Registration Dossier

Administrative data

Description of key information

Key study: Test method OECD 422. GLP study. The NOAEL of the test substance Pine oil 50% after a repeated oral exposure of 36 days in males and approximately 63 days in females was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Supporting study: Dose range finding study according to test method OECD 407. The NOEL of the test substance Pine oil 50% after an oral exposure of 14 days was determined to be 100 mg/kg bw/day in male and female rats.

Supporting studies on some components of the test substance: Repeated dose toxicity studies are available on the components terpineol, d-limonene, cineole and camphene by oral route, and alpha pinene and analogue cumene 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 terpineol, d-limonene, alpha pinene, camphene, cineole or analogue cumene 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. Based on this reasoning the more representative study was selected to be the 180-d toxicity study by oral route in dogs (Webb, 1990). The LOAEL was established 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
Study period:
16 November 2018 – 05 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
(The study period was only 14 days as it was conducted as a dose range finding study for a subsequent more extensive toxicity study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 258.22-263.41 g (range of average values of each group of males); 239.22-242.03 g (range of average values of each group of females)
- Housing: Maximum of 3 animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.5ºC
- Humidity (%): 42-68 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantity of test item was weighed as per the dose. The weighed test item was mixed well using glass rod by adding a small quantity of vehicle and then transferred to a measuring cylinder. Again, a small quantity of vehicle was added and transferred to the measuring cylinder. The procedure was repeated until the complete transfer of the test item into the measuring cylinder. The final volume was made up with vehicle to get the desired concentration as per the dose requirement.
The freshly prepared test item formulation was used for administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in dose formulations was not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once a day
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were decided based on the US EPA (United States Environmental Protection Agency), Prevention, Pesticides and Toxic Substances (7510P), Reregistration Eligibility Decision for Pine oil (case 3113), where the LOAEL for Pine oil 80% was established in 600mg/kg/day and NOAEL was 50 mg/kg/day.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on the day of randomization, on day of treatment (Day 1) prior to treatment and weekly thereafter.

FOOD CONSUMPTION: yes
-feed consumption was measured at weekly intervals. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimatization (Pre-treatment) and at week 2 of treatment.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of necropsy (day 15)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all surviving animals
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (day 15)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all surviving animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Triglycerides, Phosphorous, Calcium, Blood Urea Nitrogen, Globulin, Albumin/Globulin ratio. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy (day 15)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All the surviving animals and animals found dead were subjected to necropsy and detailed gross pathological examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, as well as organs and tissues of each animal with special emphasis on reproductive organs.
The following organs from all animals at the scheduled sacrifices were weighed wet as soon as possible to avoid drying: Kidneys, Adrenals, Spleen, Heart, Liver, Thymus, Brain, Lungs Testes/Ovaries, Epididymides/Uterus, Prostate along with seminal vesicles with coagulating gland. Relative organ weights were calculated against fasting body weight.

-HISTOPATHOLOGY: No
Statistics:
After verification, the data was subjected to statistical analysis using SPSS software, version 22. Body weight, percent change in body weight with respect to day 1, hematological, clinical chemistry estimations, urinalysis parameters (whichever applicable), absolute and relative organ weights were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity at vehicle control group and low dose group.
The mid dose group males did not reveal any clinical signs of toxicity till treatment day 3. However, one male was found with slight wet perineum on day 4, with slight wet perineum, slight nasal discharge and slight piloerection on day 5 and found recovered on day 6. The same male revealed slight nasal discharge from day 11 to 13 and slight wet perineum on day 13 which was found to be normal on day 14. Two males from mid dose group revealed slight nasal discharge from day 11 to 13 and found recovered on day 14. All the mid dose group females did not reveal any clinical signs of toxicity during the experimental period, except one female was found with slight wet perineum on day 4, recovered on day 5.
The high dose group males did not reveal any clinical signs of toxicity till day 3, one male found with slight salivation on day 5, found recovered on day 7, another male was found with slight wet perineum on days 4 and 5, found recovered on day 6. All the three males were found with slight nasal discharge from day 10 to 14 along with lethargy on day 14. All the high dose group females did not reveal any clinical signs of toxicity till day 2, all the three females were found with slight wet perineum from day 3 to 5 and recovered on day 6. Two females from this dose level revealed slight chromodacryorrhea and slight wet perineum during day 10 to 14. One female found with slight to moderate wet perineum, moderate piloerection and slight nasal discharge on days 10 and 11, found dead on day 12.

The detailed clinical examination of all the low and mid dose group animals of either sex did not reveal any treatment related changes during week 1 and 2. The detailed clinical examination of all the high dose group animals of either sex did not reveal any treatment related changes during week 1. However, all the three males and two survived females were found with rough hair coat, slight nasal discharge with reduced activities on day 15 (week 2).
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality observed at vehicle control, low dose and mid dose groups. In the high dose group one female was found dead on day 12.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant changes in mean body weight and percent change in body weight with respect to day 1 at any of the tested dose group animals of either sex. However, reduction in mean body weight and percent change in body weight with respect to day 1 at mid and high dose group males and reduction in mean body weight and percent change in body weight with respect to day 1 at high dose group females were noted when compared with concurrent control group animals. These changes are considered as treatment related due to occurences of clinical signs of toxicity at both the mid and high dose groups animals, reduction in feed consumption and occurrence of motality at high dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant changes in mean feed consumption at any of the tested dose group animals of either sex. However, a slight reduction in mean feed consumption during week 1 at high dose group males, during week 2 at mid and high dose group males; a slight reduction during week 1 and 2 at high dose group females was noted when compared with concurrent control group animals. These changes are considered as treatment related due to occurences of clinical signs of toxicity at both the mid and high dose groups animals, reduction in mean body weight and occurrence of motality at high dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed during the opthalmoscopic examination carried out during week 2 (Day 14) for all control and treated dose group animals of either sex.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in haematology at any of the tested dose group animals of either sex. However, a statistical significant increase in mean platelet volume at all the tested dose group males was noted when compared with vehicle control group males. This observed change is considered as incidental and not treatment related because of the lack of dose dependency and/or due to random biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in clinical chemistry at any of the tested dose group animals of either sex. However, in males, a statistical significant decrease in total bilirubin (low and mid dose groups), decrease in glucose (high dose group), decrease in creatinine and calcium levels (low dose group) and increase in phosphorous (high dose group) were noted when compared with control group animals. These observed changes are considered as incidental and not treatment related due to lack of dose dependency and/or due to random biological variation and also no significant changes noted in clinical chemistry parameters at any of the tested dose group females.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in the urinalysis parameters at any of the tested dose group animals of either sex.
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):
There were no treatment related changes observed in the absolute and relative organ weights at any of the tested dose group animals of either sex. However, statistical significant increase in absolute and relative adrenals weight at low and mid dose group males, increase in absolute and relative liver weight at high dose group males, increase in relative liver weight at high dose group females, decrease in absolute lungs weight at high dose group females and increase in relative kidneys weight at high dose group males were noted when compared with concurrent control group animals.

These observed changes are considered as incidental and not treatment related due to lack of dose dependency and no gross pathological changes noted in these organs during necropsy.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment gross pathological changes observed at any of the tested dose group animals of either sex during gross pathological examination. However, external gross pathological finding like, wet perineum was observed in one male at high dose group and one female which was found dead on day 12.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
LOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Effects observed at the dose tested of 300 mg/kg bw/day.
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Critical effects observed:
no

Table 1.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males

Group, Sex & Dose
(mg/kg body weight/day)

No. of

Animals

Clinical Signs of Toxicity
(No. of Animal Revealed)

Mortality (No. of incidence/

No. of Animals)

G1, M

&

0

3

N (3)

0/3

G2, M

&

100

3

N (3)

0/3

G3, M

&

300

3

73+(3), 110+(1), 117+(1)

0/3

G4, M

&

1000

3

73+(3), 110+(1), 25 (1), 1 (1)

0/3

N: Normal; M: Male

1: Lethargy; 117: Piloerection; 110: Slight wet perineum; 73: Slight nasal discharge; 25: Aggression; +: Slight

Table 1.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females

Group, Sex & Dose
(mg/kg body weight/day)

No. of

Animals

Clinical Signs of Toxicity/Detailed Clinical Examination

Mortality (No. of incidence/

No. of animals)

G1, F

&

0

3

N (3)

0/3

G2, F

&

100

3

N (3)

0/3

G3, F

&

300

3

N (2), 110+(1)

0/3

G4, F

&

1000

3

73+(2), 110+(3), 82+(2)

1/3

N: Normal; F: Female

110: Slight wet perineum; 73: Slight nasal discharge; 82: chromodacryorrhea; +: Slight

Table 2.1. Summary of body weight (g) record. Males

Group, Sex & Dose
(mg/kg body weight/day)

 

Body Weight (g) on Days

1

7

14

G1, M & 0

Mean

258.22

291.74

319.06

±SD

25.75

21.45

14.54

n

3

3

3

G2, M & 100

Mean

263.15

291.93

322.87

±SD

19.42

8.07

15.17

n

3

3

3

G3, M & 300

Mean

262.48

277.67

299.21

±SD

13.91

17.65

19.73

n

3

3

3

G4, M & 1000

Mean

263.41

274.88

295.79

±SD

14.46

14.36

15.47

n

3

3

3

Table 2.2. Summary of body weight (g) record. Females

Group, Sex & Dose
(mg/kg body weight/day)

 

Body Weight (g) on Days

1

7

14

G1, F & 0

Mean

240.31

250.00

258.68

±SD

12.54

18.60

15.78

n

3

3

3

G2, F & 100

Mean

239.22

249.31

262.99

±SD

18.91

23.59

22.23

n

3

3

3

G3, F & 300

Mean

241.06

251.51

259.58

±SD

5.62

3.95

12.87

n

3

3

3

G4, F & 1000

Mean

242.03

236.03

230.60

±SD

8.39

22.43

32.54

n

3

3

2#

#: One female found dead on Day 12

Table 3.1. Summary of percent change in body weight (%) with respect to day 1. Males

Group, Sex & Dose
(mg/kg body weight/day)

Percent Change in Body Weight (%) during Days

1 to 8

1 to 15

G1, M & 0

Mean

13.19

24.13

±SD

3.28

9.54

n

3

3

G2, M & 100

Mean

11.18

22.92

±SD

4.99

5.94

n

3

3

G3, M & 300

Mean

5.75

13.94

±SD

1.40

1.61

n

3

3

G4, M & 1000

Mean

4.44

12.30

±SD

4.83

0.55

n

3

3

Table 3.2. Summary of percent change in body weight (%) with respect to day 1. Females

Group, Sex & Dose
(mg/kg body weight/day)

Percent Change in Body Weight (%) during Days

1 to 8

1 to 15

G1, F & 0

Mean

3.95

7.61

±SD

2.44

1.42

n

3

3

G2, F & 100

Mean

4.16

9.95

±SD

3.68

4.21

n

3

3

G3, F & 300

Mean

4.35

7.66

±SD

1.35

4.02

n

3

3

G4, F & 1000

Mean

-2.48

-6.61

±SD

8.45

12.09

n

3

2#

#: One female found dead on Day 12

Table 4.1. Summary of average feed consumption (g/animal/day) record. Males

Group, Sex & Dose
(mg/kg body weight/day)

 

Feed Consumption (g/animal/day)

Week 1

Week 2

G1, M & 0

Mean

19.5

21.4

±SD

-

-

n

3

3

G2, M & 100

Mean

18.8

21.6

±SD

-

-

n

3

3

G3, M & 300

Mean

18.6

20.8

±SD

-

-

n

3

3

G4, M & 1000

Mean

17.1

20.3

±SD

-

-

n

3

3

Table 4.2. Summary of average feed consumption (g/animal/day) record. Females

Group, Sex & Dose
(mg/kg body weight/day)

 

Feed Consumption (g/rat/day)

Week 1

Feed Consumption

Week 2

Feed Consumption

G1, F & 0

Mean

13.3

18.1

±SD

-

-

n

3

3

G2, F & 100

Mean

11.4

17.5

±SD

-

-

n

3

3

G3, F & 300

Mean

11.3

17.2

±SD

-

-

n

3

3

G4, F & 1000

Mean

10.8

15.9

±SD

-

-

n

3

3

Table 5.1. Summary of haematology record. Males

Group, Sex & Dose
(mg/kg body weight/day)

 

Total Leucocyte Count

Total Erythrocyte Count 

Hemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Hemoglobin

Mean Corpuscular Hemoglobin Concentration 

Platelet Count

 (WBC)        

 (RBC)    

(HGB)      

 (HCT)   

 (MCV)     

(MCH)

(MCHC)      

 (PLT)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

G1, M & 0

Mean

11.92

7.31

13.53

41.90

57.30

18.53

32.37

784.00

±SD

5.61

0.23

0.50

1.41

0.53

0.12

0.31

127.43

n

3

3

3

3

3

3

3

3

G2, M & 100

Mean

9.08

7.37

13.87

43.33

58.77

18.83

31.97

880.33

±SD

1.38

0.55

1.12

3.19

0.68

0.25

0.15

90.52

n

3

3

3

3

3

3

3

3

G3, M & 300

Mean

10.38

7.61

14.17

43.30

56.93

18.60

32.67

930.33

±SD

1.06

0.47

1.14

2.72

0.47

0.36

0.67

46.46

n

3

3

3

3

3

3

3

3

G4, M & 1000

Mean

11.81

7.51

13.73

43.13

57.57

18.30

31.83

891.67

±SD

0.90

0.59

0.72

1.70

2.48

0.70

0.57

135.74

n

3

3

3

3

3

3

3

3

Group, Sex & Dose
(mg/kg body weight/day)

 

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(MPV)   

(Retic)

Neut

Lymph

Mono

Eos

Baso

(fL)

(%)

(%)

(%)

(%)

(%)

(%)

G1, M & 0

Mean

6.40

2.23

22.80

73.03

2.63

0.40

0.20

±SD

0.17

0.45

3.16

1.68

1.29

0.17

0.10

n

3

3

3

3

3

3

3

G2, M & 100

Mean

6.00*

2.25

29.43

65.33

2.83

0.97

0.17

±SD

0.17

0.82

1.35

2.30

0.97

0.40

0.06

n

3

3

3

3

3

3

3

G3, M & 300

Mean

5.93*

2.35

23.90

70.60

3.80

0.43

0.27

±SD

0.06

0.65

0.78

1.23

1.05

0.25

0.12

n

3

3

3

3

3

3

3

G4, M & 1000

Mean

6.03*

2.82

20.90

74.13

2.73

0.73

0.27

±SD

0.12

1.02

6.28

7.40

0.64

0.31

0.06

n

3

3

3

3

3

3

3

*: Statistically significant (P<0.05)

Group, Sex &

Dose
(mg/kg body

weight/day)

 

Absolute Reticulocyte

Count  

Absolute

Neutrophils   

Absolute

Lymphocytes  

Absolute

Monocytes

Absolute

Eosinophils

Absolute 

Basophils       

Prothrombin

Time  

Activated Prothrombin

Time       

(Retic)

Neut

Lymph

Mono

Eos

Baso

PT

APTT

(109cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

162.50

2.62

8.74

0.36

0.05

0.03

25.27

28.33

±SD

29.88

0.90

4.27

0.34

0.05

0.03

1.29

9.05

n

3

3

3

3

3

3

3

3

G2, M & 100

Mean

163.17

2.66

5.94

0.25

0.09

0.02

25.20

25.53

±SD

47.98

0.35

1.05

0.06

0.04

0.01

6.71

7.71

n

3

3

3

3

3

3

3

3

G3, M & 300

Mean

177.23

2.48

7.34

0.39

0.05

0.03

24.33

21.13

±SD

38.93

0.18

0.86

0.10

0.03

0.02

6.97

2.05

n

3

3

3

3

3

3

3

3

G4, M & 1000

Mean

208.23

2.45

8.78

0.32

0.08

0.03

33.07

27.70

±SD

65.06

0.68

1.42

0.06

0.03

0.00

20.56

11.00

n

3

3

3

3

3

3

3

3

Table 5.2. Summary of haematology record. Females

Group, Sex & Dose
(mg/kg body weight/day)

 

Total Leucocyte Count

Total Erythrocyte Count 

Hemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Hemoglobin

Mean Corpuscular Hemoglobin Concentration 

Platelet Count

 (WBC)        

 (RBC)    

(HGB)      

 (HCT)   

 (MCV)     

(MCH)

(MCHC)      

 (PLT)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

G1, F & 0

Mean

9.95

7.30

13.40

40.87

56.00

18.33

32.70

936.33

±SD

2.17

0.04

0.53

0.84

1.39

0.83

0.70

44.74

n

3

3

3

3

3

3

3

3

G2, F & 100

Mean

10.02

7.22

13.37

40.73

56.57

18.53

32.80

1044.33

±SD

1.48

0.35

0.23

0.23

2.50

1.15

0.70

118.29

n

3

3

3

3

3

3

3

3

G3, F & 300

Mean

13.14

7.31

13.53

41.43

56.73

18.53

32.60

918.00

±SD

1.44

0.19

0.15

0.25

1.12

0.32

0.00

74.75

n

3

3

3

3

3

3

2

3

G4, F & 1000

Mean

14.22

7.53

13.75

41.70

55.40

18.25

33.00

1122.00

±SD

0.83

0.27

0.64

0.14

1.84

0.21

1.56

25.46

n#

2

2

2

2

2

2

2

2

#: One female was found dead on Day 12.

Group, Sex & Dose
(mg/kg body weight/day)

 

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(MPV)   

(Retic)

Neut

Lymph

Mono

Eos

Baso

(fL)

(%)

(%)

(%)

(%)

(%)

(%)

G1, F & 0

Mean

6.07

2.15

16.90

78.97

1.97

0.83

0.23

±SD

0.12

0.73

3.70

3.92

0.29

0.25

0.12

n

3

3

3

3

3

3

3

G2, F & 100

Mean

6.10

2.15

17.13

78.77

1.87

0.93

0.27

±SD

0.10

0.31

4.42

5.80

1.24

0.38

0.06

n

3

3

3

3

3

3

3

G3, F & 300

Mean

6.17

1.97

15.77

80.30

1.57

0.83

0.33

±SD

0.21

0.61

4.25

5.19

0.42

0.60

0.15

n

3

3

3

3

3

3

3

G4, F & 1000

Mean

5.85

1.30

20.70

68.70

6.65

1.25

0.40

±SD

0.07

0.55

11.17

4.38

5.87

0.07

0.00

n#

2

2

2

2

2

2

2

#: One female was found dead on Day 12.

Group, Sex & Dose
(mg/kg body weight/day)

 

Absolute Reticulocyte Count  

Absolute Neutrophils   

Absolute Lymphocytes  

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils       

Prothrombin Time  

Activated Prothrombin Time       

(Retic)

Neut

Lymph

Mono

Eos

Baso

PT

APTT

(109cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

157.17

1.74

7.81

0.20

0.09

0.03

31.10

13.80

±SD

53.90

0.71

1.41

0.06

0.04

0.02

14.30

2.96

n

3

3

3

3

3

3

3

3

G2, F & 100

Mean

155.70

1.68

7.95

0.18

0.09

0.02

19.07

20.07

±SD

25.95

0.25

1.69

0.08

0.04

0.01

6.80

3.76

n

3

3

3

3

3

3

3

3

G3, F & 300

Mean

143.10

2.10

10.51

0.20

0.11

0.04

14.50

22.80

±SD

40.56

0.76

0.75

0.06

0.07

0.03

4.20

5.60

n

3

3

3

3

3

3

3

3

G4, F & 1000

Mean

98.65

2.99

9.75

0.92

0.18

0.05

22.10

15.60

±SD

45.33

1.77

0.06

0.78

0.01

0.00

4.67

0.71

n#

2

2

2

2

2

2

2

2

#: One female was found dead on Day 12.

Table 6.1. Summary of clinical chemistry record. Males

Group, Sex & Dose
(mg/kg body weight/day)

 

Glucose

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

 (GLU)

 (CRE)

(CHO)

(TRI)

(TPR)

 (ALB)

(ALT)

(AST)

mg/dL

 mg/dL

mg/dL

 mg/dL

 g/dL

g/dL

 U/L

 U/L

G1, M & 0

Mean

126.00

0.48

49.00

39.67

6.10

2.83

41.67

79.67

±SD

10.58

0.01

4.36

14.05

0.00

0.05

7.51

11.50

n

3

3

3

3

3

3

3

3

G2, M & 100

Mean

110.67

0.42*

44.00

33.67

6.13

2.87

34.33

68.33

±SD

18.72

0.01

3.00

6.43

0.06

0.10

2.52

3.21

n

3

3

3

3

3

3

3

3

G3, M & 300

Mean

115.33

0.48

43.00

42.33

6.23

2.86

40.00

75.00

±SD

5.03

0.03

12.12

13.80

0.15

0.03

5.57

7.00

n

3

3

3

3

3

3

3

3

G4, M & 1000

Mean

94.67*

0.47

39.00

32.00

6.27

2.92

52.67

99.00

±SD

6.11

0.02

1.73

11.79

0.31

0.18

12.66

8.89

n

3

3

3

3

3

3

3

3

*: Statistically significant (p<0.05)

Group, Sex & Dose
(mg/kg body weight/day)

 

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Albumin/Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(A/G Ratio)

(BUN)

 (Na)

 (K)  

 (CLO)

 U/L

 mg/dL

 mg/dL

mg/dL

mg/dL

mg/dL

mg/dL

 mmol/L

 mmol/L

mmol/L

G1, M & 0

Mean

166.33

0.05

8.73

5.30

3.27

0.87

10.76

150.40

3.45

105.97

±SD

34.08

0.01

0.06

0.36

0.05

0.03

0.82

1.41

0.20

0.86

n

3

3

3

3

3

3

3

3

3

3

G2, M & 100

Mean

206.33

0.03*

9.27*

5.87

3.26

0.88

9.75

150.83

3.75

105.00

±SD

42.25

0.01

0.21

0.25

0.16

0.07

2.15

3.24

0.03

0.60

n

3

3

3

3

3

3

3

3

3

3

G3, M & 300

Mean

174.33

0.02*

9.10

5.30

3.38

0.85

10.27

149.03

3.78

104.70

±SD

4.62

0.00

0.20

0.40

0.12

0.03

0.75

0.15

0.19

0.82

n

3

3

3

3

3

3

3

3

3

3

G4, M & 1000

Mean

136.67

0.04

8.97

6.30*

3.35

0.87

10.97

148.80

3.62

107.00

±SD

7.64

0.01

0.23

0.30

0.18

0.06

1.20

0.70

0.09

0.10

n

3

3

3

3

3

3

3

3

3

3

*: Statistically significant (p<0.05)

Table 6.2. Summary of clinical chemistry record. Females

Group, Sex & Dose
(mg/kg body weight/day)

 

Glucose

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

 (GLU)

 (CRE)

(CHO)

(TRI)

(TPR)

 (ALB)

(ALT)

(AST)

mg/dL

 mg/dL

mg/dL

 mg/dL

 g/dL

g/dL

 U/L

 U/L

G1, F & 0

Mean

119.33

0.51

45.33

31.67

6.50

3.06

34.33

73.00

±SD

18.01

0.04

5.13

4.16

0.26

0.09

9.50

3.46

n

3

3

3

3

3

3

3

3

G2, F & 100

Mean

107.00

0.48

54.67

26.67

6.47

3.14

29.00

70.00

±SD

11.53

0.04

10.21

8.14

0.06

0.08

3.61

9.17

n

3

3

3

3

3

3

3

3

G3, F & 300

Mean

126.00

0.49

60.67

26.33

6.30

2.99

41.67

78.33

±SD

1.73

0.04

7.02

3.79

0.44

0.19

13.28

13.28

n

3

3

3

3

3

3

3

3

G4, F & 1000

Mean

102.50

0.58

56.50

25.50

6.15

3.08

51.50

77.50

±SD

19.09

0.09

10.61

6.36

0.49

0.28

16.26

7.78

n#

2

2

2

2

2

2

2

2

#: One female was found dead on Day 12.

Group, Sex & Dose
(mg/kg body weight/day)

 

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Albumin/

Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(A/G Ratio)

(BUN)

 (Na)

 (K)  

 (CLO)

 U/L

 mg/dL

 mg/dL

mg/dL

mg/dL

mg/dL

mg/dL

 mmol/L

 mmol/L

mmol/L

G1, F & 0

Mean

109.67

0.04

9.07

4.60

3.44

0.89

8.96

146.43

3.52

106.20

±SD

39.72

0.01

0.06

0.72

0.24

0.07

0.37

0.72

0.17

0.80

n

3

3

3

3

3

3

3

3

3

3

G2, F & 100

Mean

98.00

0.02

9.13

4.37

3.33

0.95

8.23

146.23

3.59

106.57

±SD

14.93

0.01

0.21

0.76

0.03

0.03

0.96

0.50

0.10

1.72

n

3

3

3

3

3

3

3

3

3

3

G3, F & 300

Mean

110.00

0.02

9.03

4.73

3.31

0.90

11.73

146.17

3.69

106.27

±SD

53.36

0.00

0.55

0.29

0.24

0.01

2.57

1.07

0.56

0.95

n

3

3

3

3

3

3

3

3

3

3

G4, F & 1000

Mean

97.00

0.05

8.70

4.60

3.08

1.00

13.56

145.40

3.11

106.75

±SD

7.07

0.01

0.71

0.14

0.22

0.01

6.30

2.55

0.45

2.90

n#

2

2

2

2

2

2

2

2

2

2

#: One female was found dead on Day 12.

Table 7.1. Summary of absolute organ weights (g) record. Males

Group, Sex & Dose
(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Epididymes

Testes

Heart

Kidneys

Brain

Liver

PSC

Lungs

G1, M & 0

Mean

0.0533

0.3939

0.6615

1.1405

3.4398

1.2664

2.6892

2.1170

10.8868

2.4154

2.5611

±SD

0.0033

0.0749

0.0502

0.0571

0.2415

0.1716

0.2351

0.1906

1.0098

0.3625

0.4313

n

3

3

3

3

3

3

3

3

3

3

3

G2, M & 100

Mean

0.0696*

0.5771

0.6654

1.2033

3.3983

1.3397

3.1196

2.0742

11.5421

2.6900

2.8954

±SD

0.0031

0.0522

0.1348

0.1277

0.2410

0.1489

0.2602

0.1081

1.4462

0.3132

0.5263

n

3

3

3

3

3

3

3

3

3

3

3

G3, M & 300

Mean

0.0659

0.5643

0.6782

1.1154

3.4666

1.1658

2.8486

2.1140

11.0042

2.4310

2.4830

±SD

0.0038

0.0492

0.0340

0.0651

0.1936

0.0883

0.5473

0.1356

0.9703

0.1533

0.3730

n

3

3

3

3

3

3

3

3

3

3

3

G4, M & 1000

Mean

0.0639

0.4349

0.6730

1.1609

3.3178

1.1664

3.3758

2.2839

14.7867*

2.9496

2.6389

±SD

0.0099

0.1499

0.0652

0.0919

0.2031

0.0807

0.1692

0.1420

1.2961

0.4687

0.3236

n

3

3

3

3

3

3

3

3

3

3

3

PSC: Prostate+Seminal vesicles with coagulating glands

*: Statistically significant (P<0.05)

Table 7.2. Summary of absolute organ weights (g) record. Females

Group, Sex & Dose
(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Uterus

Ovaries

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

0.0955

0.6085

0.6788

0.5831

0.2129

1.0115

2.6268

2.4659

9.4801

2.6740

±SD

0.0127

0.1084

0.0535

0.0888

0.0232

0.0626

0.3510

0.0625

0.9003

0.1106

n

3

3

3

3

3

3

3

3

3

3

G2, F & 100

Mean

0.1144

0.5597

0.7401

0.7950

0.2709

1.0814

2.5443

2.4417

10.9313

2.2571

±SD

0.0060

0.1446

0.0827

0.0542

0.0537

0.1494

0.3340

0.0392

1.7597

0.2692

n

3

3

3

3

3

3

3

3

3

3

G3, F & 300

Mean

0.0950

0.6081

0.7264

0.6463

0.2147

1.0944

2.3206

2.3258

11.2076

2.2677

±SD

0.0187

0.0933

0.1095

0.2188

0.0534

0.1176

0.0929

0.0931

1.0288

0.1572

n

3

3

3

3

3

3

3

3

3

3

G4, F & 1000

Mean

0.1051

0.4002

0.5380

0.4603

0.2182

0.9071

2.7250

2.3067

11.3479

2.0137*

±SD

0.0072

0.1550

0.1348

0.0103

0.0501

0.1858

0.3120

0.1549

2.8030

0.0391

n#

2

2

2

2

2

2

2

2

2

2

#: One female was found dead on Day 12.

*: Statistically significant (P<0.05)

Table 8.1. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Males

Group, Sex & Dose
(mg/kg body weight/day)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Epididymes

Testes

Heart

Kidneys

Brain

Liver

PSC

Lungs

G1, M & 0

Mean

292.59

0.0183

0.1341

0.2259

0.3899

0.9181

0.4320

0.9183

0.7231

3.7174

0.8286

0.8720

±SD

15.73

0.0017

0.0194

0.0066

0.0095

0.4490

0.0420

0.0419

0.0413

0.1948

0.1445

0.0995

n

3

3

3

3

3

3

3

3

3

3

3

3

G2, M & 100

Mean

292.55

0.0239*

0.1975

0.2265

0.4105

0.9116

0.4572

1.0651

0.7110

3.9360

0.9187

0.9880

±SD

14.23

0.0022

0.0185

0.0374

0.0245

0.4278

0.0331

0.0384

0.0663

0.3243

0.0838

0.1557

n

3

3

3

3

3

3

3

3

3

3

3

3

G3, M & 300

Mean

279.24

0.0236*

0.2018

0.2431

0.4004

1.2423

0.4175

1.0152

0.7570

3.9362

0.8730

0.8952

±SD

15.41

0.0008

0.0068

0.0122

0.0345

0.0563

0.0224

0.1488

0.0244

0.1369

0.0817

0.1822

n

3

3

3

3

3

3

3

3

3

3

3

3

G4, M & 1000

Mean

273.49

0.0233

0.1584

0.2457

0.4259

1.2161

0.4262

1.2369*

0.8370

5.4008*

1.0791

0.9661

±SD

11.22

0.0034

0.0508

0.0138

0.0506

0.1188

0.0141

0.1044

0.0813

0.2732

0.1731

0.1258

n

3

3

3

3

3

3

3

3

3

3

3

3

PSC: Prostate+Seminal vesicles with coagulating glands

*: Statistically significant (P<0.05)

Table 8.2. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Females

Group, Sex & Dose
(mg/kg body weight/day)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Uterus

Ovaries

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

242.57

0.0393

0.2505

0.2803

0.2432

0.1312

0.4188

1.0796

1.0200

3.9043

1.1072

±SD

18.41

0.0024

0.0351

0.0189

0.0571

0.0629

0.0428

0.0667

0.0707

0.0782

0.1041

n

3

3

3

3

3

3

3

3

3

3

3

G2, F & 100

Mean

249.96

0.0461

0.2218

0.2960

0.3205

0.1707

0.4312

1.0156

0.9813

4.3557

0.9013

±SD

19.15

0.0061

0.0428

0.0226

0.0482

0.1024

0.0278

0.0686

0.0877

0.4035

0.0501

n

3

3

3

3

3

3

3

3

3

3

3

G3, F & 300

Mean

243.63

0.0389

0.2507

0.3004

0.2641

0.0892

0.4502

0.9566

0.9594

4.6072

0.9363

±SD

17.03

0.0065

0.0457

0.0617

0.0815

0.0276

0.0533

0.0940

0.1031

0.4230

0.1246

n

3

3

3

3

3

3

3

3

3

3

3

G4, F & 1000

Mean

220.90

0.0480

0.1778

0.2418

0.2118

0.0983

0.4094

1.2393

1.0533

5.1027*

0.9232

±SD

37.19

0.0048

0.0402

0.0203

0.0403

0.0061

0.0152

0.0674

0.1072

0.4099

0.1377

n

2

2

2

2

2

2

2

2

2

2

2

#: One female was found dead on Day 12.

*: Statistically significant (P<0.05)

Table 9. Summary of gross pathology record

Organs/ Lesions

Group

G1

G2

G3

G4

Dose (mg/kg body

weight/day)

0

100

300

1000

Sex

M

F

M

F

M

F

M

F

No. of Animals Terminally Sacrificed

3

3

3

3

3

3

3

2

No. of Animals Found Dead

0

0

0

0

0

0

0

1

No Abnormality Detected [External & Internal]

3

3

3

3

3

3

2

2

Wet Perineum [External]

0

0

0

0

0

0

1

1

Autolyzed [Internal]

0

0

0

0

0

0

0

1

Conclusions:
Based on the findings of this study, the NOEL of the test substance after an oral exposure of 14 days was determined to be 100 mg/kg bw/day in male and female rats.




Executive summary:

A dose range finding study was performed for the test substance pine oil 50% in accordance with OECD guideline 407 in order to evaluate its toxic potential after repeated exposure and select the appropriate doses for a subsequent toxicity study. The test item was diluted in corn oil (vehicle) and added to Sprague-Dawley rats (3 per sex per dose) at doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day for a period of 14 days. The vehicle and test item formulations were administered orally (gavage) at a dose volume of 10 mL/kg body weight. Freshly prepared test item formulations were administered to the animals as soon as possible after preparation.

All the dose group animals were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, detailed clinical examination, body weight and feed consumption weekly. Ophthalmological examination was carried out during week 2 (day 14) for all the survived animals from all the dose groups of either sex. Clinical pathology (haematology, clinical chemistry analysis and urinalysis) and gross pathological examination was carried out for all survived animals on the day of necropsy.

The low dose group did not reveal any clinical signs of toxicity and no mortality or morbidity was observed throughout the experimental period. The mid dose and high dose groups revealed treatment related clinical signs of toxicity like wet perineum, nasal discharge and lethargy and one female from high dose group was found dead on day 12 of treatment period. The detailed clinical examination carried out on during week 2 revealed treatment related changes like rough hair coat, nasal discharge and reduced activities in high dose group animals.

There were no treatment related changes noted in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, clinical pathology and gross pathological examination at low dose group animals of either sex. The mid and high dose group animals revealed treatment related reduction in body weight, percent change in body weight and feed consumption. There were no treatment related changes noted in ophthalmoscopic examination, clinical pathology and gross pathological examination at mid and high dose group animals. Some statistically significant effects were found on haematology, clinical chemistry parameters and absolute and relative organ weights in the low, mid or high dose groups, although they were considered as incidental and not treatment related because of the lack of dose dependency, due to random biological variation and/or no gross pathological changes noted in the affected organs during necropsy.

Based on these results, it can be concluded that the No Observed Effect Level (NOEL) is 100 mg/kg bw/day and Low Observed Effect Level (LOEL) is 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 January 2019 – 21 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 377.18-381.03 g (range of average values of each group of males at first day of dosing); 271.24-272.21 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 3 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 3 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.1ºC
- Humidity (%): 47-67 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.5 mg/mL and 200 mg/mL in corn oil. However, freshly prepared test item formulations were administered to the animals.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1305). Sampling and analysis of formulations were performed during week 1 (13-02-2019) and week 5 (08-03-2019) of the treatment. Approximately 10 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.
Duration of treatment / exposure:
Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.
Frequency of treatment:
Once a day
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 - Vehicle control + G1R - Vehicle Control Recovery Group
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
G2 - Low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
G3 - Mid dose
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
G4 - High dose + G4R - High dose Recovery Group
No. of animals per sex per dose:
Main Group: 12
Recovery Group: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were decided based on the results of a dose range finding study in which the No Observed Effect Level (NOEL) was found to be 100 mg/kg bw/day and Low Observed Effect Level (LOEL) was found to be 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, during end of the dosing period for main group males (on day 34) and during lactation period for main group females (on lactation day 13) of vehicle control and high dose main group animals and during last week (day 64) for all recovery group animals (both males and females).
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 36) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 66) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

OTHER:

Oestrous Cyclicity: Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.

Litter Observation: The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.

Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 36 days of treatment).






Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1)

HISTOPATHOLOGY: Yes (see table 1)
Other examinations:
Indices calculation (see table 2)

Statistics:
After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity observed at groups G1/G1R and G2 animals of either sex throughout the experimental period.
The groups G3 and G4/G4R animals of either sex did not reveal any clinical signs of toxicity for the first three days of the treatment period. Thereafter, slight wet perineum was noted during several occasions in animals of either sex of these dose groups and found recovered later during the treatment period.
The detailed clinical examination revealed treatment related wet perineum and perinasal staining at groups G3 and G4/G4R during treatment period and found recovered later in the study.
These observed clinical signs at both the dose group animals of G3 and G4/G4R are considered as treatment related due to reduced mean body weight, percent change in mean body weight in either sex during these periods and also the observations are dose dependant when compared with control group.
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any dose group during the experimental period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at group G2 when compared with vehicle control group animals of either sex during the experimental period. However, statistically significant reduction in percent change in mean body weight with respect to day 1 at group G2 animals of either sex was noted. This change is considered as incidental due to lack of consistency and no clinical signs were noted and no effects were noted in feed consumption during this period when compared with control group.
In groups G3 and G4 animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 21, 28 and 35 in males and days 7 and 14 in females was noted.
In group G4R animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 28 and 35 in males, and reduction in mean body weight on day 28 and 35 and percent change in body weight with respect to day 1 during the entire experimental period in females, was noted.
The observed changes at these dose levels [300 mg/kg and 600 mg/kg] are considered as treatment related due to occurrence of treatment related clinical signs of toxicity and also the observed changes are dose dependant and consistent during the experimental period.

There were no changes observed in the gestation body weight and percent change in body weight during gestation period at G2 and G3 dose group animals. A slight reduction in mean gestation body weight was noted on GD 0, 7, 14 and 20 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.

There were no changes observed in the lactation body weight and percent change in body weight during lactation period at groups G2 and G3 when compared with control group animals. A slight reduction in mean lactation body weight was noted throughout the lactation period and the reduction is statistically significant during lactation day 1 to 4 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period, gestation period and also due to occurrence of clinical signs of toxicity during treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in mean feed consumption was noted during pre-mating period (week 1) of main group animals in either sex at all the tested dose groups when compared with vehicle control group. This occurrence is considered as incidental at group G2 and G3 of either sex as the mean feed consumption was comparable with vehicle control group during week 2. However, the reduction at group G4 can be considered as treatment related as the reduction was continued during week 2 also and this effect is correlated with reduction in percent change in body weight at this dose level.

There were no treatment related changes observed in the mean gestation feed consumption at group G2 and G3 when compared with vehicle control group. A slight reduction in mean gestation feed consumption was noted during GD 0 to 7, 7 to 14 and 14 to 20 at group G4 when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to consistency in reduction of feed consumption at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.

There were no treatment related changes observed in the feed consumption during lactation period at all the tested dose groups when compared with control group animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed in G1 and G4 group animals of either sex during the ophthalmological examination conducted towards end of the dosing period and no ocular changes observed in G1R and G4R group animals of either sex during the ophthalmological examination conducted at the end of recovery period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in haematology parameters when compared with their concurrent control groups: increase in activated prothrombin time at group G4 males; decrease in haemoglobin at group G2 females; decrease in total leucocyte count, absolute lymphocytes and activated prothrombin time at group G4R males and increase in mean platelet volume at group G4R males. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in total bilirubin at group G3 males and statistically significant decrease in triglycerides at group G4R males were noted when compared with their concurrent control groups. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no changes observed in urinalysis parameters at any of the tested dose main group males and tested dose group recovery group animals of either sex conducted at termination.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular, physiological observations and assessment of grip strength and motor activity at all the tested dose group animals of either sex from both main and recovery group animals when compared with concurrent vehicle control group animals.
However, statistically significant increase in mean number of rearing during open field examination was noted at groups G2 and G4 females when compared with vehicle control group females. This change is considered as incidental but not treatment related due to lack of dose dependency and no effects were noted in other functional observation battery parameters at these dose levels.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in the absolute and relative organ weights at any of the tested dose group animals of either sex in both main and recovery group animals.
However, statistically significant increase in absolute and relative liver weight at group G4, statistically significant decrease in absolute seminal vesicles with coagulating glands at group G3 males; statistically significant decrease in absolute heart weight at group G4R females were noted when compared with concurrent vehicle control group. These changes are considered as incidental and not treatment related as the observations do not occur in dose dependant manner, are not found in both sexes and also no gross or histopathological changes were noted in these organs at this dose level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed during necropsy at any of the main and recovery group animals of either sex. Also, there were no gross pathological changes noted in any of the pups sacrificed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related histopathological findings were noticed in this study.
Lesions considered to be spontaneous and incidental were observed in treated group and control group animals. These lesions consisted of interstitial mononuclear cell infiltrate and concretions in prostate gland, and the presence of calculi in kidney.
1 female from high dose group G4 revealed mononuclear cells infiltration at sub-urothelium of kidney. This lesion was considered spontaneous because of lack of consistency.
3 males from G4 group revealed presence of concretions in prostate gland. Inflammation and concretions of the prostate gland is common background finding in rats and mice, which increases with age (Dianne C., et.al, 2012).
Females from vehicle control group and high dose group revealed presence of placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary. The presence of discrete pigmented nodules along the uterine-mesometrial boundary was reported. Each nodule overlies a site of placental detachment at parturition, or the site of the resorption of a feto-placental unit or decidualized implantation site. These nodules were often called placental scars and may persist for many months postpartum, perhaps even the lifespan of a rodent (Janet M., 1990).
Presences of ultimobranchial cyst or ectopic thymus in thyroid were congenital lesions and it does not have toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Oestrus cycle: There were no changes (irregularities) observed in the oestrus cyclicity of females at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14 of dams.
Litter observations: There were no treatment related changes observed in litter observation parameters at all the tested dose groups when compared with vehicle control group animals.
Serum Thyroxine (T4) Levels: There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males and lactation day 13 pups. However, statistically significant decrease in T4 levels of males at group G4 was noted when compared with vehicle control group. This change is considered as incidental and not treatment related as the observations do not occur in dose dependant manner and also the values obtained are within historical control range. As no treatment related changes were noted in males and lactation day 13 pups the assessment of T4 in blood samples from the dams and day 4 pups was not performed.
Key result
Dose descriptor:
LOEL
Effect level:
300 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
food consumption and compound intake
Remarks on result:
other: Effects observed at the dose tested of 300 mg/kg bw/day.
Key result
Dose descriptor:
NOEL
Effect level:
100 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
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No adverse effects were found at any dose tested.
Key result
Critical effects observed:
no

Table 4.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males

Group, Sex & Dose

(mg/kg body weight)

No. of Animals

Clinical Signs of Toxicity/
Detailed Clinical Examination
(No. of Animals)

Mortality

(No. of Mortality /

No. of Animals dosed)

G1, M & 0

12

N (12)

0/12

G2, M & 100

12

N (12)

0/12

G3, M & 300

12

N (8); 110 (3); 82 (1)

0/12

G4, M & 600

12

N (6); 110 (6)

0/12

M: Male; N: Normal; 110: Wet perineum; 82: Perinasal staining

Table 4.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females

Group, Sex & Dose

(mg/kg body weight)

No. of Animals

Clinical Signs of Toxicity/
Detailed Clinical Examination
(No. of Animals)

Mortality

(No. of Mortality /

No. of Animals dosed)

G1, F & 0

12

N (12)

0/12

G2, F & 100

12

N (12)

0/12

G3, F & 300

12

N (8); 110 (4)

0/12

G4, F & 600

12

N (5); 110 (7)

0/12

F: Female; N: Normal; 110: Wet perineum

Table 4.3. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Recovery groups

Group, Sex & Dose

(mg/kg body weight)

No. of Animals

Clinical Signs of Toxicity/
Detailed Clinical Examination
(No. of Animals)

Mortality

(No. of Mortality /

No. of Animals dosed)

G1R, M & 0

5

N (5)

0/5

G4R, M & 600

5

N (3); 110 (2)

0/5

G1R, F & 0

5

N (5)

0/5

G4R, F & 600

5

N (3); 110 (2)

0/5

M: Male; F: Female; R: Recovery; N: Normal; 110: Wet perineum

Table 5.1. Summary of body weight (g) record. Males

Group, Sex & Dose

(mg/kg body weight)

Body Weight (g) on Day

1

7

14

21

28

35

G1, M & 0

Mean

381.03

389.68

399.42

410.44

421.01

430.64

±SD

35.22

36.88

38.54

39.75

41.60

40.79

n

12

12

12

12

12

12

G2, M & 100

Mean

377.18

382.56

390.29

401.50

409.69

420.12

±SD

34.26

34.69

35.09

34.76

33.37

33.81

n

12

12

12

12

12

12

G3, M & 300

Mean

378.65

383.40

390.25

397.82

405.35

416.56

±SD

33.89

34.48

36.34

36.53

37.42

37.36

n

12

12

12

12

12

12

G4, M & 600

Mean

379.39

378.29

380.29

386.13

392.89

403.02

±SD

32.03

31.84

31.22

33.21

34.10

34.33

n

12

12

12

12

12

12

Table 5.2. Summary of body weight (g) record. Females

Group, Sex & Dose

(mg/kg body weight)

Body Weight (g) on Days

1

7

14

21#

28#

G1, F & 0

Mean

271.32

277.65

284.52

294.48

303.51

±SD

14.70

15.30

15.77

18.17

19.76

n

12

12

12

6

3

G2, F & 100

Mean

271.24

276.96

278.30

283.87

296.55

±SD

15.07

15.77

15.75

17.32

9.49

n

12

12

12

7

3

G3, F & 300

Mean

272.21

271.34

273.93

267.49

274.21

±SD

16.11

16.02

15.95

18.29

20.14

n

12

12

12

5

4

G4, F & 600

Mean

271.70

272.07

270.96

266.87

265.26

±SD

15.28

15.58

16.05

12.03

10.62

n

12

12

12

7

5

#: The data of Day 21 and 28 body weight was not subjected to statistical analysis due to uneven number of variables

Table 5.3. Summary of body weight (g) record. Recovery groups

Group, Sex & Dose (mg/kg body weight)

Body Weight (g) on Day

1

7

14

21

28

35

42

49

56

63

G1R, M & 0

Mean

375.04

387.85

396.37

407.72

419.05

427.32

438.02

449.49

455.22

462.27

±SD

27.86

27.90

26.43

26.46

28.44

26.52

33.26

37.89

37.83

36.57

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

375.02

376.48

378.88

391.20

397.15

407.68

420.85

436.91

441.32

450.62

±SD

24.55

29.70

32.63

35.14

34.76

32.71

40.13

47.18

46.16

42.45

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

270.17

276.46

283.02

289.28

295.46

300.34

302.72

305.75

311.49

319.96

±SD

11.70

11.50

11.66

11.90

9.99

7.71

7.29

6.66

8.64

6.48

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

269.74

270.61

275.06

277.47

277.96*

283.45*

288.21

290.14

294.73

301.14

±SD

11.53

12.03

10.63

13.27

11.26

10.17

12.72

17.39

16.26

17.58

n

5

5

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group

Table 6.1. Summary of percent change in body weight (%) with respect to day 1. Males

Group, Sex & Dose (mg/kg body weight)

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21

1 to 28

1 to 35

G1, M & 0

Mean

2.26

4.79

7.68

10.44

13.01

±SD

1.06

1.27

1.82

2.40

2.46

n

12

12

12

12

12

G2, M & 100

Mean

1.43

3.49*

6.50

8.72

11.50

±SD

0.52

1.00

1.30

1.98

2.31

n

12

12

12

12

12

G3, M & 300

Mean

1.25*

3.03*

5.04*

7.02*

10.01*

±SD

0.45

0.65

1.14

1.20

1.43

n

12

12

12

12

12

G4, M & 600

Mean

-0.28*

0.27*

1.81*

3.59*

6.28*

±SD

1.22

1.66

3.00

3.20

3.38

n

12

12

12

12

12

* Statistically significant (P<0.05) change than the vehicle control group.

Table 6.2. Summary of percent change in body weight (%) with respect to day 1. Females

Group, Sex & Dose (mg/kg

body weight)

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21#

1 to 28#

G1, F & 0

Mean

2.33

4.86

6.94

9.92

±SD

0.58

1.05

1.30

1.82

n

12

12

5

3

G2, F & 100

Mean

2.11

2.61*

4.48

5.99

±SD

0.72

1.48

0.73

0.73

n

12

12

7

3

G3, F & 300

Mean

-0.31*

0.66*

2.23

3.66

±SD

1.34

1.97

2.51

2.67

n

12

12

5

4

G4, F & 600

Mean

0.15*

-0.27*

0.62

0.14

±SD

1.88

1.68

2.07

2.44

n

12

12

7

5

#: The data of Day 21 and 28 body weight was not subjected to statistical analysis due to uneven number of variables

* Statistically significant (P<0.05) change than the vehicle control group.

Table 6.3. Summary of percent change in body weight (%) with respect to day 1. Recovery groups

Group, Sex & Dose (mg/kg

body weight)

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21

1 to 28

1 to 35

1 to 42

1 to 49

1 to 56

1 to 63

G1R, M & 0

Mean

3.44

5.74

8.78

11.79

14.03

16.79

19.80

21.35

23.26

±SD

1.59

1.48

1.80

2.36

2.12

1.55

2.75

3.02

3.02

n

5

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

0.32*

0.98*

4.23

5.84*

8.68*

12.11

16.33

17.52

20.06

±SD

2.01

4.14

4.47

4.67

4.04

5.42

7.07

6.87

6.06

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

2.33

4.77

7.09

9.41

11.25

12.13

13.28

15.40

18.55

±SD

0.94

1.21

1.95

2.32

2.66

2.82

3.68

4.29

4.06

n

5

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

0.32*

2.00*

2.86*

3.08*

5.14*

6.87*

7.53*

9.24*

11.61*

±SD

1.19

1.69

2.15

2.83

3.00

2.95

3.70

3.18

3.50

n

5

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7.1. Summary of average feed consumption (g/animal/day) record. Males

Group, Sex & Dose
(mg/kg body weight)

Feed Consumption (g/animal/day)

Week 1

(Day 1 to 7)

Week 2

(Day 7 to 14)

G1, M & 0

Mean

19.41

22.53

±SD

1.01

2.12

n

12

12

G2, M & 100

Mean

17.96*

23.03

±SD

0.73

1.78

n

12

12

G3, M & 300

Mean

19.36

22.14

±SD

0.50

1.24

n

12

12

G4, M & 600

Mean

17.97*

20.47

±SD

0.28

1.15

n

12

12

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7.2. Summary of average feed consumption (g/animal/day) record. Females

Group, Sex & Dose
(mg/kg body weight)

Feed Consumption (g/animal/day)

Week 1 (Day 1 to 7)

Week 2 (Day 7 to 14)

G1, F & 0

Mean

15.79

16.65

±SD

0.32

0.74

n

6

6

G1, F & 100

Mean

14.14*

16.81

±SD

0.37

1.83

n

6

6

G3, F & 300

Mean

13.77*

16.00

±SD

0.38

0.80

n

6

6

G4, F & 600

Mean

13.64*

15.77

±SD

0.34

1.12

n

6

6

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7.3. Summary of average feed consumption (g/animal/day) record. Recovery groups

Group, Sex & Dose
(mg/kg body weight)

Feed Consumption (g/animal/day)

Week 1

(Day 1 to 7)

Week 2

(Day 7 to 14)

Week 3

(Day 14 to 21)

Week 4

(Day 21 to 28)

Week 5

(Day 28 to 35)

Week 6

(Day 35 to 42)

Week 7

(Day 42 to 49)

Week 8

(Day 49 to 56)

Week 9

(Day 56 to 63)

Week 10

(Day 63 to 67)

G1R, M & 0

Mean

19.00

20.97

21.80

22.95

23.37

23.78

24.10

24.64

25.32

24.10

±SD

0.63

2.36

1.32

0.49

0.55

0.28

0.14

0.13

0.46

0.14

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

18.68

20.75

20.91

21.28

22.72

23.35

23.63

24.08

24.74

23.63

±SD

1.66

1.55

1.48

1.82

0.65

0.39

0.28

0.12

0.23

0.28

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

15.05

16.48

16.81

17.43

17.88

18.69

20.42

21.49

22.43

20.42

±SD

1.66

0.07

0.08

0.32

0.30

0.65

0.17

0.13

0.46

0.17

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

14.34

16.10

16.46

16.76

17.43

18.15

19.88

20.73

22.07

19.88

±SD

0.09

0.42

0.39

0.27

0.05

0.30

0.08

0.37

0.24

0.08

n

5

5

5

5

5

5

5

5

5

5

Table 8.1. Neurological/functional observation battery record. Open field and sensory observations. Males

Day 36

Parameters↓

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

Dose (mg/kg body weight)

0

100

300

600

Number of Randomly selected Animals

5

5

5

5

                                                         Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

3.8

3.4

3.0

3.0

±SD

0.8

1.1

1.2

0.7

Numbers of Urination

Mean

2.8

2.4

1.6

2.8

±SD

0.8

1.1

0.5

0.8

Number of Defecation

Mean

2.8

2.2

2.4

3.2

±SD

1.3

1.3

0.5

1.3

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

2.8

2.4

3.2

3.0

±SD

0.8

0.5

0.8

0.7

                                                           Sensory Observations

Approach Response

1

1

1

1

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

Body temperature (°F)

Mean

98.6

98.8

98.4

98.2

±SD

0.8

0.8

0.8

0.9

Table 8.2. Neurological/functional observation battery record. Open field and sensory observations. Females

Lactation Day 13

Parameters↓

Group & Sex

G1 & F

G2 & F

G3 & F

G4 & F

Dose (mg/kg body weight)

0

100

300

600

Number of Randomly selected Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

2.4

3.6*

2.8

3.6*

±SD

0.5

0.5

0.8

0.5

Numbers of Urination

Mean

3.6

3.6

3.4

3.6

±SD

0.5

1.1

0.9

1.1

Number of Defecation

Mean

3.4

3.0

2.8

3.2

±SD

0.9

0.7

0.8

0.8

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

2.8

2.8

2.8

3.4

±SD

0.8

0.8

0.8

0.5

Sensory Observations

Approach Response

1

1

1

1

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

Body temperature (°F)

Mean

98.7

98.9

98.5

99.0

±SD

0.5

0.4

0.7

0.3

Table 8.3. Neurological/functional observation battery record. Open field and sensory observations. Recovery groups

Day 66

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/kg body weight)

0

600

0

600

Number of Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

3.0

2.8

2.8

3.0

±SD

0.7

0.8

0.8

0.7

Numbers of Urination

Mean

3.0

2.6

3.0

3.0

±SD

0.7

0.5

0.7

0.7

Number of Defecation

Mean

2.8

3.0

2.8

3.0

±SD

0.8

0.7

0.8

1.0

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

2.8

2.8

3.0

3.0

±SD

0.8

0.8

0.7

0.7

Sensory Observations

Approach Response

1

1

1

1

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

Body temperature (°F)

Mean

98.9

98.5

98.8

98.6

±SD

0.5

0.7

0.6

1.0

Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling - 1= Absent, 2=Present, Sensory Observations:

a. Startle Response - 2=Normal, b. Touch Response - 2=Normal, c. Pupil Response 2=Normal, d. Response to Nociceptive stimuli - 2=Normal, e. Righting Reflex - 1=Present, 2=Slow, 3=Absent  

* Statistically significant (P<0.05) change than the vehicle control group.

Table 9.1. Summary of haematology record. Males

Group, Sex & Dose

(mg/kg body weight)

Total

Leucocyte Count

Total

Erythrocyte Count

Hemoglobin

Haematocrit

Mean

Corpuscular Volume

Mean

Corpuscular Hemoglobin

Mean

Corpuscular Hemoglobin Concentration

Platelet

Count

Mean

Platelet Volume

Reticulocyte

Count

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(Retic)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

(%)

G1, M & 0

Mean

7.90

8.04

13.38

43.22

53.86

16.66

30.94

831.00

5.76

1.55

±SD

1.57

0.58

0.86

2.27

1.14

0.41

0.70

95.01

0.30

0.13

n

5

5

5

5

5

5

5

5

5

5

G2, M & 100

Mean

7.20

7.53

12.44

41.10

54.64

16.52

30.26

797.00

5.90

1.68

±SD

0.60

0.57

1.05

2.52

1.71

0.68

1.77

94.89

0.29

0.52

n

5

5

5

5

5

5

5

5

5

5

G3, M & 300

Mean

7.11

7.71

13.18

42.26

54.80

17.10

31.18

783.20

5.92

1.44

±SD

0.90

0.26

0.59

0.94

1.99

0.92

0.72

65.48

0.26

0.74

n

5

5

5

5

5

5

5

5

5

5

G4, M & 600

Mean

8.58

7.74

12.92

42.88

55.56

16.66

30.08

830.80

6.00

2.33

±SD

1.14

0.71

1.55

3.16

2.35

0.67

2.27

152.49

0.25

1.60

n

5

5

5

5

5

5

5

5

5

5

Group, Sex & Dose

(mg/kg body weight)

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

G1, M & 0

Mean

24.34

70.60

2.86

1.80

0.20

±SD

6.17

6.81

0.98

0.66

0.10

n

5

5

5

5

5

G2, M & 100

Mean

27.48

66.72

3.66

1.68

0.22

±SD

2.57

2.34

2.08

0.25

0.11

n

5

5

5

5

5

G3, M & 300

Mean

27.00

66.68

3.72

2.14

0.22

±SD

5.31

4.48

0.74

1.03

0.08

n

5

5

5

5

5

G4, M & 600

Mean

23.38

71.96

2.98

1.32

0.24

±SD

3.67

5.22

1.84

0.36

0.09

n

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

 

Absolute Reticulocyte Count

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

124.26

1.91

5.59

0.22

0.15

0.01

26.70

13.36

±SD

3.05

0.51

1.37

0.08

0.07

0.01

0.97

2.24

n

5

5

5

5

5

5

5

5

G2, M & 100

Mean

124.68

1.99

4.79

0.27

0.12

0.02

28.30

16.16

±SD

29.57

0.33

0.23

0.15

0.03

0.01

2.31

2.22

n

5

5

5

5

5

5

5

5

G3, M & 300

Mean

111.26

1.94

4.72

0.27

0.15

0.02

26.20

17.40

±SD

56.81

0.53

0.54

0.07

0.05

0.01

1.29

4.09

n

5

5

5

5

5

5

5

5

G4, M & 600

Mean

176.02

2.03

6.14

0.26

0.12

0.02

25.68

17.90*

±SD

109.06

0.54

0.59

0.20

0.04

0.01

1.38

1.42

n

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 9.2. Summary of haematology record. Females

Group, Sex & Dose

(mg/kg body weight)

Total

Leucocyte Count

Total

Erythrocyte Count

Hemoglobin

Haematocrit

Mean

Corpuscular Volume

Mean

Corpuscular Hemoglobin

Mean

Corpuscular Hemoglobin Concentration

Platelet

Count

Mean

Platelet Volume

Reticulocyte

Count

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(Retic)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

(%)

G1, F & 0

Mean

12.44

7.68

14.78

44.32

57.78

19.26

33.34

604.20

7.40

1.76

±SD

3.97

0.57

0.86

3.30

1.85

0.67

0.62

247.85

0.76

1.01

n

5

5

5

5

5

5

5

5

5

5

G2, F & 100

Mean

9.89

6.89

13.36*

40.82

59.28

19.46

32.78

671.40

7.16

2.07

±SD

2.84

0.51

0.57

2.29

1.13

0.73

0.62

197.31

0.66

0.82

n

5

5

5

5

5

5

5

5

5

5

G3, F & 300

Mean

10.38

7.70

14.74

45.38

59.00

19.14

32.48

610.40

7.50

2.18

±SD

2.05

0.29

0.44

1.37

1.07

0.44

0.87

232.46

1.07

1.12

n

5

5

5

5

5

5

5

5

5

5

G4, F & 600

Mean

9.17

7.22

14.14

43.16

59.86

19.62

32.78

801.40

7.06

1.92

±SD

2.17

0.65

1.05

3.08

2.55

0.76

0.69

145.93

0.58

0.98

n

5

5

5

5

5

5

5

5

5

5

Group, Sex & Dose

(mg/kg body weight)

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

G1, F & 0

Mean

27.94

66.88

2.96

1.78

0.24

±SD

7.71

7.63

1.43

0.82

0.05

n

5

5

5

5

5

G2, F & 100

Mean

31.52

63.44

2.52

2.16

0.20

±SD

12.12

12.91

1.47

0.62

0.07

n

5

5

5

5

5

G3, F & 300

Mean

24.40

71.14

1.62

2.38

0.26

±SD

5.35

6.68

1.06

1.07

0.09

n

5

5

5

5

5

G4, F & 600

Mean

22.38

71.52

3.92

1.80

0.24

±SD

6.45

7.33

2.34

0.37

0.05

n

5

5

5

5

5

Group, Sex &

Dose (mg/kg body weight)

Absolute

Reticulocyte Count

Absolute

Neutrophils

Absolute

Lymphocytes

Absolute

Monocytes

Absolute

Eosinophils

Absolute 

Basophils

Prothrombin Time

Activated

Prothrombin Time

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

131.92

3.47

8.33

0.38

0.21

0.04

23.02

14.64

±SD

69.35

1.62

2.65

0.24

0.10

0.01

4.81

2.57

n

5

5

5

5

5

5

5

5

G2, F & 100

Mean

140.98

3.11

6.28

0.25

0.22

0.02

21.80

16.98

±SD

50.35

1.37

2.14

0.15

0.11

0.01

3.93

4.20

n

5

5

5

5

5

5

5

5

G3, F & 300

Mean

167.44

2.62

7.28

0.18

0.26

0.03

21.94

15.46

±SD

86.60

1.04

0.76

0.16

0.16

0.02

3.01

3.26

n

5

5

5

5

5

5

5

5

G4, F & 600

Mean

134.16

2.16

6.46

0.35

0.17

0.02

25.00

19.90

±SD

51.84

1.21

0.99

0.21

0.06

0.00

8.24

9.87

n

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 9.3. Summary of haematology record. Recovery groups

Group, Sex & Dose

(mg/kg body weight)

Total

Leucocyte Count

Total

Erythrocyte Count

Hemoglobin

Haematocrit

Mean

Corpuscular Volume

Mean

Corpuscular Hemoglobin

Mean

Corpuscular Hemoglobin Concentration

Platelet

Count

Mean

Platelet Volume

Reticulocyte

Count

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(Retic)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

(%)

G1R, M & 0

Mean

8.71

8.19

14.48

42.66

52.14

17.70

33.96

962.60

6.92

1.47

±SD

0.61

0.43

0.41

1.58

1.65

0.49

0.44

140.30

0.15

0.23

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

7.51*

8.07

14.68

42.50

52.66

18.18

34.50

794.00

7.58*

1.31

±SD

0.59

0.34

0.46

1.31

0.96

0.52

0.46

85.12

0.44

0.16

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

5.93

7.34

13.78

40.00

54.58

18.76

34.38

867.60

7.26

1.51

±SD

1.05

0.25

0.36

0.78

1.38

0.22

0.58

150.83

0.32

0.34

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

5.10

7.47

14.12

40.42

54.12

18.94

35.02

844.80

7.56

1.55

±SD

1.74

0.39

0.44

2.18

1.48

0.96

1.63

153.15

0.41

0.28

n

5

5

5

5

5

5

5

5

5

5

Group, Sex & Dose

(mg/kg body weight)

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

G1R, M & 0

Mean

20.92

74.34

2.96

1.32

0.20

±SD

2.23

3.30

1.56

0.52

0.00

n

5

5

5

5

5

G4R, M & 600

Mean

26.32

69.66

2.14

1.48

0.22

±SD

7.17

7.97

1.15

0.40

0.11

n

5

5

5

5

5

G1R, F & 0

Mean

24.28

70.96

2.46

1.80

0.22

±SD

7.22

8.79

1.24

0.78

0.13

n

5

5

5

5

5

G4R, F & 600

Mean

24.40

70.32

2.26

2.72

0.16

±SD

5.54

6.18

0.30

0.89

0.09

n

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Absolute

Reticulocyte Count

Absolute

Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute 

Basophils

Prothrombin Time

Activated

Prothrombin Time

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1R, M & 0

Mean

120.24

1.82

6.48

0.26

0.11

0.02

25.14

21.48

±SD

16.86

0.20

0.61

0.14

0.03

0.01

3.32

4.44

n

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

105.48

1.97

5.24*

0.16

0.11

0.02

22.26

14.92*

±SD

13.61

0.55

0.76

0.08

0.04

0.01

2.59

3.67

n

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

110.42

1.38

4.27

0.14

0.10

0.01

26.70

20.06

±SD

23.36

0.19

1.19

0.05

0.03

0.01

2.11

4.15

n

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

114.88

1.24

3.60

0.11

0.13

0.01

27.28

17.46

±SD

16.01

0.54

1.35

0.04

0.03

0.01

5.16

2.20

n

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10.1. Summary of clinical chemistry record. Males

Group, Sex & Dose

(mg/kg body weight)

Glucose

Urea

Creatinine

Total

Cholesterol

Triglycerides

Total Protein

Albumin

Alanine

aminotransferase

Aspartate

aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, M & 0

Mean

77.60

31.70

0.47

44.40

39.40

6.12

3.04

43.40

109.00

±SD

8.32

1.02

0.17

10.16

10.74

0.22

0.13

12.70

40.98

n

5

5

5

5

5

5

5

5

5

G2, M & 100

Mean

81.60

30.26

0.51

45.80

48.60

5.88

3.04

51.40

116.80

±SD

12.97

5.31

0.02

4.92

13.09

0.22

0.11

7.60

27.60

n

5

5

5

5

5

5

5

5

5

G3, M & 300

Mean

83.40

37.84

0.53

53.80

33.60

6.78

3.06

45.40

111.00

±SD

11.28

6.95

0.05

24.47

7.27

1.31

0.19

7.16

10.98

n

5

5

5

5

5

5

5

5

5

G4, M & 600

Mean

97.60

31.84

0.56

42.00

27.20

5.78

2.75

43.60

101.00

±SD

28.01

3.36

0.06

5.70

6.14

0.46

0.35

4.88

13.51

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg

body weight)

Alkaline

phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea

Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

116.20

0.05

9.36

5.12

3.08

14.79

150.98

3.96

112.58

±SD

34.17

0.01

0.23

0.57

0.10

0.47

1.72

0.51

1.46

n

5

5

5

5

5

5

5

5

5

G2, M & 100

Mean

101.60

0.03

9.46

5.36

2.84

14.12

149.48

4.15

112.34

±SD

21.15

0.02

0.38

0.36

0.12

2.48

1.70

0.63

1.84

n

5

5

5

5

5

5

5

5

5

G3, M & 300

Mean

141.20

0.03*

9.76

5.30

3.72

17.66

148.70

3.87

110.82

±SD

60.95

0.01

0.59

1.41

1.41

3.25

4.53

0.47

1.51

n

5

5

5

5

5

5

5

5

5

G4, M & 600

Mean

91.20

0.03

9.48

6.54

3.03

14.86

151.38

4.46

112.28

±SD

23.76

0.02

0.35

0.76

0.48

1.57

2.14

0.36

3.23

n

5

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10.2. Summary of clinical chemistry record. Females

Group, Sex & Dose (mg/kg body weight)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, F & 0

Mean

80.60

48.44

0.48

67.60

106.40

6.68

3.03

110.80

143.40

±SD

18.90

5.74

0.06

11.70

47.83

0.36

0.24

23.34

43.36

n

5

5

5

5

5

5

5

5

5

G2, F & 100

Mean

76.80

45.38

0.45

67.80

292.80

6.26

2.81

112.80

143.00

±SD

24.14

8.53

0.07

10.33

238.17

0.42

0.24

22.44

69.42

n

5

5

5

5

5

5

5

5

5

G3, F & 300

Mean

87.80

50.10

0.42

72.80

151.00

6.88

3.12

96.20

89.00

±SD

13.81

16.09

0.06

10.21

56.63

0.26

0.29

14.50

30.51

n

5

5

5

5

5

5

5

5

5

G4, F & 600

Mean

85.80

47.94

0.51

77.00

76.80

7.20

3.37

90.20

105.40

±SD

24.79

13.82

0.07

15.87

45.38

0.68

0.35

33.87

36.62

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg

body weight)

Alkaline

phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea

Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, F & 0

Mean

170.40

0.02

10.28

9.25

3.65

22.60

154.46

3.90

122.36

±SD

64.28

0.00

0.75

1.40

0.24

2.68

16.36

0.98

15.63

n

5

5

5

4

5

5

5

5

5

G2, F & 100

Mean

240.60

0.02

10.00

6.54

3.45

21.18

157.82

3.58

121.80

±SD

93.37

0.00

1.08

2.93

0.27

3.98

17.56

0.17

14.22

n

5

5

5

5

5

5

5

5

5

G3, F & 300

Mean

296.80

0.02

10.30

6.58

3.76

23.38

140.22

3.78

107.34

±SD

141.57

0.00

1.06

2.78

0.10

7.51

1.08

0.30

5.50

n

5

5

5

5

5

5

5

5

5

G4, F & 600

Mean

183.80

0.03

10.52

6.62

3.83

22.37

150.82

3.68

111.00

±SD

85.44

0.02

0.98

2.75

0.55

6.45

6.51

0.62

7.02

n

5

5

5

5

5

5

5

5

5

Table 10.3. Summary of clinical chemistry record. Recovery groups

Group, Sex & Dose (mg/kg

body weight)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine

aminotransferase

Aspartate

aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1R, M & 0

Mean

118.20

26.78

0.62

51.00

52.20

6.64

3.12

51.00

42.00

±SD

28.34

6.15

0.05

8.46

15.07

0.43

0.20

7.68

7.52

n

5

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

103.60

30.60

0.60

45.00

33.60*

6.72

3.10

49.60

49.80

±SD

8.17

4.38

0.06

10.77

7.09

0.45

0.22

12.74

13.31

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

107.80

29.74

0.66

59.80

48.40

7.00

3.36

40.20

42.20

±SD

17.54

4.93

0.03

13.77

19.09

0.19

0.09

7.40

4.38

n

5

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

102.60

28.54

0.64

51.20

28.60

6.78

3.20

44.20

43.40

±SD

13.39

4.21

0.05

11.23

7.86

0.43

0.22

3.11

3.91

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Alkaline

phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1R, M & 0

Mean

116.40

0.02

9.84

7.56

3.52

12.50

147.54

3.06

111.76

±SD

29.87

0.00

0.36

0.64

0.25

2.87

3.55

0.15

1.55

n

5

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

93.40

0.02

10.12

7.30

3.62

14.28

151.54

3.01

111.42

±SD

34.30

0.00

0.33

0.28

0.30

2.04

1.64

0.10

1.33

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

49.80

0.02

9.90

7.48

3.64

13.88

152.42

3.11

112.38

±SD

14.58

0.00

0.14

0.13

0.25

2.30

0.72

0.30

1.28

n

5

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

61.80

0.02

9.82

7.22

3.58

13.32

151.46

3.15

112.10

±SD

23.57

0.00

0.25

0.37

0.30

1.96

1.18

0.27

0.80

n

5

5

5

5

5

5

5

5

5

Table 11. Summary of vaginal smear examination for determination of oestrus cyclicity

Group & Dose
(mg/kg body weight/day)

Total No. of Females

No. of Females with Regular Oestrus Cyclicity during

Pre-mating and Mating

No. of Females confirmed with pregnancy

 

No. of Females with Regular Oestrus Cyclicity on Lactation Day 14

No. of Females with Irregular Oestrus Cyclicity during

Pre-mating, Mating and on Lactation day 14

G1 & 0

12

12

10

10

0

G2 & 100

12

12

11

11

0

G3 & 300

12

12

10

10

0

G4 & 600

12

12

10

10

0

Table 12. Summary record of gestation body weight (g)

Group & Dose
(mg/kg body weight)

Body weight (g) on Gestation Days

0

7

14

20

G1 & 0

Mean

293.54

312.05

346.33

411.36

±SD

18.46

20.26

19.17

18.87

n

10

10

10

10

G2 & 100

Mean

285.12

300.22

338.92

404.73

±SD

18.74

20.01

18.03

20.92

n

11

11

11

11

G3 & 300

Mean

280.85

300.22

336.08

406.54

±SD

15.00

15.55

15.71

26.97

n

10

10

10

10

G4 & 600

Mean

276.42

294.72

327.07

389.37

±SD

16.05

12.25

15.04

14.98

n

10

10

10

10

n: Number of dams (the data of non-pregnant animals is excluded for mean calculations)

Table 13. Summary record of percent change in body weight (%) during gestation period

Group & Dose
(mg/kg body weight)

Change in body weight (%) during Gestation

0 to 7

7 to 14

14 to 20

G1 & 0

Mean

6.30

11.13

18.93

±SD

1.87

4.58

5.33

n

10

10

10

G2 & 100

Mean

5.30

13.02

19.45

±SD

1.74

3.22

2.49

n

11

11

11

G3 & 300

Mean

6.91

11.98

20.96

±SD

0.80

1.64

5.69

n

10

10

10

G4 & 600

Mean

6.72

10.99

19.20

±SD

2.27

2.64

5.69

n

10

10

10

n: Number of dams (the data of non-pregnant animals is excluded for mean calculations)

Table 14. Summary of lactation body weight (g) record

Group, Sex & Dose
(mg/kg body weight)

Body weight (g) on Lactation Days

1

4

7

13

G1 & 0

Mean

327.10

330.97

334.42

348.86

±SD

16.26

16.38

15.95

16.17

n

10

10

10

10

G2 & 100

Mean

322.50

326.63

331.30

348.67

±SD

18.78

18.07

17.20

15.61

n

11

11

11

11

G3 & 300

Mean

310.79

315.83

320.05

337.45

±SD

18.55

17.65

18.21

18.54

n

10

10

10

10

G4 & 600

Mean

311.35

314.75

318.00

336.09

±SD

18.53

18.37

18.57

19.69

n

10

10

10

10

n: Number of dams

Table 15. Summary of percent change in body weight (%) during lactation

Group & Dose
(mg/kg body weight)

Change in body weight (%) during Lactation Day (LD)

1 to 4

4 to 7

7 to 13

G1 & 0

Mean

1.18

1.05

4.35

±SD

0.45

0.55

2.19

n

10

10

10

G2 & 100

Mean

1.30

1.45

5.29

±SD

0.66

0.59

1.70

n

11

11

11

G3 & 300

Mean

1.65

1.33

5.46

±SD

0.99

0.72

1.59

n

10

10

10

G4 & 600

Mean

1.10*

1.03

5.70

±SD

0.35

0.47

2.20

n

10

10

10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 16.1. Summary of absolute organ weights (g) record. Males

Group, Sex & Dose

(mg/kg body weight)

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Brain

Liver

Prostate

Seminal vesicles with

coagulating glands

Thyroid along with

parathyroid#

G1, M & 0

Mean

0.0778

0.3166

0.8525

3.5521

1.5253

1.4954

3.2095

2.1084

12.2106

1.5663

1.7826

0.0284

±SD

0.0230

0.0685

0.0981

0.3053

0.2003

0.2606

0.5003

0.1619

1.6959

0.2473

0.3736

0.0062

n

5

5

5

12

12

5

5

5

5

12

12

12

G2, M &

100

Mean

0.0908

0.3110

0.8461

3.5704

1.5355

1.4677

3.2837

2.0750

12.9020

1.7886

1.5368

0.0291

±SD

0.0067

0.0833

0.1153

0.3160

0.1338

0.0619

0.4710

0.3149

0.9458

0.3161

0.2519

0.0054

n

5

5

5

12

12

5

5

5

5

12

12

12

G3, M &

300

Mean

0.0718

0.4086

0.7552

3.4894

1.4944

1.4913

3.4137

2.2140

12.8520

1.7174

1.3662*

0.0271

±SD

0.0118

0.1711

0.1110

0.2024

0.1371

0.2276

0.3343

0.0545

1.3223

0.3604

0.4805

0.0055

n

5

5

5

12

12

5

5

5

5

12

12

12

G4, M &

600

Mean

0.0831

0.2943

0.8536

3.5823

1.4279

1.4830

3.5043

2.1889

15.3838*

1.6183

1.5367

0.0270

±SD

0.0158

0.0744

0.2004

0.3689

0.1220

0.1331

0.3005

0.1384

1.7056

0.3435

0.5163

0.0051

n

5

5

5

12

12

5

5

5

5

12

12

12

G1R, M & 0

Mean

0.0773

0.3618

0.7242

3.6039

1.6468

1.9188

3.5834

2.2569

13.2972

1.7121

1.8645

0.0261

±SD

0.0092

0.0263

0.0591

0.3635

0.0920

0.2272

0.4585

0.0468

1.4254

0.1573

0.0884

0.0063

n

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M &

600

Mean

0.0789

0.3709

0.7171

3.6500

1.6955

1.7452

3.3656

2.2167

12.9538

1.6941

1.8200

0.0260

±SD

0.0065

0.0291

0.0499

0.2753

0.1809

0.2607

0.0824

0.0687

1.0505

0.2033

0.2601

0.0061

n

5

5

5

5

5

5

5

5

5

5

5

5

#: Weighed post fixation

*: Statistically significant than the control group (p<0.05)

Table 16.2. Summary of absolute organ weights (g) record. Females

Group, Sex & Dose

(mg/kg body weight)

Adrenals

Thymus

Spleen

Heart

Kidneys

Brain

Liver

Thyroid along with parathyroid#

G1, F & 0

Mean

0.1048

0.2882

0.6049

1.2601

2.5891

2.0211

14.5537

0.0349

±SD

0.0236

0.0253

0.0295

0.0594

0.3982

0.0756

1.2896

0.0031

n

5

5

5

5

5

5

5

12

G2, F & 100

Mean

0.1010

0.3114

0.6619

1.3266

2.7373

2.0318

15.2112

0.0352

±SD

0.0207

0.0316

0.0991

0.0908

0.4297

0.0535

0.6016

0.0031

n

5

5

5

5

5

5

5

12

G3, F & 300

Mean

0.0879

0.2689

0.6149

1.3114

2.5901

2.0801

15.7306

0.0350

±SD

0.0104

0.0410

0.1610

0.1273

0.4058

0.1362

1.0902

0.0040

n

5

5

5

5

5

5

5

12

G4, F & 600

Mean

0.0834

0.3016

0.6075

1.3731

2.4364

2.0034

15.5934

0.0350

±SD

0.0139

0.0282

0.0727

0.1828

0.3454

0.2747

0.8368

0.0018

n

5

5

5

5

5

5

5

12

G1R, F & 0

Mean

0.0985

0.3394

0.6029

1.1973

2.7865

2.4941

9.4607

0.0277

±SD

0.0077

0.0363

0.0939

0.0312

0.0626

0.1286

0.5923

0.0008

n

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

0.0967

0.3621

0.6393

1.1068*

2.8554

2.4116

9.2773

0.0273

±SD

0.0068

0.0448

0.0296

0.0372

0.2076

0.0840

0.3590

0.0047

n

5

5

5

5

5

5

5

5

#: Weighed post fixation

*: Statistically significant than the control group (p<0.05)

Table 17.1. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Males

Group, Sex &

Dose (mg/kg body weight)

Fasting

Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Brain

Liver

Prostate

Seminal

vesicles with coagulating glands

Thyroid

along with

parathyroid

G1, M & 0

Mean

416.86

0.0199

0.0804

0.2185

0.8611

0.3676

0.3762

0.8114

0.5349

3.0979

0.3771

0.4286

0.0069

±SD

44.25

0.0062

0.0170

0.0422

0.1152

0.0474

0.0280

0.0895

0.0299

0.4111

0.0559

0.0834

0.0016

n

12

5

5

5

12

12

5

5

5

5

12

12

12

G2, M & 100

Mean

410.05

0.0228

0.0770

0.2118

0.8756

0.3767

0.3696

0.8248

0.5228

3.2457

0.4377

0.3737

0.0072

±SD

34.57

0.0014

0.0154

0.0203

0.0980

0.0423

0.0264

0.1062

0.0845

0.2526

0.0783

0.0444

0.0017

n

12

5

5

5

12

12

5

5

5

5

12

12

12

G3, M & 300

Mean

404.28

0.0180

0.1010

0.1882

0.8730

0.3725

0.3697

0.8498

0.5523

3.1965

0.4278

0.3395

0.0067

±SD

43.34

0.0035

0.0402

0.0253

0.1111

0.0445

0.0333

0.0672

0.0248

0.2120

0.0867

0.1157

0.0014

n

12

5

5

5

12

12

5

5

5

5

12

12

12

G4, M & 600

Mean

395.73

0.0208

0.0739

0.2141

0.9181

0.3649

0.3641

0.8614

0.5383

3.7942*

0.4115

0.3907

0.0069

±SD

39.18

0.0061

0.0263

0.0753

0.1657

0.0561

0.0300

0.0834

0.0455

0.5655

0.0858

0.1405

0.0013

n

12

5

5

5

12

12

5

5

5

5

12

12

12

G1R, M & 0

Mean

457.78

0.0169

0.0792

0.1583

0.7890

0.3607

0.4224

0.7818

0.4951

2.9004

0.3762

0.4093

0.0057

±SD

32.41

0.0015

0.0055

0.0091

0.0796

0.0244

0.0746

0.0774

0.0387

0.1774

0.0492

0.0395

0.0016

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 600

Mean

446.24

0.0178

0.0835

0.1621

0.8226

0.3812

0.3916

0.7600

0.5008

2.9084

0.3805

0.4147

0.0058

±SD

43.73

0.0022

0.0077

0.0215

0.0841

0.0388

0.0467

0.0763

0.0534

0.1148

0.0403

0.0937

0.0012

n

5

5

5

5

5

5

5

5

5

5

5

5

5

*: Statistically significant than the control group (p<0.05)

Table 17.2. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Females

Group, Sex & Dose (mg/kg body weight)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Heart

Kidneys

Brain

Liver

Thyroid along with parathyroid

G1, F & 0

Mean

335.46

0.0323

0.0890

0.1870

0.3894

0.7987

0.6248

4.4983

0.0103

±SD

17.75

0.0072

0.0080

0.0128

0.0225

0.1160

0.0376

0.4458

0.0007

n

10

5

5

5

5

5

5

5

10

G2, F & 100

Mean

335.68

0.0300

0.0927

0.1971

0.3952

0.8142

0.6053

4.5315

0.0105

±SD

14.74

0.0056

0.0086

0.0294

0.0293

0.1169

0.0242

0.2128

0.0011

n

11

5

5

5

5

5

5

5

11

G3, F & 300

Mean

321.23

0.0274

0.0843

0.1906

0.4083

0.8052

0.6491

4.8949

0.0111

±SD

19.34

0.0032

0.0171

0.0441

0.0370

0.1097

0.0618

0.2250

0.0016

n

10

5

5

5

5

5

5

5

10

G4, F & 600

Mean

322.66

0.0261

0.0950

0.1913

0.4327

0.7671

0.6322

4.8979

0.0109

±SD

19.22

0.0047

0.0157

0.0346

0.0869

0.1540

0.1329

0.5877

0.0010

n

10

5

5

5

5

5

5

5

10

G1R, F & 0

Mean

309.32

0.0318

0.1101

0.1955

0.3875

0.9013

0.8070

3.0633

0.0090

±SD

9.19

0.0020

0.0149

0.0342

0.0188

0.0268

0.0491

0.2507

0.0005

n

5

5

5

5

5

5

5

5

5

G4R, F & 600

Mean

291.01

0.0333

0.1249

0.2202

0.3805

0.9828

0.8303

3.1921

0.0094

±SD

13.04

0.0029

0.0185

0.0167

0.0061

0.0846

0.0516

0.1624

0.0015

n

5

5

5

5

5

5

5

5

5

Table 18. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - adult males

Group, Sex & Dose
(mg/kg body weight)

Serum T4 Levels

(ng/mL)

G1, M & 0

Mean

68.214

±SD

1.654

n

12

G2, M & 100

Mean

66.284

±SD

3.473

n

12

G3, M & 300

Mean

64.914*

±SD

2.161

n

12

G4, M & 600

Mean

65.095

±SD

3.031

n

12

*: Statistically significant than the control group (p<0.05)

Table 19. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - lactation day 13 pups

Group & Dose
(mg/kg body weight/day)

Serum T4 Levels (ng/mL)

G1 & 0

Mean

70.035

±SD

5.040

n

10

G2 & 100

Mean

70.175

±SD

7.644

n

11

G3 & 300

Mean

68.226

±SD

4.748

n

10

G4 & 600

Mean

70.150

±SD

5.715

n

10

n: Number of dams (pooled sample from two pups were selected for analysis from each dam)

Table 20. Summary of gross pathology findings: sire and dam

 

 

Parameters

Sex

Male

Female

Group

G1

G1R

G2

G3

G4

G4R

G1

G1R

G2

G3

G4

G4R

Dose

(mg/kg body weight)

0

0

100

300

600

600

0

0

100

300

600

600

No of rats

12

5

12

12

12

5

12

5

12

12

12

5

No. of dead animals during treatment period or recovery period

0

0

0

0

0

0

0

0

0

0

0

0

No. of moribund sacrificed animals

0

0

0

0

0

0

0

0

0

0

0

0

No. of terminally sacrificed

12

5

12

12

12

5

12

5

12

12

12

5

No of showing gross pathology

  1. No of animals showing external pathology

0

0

0

0

0

0

0

0

0

0

0

0

  1. No of animals showing visceral pathology

0

0

0

0

0

0

0

0

0

0

0

0

Table 21. Summary of gross pathology findings :pups

Group

G1

G2

G3

G4

Dose (mg/kg body weight/day)

0

100

300

600

Sex

Male

Female

Male

Female

Male

Female

Male

Female

No. of dead pups

0

0

0

0

0

0

0

0

No. of showing gross pathology

v No. of pups showing external pathology

0

0

0

0

0

0

0

0

v No. of pups showing visceral pathology

0

0

0

0

0

0

0

0

No. of pups sacrificed on lactation day 4

0

13

0

11

0

15

0

8

No. of showing gross pathology

v No. of pups showing external pathology

0

0

0

0

0

0

0

0

v No. of pups showing visceral pathology

0

0

0

0

0

0

0

0

No. of pups sacrificed on lactation day 13

45

60

67

31

61

46

57

37

No. of showing gross pathology

v No. of pups showing external pathology

0

0

0

0

0

0

0

0

v No. of pups showing visceral pathology

0

0

0

0

0

0

0

0

Table 22. Summary of histopathology findings

Sex

Male

Female

Group

G1

G4

G1

G4

Dosage (mg/kg/day)

0

600

0

600

ORGAN

SEVERITY

 

 

 

 

KIDNEYS

5

5

5

5

Basophilic tubules; unilateral

Minimal

1

1

0

0

Sub-urothelium ; Mononuclear cells

infiltrate; unilateral

Slight

0

0

0

1

THYROID

5

5

5

5

Ultimobranchial cyst

Not graded

2

0

2

2

Ectopic Tissue: Thymus

Not graded

0

2

0

1

PROSTATE GLAND

12

12

X

X

Interstitium, Mononuclear cells infiltrate

Minimal

2

1

X

X

Slight

0

1

X

X

Concretions

Minimal

0

2

X

X

Slight

0

1

X

X

UTERUS

X

X

5

5

Placental scar

Not graded

X

X

5

5

Note: The numeral indicates number of animal/ tissues; X: Organs not subjected for histopathological examination due to sex difference.

Conclusions:
Based on the findings of this study, the NOAEL of the test substance after a repeated oral exposure of 36 days in males and approximately 63 days in females was determined to be 600 mg/kg bw/day.




Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance Pine oil 50% by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight based on the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The test item was administered to the main group males for a period of 36 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and PND 13 pups.

There were no clinical signs, no mortality/morbidity, no effects of test item on mean body weight, feed consumption, no changes in ophthalmological examination and neurological/functional examination, clinical pathology, organ weights and gross pathology noted at group G2 of either sex. The group G3 and G4/G4R animals of either sex revealed treatment related wet perineum and recovered later in the study. A treatment related reduction in body weight was noted at both G3 and G4/G4R groups of either sex. No treatment related changes were noted in clinical pathology and organ weights and no gross pathological changes were noted at group G3 and G4/G4R. There were no treatment related changes in oestrus cyclicity, maternal and lactation body weight and feed consumption, mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter at group G2 and G3. A treatment related reduction in mean body weight and feed consumption during gestation and lactation periods and increased gestation length was noted at group G4. There were no treatment related changes occurred in histopathological examination conducted for group G4 animals in either sex. There were no treatment related changes noted in serum thyroxine hormone (T4) levels.

It can be concluded that the No Observed Effect Level (NOEL) is 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day, and the No Observed Adverse Effect Level (NOAEL) is 600 mg/kg bw/day as no adverse effects were found at any dose level tested.

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:
supporting study
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:
supporting study
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:
supporting study
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:
supporting study
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:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
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:
supporting study
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:
supporting study
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:
supporting study
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:
supporting study
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:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study although predating current guidelines was performed similarly to OECD Guideline 408.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
(no data on details of test animals and environmental conditions)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: The animals were housed individually in wire cages
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
7% loss of test compound from lab animal diet during a 7-day period.
Duration of treatment / exposure:
20 weeks
Frequency of treatment:
Fresh diets were made and distributed weekly.
Dose / conc.:
10 000 ppm
Remarks:
Equivalent to 400 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the Guidance on information requirements and chemical safety assessment R8, table R 8 -17)
Dose / conc.:
2 500 ppm
Remarks:
Equivalent to 100 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the Guidance on information requirements and chemical safety assessment R8, table R 8 -17)
Dose / conc.:
1 000 ppm
Remarks:
Equivalent to 40 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the Guidance on information requirements and chemical safety assessment R8, table R 8 -17)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Tissues from rats dying during the experiment were examined for gross changes and were preserved if autolysis was not advanced. Organs were not weighed but abnormalities and the suspected reason for death were noted.

HISTOPATHOLOGY: Yes; Organs weighed at termination, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10% buffered formalin-saline solution for histopathological examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Detailed microscopic examinations were done on 6 or 8 rats, evenly divided by sex, from the high dose group and the control group. If changes attributable to the test compound were found in the high dose group, additional animals on lower dosage levels were examined as indicated.
Clinical signs:
no effects observed
Mortality:
no mortality observed
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:
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
Description (incidence and severity):
No macroscopic change in the tissues in the 10000, 2500 and 1000 ppm exposure groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic change in the tissues in the 10000 ppm exposure group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Equivalent to NOAEL > 400 mg/kg bw/day.
Key result
Critical effects observed:
no
Conclusions:
No effects were observed in a 20-weeks repeated dose toxicity study in rats at the tested concentrations (up to 10000 ppm).
Executive summary:

In a 20 -weeks oral exposure study, Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information requirements and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 g/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg Terpinyl-Acetate-Alpha per kg bodyweight per day. Therefore, the NOAEL was calculated to be higher than 400 mg/kg bw/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 September 2012 – 28 March 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD guideline 422 and GLP
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(mating females were only administered test chemical up to 4 days of lactation instead of 13 days)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River. Atsugi Breeding Center.
- Age at study initiation: 10 weeks
- Weight at study initiation: 350-420 g (378 g average in males); 215-257 g (231 g average in females)
- Housing: bracket-type metal net cage (W 254 × D 350 × H 170 mm). During mating (1 male: 1 female) and from 17th day of pregnancy to 4th day of lactation a plastic cage (W340 × D400 × H185 mm) was used.
- Diet (e.g. ad libitum): NMF solid form (radiation sterilization, Oriental Yeast Co., Ltd.), ad libitum.
- Water (e.g. ad libitum): Tap water (metal cage) and from bottle (during time of lactation in plastic cage), ad libitum.
- Acclimation period: 20 days (including 3 days of quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24ºC
- Humidity (%): 39-65 %
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation of the test solutions was conducted by taking a required amount of the test substance with a glass syringe for each concentration and accurately weighing it in a beaker. The weighed test substance was transferred to a measuring cylinder. The beaker was washed several times with a small amount of solvent, and the liquid was also added to the measuring cylinder. The mixture was shaken by hand and mixed, and it was visually confirmed that it was completely dissolved. Next, the solvent was added to the measuring cylinder to the required amount of test material.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample of 10 mL of each formulation prepared for administration in the first and last week of the dosing period was analysed for achieved concentration of the test substance. The mean concentrations of terpineol in test formulations analysed for the study were within applied limits (99.5 to 103.0% of nominal concentrations), confirming accurate formulation.
The determination of the concentrations was performed by HPLC.
Duration of treatment / exposure:
Males: From 14th day before mating up to 30 days after mating (total 44 days).
Females: From 14th day before mating up to 4 days of lactation (total 41-51 days).
Non-mating female group: 44 days
Frequency of treatment:
Once a day, 7 days a week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Mating male group: 12 per sex per dose (including 5 in the control and high dose group for 14-day observation after treatment)
Mating female group: 12 per sex per dose
Non-mating female group: 10 (control group, including 5 for 14-day observation after treatment) and 10 (high dose group, including 3 for 14-day observation after treatment)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary range finding study was conducted with doses of 250, 500 and 1000 mg/kg bw/ day. One male in the 1000 mg/kg bw/day dose group showed a decrease in body weight and food intake, a decrease in feces volume, a decrease in locomotor activity and a decrease in respiratory rate, and died on the 12 th day of administration. A high value of liver and kidney weight and a high value of urea nitrogen and total protein were observed in autopsy of surviving males of high dose group. In each female of 500 and 1000 mg/kg bw/day group, a significant reduction in walking and muscle tone and a licking gait were observed only on 1 day of administration. Therefore, in this study, 1000 mg/kg bw/day at which obvious signs of toxicity is expected was set as high dose, and 300 and 100 mg/kg bw/day were set as medium and low dose, respectively, divided by the common ratio of about 3.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during the quarantine period, three times daily for the administration period (before administration, immediately after administration and 1 to 3 hours after administration), twice daily during the recovery period (morning, afternoon)
- Cage side observations: The presence or absence of abnormalities in general conditions such as life and death, appearance of the body, posture, behavior and emissions (urine, feces) were observed. Regular observation accompanied by handling of animals after 2 weeks of administration was also carried out as observation of detailed general condition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed general condition observation was performed once for all individuals, before administration. Also, males and non-mating females were observed once a week during the administration period and during the recovery period. Females in the mating group were observed once a week during the pre-mating period, during the mating period, during the pregnancy period and during the lactation period.

BODY WEIGHT: Yes
- Time schedule for examinations: For all individuals, body weight was measured on acclimated days 1, 3, 8, 13 and 20 and on the day of administration 1, 8, 15, 22, 29, 36, 42, 44 and on the day of necropsy. For the recovered animals further recovered the body weight was measured on days 1, 8 and 14 and on the day of necropsy. Body weights of female mating group were measured on day of administration 1, 8, 15, 22, on day of gestation 0, 7, 14, and on day 0 and 4 of lactation.

FOOD CONSUMPTION: yes
-Food consumption was determined daily by weighing the residue. Measurements were not made during the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The day before necropsy after administration period and after 14-day observation period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals per sex and per group
- Parameters checked: Erythrocyte count (RBC), Haemoglobin concentration (HGB), Haematocrit (HCT), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Reticulocyte count (Retic), Platelet count (PLT), White blood cell count (WBC), differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC)), Prothrombin time (PT), Activated partial thromboplastin time (APTT) and Fibrinogen (FIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The day before necropsy after administration period and after 14-day observation period.
- Animals fasted: Yes
- How many animals: 5 animals per sex and per group
- Parameters checked: Serum was analysed for: Alkaline phosphatase (ALP), Total bile acid (TBA), Total cholesterol (T-CHO), Triglyceride (TG), Phospholipids (PL), Total bilirubin (T-BIL), Glucose (GLU), Urea nitrogen (BUN), Creatinine (CRNN), Sodium (Na), Potassium (K), Chlorine (Cl), Canoesium (Ca), Inorganic phosphorus (P), Total protein (TP) and Anolebumin (ALB). Heparinized blood was examined for Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Lactate dehydrogenase (LDH) and Gamma-glutamyl transpeptidase (GTP).

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of the administration period and during the recovery period urine was collected for 4 and 20 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: For the first 4 hours urine collected pH and urine volume were taken. Osmotic pressure, electrolytes (sodium, potassium and chlorine) and urine volume were measured for the 20 hours urine obtained.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy, which included a visual examination of the external tissues and organs of the head, thorax and abdomen.

HISTOPATHOLOGY: Yes

- ORGAN WEIGHTS:
The absolute weight and relative weight of the following organs were measured: Brain, pituitary, thyroid (including parathyroid gland), adrenal gland, spleen, thymus, tongue, heart, liver, Kidney, testis, epididymis, prostate, seminal vesicle (including coagulated gland), ovary and uterus.

-HISTOPATHOLOGY:
For all individuals, the following organs / tissues were fixed for histopathology examination: cerebrum, cerebellum, sciatic nerve, spinal cord (chest), the eyeball, optic nerve, pituitary, thyroid, parathyroid, adrenal gland, spleen, thymus, submandibular lymph node, mesenteric lymph node, heart, aortic aorta, trachea, lung (including bronchus), tongue, larynx, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, kidney, testis, ovary, epididymis, uterus, prostate, seminal vesicle (coagulating glands mate), skin (inguinal region), mammary gland (inguinal region), sternum, femur (including bone marrow) and femoral skeletal muscle. Samples of any abnormal tissues were also retained and processed for examination. Testis and epididymis were fixed with Bouin's fixative.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
CAGE SIDE OBSERVATIONS: There was no abnormality in any animal.
SIGNS AND ARENA OBSERVATIONS: Wiggling was observed on the first day of pregnancy in one female in the mating group of 1000 mg/kg/day administration group.
In addition, two animals were observed gait abnormality and jumping respectively but they were temporary findings and it was judged that there was no relation with the administration of the test substance because it was not related to the dose. Furthermore, facial cleansing behavior was observed in another animal, which is a normal behavior observed when the animal calms down, and is not related to administration of the test substance.

General condition of surviving animals: In the 1000 mg/kg/day dose group, 4 males had nursing behaviour and 1 male a decrease in feces volume, and in females, a decrease in feces volume was observed. In 3 females of the non - mating group, decrease in fecal volume, scaling, decreased motor activity, wiggling gait or decrease in respiration rate were observed.
These symptoms were not seen during the recovery period, thus it was judged as no treatment related.

Gripping strength: There was no significant difference between the control group and each test substance administration group.

Motor activity: There was no significant difference between the control group and each test substance administration group. Meanwhile, in the females of the non-mating group, a significant low value was observed in the measurement values of 0 - 10 and 10 - 20 intervals in the 1000 mg/kg/day dose group compared with the control group, but there was no significant difference in the total measurement value (0-60 interval) and it was equivalent to the measurement value of the control group at the end of the recovery period week. Thus it was judged to be a change within the variation range.
Mortality:
mortality observed, treatment-related
Description (incidence):
6 females in the 1000 mg/kg/day dose group were found dead or euthanized. These animals showed worsening status before moribund and death, suppression and reduction in body weight gain, low food consumption, decreased fecal volume, scraping, wiggling, decreased motor activity, decreased respiratory rate, abdominal drainage and decrease in body temperature. In addition, it was mainly characterized by malnutrition, reduction of spleen and thymus, downsizing of kidney (renal tubular dilation, vacuolization of proximal renal tubule, vacuolation of distal tubule / collecting duct, papillary necrosis, single cell necrosis of teat canal, regeneration of the papillary collecting duct, papillary cell infiltration), single cell necrosis of the transitional epithelial cell, hypertrophy / hyperplasia of the transitional epithelial cell, adrenal gland cell vacuolation, eosinophilic droplets and vacuolation of hepatocytes in the liver. Other secondary changes accompanying state deterioration include atrophy of various lymphoid tissues, atrophy of colonic mucosa, land mucosa and uterine atrophy or reduction of zymogen granules of pancreas, mesenterium lymph nodes, submandibular lymph nodes and thymus.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males, suppression of body weight gain was observed in the 1000 mg/kg/day dose group, and a significant lower value was observed in body weight gain during the administration period. In the group administered with 300 mg/kg/day or less, the body weight transition was similar to that of the control group, and no significant difference was observed.
In the mating group females, body weight was lower than that of the control group through premating, pregnancy and lactation period in the 1000 mg/kg/day dose group, but no significant difference was observed. A significant low value was observed on the 0th day of lactation compared to the control group in the 300 mg/kg/day administration group, but it was a temporary change, and the weight gain increase in the subsequent lactation period was found to be a significant high value. It was judged that it was not related to administration of the test substance. In the 100 mg/kg/day administration group, no significant difference was observed between the control group and the control group.
In the non-mating group females, mean body weight decreased due to the moribund / death cases on day 4 of administration in the 1000 mg/kg/day dose group, and a significant lower value was observed than in the control group. However, it is a transient fluctuation due to the moribund / death cases, not an influence on the body weight by administration of the test substance.
In the 1000 mg/kg/day dose group, both sexes showed weight gain above the control group during the recovery period, but no significant difference was found between the treated group and the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In males, a significant high value was observed on the 30th and 44th days of administration in the 1000 mg/kg/day dose group, but it was judged as a change within the variation range because it was temporary. In the group administered with 300 mg/kg/day or less, the food consumption was equivalent to that of the control group, and no significant difference was observed. In the mating group females, there was no significant difference between the control groups and the test substance administration group in the pre-mating