule epithelial cells, and the severity ranged from minimal in males exposed to 25 ppm to moderate in males exposed to 400 ppm.
With the exception of one chamber control rat, nephropathy occurred in all males, with the mean severity increasing from minimal in chamber control males to moderate in males exposed to 400 ppm.
There were no exposure-related microscopic findings in females, including those that died before the end of the study.
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 (Swenberg, 1993). 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.
However, measures of α2μ-globulin were not performed in the current study. While it is possible that the observed kidney lesions are secondary to α2μ-globulin nephropathy, the increases in kidney weights in both male and female rats suggest that another independent mechanism of toxicity may have played a role in the lesion development.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
SPERM MOTILITY AND VAGINAL CYTOLOGY:
There were significantly decreased sperm count in cauda epididymis in 200 and 400 ppm males compared to 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.
The minor changes in cycle length observed only in the 400 ppm group, combined with a lack of ovarian histopathology findings, did not provide sufficient evidence for female reproductive toxicity potential under the conditions of the study.
Thus, the test item exposure by inhalation exhibits the potential to be a reproductive toxicant in male rats, but not in female rats.
Key result
Dose descriptor:
LOEL
Effect level:
25 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Significant decrease of sperm per cauda in 200 and 400 ppm.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 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 BodyWeight (g)

Final BodyWeight (g)

Change in BodyWeight (g)

Final Weight Relativeto 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 unnett’s test

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 14 weeks/number initially in group

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

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

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

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 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 4: clinical 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

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*

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

** P≤0.01

Table 5: 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 6: Epididymal Spermatozoal Measurements for Male Rats

 

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.

a Data are presented as mean ± standard error.

Conclusions:
In a repeated dose toxicity study with alpha pinene after 14 weeks of vapour inhalation exposure to rats, increases of incidences of kidney lesions at 25 ppm and decrease of sperm per cauda at 200 ppm in male rats were reported, thus the LOEL was determined to be 25 ppm.
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 rats 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, clinical chemistry, macropathology, histopathology and sperm motility and vaginal cytology investigations were undertaken.

All exposed male rats survived to the end of the studies, while six 400 ppm female rats died before the end of the study. The major targets for test item toxicity were the liver, urinary system, and male reproductive system.

Relative liver weights were significantly greater compared to controls in males at 100 ppm and greater and in all females treated groups (LOEL=25 ppm), however, without accompanying histopathologic changes. Increased liver weight is a common finding in toxicity studies and can be associated with induction of liver metabolizing enzymes.

Absolute kidney weights were increased in male rats exposed to 100 ppm or greater and 50 and 200 ppm female rats; in males, these increases were accompanied by histopathologic lesions including granular casts and hyaline droplet accumulation at all exposure concentrations, as well as exposure concentration-dependent increases in the severity of nephropathy (LOEL=25 ppm), which is a common spontaneous lesion observed in male rats (α2μ-globulin nephropathy). 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. However, measures of α2μ-globulin were not performed in the current study. While it is possible that the observed kidney lesions are secondary to α2μ-globulin nephropathy, the increases in kidney weights in both male and female rats suggest that another independent mechanism of toxicity may have played a role in the lesion development.

There were also significantly lower numbers of sperm per cauda compared to controls in 200 and 400 ppm male rats (LOEL=200 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 the minor changes in cycle length observed only in the 400 ppm group, combined with a lack of ovarian histopathology findings, does not provide sufficient evidence for reproductive toxicity.

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
Reason / purpose:
reference to other study
Remarks:
of which it is a range-finding study
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
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals 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

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
Dose / conc.:
0 ppm
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)
Dose / conc.:
800 ppm
Remarks:
(4.53 mg/L)
Dose / conc.:
1 600 ppm
Remarks:
(9.06 mg/L)
No. of animals per sex per dose:
5
Control animals:
yes
Positive control:
no
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).
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.
Key result
Dose descriptor:
LOAEL
Effect level:
800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
histopathology: non-neoplastic
Critical effects observed:
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

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.

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.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29 November 2004 - 15 December 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to other study
Remarks:
of which it is a range-finding study
Principles of method if other than guideline:
- Principle of test: Rats were exposed to alpha pinene via whole body inhalation 5 days per week for 16 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
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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: 100-102 g (male) and 90-92 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

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: 99.6 ± 1.4 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 ± 130 ppm for the 1600 ppm group.
Duration of treatment / exposure:
16 days; 6 hours plus T90 (12 minutes) per day.
Frequency of treatment:
Five times per week, weekdays only
Dose / conc.:
0 ppm
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)
Dose / conc.:
800 ppm
Remarks:
(4.53 mg/L)
Dose / conc.:
1 600 ppm
Remarks:
(9.06 mg/L)
No. of animals per sex per dose:
5
Control animals:
yes
Positive control:
no
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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Abnormal breathing, ataxia, lethargy, and nasal/eye discharge occurred in one 1,600 ppm male. In rats exposed to 800 ppm, nasal/eye discharge was observed in two males and five females, ataxia was observed in two males and two females, and tremors and abnormal breathing were observed in one male. Nasal/eye discharge and tremors were observed in three females exposed to 400 ppm.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats exposed to 1600 ppm were found dead by day 2. All rats exposed to 800 ppm were found dead by day 16.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights of all exposed rats that survived to the end of the study were similar to those of the chamber controls; the mean body weight gain of 400 ppm females was significantly less than that of the chamber control group.
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 liver weights of 400 ppm males and 200 ppm females were significantly greater than those of the chamber controls. The relative liver weights of 400 ppm males and all surviving groups of exposed females were significantly greater than those of the chamber controls. The absolute kidney weight of 200 ppm females was significantly greater than that of the chamber controls, and the relative kidney weights of all surviving groups of exposed males and 200 and 400 ppm females were significantly greater than those of the chamber controls. In females, the absolute lung weights of the 100 and 200 ppm groups and the relative lung weight of the 100 ppm group were significantly greater than those of the chamber controls.
There were no exposure-related microscopic findings.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no exposure-related microscopic findings
Histopathological findings: neoplastic:
not examined
Details on results:
Based on the mortality of 800 and 1600 ppm rats and a lack of significant histopathologic findings in rats exposed to 400 ppm or less, exposure concentrations of 0, 25, 50, 100, 200, and 400 ppm alpha pinene were selected for the 3-month rat study.
Key result
Dose descriptor:
LOAEL
Effect level:
800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
histopathology: non-neoplastic
Critical effects observed:
not specified

Table 1: Survival and Body Weights

Concentration

(ppm)

Survivalb

Initial BodyWeight (g)

Final BodyWeight (g)

Change in Body Weight (g)

Final Weight Relative to Controls (%)

Male

 

0

5/5

101± 3

172 ± 3

74 ± 4

 

100

5/5

101± 3

171 ± 2

70 ± 1

99

200

5/5

102 ± 3

173 ± 7

71 ± 5

100

400

5/5

100 ± 3

176 ± 4

75 ± 2

102

800

0/5 c

101± 3

-

-

-

1600

0/5 d

102 ± 4

-

-

-

Female

0

 5/5

91± 2

125 ± 3

34 ± 2

 

100

 5/5

90 ± 2

130 ± 3

40 ± 2

104

200

 5/5

91± 2

129 ± 2

38 ± 2

103

400

5/5

92 ± 1

118 ± 2

26 ± 2*

94

800

0/5 e

92 ± 2

-

-

-

1600

0/5 f

91 ± 1

-

-

-

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

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 17/number initially in group

c Days of death: 8, 8, 8, 8, 16

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

e Day of deaths: 8

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

Male

 

n

5

5

5

5

 

Necropsy body wt

172 ± 3

171 ± 2

173 ± 7

176 ± 4

Heart

Absolute

0.620 ± 0.008

0.614 ± 0.006

0.592 ± 0.028

0.620 ± 0.018

 

Relative

3.602 ± 0.098

3.584 ± 0.033

3.418 ± 0.039

3.534 ± 0.059

R. Kidney

Absolute

0.714 ± 0.014

0.758 ± 0.020

0.790 ± 0.044

0.796 ± 0.026

Relative

4.142 ± 0.075

4.425 ± 0.117*

4.556 ± 0.098**

4.535 ± 0.069**

Liver

Absolute

7.988 ± 0.154

8.064 ± 0.196

8.284 ± 0.410

9.668 ± 0.422**

Relative

46.331 ± 0.589

47.091 ± 1.287

47.824 ± 0.791

55.069 ± 1.780**

Lung

Absolute

1.294 ± 0.119

1.216 ± 0.067

1.228 ± 0.060

1.566 ± 0.075

Relative

7.504 ± 0.660

7.081 ± 0.309

7.100 ± 0.245

8.971 ± 0.568

R. Testis

Absolute

0.994 ± 0.015

0.984 ± 0.016

0.981 ± 0.034

0.991 ± 0.016

Relative

5.770 ± 0.130

5.744 ± 0.064

5.681 ± 0.157

5.654 ± 0.085

Thymus

Absolute

0.403 ± 0.012

0.439 ± 0.016

0.427 ± 0.016

0.426 ± 0.007

Relative

2.344 ± 0.095

2.565 ± 0.106

2.477 ± 0.096

2.431 ± 0.037

Female

 

Necropsy body wt wtwwt

125 ± 3

130 ± 3

129 ± 2

118 ± 2

Heart

 

Absolute

0.466 ± 0.012

0.484 ± 0.022

0.480 ± 0.008

0.444 ± 0.017

Relative

3.728 ± 0.089

3.727 ± 0.080

3.736 ± 0.073

3.752 ± 0.091

R. Kidney

Absolute

0.530 ± 0.021 b

0.586 ± 0.016

0.602 ± 0.007*

0.578 ± 0.017

Relative

4.220 ± 0.058 b

4.521 ± 0.038

4.686 ± 0.071**

4.896 ± 0.166**

Liver

Absolute

4.854 ± 0.194

5.404 ± 0.260

5.764 ± 0.051**

5.244 ± 0.138

Relative

38.750 ± 0.728

41.602 ± 1.031*

44.888 ± 0.926**

44.379 ± 1.012**

Lung

Absolute

0.850 ± 0.022

1.066 ± 0.041**

1.042 ± 0.046*

0.862 ± 0.060

Relative

6.808 ± 0.233

8.221 ± 0.223*

8.114 ± 0.371

7.315 ± 0.572

Thymus

Absolute

0.317 ± 0.003

0.335 ± 0.006

0.361 ± 0.011*

0.285 ± 0.017

Relative

2.535 ± 0.052

2.590 ± 0.059

2.807 ± 0.062

2.413 ± 0.149

* 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.

b n=4

Conclusions:
In the 2-week study with alpha-pinene, the two highest exposure concentrations (800 and 1600 ppm) were overtly toxic to rats, 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 rats 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 16 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 rats, 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 rats of both sexes, but not considered to be life threatening. There were no significant histopathologic findings in rats exposed to 400 ppm or less. Therefore, 400 ppm was selected as the high concentration for the 3-month studies in rats.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Weight of evidence from experimental results with individual components:

Camphene (28 days, rats, oral):

The test item was daily administered to SPF Wistar rats (male and female) for 28 days at doses of 0, 62.5, 250 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. Based on the results of this study, for female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day). The renal toxic effects found in all dose levels groups in male rats are interpreted as uniquely specific for male rats, and as having no relevance for other animal species and humans.

d-limonene (90 days, rats, oral):

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.

In a subchronic toxicity study, d-limonene was administered through gavage to groups of 5 or 10 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 2, 5, 10, 30 and 75 mg/kg bw/day for 13 weeks (5 days/week). The NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

In a subchronic toxicity study, d-limonene was administered through gavage to groups of male and female rats at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 91 days. As chronic nephrosis and granular casts formation were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

d-limonene (28 days, rats, oral):

In a subacute toxicity study, d-limonene was administered through gavage to groups of 5 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 75, 150 and 300 mg/kg bw/day for 1 or 4 weeks (5 days/week). As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

d-limonene (90 days, mouse, oral):

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.

d-limonene (180 days, dogs, oral):

In a 6-month subchronic toxicity study performed similarly to OECD Guideline 409, d-limonene was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (0, 100 or 1000 mg/kg bw/day) in two divided doses for 180 days. The NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.

In a 6-month subchronic toxicity study, three dogs per sex and per dose group were administered d-limonene by gavage once per day for 6 months at the dose level of 0, 0.4, 1.2 or 3.6 mL/kg bw/day. The NOAEL in this study is 1.2 mL/kg bw/day (equivalent to 1000 mg/kg bw/day) in males and 0.4 mL/kg bw/day (equivalent to 340 mg/kg bw/day) in females on the basis of decreased body weight and protein casts observed in the renal tubule at the next dose level. Food consumption was also decreased in the intermediate dose females.

alpha pinene (90 days, rats, inhalation):

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 rats 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. Relative liver weights were significantly greater compared to controls in males at 100 ppm and greater and in all females treated groups (LOEL=25 ppm), however, without accompanying histopathologic changes. Increased liver weight is a common finding in toxicity studies and can be associated with induction of liver metabolizing enzymes. Absolute kidney weights were increased in male rats exposed to 100 ppm or greater and 50 and 200 ppm female rats; in males, these increases were accompanied by histopathologic lesions including granular casts and hyaline droplet accumulation at all exposure concentrations, as well as exposure concentration-dependent increases in the severity of nephropathy (LOEL=25 ppm), which is a common spontaneous lesion observed in male rats (α2μ-globulin nephropathy). There were also significantly lower numbers of sperm per cauda compared to controls in 200 and 400 ppm male rats (LOEL=200 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. In females the minor changes in cycle length observed only in the 400 ppm group, combined with a lack of ovarian histopathology findings, does not provide sufficient evidence for reproductive toxicity.

alpha pinene (90 days, mouse, inhalation):

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. 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.

cineole (30 days, mouse, oral):

A short term repeated dose toxicity study (oral route) was performed on 1,8-cineole according to OECD guideline 407. The substance was emulsified in 2% Tween 80 and administered daily to mice by oral gavage in doses of 0 (control, only 2% Tween 80), 21.38, 64.15 and 192.45mg/kg bw/day during 30 days.Animal behavior, body weight and mortality were recordedduring the study. After being sacrificed on day 31,organs/tissues were examined macroscopically and gross lesions were recorded. Relative weight of liver, spleen and double kidneys was calculated.Heart, liver, spleen, lungs, kidney, testis and ovary of treatment groups and control group were examined microscopically. Also, ultrastructural observation by electron microscopy was performed on those organ cells with effects. There were no significant differences in body weight and relative organ weight between the control group and treatment groups. The histopathological examinations showed that granular degeneration and vacuolar degeneration appeared in liver and kidney tissue after administration of high dose (192.45 mg/kg bw/day). Under electron microscopy, a series of ultrastructural changes were observed. At the subcellular level the influence of test substance was found on the mitochondria, endoplasmic reticulum and other membrane type structure of liver and kidney. Based on these results, the NOAEL of the test substance was determined to be 64.15 mg/kg bw/day.

cineole (50 days, rats, oral):

1,8-cineole was daily administered to Wistar rats (male and female) for 50 days at doses of 0, 100, 500 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. In all experimental groups, animals were observed for signs of toxicity, such as piloerection, diarrhea, changes in locomotor activity or mortality. Body weight, food and water consumption were determined once a week. Haematological and clinical tests were also carried out. At the end of the study, animals were macroscopically examined. Alterations in organs were determined, organs were weighed and their relative weights calculated. Histological preparations from the main organs were examined for microscopic changes. Body weight, haematological and clinical tests, and organ weights (absolute and relative) were statistically compared with the control group. Occasional alterations in rats of both sexes were observed, as evidenced in hematological and biochemical parameters and histopathological analysis. However, these changes showed low clinically relevant, since they occurred in a non-generalized manner between animals of the treated groups. Since the lower dose (100 mg/kg) induced reduction of water consumption and mean platelet volume and alkaline phosphatase, it was not possible to establish the NOAEL.

Weight of evidence: conclusion:

The absence of any d-limonene-induced renal lesions in the study with dogs provides evidence that hydrocarbon-induced nephropathy in the male rat is species- and sex-specific. Therefore, the male rat response to d-limonene, alpha pinene, camphene or cineole may not be appropriate for assessing the potential risk of a similar nephrotoxic response in any other species, including humans. According to CLP annex I 3.9.2.8.1. (e), substance-induced species-specific mechanisms of toxicity, i.e. demonstrated with reasonable certainty to be not relevant for human health, shall not justify classification.

The key study was then selected to be the 180-d toxicity study by oral route in dogs (Webb, 1990) for DNEL derivation since mammalian exposure is more relevant than rodent exposure regarding human health effect assessment. The key value has been then selected to be the LOAEL at 1000 mg/kg bw/d.

Justification for classification or non-classification

Based on the available data, the substance is not classified for specific target organ toxicity by repeated exposure (STOT-RE) according to CLP Regulation (EC) no 1272/2008.