Registration Dossier

Administrative data

Description of key information

OECD 422 (gavage): there was some adaptive liver enlargement, especially in females associated with minimal centrilobular hepatocyte

hypertrophy and biochemical findings. Histopathological changes associated with hyaline droplets were observed in the kidneys of male rats receiving 250 or 750 mg/kg/day but such well-known changes are considered not relevant to human health. Testicular and epididymal toxicity was evident in males at 750 mg/kg/day leading to infertility. The NOAEL was established at 250 mg/kg/day for male rats based on testicular/epididymal toxicity (gavage administration was considered to be a worst case for this effect), and at 250 mg/kg/day in female rats based on the effect reported in the liver at 750 mg/kg/day.
90-day toxicity study (diet): male rats were exposed at 12000 ppm, corresponding to 623 mg/kg/day (i.e. an exposure level similar to the 28-day toxicity study by gavage). Increased liver weights (absolute and relative) were noted but no significant testicular and epididymal toxicity was evidenced in organ weight, spermiological parameters and at histopathological level.
OECD 413 (inhalation): treatment related mucous cell hyperplasia of the respiratory epithelium of the nasal turbinates was evident in almost all males exposed to terpineol multiconstituent. This finding was also observed in the nasal pharynx, but at a lower incident and severity.
Complete recovery of the degeneration and inflammation in the respiratory epithelium was observed. Only partial recovery of the degeneration of the olfactory epithelium was observed, although due to the capacity of the tissue to regenerate, complete recovery may occur following a longer recovery period. These changes were not considered adverse at the incidence and severity seen.
The No Observed Adverse Effect Concentration (NOAEC) for this study was considered to be 2.23 mg/L.

In a toxicokinetic study, six groups of male Sprague-Dawley rats were administered Terpineol multiconstituent via oral (gavage) at doses of 0 (vehicle), 100, 250, 400, 600 or 750 mg/kg bw/day.

The test item was clinically well tolerated up to 750 mg/kg bw/day. Clinical signs were limited to ptyalism. Slightly lower mean body weight and mean food consumption were recorded at the dose levels of 600 and 750 mg/kg bw/day and were not considered adverse. At microscopic examination, effects were observed on male genital tract at 600 and 750 mg/kg/day and were considered adverse at 750 mg/kg bw/day only, as degeneration/necrosis was noted at this dose.

Repeated dose toxicity studies were conducted in rabbits in order to investigate the adequacy of the vehicle for gavage administration and the tolerance of this species to the registered substance after repeated exposure. It was concluded that, due to the steep dose response between 400 and 600 mg/kg bw/day in methylcellulose, and the potential for the toxicity profile to be different between the non-pregnant and pregnant female rabbit, a cautious approach should be taken in the preliminary embryo fetal development study, with the study conducted in two phases, in order to establish a high dose level suitable for repeated administration for the required 23-day dosing period (Day 6 to Day 28 after mating).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 August 2011 to 04 February 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP study; haematology, clinical chemistry and ophthalmological examination not performed
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
not applicable
GLP compliance:
no
Remarks:
The study was conducted in a GLP laboratory.
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore, India.
- Age at study initiation: 12 weeks
- Weight at study initiation: G1: 371.07 ± 12.60 g; G2: 369.06 ± 10.11 g
- Housing: one rat per cage was housed in sterilized suspended polysulfone cages (size: approximately L 425 mm x W 266 mm x H 175 mm) with stainless steel top
- Diet: Ssniff rats/mice pellet food and powder food – maintenance meal - manufactured by Ssniff spezialdiäten GmbH, Ferdinand-Gabriel-Weg 16, D-59494 SÖest, Germany was provided ad libitum.
- Water: deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 60-67 %
- Air changes: 12-14 air changes/hour
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
- The required quantities of test item (G2- 12.0 g) was weighed, dissolved in 30 mL corn oil and mixed with approximately 300 g of Ssniff powder food for 2 minutes in stainless steel drum using a stainless steel spoon to prepare the premix. The premix was added in portions to the remaining bulk food (approximately 700 g) and mixed in a stainless steel drum using spoon for approximately 5 to 8 minutes.
- For the negative control group (G1), 1000 g of Ssniff powder food was mixed with 30 mL corn oil and mixed manually in a stainless steel drum using a spoon for 7-10 minutes.
A similar procedure was followed for the subsequent mixing sessions. The leftover formulated diet was sent for incineration.
- Rate of preparation of diet (frequency): test item fortified food was prepared daily.

Stability and homogeneity of the test item
- The Sponsor confirmed 2 days ambient storage stability and 8 days frozen at 100 and 20000 ppm, when the test material was first dissolved in corn oil to improve stability (Thacker C., 2010).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Test item fortified diet was fed daily ad libitum throughout the treatment period (13 weeks)
Remarks:
Doses / Concentrations:
12000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 males/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: a dose level of 12000 ppm for terpineol multiconstituent No. 2 was selected by the Sponsor. In addition to test dose, a concurrent negative control group (0 ppm) was included.
- Rationale for animal assignment: rats were randomly distributed to different groups by body weight stratification method using ProvantisTM software. Grouping was done one day prior to initiation of treatment.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
Time schedule: general clinical signs were observed once daily. All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: yes
Time schedule: detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment period.

BODY WEIGHT:yes
Time schedule for examinations: individual body weights were recorded before the start of treatment and at weekly intervals thereafter except for Week 13 wherein the animals were weighed on Day 6 of that week. Fasting body weights were recorded on Day 91.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured daily during the treatment period. The average food consumption (g/rat/day) was calculated.
Mean food intake (g/kg bw/day) and test item intake (mg/kg bw/day) were calculated from food intake and body weight data.
Food Intake (g/kg bw/day):
Net food intake (g/kg bw/day) = (Mean food intake / Mean Body weight) x 1000
Test Item Intake (mg/kg bw/day) = (Dose (ppm) / 1000) x Net food intake

WATER CONSUMPTION: no

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: no

CLINICAL CHEMISTRY:no

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: no
Sacrifice and pathology:
GROSS PATHOLOGY: yes
Rats sacrificed by design (terminal necropsy) were euthanized by exsanguination under isoflurane anaesthesia. All rats in the study were subjected to necropsy after overnight fasting (water allowed).

HISTOPATHOLOGY: yes
Tissue collection: on completion of the gross pathology examination, the tissues listed in the table 7.5.1/1 were collected and weighed from all the animals. The organ weight ratios as percentage of body weight were also determined. All the collected tissues were preserved in 10 % neutral buffered formalin.
Histopathological examination of testes and epididymides were performed for all the animals. The tissues were processed for routine paraffin embedding and 5 µm sections stained with Mayer’s Haematoxylin and Eosin stain.
Other examinations:
Sperm evaluation: sperm evaluation was carried out at termination for all males. At necropsy, right testes and corresponding epididymis were collected and frozen for sperm count and the right vas deferens was collected for evaluation of sperm motility and sperm morphology. Sperm motility was evaluated for all the groups using Hamilton- Thorne TOX-IVOS sperm analyzer.
Statistics:
Data were captured using ProvantisTM for the parameters namely body weights and organ weights, and were analysed using built-in statistical tests.
The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package version 12.0. All quantitative variables like body weight, sperm parameters, organ weights and organ weight ratios were tested for homogeneity of variances (Levene’s test) within the group before performing one-way analysis of variance (ANOVA).
For the non-optimal data (non-normal or heteroschedastic), ANOVA was done after suitable transformation of data.
Comparison of means between the treatment group and the control group was done using Dunnett’s test, where ‘F’ test found significant.
All analyses and comparisons were evaluated at the 5% (P≤0.05) level.
Statistically significant differences (P≤0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below:
+/-: significantly higher/lower than the control group
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increase in liver weight
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment related mortality or signs of toxicity were noted.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight on Day 8 was lower when compared to the initial body weight (3.6 % of mean body weight loss) in the treated group. After Day 15 the mean body weights were increased when compared to the initial body weight but remained significantly lower when compared to control group (5-6 % lower than controls). The net weight gain was markedly reduced during the first week of treatment and during the Days 57-64.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake was markedly reduced during the first week of treatment in animal receiving 12000 ppm of test item (up to 80-90% decrease during the first 3 days when compare to control group) indicating the non-palatability of the test item, and then remained significantly lower during most of treatment period (13% decrease in mean food intake when compare to controls).
Thus, the decreased body weights at 12000 ppm dose group were associated with the decrease in the food intake throughout the treatment period.

ORGAN WEIGHTS
There were no test item-related changes in the terminal fasting body weights. The lower terminal fasting body weight (6%) noted in the test item treated group was considered incidental.
Increased liver weights (absolute-13 % and relative-20 %) noted in the treatment group was considered to be test item-related. Slight increase in relative weights (paired and unpaired) of testes and epididymides observed in the treatment group were considered to be incidental, as it was not associated with any microscopic change.
All other weight changes noted in treated males which attained statistical significance were of low magnitude and/or were comparable to historical data, hence considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test item-related histological changes observed in testis and epididymis.

OTHER FINDINGS
A slight significant increase in the percentage of abnormal (4.8%) sperms was noted in treatment group as compared to the control group. However, the change was considered incidental as it was well within range of normal biological variation noted among male rats [the range of the in-house historical control data for mean percentage of abnormal sperms: 0.1- 7.4%] . The sperm motility remained unaffected by dietary administration of test item. There were no test item-related changes observed in cauda epididymal weight/sperm count and testicular weight/spermatid count.
Key result
Dose descriptor:
other: NOAEL male fertility
Effect level:
>= 12 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No testicular and epididymal toxicity was evidenced in animals receiving terpineol multi-constituent No. 2 at 12000 ppm for 90 days to Sprague-Dawley rats
Critical effects observed:
not specified

Table 7.5.1/2: Calculated mean food and test item intake

Group No.

Dose (ppm)

G1

0

G2

12000

Food Intake

g/animal/day

23.99

20.97

g/animal/91 days

2183.34

1908.63

Net Food Intake

g/kg bw/day

55.94

51.89

g/kg bw/91 days

5090.69

4721.76

Test Item Intake

mg/kg bw/day

0.00

622.65

mg/kg bw/91 days

0.00

56661.15

Table 7.5.1/3: Summary of Terminal Body Weights and Organ Weights (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Heart

(g)

Liver

(g)

Prostate

(g)

Seminal vesicles

& coagulating

glands

(g)

Spleen

(g)

Adrenals

(g)

Epididymides

(g)

Kidneys

(g)

Testes

(g)

G1

0

449.12 ± 22.56

1.415 ± 0.108

12.850 ± 0.999

1.399 ± 0.170

1.9188 ± 0.1937

0.845 ± 0.095

0.053 ± 0.007

1.437 ± 0.072

2.666 ± 0.161

3.977 ± 0.225

G2

12000

422.26 ± 14.55 (-)

1.311 ± 0.082 (-)

14.468 ± 0.894 (+)

1.246 ± 0.163

1.7579 ± 0.2106

0.858 ± 0.091

0.057 ± 0.009

1.464 ± 0.089

2.676 ± 0.110

4.072 ± 0.186

%Diff

6

7

13

$

$

$

$

$

$

$

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/4: Summary of Terminal Body Weights and Organ to Body Weight Ratios (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Heart

(%)

Liver

(%)

Prostate

(%)

Seminal vesicles

& coagulating

glands

(%)

Spleen

(%)

Adrenals

(%)

Epididymides

(%)

Kidneys

(%)

Testes

(%)

G1

0

449.12 ± 22.56

0.3152 ± 0.0203

2.8592 ± 0.1207

0.3123 ± 0.0415

0.4281 ± 0.0481

0.1884 ± 0.0225

0.012 ± 0.002

0.321 ± 0.022

0.5944 ± 0.024

0.887 ± 0.052

G2

12000

422.26 ± 14.55 (-)

0.3103 ± 0.0136 

3.4271 ± 0.1922 (+)

0.2951 ± 0.0382

0.4165 ± 0.0491

0.2033 ± 0.0216

0.013 ± 0.002

0.347 ± 0.027 (+)

0.634 ± 0.017 (+)

0.965 ± 0.049 (+)

%Diff

6

$

20

$

$

$

$

8

7

9

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/5: Summary of Terminal Body Weight and Paired Organ Weights (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Left Adrenal

(g)

Right

Adrenal

(g)

Left

Epididymis

(g)

Right

Epididymis

(g)

Left Kidney

(g)

Right Kidney

(g)

Left Testis

(g)

Right Testis

(g)

G1

0

449.12 ± 22.56

0.027 ± 0.004

0.027 ± 0.004

0.725 ± 0.049

0.712 ± 0.034

1.327 ± 0.099

1.339 ± 0.075

1.974 ± 0.115

2.004 ± 0.115

G2

12000

422.26 ± 14.55 (-)

0.028 ± 0.005

0.029 ± 0.004

0.743 ± 0.044

0.721 ± 0.052

1.324 ± 0.072

1.352 ± 0.065

2.042 ± 0.081

2.029 ± 0.109

%Diff

6

$

$

$

$

$

$

$

$

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/6: Summary of Terminal Body Weight and Paired Organ to Body Weight Ratios (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Left Adrenal

(%)

Right

Adrenal

(%)

Left

Epididymis

(%)

Right

Epididymis

(%)

Left Kidney

(%)

Right Kidney

(%)

Left Testis

(%)

Right Testis

(%)

G1

0

449.12 ± 22.56

0.006 ± 0.001

0.006 ± 0.001

0.162 ± 0.013

0.159 ± 0.011

0.295 ± 0.018

0.298 ± 0.008

0.440 ± 0.028

0.447 ± 0.024

G2

12000

422.26 ± 14.55 (-)

0.007 ± 0.001

0.007 ± 0.001(+)

0.176 ± 0.013 (+)

0.171 ± 0.015

0.313 ± 0.012 (+)

0.320 ± 0.014 (+)

0.484 ± 0.020 (+)

0.481 ± 0.029 (+)

%Diff

6

$

14

9

$

6

7

10

8

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/7:Summary of Vas Deferns Sperm Motility, Cauda Epididymidal Count and Detergent and Homogenization Resistant Testicular Spermatid Counts

 

Group No.

Dose (ppm)

Motility

Cauda Epididymal Sperm Counts

Detergent and Homogenization Resistant Testicular Spermatid Counts

Percentage of

progressive

motile sperms

Percentage

of motile

sperms

Cauda

epididymis

weight (g)

No. of sperms

per cauda

epididymis

(x 106)

No. of sperms

per gram of

cauda

epididymis

(x 106)

Parenchyma

weight (g)

No. of

spermatids

per testis

(x 106)

No. of

spermatids per

gram of

parenchyma (x 106)

G1

0

88.3 ± 4.00

66.3 ± 5.98

0.265 ± 0.022

224.73 ± 22.70

850.74 ± 69.17

1.792 ± 0.107

233.65 ± 19.72

130.58 ± 11.24

G2

12000

87.8 ± 4.16

65.3 ± 5.81

0.280 ± 0.010

230.20 ± 25.31

822.50 ± 79.23

1.815 ± 0.102

224.63 ± 20.28

123.89 ± 10.89

%Diff

$

$

$

$

$

$

$

$

N = 10

$: Not calculated as difference is not statistically significant.

Conclusions:
No testicular and epididymal toxicity was evidenced in Sprague-Dawley rats receiving terpineol multiconstituent No. 2 at 12000 ppm for 90 days.
Executive summary:

In a repeated dose oral dietary toxicity study terpineol multiconstituent No. 2 was administered to Sprague-Dawley rats for 90 days. The test item was dissolved in corn oil, mixed in Ssniff powder feed at the dose level of 12000 ppm and fed to male Sprague-Dawley rats (10/dose) daily ad libitum for 13 weeks. Rats in the control group were fed basal diet only without any test item admixtures. All rats were observed for clinical signs, mortality, and changes in the body weights and food intake. Sperm evaluations were conducted at termination for all the males from each group. Sperm motility, count and morphology were evaluated for all the groups. All rats were subjected to detailed necropsy at termination and organs were weighed. Histopathological examination of the testes and the epididymides were carried out.

 

No treatment related mortality or signs of toxicity were noted. The body weights were significantly reduced in rats receiving test item at 12000 ppm. This decrease was associated with a decrease in the food intake throughout the treatment period. The Food consumption was significantly reduced in males receiving test item at 12000 ppm during the treatment period. The calculated mean daily test item consumption was 0 and 622.65 mg/kg bw/day corresponding to 0 and 12000 ppm, respectively.

 

A slight significant increase in the percentage of abnormal sperms (4.8%) was noted at 12000 ppm as compared to the control group. However, the change was considered incidental as it was well within the range of normal biological variation noted among male rats [the range of the in-house historical control data for mean percentage of abnormal sperms: 0.1- 7.4%]. The sperm motility remained unaffected by dietary administration of test item. There were no test item-related changes observed in cauda epididymal weight/sperm count and testicular weight/spermatid count.

 

There were no test item-related changes in the terminal fasting body weights. Increased liver weights (absolute-13 % and relative-20 %) were noted in the treatment group. Increased relative weights (paired and unpaired) of testes and epididymides were observed in the treatment group. There were no test item-related histological changes observed in the testis and the epididymis.

No testicular and epididymal toxicity was evidenced in Sprague-Dawley rats receiving terpineol multiconstituent No. 2 at 12000 ppm for 90 days. 

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 28 to September 03, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD guideline 422 without deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
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, UK
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 303 to 375 g for males and 198 to 253 g for females
- Housing: Up to 5 during pre-mating for all animals and after mating for males and during toxicity phase for unmatted females, individually with litter for females during gestation and lactation.
- Diet (e.g. ad libitum): Standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Main phase males, Toxicity phase females and Recovery phase animals.
- Water (e.g. ad libitum): Potable water taken from the public supply, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 April 2010 To: 29 June 2010
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Approximately 50% of the final volume of corn oil was added to the required amount of test material. The formulation was mixed using a magnetic stirrer until all of the test material had dissolved and more corn oil was added to make up the required volume. The formulation was then mixed using a magnetic stirrer until homogeneous.
Initially all formulations were prepared freshly on the day of use and used within two hours of completion of preparation. However, following confirmation of the results from a homogeneity and stability, formulations were prepared weekly, subdivided into daily aliquots and used within 8 days of preparation.

VEHICLE
- Concentration in vehicle: 12, 50 and 150 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last weeks of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two assays from each group were analysed. The mean concentrations of Terpineol multiconstituent in test formulations analysed for the study were within applied limits, +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. An additional five males and five females were dosed with the vehicle or at 750 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. Offspring were not dosed.
Frequency of treatment:
Once a day, 7 days a week
Remarks:
Doses / Concentrations:
60, 250 and 750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- Reproductive subgroup (main phase): 10 males and 10 females/dose (except for control males and at top dose: 5 males/dose)
- Toxicity subgroup: 5 females/dose and same males as for reproductive subgroup
- Recovery subgroup: 5 males and 5 females /dose (control and top dose); Recovery phase males also used for pairing with Main reproductive phase females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a two week preliminary study (Huntingdon Life Sciences Study No. OAD0003) which tested dose levels of 150, 600 and 1000 mg/kg bw/day. In that study animals dosed at 600 and 1000 mg/kg bw/day showed post dose observations of salivation and chin rubbing and some females at 1000 mg/kg bw/day also showed isolated incidences of reduced activity, reduced body tone and unsteady gait. An initial reduction in bodyweight was recorded in males at 600 and 1000 mg/kg bw/day. At 1000 mg/kg bw/day increased water consumption was recorded and at necropsy liver weights was increased whilst the testis and epididymal weight were reduced (67 and 76% of control, respectively). The dose levels selected for the study included a high dose of 750 mg/kg bw/day which was expected to generate some toxic reaction but any effect on testes was expected to be minimal and to not impair the mating performance of these animals. The low and intermediate dose levels were selected to establish a no observed adverse effect level to give suitable safety margins and establish dose response relationships.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Animals and cages were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded in relation to dose administration. For the Main phase males and Toxicity phase females dosing observations were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For Main phase females these were recorded daily during the first week of dosing, twice weekly during Week 2 of dosing, on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation. Observations were recorded at the following times in relation to dose administration:
Pre-dose
On return of the animal to its home cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal (physical condition and behaviour during handling with particular attention to possible signs of neurotoxicity). For the Reproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation. A weekly physical examination including arena observations was performed during the recovery period.

BODY WEIGHT:
The weight of the Main phase males and Toxicity phase females was recorded on the day that dosing commenced (Week 0), weekly throughout the dosing and recovery periods and before necropsy. Main phase females were weighed on the day that dosing commenced (Week 0), weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the start of study for Main phase males and Toxicity phase females and Main phase females until the animals were paired for mating. Food consumption was recorded weekly (g/animal/week) during the recovery period. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage. Food consumption was not recorded for Main phase males and females during pairing.
For each Main phase female after mating, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-6, 7-13 and 14-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation.

OTHER:
- SENSORY REACTIVITY:
Sensory reactivity and grip strength assessments were performed (before dosing) on the second 5 main phase (group 5 and 6)/recovery group (control and group 7) males and on toxicity phase (Groups 5 and 6)/recovery phase (Control and Group 7) females during Week 5 of study. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle reflex, tail pinch response and grip strength.

- MOTOR ACTIVITY:
During Week 5 of study (before dosing), the motor activity of the second five main phase (Groups 5 and 6)/recovery phase (Control and Group 7) males and on toxicity phase (Groups 5 and 6)/recovery phase (Control and Group 7) females was measured using a Rodent Activity Monitoring System (Version 2.0.3). Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).

- HAEMATOLOGY:
During Week 5 of treatment (before dosing on each occasion) and after 2 weeks of recovery, blood samples were obtained from the first five main phase males and on the toxicity phase females after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein. The following were measured using a Bayer Advia 120 haematology analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)) and Platelet count (Plt). The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded. Prothrombin time (PT) (using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent) and Activated partial thromboplastin time (APTT) (using an ACL 3000 Plus Analyser and IL APTT reagent) were also measured.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected and the plasma was examined using a Roche P Modular Analyser for: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile Acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb) (by chemical assay).
Sacrifice and pathology:
SACRIFICE:
Main phase males and Toxicity phase females were killed in Week 6 after completion of the Week 5 investigations. The recovery phase animals were killed after 2 weeks of recovery and after haematology and blood chemistry sampling.
Main phase females (Groups 1, 5 and 6) were killed on Day 7 of lactation. Main phase females that did not litter (Group 7) were killed on Day 25 after mating. Offspring were killed on Day 7 of age.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For Reproductive subgroup females, the number of uterine implantation sites was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 1 were weighed.
The following tissues from Main phase male, Toxicity phase female, all recovery phase animals, Main phase females that did not litter (Group 7) and those animals killed or dying prematurely were fixed for histopathology: Adrenal glands, Brain, Pituitary, Prostate, Caecum, Colon, Rectum, Sciatic nerves, Duodenum, Seminal vesicles and coagulation gland, Epididymides (L&R), Skeletal muscle, Skin, Mammary glands (inguinal area), Heart, Spinal cord, Ileum, Spleen, Jejunum, Sternum with marrow, Kidneys, Stomach, Liver, Testes (L&R), Lungs, Thymus, Lymph nodes (mandibular and mesenteric), Thyroid with parathyroids, Trachea, Urinary bladder, Oesophagus, Uterus with cervix and oviducts, Peyer’s patch, Ovaries (L&R) and Vagina.
The following tissues from each Main phase female that did litter (Groups 1, 5 and 6) were fixed for histopathology: Ovaries (L&R), Uterus with cervix and oviducts and vagina. Samples of any abnormal tissues were also retained and processed for examination.

POSTMORTEM EXAMINATIONS (OFFSPRING):
All offspring killed or dying prior to scheduled termination, and of those killed at the end of the study were subjected to detailed necropsy.
Other examinations:
ESTROUS CYCLICITY (PARENTAL ANIMALS):
For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all Reproductive subgroup females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.

LITTER OBSERVATIONS:
PARAMETERS EXAMINED:
All litters were examined at approximately 24 hours after birth and then daily thereafter for clinical signs (evidence of ill health or reaction to treatment), litter size (mortality and consequent changes in litter size from Days 1-7 of age), sex ratio of each litter (recorded on Days 1, 4 and 7 of age) and individual bodyweight (recorded on Days 1, 4 and 7 of age).

GROSS EXAMINATION OF PUPS:
All offspring killed or dying prior to scheduled termination, and of those killed at the end of the study were subjected to detailed necropsy.
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, litter size and survival indices and clinical pathology data:
1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For comparisons involving two groups only t-tests were used, for all other comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For comparisons involving two groups only, Wilcoxon’s rank sum tests (Wilcoxon 1945) were used. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.
For gestation length and sperm count estimates an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No death attributed to the test material. Underactive behaviour and unsteady reactions, in males and females receiving 750 mg/kg/day (during week 1). Dose related increases in post dosing salivation and chin rubbing were seen.
Mortality:
mortality observed, treatment-related
Description (incidence):
No death attributed to the test material. Underactive behaviour and unsteady reactions, in males and females receiving 750 mg/kg/day (during week 1). Dose related increases in post dosing salivation and chin rubbing were seen.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 750 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. No body weight effects in unmatted females receiving up to 750 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Visual assessment of water consumption indicated that males and females receiving 750 mg/kg/day were consuming more water than the Controls during the dosing period
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day:urea and creatinine levels were significantly higher in females and in males. At 250 and 750 mg/kg/day: glucose plasma levels were significantly higher in females and marginally high in males. No effects remained after recovery period
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
at 750 mg/kg/day: Bodyweight adjusted liver weights were significantly higher than Control in males and females and bodyweight adjusted kidney weights were significantly higher than Control in males; testis weight was markedly low in males.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 750 mg/kg/day showed a range of testicular findings including small, blue and flaccid testes. Epididymides of males receiving 750 mg/kg/day were observed generally small but some contained masses or appeared enlarged.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with CAS 8000-41-7 at 750 mg/kg/day. Histopathological findings in the kidneys of males receiving 250 and 750 mg/kg/day. Effects resolved after recovery period.
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY (PARENTAL ANIMALS):
In the 60 mg/kg/day dose group, one male was found dead on Day 31 of study and one female was killed because of parturition difficulties. This female had given birth to three pups and but still had 15 live pups and one early resorption in utero. The difficulty during parturition may be associated with the presence of an abnormally enlarged placenta as maternal necropsy findings and microscopic evaluation of the organs did not identify any other factors. In the absence of any other death in the intermediate and high dose groups these deaths are not attributed to the test material.

CLINICAL SIGNS:
On the first two days of dosing most of the females and a few males receiving 750 mg/kg/day were recorded as having an unsteady gait and some animals were underactive, but all findings resolved within the working day. Over activity was also observed as a post dosing sign during Week 1 in females dosed at 60 mg/kg/day and males and females at 250 mg/kg/day. Signs of salivation and/or chin rubbing were recorded, largely at 750 mg/kg/day, but these are common reactions to the dosing process where the material might be distasteful and probably unrelated to systemic toxicity.

BODY WEIGHT AND FOOD CONSUMPTION:
There were no statistically significant effects of Terpineol multiconstituent on bodyweight or bodyweight gain.
Overall weight gain (Weeks 1-5) was slightly, but not significantly reduced for males dosed at 750 mg/kg/day (83% of Control). Much of this effect occurred during pairing and there were minimal or no effects on bodyweight in males receiving 250 or 60 mg/kg/day.
Weight gain of females from Week 0-5 were slightly lower than Control at all dose levels, but in the absence of any consistent trends it was considered to be unaffected by the test material.
During gestation there was no clear effect on bodyweight although gains were slightly lower than Control, and during lactation bodyweight gain of females receiving 250 mg/kg/day were lower than Control.

There were no test material related effects on food consumption. The increase in food consumption observed in all animals during the recovery period was due to cessation of dose administration which used corn oil as the vehicle thereby supplying a portion of the required nutrients.

WATER CONSUMPTION:
Qualitative assessment of water consumption by checking residual levels in the water bottles indicated that animals receiving 750 mg/kg/day consumed more water than the Controls.

REPRODUCTIVE FUNCTION (ESTROUS CYCLE) AND REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
There was no effect of Terpineol multiconstituent on oestrous cycles or precoital interval. All females dosed at 750 mg/kg/day failed to litter and were found to have no implantation sites and not to have been pregnant when examined at necropsy on Day 25 after mating. It is considered that the testicular and epididymal effects observed in males receiving 750 mg/kg/day would have been sufficient to prevent fertilisation.
There was no effect of Terpineol multiconstituent on mating performance or fertility at dose levels of 250 mg/kg/day or below.
All females that littered had normal length gestation periods (22 – 23 days duration) but a slightly higher proportion of females at 250 mg/kg/day gave birth after 23 days.

ORGAN WEIGHTS (tables 6 and 7):
After 5 weeks of dosing bodyweight adjusted mean liver weights of males and females receiving 750 mg/kg/day were significantly increased, kidney weights of males were also significantly increased. Testis weight was markedly lower in males receiving 750 mg/kg/day (58% of Control) and there was also a suggestion of slightly lower epididymal weights for these males. Two males at 250 mg/kg/day had combined testis weight below the background range (90 percentile range – males: 2.97-4.07 (n=155)) associated to lower epididymal weights, group mean values for this group were similar to Control.
After two weeks without dosing liver and kidney weights were no longer enlarged but testis and epididymal weights showed no evidence of recovery.

GROSS PATHOLOGY (Table 8):
Males receiving 750 mg/kg/day showed a range of testicular findings including small, blue and flaccid testes. Epididymides of males receiving 750 mg/kg/day were observed generally small but some contained masses or appeared enlarged. Two males receiving 250 mg/kg/day also had small testes. Similar findings were still apparent in males killed after two weeks of recovery. There were no significant necropsy findings for females on Day 7 of lactation.

HISTOPATHOLOGY (Table 9):
Liver:
Minimal centrilobular hepatocyte hypertrophy was seen in the liver of three toxicity phase females dosed with Terpineol multiconstituent at 750 mg/kg/day. However, these histopathology findings showed complete recovery after 2 weeks without dosing.
Kidney:
Histopathological changes associated with hyaline droplets were observed in the kidneys of male rats receiving 250 or 750 mg/kg/day but such changes are commonly associated with administration of volatile hydrocarbons and are of no consequence to human risk assessment.
Epididymes:
Histopathological assessment of the epididymides revealed reduced numbers, or a complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) of males receiving 750 mg/kg/day.Similar changes were still evident following the 2 week recovery period. Spermatocele granuloma(ta) were observed in two males receiving 750 mg/kg/day and one receiving 60 mg/kg/day. However the significance of this change in the single male receiving 60 mg/kg/day is uncertain as spermatocele granuloma(ta) can occur spontaneously in rats of this age.
testes:
Moderate to severe seminiferous tubular atrophy/degeneration was seen in the testes of all animals dosed with Terpineol multiconstituent at 750 mg/kg/day, accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation. The same changes and reduced organ weights were still evident following the 2 week recovery period but at a lower incidence and severity, indicating a degree of recovery from these changes.


OTHER FINDINGS:
SIGNS AND ARENA OBSERVATIONS:
There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment.

SENSORY REACTIVITY OBSERVATIONS AND GRIP STRENGTH:
Sensory reactivity observations and grip strength values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.

MOTOR ACTIVITY:
Motor activity scores for males and females showed considerable inter-group variation but no clear dose related trends such that an association with test material was considered unlikely.

HAEMATOLOGY (Tables 2 and 3):
There were no marked effects of CAS 8000-41-7 upon haematology parameters.

BLOOD CHEMISTRY (Tables 4 and 5):
During Week 5 of study, urea and creatinine levels were significantly higher in females receiving 750 mg/kg/day and slightly, but not significantly, high in males at the same dose level. By Week 2 of recovery urea and creatinine levels were still higher than Control for males and females previously receiving 750 mg/kg/day, attaining statistical significance for females only.
Glucose plasma levels were significantly higher than Control in females dosed at 250 and 750 mg/kg/day and marginally high in the males at the same doses but were similar to Control after dosing ceased.
Potassium levels were significantly higher than Control in males and females receiving 750 mg/kg/day, visual inspection of the blood samples did not indicate that they were haemolysed, and levels were similar to respective Controls during the recovery phase.
Bile acid plasma levels for females at all dose levels were higher than the concurrent Control attaining significance at 750 mg/kg/day. A dose related trend was apparent, however, individual values were all within the Historical Control range (90 percentile range – females: 8.7-49.7 (n=38)).
.

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Males receiving 750 mg/kg/day showed evidence of testicular and epididymal toxicity leading to infertility. NOAEL was set at 250 mg/kg/day where fertility of male was unaffected by the test substance and no other effects were reported.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Table 2: Haematology - group mean values during Week 5 of treatment

Dose (mg/kg/day)

Hct

Hb

RBC

MCH

MCHC

MCV

WBC

N

L

E

B

M

LUC

Plt

PT

APTT

L/L

g/dL

x1012/L

pg

g/dL

fL

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

sec

sec

 

Males

Control (n=5)

0.454 ± 0.0185

15.6 ± 0.68

8.44 ± 0.280

18.5 ± 0.90

34.5 ± 0.23

53.7 ± 2.54

13.18 ± 2.573

1.56 ± 0.584

11.08 ± 2.316

0.11 ± 0.021

0.05 ± 0.018

0.27 ± 0.063

0.10 ± 0.032

1228 ± 113.6

13.9 ± 0.37

16.6 ± 1.17

60 (n=4)

0.437 ± 0.0052

14.9 ± 0.45

8.30 ± 0.245

18.0 ± 0.84

34.1 ± 0.66

52.7 ± 1.82

16.30 ± 5.547

3.78 ± 1.968*

11.79 ± 3.309

0.16 ± 0.082

0.08 ± 0.032

0.39 ± 0.219

0.12 ± 0.044

1119 ± 109.7

14.1 ± 0.47

18.6 ± 2.42

250 (n=5)

0.443 ± 0.0161

15.1 ± 0.57

8.34 ± 0.334

18.2 ± 0.33

34.2 ± 0.37

53.1 ± 1.38

12.57 ± 0.972

1.33 ± 0.237

10.82 ± 0.760

0.11 ± 0.023

0.06 ± 0.009

0.17 ± 0.029

0.09 ± 0.016

1193 ± 130.8

13.8 ± 0.30

16.3 ± 3.97

750 (n=5)

0.434 ± 0.0174

14.8 ± 0.68

8.35 ± 0.424

17.8 ± 0.21

34.2 ±0.67

51.9 ± 0.98

14.64 ± 3.813

2.10 ± 0.869

11.94 ± 3.192

0.09 ± 0.057

0.08 ± 0.029

0.32 ± 0.055

0.11 ±0.054

1123 ± 199.7

13.8 ± 0.38

16.2 ± 2.75

 

Females

Control (n=5)

0.414 ± 0.0165

14.6 ± 0.56

7.85 ± 0.347

18.6 ± 0.48

35.2 ± 0.36

52.7 ± 0.93

9.36 ± 2.826

0.87 ± 0.638

8.15 ± 2.166

0.10 ± 0.016

0.03 ± 0.016

0.14 ± 0.045

0.08 ± 0.040

1251 ± 75.2

14.4 ± 0.40

16.2 ± 1.99

60 (n=4)

0.400 ± 0.0037

13.9 ± 0.13*

7.73 ± 0.163

18.0 ± 0.54

34.8 ± 0.58

51.7 ± 0.97

8.69 ± 2.306

0.96 ± 0.897

7.39 ± 1.531

0.11 ± 0.075

0.04 ± 0.006

0.15 ± 0.074

0.05 ± 0.021

1169 ± 69.9

14.6 ± 0.92

13.2 ± 3.22

250 (n=5)

0.403 ± 0.0150

14.0 ± 0.33*

7.66 ± 0.273

18.3 ± 0.34

34.8 ± 0.57

52.5 ± 1.08

8.89 ± 3.411

1.06 ± 0.955

7.48 ± 2.324

0.06 ± 0.031

0.02 ± 0.012

0.20 ± 0.158

0.07 ± 0.044

1219 ± 130.2

13.8 ± 0.18

15.2 ± 1.31

750 (n=5)

0.391 ± 0.0121*

13.5 ± 0.42**

7.31 ± 0.382*

18.5 ± 0.80

34.6 ± 0.64

53.6 ± 1.85

7.54 ± 1.684

0.80 ± 0.462

6.47 ± 1.662

0.07 ± 0.035

0.02 ± 0.016

0.11 ± 0.025

0.05 ± 0.018

1013 ± 297.5

14.4 ± 0.45

13.8 ± 1.99

Table 3: Haematology - group mean values for females after 2 weeks of recovery

Dose (mg/kg/day)

Hct

Hb

RBC

MCH

MCHC

MCV

WBC

N

L

E

B

M

LUC

Plt

PT

APTT

L/L

g/dL

x1012/L

pg

g/dL

fL

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

sec

sec

 

Males

Control (n=5)

0.447 ± 0.0137

15.0 ± 0.52

8.93 ± 0.265

16.9 ± 0.63

33.7 ± 0.16

50.0 ± 1.75

11.81 ± 1.308

1.44 ± 0.429

9.85 ± 1.261

0.11 ± 0.020

0.06 ± 0.017

0.28 ± 0.032

0.07 ± 0.010

1239 ± 245.7

13.8 ± 0.42

14.3 ± 1.98

750 (n=5)

0.415 ± 0.0166*

14.2 ± 0.56*

8.28 ± 0.145**

17.1 ± 0.57

34.2 ± 0.33*

50.2 ± 1.75

15.00 ± 3.831

1.94 ± 0.801

12.39 ± 3.435

0.14 ± 0.035

0.08 ± 0.030

0.36 ± 0.159

0.08 ± 0.044

996 ± 130.0

14.6 ± 0.57*

16.3 ± 2.82

 

Females

Control (n=4)

0.405 ± 0.0191

14.2 ± 0.50

7.87 ± 0.369

18.1 ± 0.53

35.2 ± 0.56

51.4 ± 1.38

6.69 ± 1.758

0.77 ± 0.520

5.65 ± 1.326

0.09 ± 0.026

0.02 ± 0.006

0.15 ± 0.070

0.03 ± 0.013

1120 ± 199.5

14.5 ± 0.81

13.0 ± 0.69

750 (n=5)

0.404 ± 0.0131

14.1 ± 0.27

7.83 ± 0.303

18.0 ± 0.56

34.9 ± 0.49

51.7 ± 1.77

7.57 ± 2.070

0.64 ± 0.121

6.60 ± 1.963

0.09 ± 0.018

0.03 ± 0.009

0.16 ± 0.067

0.04 ± 0.018

1031 ± 208.8

14.8 ± 0.50

12.3 ± 2.65

Table 4: Blood chemistry - group mean values taken during Week 5 of treatment

Dose (mg/kg/day)

ALP

ALT

AST

gGT

Bili

BIAC

Urea

Creat

Gluc

Chol

Na

K

Cl

Ca

Phos

Total Prot

Alb

A/G

U/L

U/L

U/L

U/L

µmol/L

umol/L

mmol/L

µmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

g/L

g/L

Ratio

 

Males

Control (n=5)

124 ± 23.0

45 ± 12.2

61 ± 3.5

0 ± 0.0

2 ± 0.0

30.6 ± 16.59

4.19 ± 1.019

25 ± 3.1

7.29 ± 1.576

1.36 ± 0.155

142 ± 1.4

4.9 ± 0.27

101 ± 1.3

2.63 ± 0.044

2.13 ± 0.152

63 ± 1.9

35 ± 1.6

1.28 ± 0.120

60 (n=4)

145 ± 22.2

51 ± 8.9

68 ± 13.5

1 ± 0.6

2 ± 0.6

25.1 ± 14.13

4.84 ± 1.262

27 ± 4.5

8.93 ± 1.254

1.41 ± 0.137

142 ± 1.7

5.5 ± 1.03

103 ± 1.4

2.60 ± 0.048

2.22 ± 0.186

63 ± 1.9

33 ± 0.5

1.14 ± 0.090

250 (n=5)

164 ± 18.5*

52 ± 4.0

59 ± 5.5

0 ± 0.0

2 ± 0.4

29.8 ± 8.41

4.90 ± 0.596

27 ± 2.2

9.63 ± 1.264

1.91 ± 0.307

142 ± 2.2

5.6 ± 0.48

102 ± 1.1

2.55 ± 0.083

2.21 ± 0.156

61 ± 2.8

34 ± 1.2

1.27 ± 0.046

750 (n=5)

146 ± 24.6*

46 ± 15.9

69 ± 17.0

0 ± 0.4

2 ± 0.4

31.3 ± 13.30

5.35 ± 1.412

29 ± 4.0

8.72 ± 2.129

1.46 ± 0.463

140 ± 1.8

5.9 ± 0.66*

102 ± 0.5

2.51 ± 0.096*

2.19 ± 0.199

62 ± 2.8

35 ± 0.7

1.29 ± 0.088

 

Females

Control (n=5)

71 ± 14.8

40 ± 7.1

66 ± 8.3

0 ± 0.0

2 ± 0.4

14.2 ± 4.01

5.23 ± 0.853

28 ± 2.1

7.12 ± 0.513

1.51 ± 0.414

142 ± 0.9

4.2 ± 0.42

103 ± 0.9

2.56 ± 0.054

1.63 ± 0.106

66 ± 2.2

39 ± 2.3

1.50 ± 0.179

60 (n=4)

71 ± 19.3

42 ± 8.6

68 ± 11.8

0 ± 0.5

2 ± 0.4

18.7 ± 7.81

5.91 ±1.008

34 ±3.3

7.57 ± 1.426

1.46 ± 0.120

141 ± 1.8

4.7 ± 0.39

103 ± 1.8

2.52 ± 0.087

1.69 ± 0.060

64 ± 6.2

37 ± 2.7

1.36 ± 0.180

250 (n=5)

85 ± 15.8

45 ± 12.7

66 ± 22.9

0 ± 0.0

2 ± 0.7

25.1 ± 11.05

5.40 ± 0.407

32 ± 5.3

8.99 ± 0.997*

1.96 ± 0.394

141 ± 1.5

4.0 ± 0.33

102 ± 1.7

2.52 ± 0.066

1.67 ± 0.208

66 ± 3.4

39 ± 2.3

1.44 ± 0.237

750 (n=5)

75 ± 20.1

40 ± 6.7

58 ± 11.9

1 ± 0.5

2 ± 0.0

31.3 ± 14.04*

7.59 ± 2.350*

41 ± 3.1**

10.00 ± 1.372**

1.91 ± 0.464

139 ± 2.6*

5.6 ± 0.74**

102 ± 2.0

2.53 ± 0.064

1.53 ± 0.212

66 ± 2.3

38 ± 1.5

1.39 ± 0.089

Table 5: Blood Chemistry - Recovery Week 2

Dose (mg/kg/day)

ALP

ALT

AST

gGT

Bili

BIAC

Urea

Creat

Gluc

Chol

Na

K

Cl

Ca

Phos

Total Prot

Alb

A/G

U/L

U/L

U/L

U/L

µmol/L

umol/L

mmol/L

µmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

g/L

g/L

Ratio

 

Males

Control (n=5)

125 ± 35.1

50 ± 13.2

69 ± 7.4

0 ± 0.4

2 ± 0.0

32.1 ± 11.48

5.49 ± 0.910

30 ± 3.0

7.79 ± 1.269

2.02 ± 0.357

142 ± 1.2

4.9 ± 0.36

102 ± 1.1

2.66 ± 0.049

2.18 ± 0.145

67 ± 4.0

36 ± 1.5

1.17 ± 0.153

750 (n=5)

98 ± 15.3

42 ± 2.8

65 ± 4.7

0 ± 0.4

2 ± 0.0

30.0 ± 13.01

7.29 ± 1.663

35 ± 5.2

8.77 ± 0.638

1.94 ± 0.668

142 ± 0.7

4.9 ± 0.24

101 ± 0.4

2.63 ± 0.074

2.26 ± 0.086

64 ± 2.4

34 ± 1.8

1.17 ± 0.111

 

Females

Control (n=5)

65 ± 17.9

39 ± 6.6

63 ± 5.2

0 ± 0.4

2 ± 0.4

32.3 ± 18.29

7.17 ± 0.447

38 ± 3.2

7.71 ± 0.735

2.06 ± 0.377

141 ± 1.1

4.2 ± 0.16

102 ± 0.9

2.61 ± 0.064

1.53 ± 0.214

71 ± 4.0

41 ± 2.3

1.35 ± 0.140

750 (n=5)

65 ± 21.7

39 ± 12.0

65 ± 7.9

0 ± 0.4

2 ± 0.0

26.3 ± 27.87

8.59 ± 1.269*

50 ± 7.1*

7.97 ± 0.402

2.51 ± 0.240

142 ± 1.5

4.2 ± 0.24

101 ± 1.8

2.57 ± 0.076

1.61 ± 0.118

67 ± 4.2

38 ± 2.3

1.31 ± 0.019

Conclusions:
Based on the findings in this study, the No-Observed-Adverse-Effect-Level (NOAEL) for males and unmated females was 250 mg/k/g/day.
Executive summary:

In a GLP study conducted according to OECD Guideline 422, three groups, each comprising of ten male and ten female rats for the Main (reproductive) phase and five female rats for the Toxicity phase received Terpineol multiconstituent by gavage at doses of 60, 250 or 750 mg/kg bw/day at a dose volume of 5 mL/kg bw/day. Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. An additional ten males and ten females were dosed with the vehicle or at 750 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

In the 60 mg/kg/day dose group, one male was found dead on Day 31 of study and one female was killed because of parturition difficulties. In the absence of any other death in the intermediate and high dose groups these deaths are not attributed to the test material. 

No significant findings were recorded for clinical signs,detailed physical examination and arena observations. Underactive behaviour and unsteady reactions, in males and females were observed briefly during Week 1 inanimals receiving 750mg/kg/day and dose‑related increases in post‑dosing salivation and chin rubbing were seen.  Behavioural testing during Week 5 of dosing, including sensory reactivity findings, grip strength values and motor activity scores showed no differences considered to be associated with exposure to the test material.

There were no clear effects on bodyweight in males or unmated females receiving up to 750 mg/kg/day. Males receiving 750 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. Bodyweight during the recovery phase was similar to Controls. Bodyweight and bodyweight gain were unaffected during gestation. During lactation females receiving 250 mg/kg/day showed lower weight gain than Controls.

There were no adverse effects on food consumption in males, unmated females or females during gestation and lactation but visual assessment of water consumption indicated that males and females receiving 750 mg/kg/day were consuming more water than the Controls during the dosing period.

Among the toxicity subgroup animals, There were no clinically significant effects of Terpineol multiconstituent upon haematology parameters. Females showed slight anaemia but males were essentially unaffected. At the end of the two week recovery period, no ntergroup differences were present in females whereas slightly affected in males.

At 750 mg.kg/day, urea and creatinine levels were significantly higher than Controls in females and slightly high in males. Glucose plasma levels were significantly higher than Control in females dosed at 250 and 750 mg/kg/day and marginally high in males. Potassium levels were significantly higher than Control in males and females receiving 750 mg/kg/day. all the above discussed parameters showed complete recovery after 2 week recovery period.

There were no effects of Terpineol multiconstituent on oestrous cycles, precoital interval or mating. Gestation length was within the normal range but there was a small increase in the numbers of animals at 250 mg/kg/day having longer (23 day) gestation periods.  At dose levels up to and including 250 mg/kg/day there were no effects of the test material on the number of implantations, post implantation survival index, live birth index, viability index and lactation index. Male and female offspring bodyweights were not adversely affected by Terpineol multiconstituent.

At 750 mg/kg/day, relative liver weights were significantly higher than Control in males and females and relative kidney weights were significantly higher than Control in males. Testis weight was markedly low in males receiving 750 mg/kg/day and there was also an indication of low epididymal weights at this dose. Liver and kidney weights returned to normal after two weeks when the animals did not receive Terpineol multiconstituent but testis and epididymal weights showed no evidence of recovery.

Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with Terpineol multiconstituent at 750 mg/kg/day was not present after 2 weeks recovery and histopathological findings in the kidneys of males receiving 250 and 750 mg/kg/day also resolved after the end of dosing.

 

At 750 mg/kg/day, reduced numbers or complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) were observed in the epididymides and were still present following the 2‑week recovery period. Spermatocele granuloma(ta) that were seen in two males receiving 750 mg/kg/day and one receiving 60 mg/kg/day were not seen at the end of the recovery period. The significance of this change in the single male receiving 60 mg/kg/day is uncertain as spermatocele granuloma(ta) can occur spontaneously in rats of this age and there was no other degenerative change in the testes or epididymides of this animal.

Moderate to severe seminiferous tubular atrophy/degeneration was seen in the testes of all animals dosed with Terpineol multiconstituent at 750 mg/kg/day, accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation. Similar findings were still evident following the 2‑week recovery period but at a lower incidence and severity suggesting a degree of recovery. However gavage is considered to be a worst case for this effect (not confirmed by diet administration).

Based on the findings in this study, the No-Observed-Adverse-Effect-Level (NOAEL) for males and unmated females was 250 mg/k/g/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 November to 04 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Guideline on repeated dose toxicity, Committee for Human Medicinal Products (CPMP/SWP/1042/99 Rev 1), European Medicines Agency, 18 March 2010.
Principles of method if other than guideline:
In a repeated dose toxicity study, male Sprague-Dawley rats were administered Terpineol multiconstituent via oral (gavage) at doses of 0 (vehicle), 100, 250, 400, 600 or 750 mg/kg bw/day for 1 or 2 weeks.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD® (SD) IGS BR
Details on species / strain selection:
The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France. Sprague-Dawley rats are the animal species (and strain) used in the previous toxicological studies performed with the Terpineol multiconstituent.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Mean body weight of 382 g (range: 341-428 g)
- Housing: Animals were housed by three from the same group and subgroup in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted maintenance diet, batch No. 4301997 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 13 November 2014 To: 04 December 2014
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since it is a route of exposure which is recommended by the authorities for the type of study.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation procedure: Test item was weighed and mixed with the required quantity of vehicle (corn oil).
The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the dosing procedure.
Frequency of preparation: On the days of treatment
Delivery conditions: At room temperature and protected from light

VEHICLE
- Concentration in vehicle: 20, 50, 80, 120 and 150 mg/mL
- Dose volume: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determination of test item concentrations in dose formulations: Once in Days 1, 7 and 14
A sample was taken from control and test item dose formulations and analyzed using the validated method.
Analytical technique: Gas Chromatography with FID detection (GC-FID)
Acceptance criterion: Measured concentration = nominal concentration ± 10%
Duration of treatment / exposure:
7 and 14 days
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males/dose (subgroup A): 7 days
9 males/dose (subgroups B and S): 14 days

* subgroup A & B: principal groups; subgroups S: Satellite animals were allocated only for toxicokinetic investigations in the testes and in blood sampling at sacrifice
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor (no data reported).
- Rationale for animal assignment: During the acclimation period, the required number of animals (72 principal and 18 satellite males) was selected according to body weight and clinical condition. The animals were allocated to groups according to a computerized stratification procedure so that the average body weight of each group was similar.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment periods, including weekends. Each animal was observed twice a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic and tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once before the beginning of the treatment period, then daily until the end of the treatment period (until Day 7 for subgroup A and until Day 14 for subgroups B and S).

FOOD CONSUMPTION:
- The quantity of food consumed by the animals in each cage was recorded daily until the end of the treatment period. After the last record for all animals (Day 7 for subgroup A or Day 14 for subgroups B and S), the feeders: were removed for the animals of subgroups A and B sacrificed 12 h post-dose (start of fasting period), but was let available until 12 h post-dose to the animals of subgroups A and B sacrificed 24 h post-dose and then removed at that time (start of fasting period).
Food consumption was calculated per animal and per day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
- Fate of subgroup S (satellite animals):
On completion of the treatment period, after at least 10 h of fasting, on Day 14, 6 h after test item administration, all satellite animals (of groups 1 to 6)) were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital. Blood was collected from the vena cava for toxicokinetics and then the animals were sacrificed by exsanguination.
Both testes were sampled, weighed (individually) and stored at -20 °C for determination of test item levels.
After testes sampling, carcasses of satellite animals were discarded without further examination or sampling.
- Fate of subgroups A and B (principal animals)
On completion of the treatment period, after at least 10 h fasting (and no more than 14 h), all principal animals of groups 1 to 6 were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital. The last blood sample for toxicokinetics was collected from the vena cava in groups 2 to 6 and then the animals were sacrificed by exsanguination.
Sacrifice was performed at the following time-points:
Subgroup A: on Day 7, 12 h after treatment. For groups 2 to 6, after the last blood sampling (three animals per group), on Day 8, 24 h after the last test item administration. For groups 2 to 6 after the last blood sampling (three animals per group).
Subgroup B: on Day 14, 12 h after treatment. For groups 2 to 6 after the last blood sampling (three animals per group), on Day 15, 24 h after the last test item administration. For groups 2 to 6, after the last blood sampling (three animals per group).
For subgroups A and B, group 1 animals were sacrificed at the same time than the other groups but were not sampled for toxicokinetics at sacrifice: three animals 12 h post-dose and three animals 24 h post-dose.

ORGAN WEIGHT
The body weight of each animal was recorded before sacrifice at the end of the treatment period. The following organs were weighed wet as soon as possible after dissection: liver, testes: for all animals, both testes were weighed separately, epididymis.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

MACROSCOPIC POST-MORTEM EXAMINATION
A complete macroscopic post-mortem examination was performed on each animal. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY
Preservation of tissues:
For all animals, the following tissues were preserved in 10% buffered formalin (except for the testes and epididymides, which were fixed in Modified Davidson's Fixative):
any macroscopic findings for all principal animals,
liver, both testes and epididymes for all animals of subgroup A in groups 1, 4, 5 and 6 (no tissues were preserved for groups 2 and 3) (see § Study plan adherence),
liver, one testis per male (the right one) and epididymes for all animals of subgroup B sacrificed 12 h post-dose (groups 1 to 6),
liver and epididymis for all animals of subgroup B sacrificed 24 h post-dose (groups 1 to 6).
Preparation of histological slides: All tissues required for microscopic examination, were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert, et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin.
This tissue processing was performed at CiToxLAB France.
Microscopic examination:
A microscopic examination was performed on the following tissues:
one testis per male (the right one) and epididymides for all animals of subgroup A in groups 1 and 6 (no microscopic examination in groups 4 and 5 in the first instance),
liver, one testis per male (the right one) and epididymes for all animals of subgroup B sacrificed 12 h post-dose in groups 1, 4, 5 and 6 (no microscopic examination in groups 2 and 3 in the first instance),
liver and epididymides for all animals of subgroup B sacrificed 24 h post-dose in groups 1, 4, 5 and 6 (no microscopic examination in groups 2 and 3 in the first instance).
Macroscopic findings were not examined in the first instance.
Summary for testes sampling (subgroups A, B and S):
Depending on the subgroups, testis sampling was performed for toxicokinetics and/or histopathology. Testis sampling was summarized as follows:
for subgroup A, no sampling for toxicokinetics. Both testes were weighed and preserved separately for histopathology,
for subgroup B, the left testis was sampled as soon as possible and weighed for determination of testis level of the test item; the right testis was weighted and preserved for histopathology,
for subgroup S, both testes were sampled and weighed separately for determination of testis level of the test item.
Other examinations:
None
Statistics:
CITOX software was used to perform the statistical analyses of body weight and food consumption data.
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Please refer "Attached background material" section for further details
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Ptyalism was observed on animals from subgroups A and B. For those from subgroups B, this clinical sign was noted at dose levels of 250, 400, 600 and 750 mg/kg bw/day. For those from subgroup A, this sign was noted at doses of 600 and 750 mg/kg bw/day.
- At 250 mg/kg bw/day from subgroup B, ptyalism appeared on Day 10 in one male, on Days 10 and 12 in another male and on Day 12 in the other four animals. The same tendency was noted when the animals were treated with 400 mg/kg bw/day. Ptyalism was observed on Days 10 and 12 in one male; in 4/5 rats, ptyalism was observed on Day 12 only.
- At 600 and 750 mg/kg bw/day, the frequency of occurrence was higher and ptyalism appeared earlier. At 600 mg/kg bw/day from subgroup B, ptyalism was seen for the first time on Day 3 in 4/6 animals. In the two others, this clinical sign was observed later (on Days 11 and 12). Ptyalism was noted in two to five occasions. Ptyalism was observed with a higher incidence for males at 750 mg/kg bw/day (4-6 occasions, from Days 3 or 4 to 12) from subgroup B than those of the lower dose level. In 5/6 animals from subgroup A, at 600 or 750 mg/kg bw/day, ptyalism was also observed in one to two occasions, between Day 3 and Day 7.
This effect was considered related to treatment with the test item but not toxicologically relevant.
- Chromodacryorrhea was noted in one male at 600 mg/kg bw/day from subgroup B from Day 2 to Day 4. This finding was not seen in any other animal or recording occasion. Therefore it was not considered to be of toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There were no statistically significant body weight differences during the study. However, a slightly lower mean body weight was recorded in animals treated with 600 or 750 mg/kg bw/day, when compared to control animals (up to -6%). It was not considered adverse in view of the low magnitude.
- In animals, from subgroup A, at 400 mg/kg bw/day, the body weight gain, between Days 6 and 7, was lower than the body weight gain of vehicle control dosed animals (-86%, p<0.05). The same tendency was observed in animals treated with 750 mg/kg bw/day (-100%, 0 g gained, p<0.01). Body weight gain was, between Days 1 and 7, lower in animals from subgroup A given 600 or 750 mg/kg bw/day in comparison to control group (-33%, p<0.05 and -39%, p<0.05, respectively). Moreover, for those from subgroup B given 600 or 750 mg/kg bw/day, between Days 1 and 14, the same tendency was observed, when compared to vehicle group (-37%, p<0.05 and -47%, p<0.01%, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- There were no statistical differences of food consumption during this study.
- During the first days of treatment, between Days 1 and 2, 2 and 3, and, 3 and 4, food consumption was lower in all rats from subgroup B, given 600 or 750 mg/kg bw/day when compared to vehicle group (-26%, 18% and -20%, respectively, at 600 mg/kg bw/day and -32%, -25% and -19%, respectively, at 750 mg/kg bw/day).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Testes, epididymides and livers of animals from subgroups A and B and sacrificed 12 or 24 h post treatment were weighed at necropsy.
- Testes: There were no test item-related changes in the testes weights either in animals from subgroup A or subgroup B sacrificed 12 or 24 h post-treatment.

- Epididymides: When compared with controls, the mean absolute and relative weights of epididymides were higher in animals from subgroup A sacrificed 12 h post-treatment and in animals from both subgroups (A and B) treated at 600 mg/kg bw/day and sacrificed 24 h post-treatment. These variations reached a statistical significance for the relative weight in subgroup B animals sacrificed 24 h post-treatment (p<0.05).

- Liver: When compared with controls, the mean absolute and relative liver weights were higher in subgroup A animals treated at 750 mg/kg bw/day and sacrificed 12 or 24 h post-treatment, reaching a statistical significance (p<0.05) for the relative weight 12 h post-treatment. In subgroup B animals, the mean absolute and relative liver weights were higher in animals treated at 750 mg/kg bw/day and sacrificed 24 h post-treatment, reaching a statistical significance for the relative weight (p<0.05).
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes at necropsy either in animals from subgroup A or B whatever the time of sacrifice (12 or 24 h post-treatment).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Right testes
Subgroup B (0, 400, 600 or 750 mg/kg bw/day then sacrificed 12 h after the last dosing):
- Macro and/or microvacuolation of germ cells, considered to be test item-related, was observed at 600 and 750 mg/kg bw/day. At 750 mg/kg bw/day, changes were more pronounced and associated with degeneration/necrosis of germ cells, (particularly in stages VII and VIII) along with multinucleated giant cells in one animal. Incomplete release of sperm (stages VII and VIII) was noted in two animals (750 mg/kg bw/day) and was considered to be secondary to the degeneration/necrosis observed at these stages.
- At 600 mg/kg bw/day, minimal microvacuolation of germ cells was seen in one animal only without any other associated changes.
- Vacuolation of Sertoli cells was observed in the testes from occasional animals including controls. This was considered to be without relationship to the test item.
- Atrophy/degeneration of tubules was also observed occasionally. As this observation is commonly seen in rats, and seen in one control animal from the subgroup A, any relationship to the test item was excluded.
Subgroup A (0 and 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Testes from one high-dose animal sacrificed 12 h post-treatment were not sampled and therefore not examined histologically. Minimal microvacuolation was observed in germ cells of both animals treated at 750 mg/kg bw/day and sacrificed 12 h post-treatment.
- Slight macrovacuolation was observed in 1/3 animals treated at 750 mg/kg bw/day and sacrificed 24 h post-treatment.

Epididymides
Subgroup B (0, 400, 600 or 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Slight to moderate hypertrophy of epithelial cells of the body of epididymides was observed at 600 and 750 mg/kg bw/day in both subgroup B animals sacrificed either 12 or 24 h post-treatment. In one male (750 mg/kg bw/day and sacrificed 12 h post-treatment), this was accompanied with a few degenerated cells of the epithelium.
- Minimal to slight decreased size of the lumen was also observed at 600 and 750 mg/kg bw/day. This change was considered to be secondary to the hypertrophy of the epithelium.
- Sloughed cells were occasionally observed in the lumen but without prevalence in a specific group. Consequently, this observation was considered to be without relationship to the test item.
- Reduced sperm count was seen in one animal (600 mg/kg bw/day, sacrificed 24 h post treatment). In the absence of dose-related incidence, this isolated observation was considered to be fortuitous.
Subgroup A (0 and 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Minimal epithelial cells hypertrophy was observed in the body of epididymides from both animals treated at 750 mg/kg bw/day then sacrificed 12 h but this was not seen in those sacrificed 24 h post-treatment.
- From one high dose animal sacrificed 12 h post-treatment, epididymides were not sampled and therefore not examined histologically.

Liver:
Subgroup B (0, 400, 600 or 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Minimal to slight centrilobular hepatocellular hypertrophy was observed in all animals treated at 750 mg/kg bw/day and sacrificed 12 h post-treatment and in 1/3 animals sacrificed 24 h post-treatment without prevalence in a specific group.
- Minimal to slight periportal/midzonal vacuolation was observed in all groups, including controls. As a consequence, any relationship to the test item was excluded.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Remarks on result:
not measured/tested

- Degenerative/necrotic germ cells in testes at 750 mg/kg bw/day were considered to be adverse.

- At 600 mg/kg bw/day, considering the low magnitude, incidence and nature of the changes (minimal microvacuolation in a single animal only), this was not considered to be adverse in the experimental conditions of the study.

- Hypertrophy of epithelial cells in epididymides was observed at 600 and 750 mg/kg bw/day and correlated with the higher weight at necropsy (at 600 mg/kg bw/day only). Due to a few degenerated cells seen in one animal at 750 mg/kg bw/day, it was considered as adverse at this dose level. The associated decreased size of the lumen was considered to be secondary to the hypertrophy of epithelial cells.

- Centrilobular hepatocellular hypertrophy correlated with the higher weight at 750 mg/kg bw/day. This was not considered as adverse.

- Periportal/midzonal vacuolation in the liver of most of the animals, including controls was suggestive of a vehicle effect (corn oil).

Conclusions:
Under the test conditions, the test item was clinically well tolerated up to 750 mg/kg bw/day. Clinical signs were limited to ptyalism. Slightly lower mean body weight and mean food consumption were recorded at the dose levels of 600 and 750 mg/kg/day and were not considered adverse.
At microscopic examination, effects were observed on male genital tract at 600 and 750 mg/kg/day and were considered adverse at 750 mg/kg/day only, as degeneration/necrosis was noted at this dose.
Executive summary:

In a repeated dose toxicity study, Terpineol multiconstituent was administered to six groups of male Sprague-Dawley rats via oral (gavage) at doses of 0 (vehicle), 100, 250, 400, 600 or 750 mg/kg bw/day. Each group was composed of three subgroups: subgroups A and B (six males in each subgroup) corresponded to principal animals and subgroup S (three males) corresponded to satellites animals, especially for toxicokinetics investigations in the testes and in blood samples. The rats from subgroup A were treated for 7 days, those from subgroups B and S for 14 days. During the study, morbidity, mortality, clinical signs, body weight, food consumption, organ weights, gross and histopathology investigations were undertaken.

 

There were no unscheduled deaths during this study. Ptyalism was observed on several occasions in animals treated from the dose level of 250 mg/kg bw/day, in both subgroups A and B. Slightly lower mean body weight, body weight gain and food consumption were recorded in animals given 600 or 750 mg/kg bw/day of test item, when compared to control group. This tendency was observed in both subgroups (A and B).

At the dose level of 750 mg/kg bw/day for 14 days (subgroup B), adverse changes were noted in testes and consisted in micro and/or macrovacuolation and degeneration/necrosis of germ cells in stages VII and VIII. Epithelial hypertrophy in epididymides was also observed along with decreased size of the lumen. At 750 mg/kg bw/day for 7 days (subgroup A), only slight vacuolated changes were seen in testes of a single animal and minimal hypertrophy of epithelial cells was seen in the epididymides. At 600 mg/kg bw/day for 14 days (subgroup B), minimal vacuolation of germ cells was seen in a single animal and hypertrophy of epithelial cells was noted in epididymides along with reduced lumen. In the liver, minimal hepatocellular hypertrophy was seen after 14 days of treatment at 750 mg/kg bw/day (subgroup B).

 

Therefore, the test item was clinically well tolerated up to 750 mg/kg bw/day.

Clinical signs were limited to ptyalism. Slightly lower mean body weight and mean food consumption were recorded at the dose levels of 600 and 750 mg/kg/day and were not considered adverse.

At microscopic examination, effects were observed on male genital tract at 600 and 750 mg/kg/day and were considered adverse at 750 mg/kg/day only, as degeneration/necrosis was noted at this dose.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 December 2017 to 19 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Pilot study was conducted to select doses for preliminary embryo-fetal toxicity study, performed in GLP laboratory.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The pilot study (14 days study) was conducted to establish suitable doses of Terpineol multiconstituent for investigation in a preliminary embryo-fetal toxicity study in the New Zealand White rabbit.
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on species / strain selection:
The female New Zealand White (sexually mature, virgin) rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS
- Females - nulliparous and non-pregnant: Yes
- Age at study initiation: Group 1: 18-22 weeks; Groups 2 and 3: 19-23 weeks
- Weight at study initiation: Group 1: 3.18-3.81 kg; Groups 2 and 3: 3.35-3.76 kg
- Housing: Animals were individually housed in suspended plastic cages fitted with perforated floor panels and mounted in batteries.
- Diet: Teklad 2930 (pelleted). The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Restricted (initially 150 g/animal/day during acclimatization up to 200 g/animal/day for all animals one week prior to the onset of treatment (Group 1). In addition, due to evidence of low intake of the dry pelleted diet in some animals, a bowl of moistened diet (50g diet with 50mL water or 100g diet with 100 mL water) was provided to each animal. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes (ad libitum). Bottles were changed at appropriate intervals. Water bowls were also provided.
- Acclimation period: Group 1: two weeks; Groups 2 and 3: three weeks

DETAILS OF FOOD AND WATER QUALITY: No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 15-21 °C
- Humidity: 45-70 %
- Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 14 h light : 10 h dark

IN-LIFE DATES: From: 13 December 2017 To: 19 January 2018
Route of administration:
oral: gavage
Details on route of administration:
The oral (gavage) route of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Vehicle (corn oil) was gradually added to the required amount of test item and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. Suspensions at the required concentrations were prepared by dilution of individual weighing of the test item. The use of plastic or rubber equipment/storage containers was avoided.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Frequency of preparation: Weekly
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 100 and 200 mg/mL
- Dose volume: 1 mL/kg bw/day
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily, at approximately the same time each day.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 2
No. of animals per sex per dose:
3 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose level selection of 100 mg/kg bw/day for Group 1 animals was selected in conjunction with the sponsor based on the results of a previous OECD 422 combined toxicity and reproductive screening study (Huntingdon Life Sciences Report No. OAD0004) in the CD rat and an OECD414 study (Huntingdon Life Sciences Report No. OAD0011) in the CD rat. In those studies, the No-Observed-Adverse Effect-Level (NOAEL) was 250 mg/kg bw/day for unmated females and 600 mg/kg bw/day for maternal toxicity and embryo-fetal survival. Therefore, in the absence of data on administration of the test item in female rabbits, the dose level of 100 mg/kg bw/day for was selected for Group 1. The dose levels for subsequent groups were selected based on the results of Group 1.
- Rationale for animal assignment: Random, avoiding allocation of more than one sibling per group; on the day of dosing.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration:
Pre-dose observation; 10 to 20 minutes after the end of dosing each group; One to two hours after completion of dosing; As late as possible in the working day
A detailed physical examination was performed on each animal to monitor general health. This was performed weekly during acclimatization and then daily from three days prior to the start of treatment (Day -3), on the day that treatment commenced and daily throughout the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded weekly during acclimatization and then daily for three days prior to the start of treatment, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily throughout the study from three days before treatment started until scheduled termination.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
NECROPSY
- All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded. Any abnormal tissues were sampled as appropriate and preserved in appropriate fixative.
Schedule: Animals were killed following 14 days of treatment by intravenous injection of sodium pentobarbitone.
Any preserved tissues were not processed but were held pending any possible future requirement for further examination.
Other examinations:
None
Statistics:
No statistical analysis was performed on the study.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In all groups, a total of 5/9 females (including two controls) showed overall body weight loss (0.5 to 11% body weight loss). In addition, one control female required premature euthanasia on Day 11 of study due to overall body weight loss of 390 g (11% body weight loss).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In all groups, some individual animals showed low intake of the pelleted diet (classified as less than 50 g/day) and the provision of moistened diet did not, in general, improve sufficiently the level of food consumption after 14 days of treatment (except for 2/3 females receiving 100 mg/kg bw/day).
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
Description (incidence and severity):
No macroscopic abnormalities were apparent.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
other: no NOAEL identified
Key result
Critical effects observed:
not specified

- One control female required premature euthanasia on Day 11 of study due to overall body weight loss of 390 g (11% body weight loss) and clinical signs of noisy/irregular respiration, reduced faecal and urine output and a reduction in diet and hay consumption. The overall poor and irregular performance of all study groups was considered to be attributable to the use of corn oil as the formulation vehicle.

- The use of corn oil as the formulation vehicle limited the dose volume to 1 mL/kg bw/day due to the known effects which corn oil can induce in rabbits (decreased food intake leading to body weight loss, changes in excreta output and a general decline in clinical condition).  As a consequence of the limits of the formulation stability (maximum concentration of 200 mg/mL) and the dose volume, the maximum dose level of Terpineol multiconstituent that could be administered was 200 mg/kg bw/day.

- It was not possible to establish the toxicity profile of Terpineol multiconstituent in this pilot study in the rabbit.

Conclusions:
It is concluded that, to assess the toxicity profile of Terpineol multiconstituent when administered to rabbits, an alternative vehicle (different from corn oil) should be employed.
Executive summary:

In a repeated dose toxicity study (pilot study), groups of New Zealand White rabbits (3 females/dose) received Terpineol multiconstituent, via oral (gavage) at the dose levels of 100 and 200 mg/kg bw/day. A similarly constituted control group received vehicle (corn oil) throughout the same treatment period. During the study, clinical condition, body weight, food consumption and macropathology investigations were undertaken. Initially three non-pregnant rabbits received Terpineol multiconstituent at a dose of 100 mg/kg bw/day for a period of 14 days. Based on the results obtained, a further six non‑pregnant rabbits were assigned to the study, with three females allocated as a control group and three females treated at a dose level of 200 mg/kg/day for 14 days.

It was not possible to establish the toxicity profile of Terpineol multiconstituent in this pilot study in the rabbit.

The use of corn oil as the formulation vehicle limited the dose volume to 1 mL/kg bw/day due to the known effects which corn oil can induce in rabbits (decreased food intake leading to bodyweight loss, changes in excreta output and a general decline in clinical condition). As a consequence of the limits of the formulation stability (maximum concentration of 200 mg/mL) and the dose volume, the maximum dose level of Terpineol multiconstituent that could be administered was 200 mg/kg bw/day.

There was no discernible difference in the performance of the treated animals compared to controls, with some indications that the control females showed the poorest performance. In all groups, some individual animals showed low intake of the pelleted diet (classified as less than 50 grams per day) and the provision of moistened diet did not, in general, improve sufficiently the level of food consumption after 14 days of treatment (except for 2/3 females receiving 100 mg/kg bw/day). As a consequence, a total of 5/9 females (including two controls) showed overall body weight loss (0.5 to 11% body weight loss). In addition, one control female required premature euthanasia on Day 11 of study due to overall body weight loss of 390 grams (11% body weight loss) and clinical signs of noisy/irregular respiration, reduced faecal and urine output and a reduction in diet and hay consumption; no macroscopic abnormalities were apparent. The overall poor and irregular performance of all study groups was considered to be attributable to the use of corn oil as the formulation vehicle.

It is concluded that, to assess the toxicity profile of Terpineol multiconstituent when administered to rabbits, an alternative vehicle (different from corn oil) should be employed.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 March to 14 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Pilot study was conducted to select doses for preliminary embryo-fetal toxicity study, performed in GLP laboratory.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The pilot study (14 days study) was conducted to establish suitable doses of Terpineol multiconstituent for investigation in a preliminary embryo-fetal toxicity study in the New Zealand White rabbit.
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on species / strain selection:
The female New Zealand White (sexually mature, virgin) rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS
- Females - nulliparous and non-pregnant: Yes
- Age at study initiation: Group 1: 17-22 weeks; Group 2: 19-24 weeks; Group 3: 20-25 weeks
- Weight at study initiation: Group 1: 2.86-3.36 kg; Group 2: 2.79-3.37 kg; Group 3: 2.70-3.25 kg
- Housing: Animals were individually housed in suspended plastic cages fitted with perforated floor panels and mounted in batteries.
- Diet: Teklad 2930 (pelleted). The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Restricted (initially 150 g/animal/day during acclimatization up to 200 g/animal/day for all animals one week prior to the onset of treatment (Group 1). In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes (ad libitum). Bottles were changed at appropriate intervals.
- Acclimation period: Group 1: 14 days; Group 2: 26 days; Group 3: 33 days

DETAILS OF FOOD AND WATER QUALITY: No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 15-21 °C
- Humidity: 45-70 %
- Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 14 h light : 10 h dark

IN-LIFE DATES: From: 28 March 2018 To: 14 May 2018
Route of administration:
oral: gavage
Details on route of administration:
The oral (gavage) route of administration was chosen to simulate the conditions of possible human exposure.
Vehicle:
methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Vehicle (1% methylcellulose) was gradually added to the required amount of test item and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. Suspensions at the required concentrations were prepared by dilution of individual weighing of the test item. The use of plastic or rubber equipment/storage containers was avoided.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8 °C).

VEHICLE
- Concentration in vehicle: 50, 80 and 120 mg/mL
- Dose volume: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily, at approximately the same time each day.
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
Group 3
No. of animals per sex per dose:
3 females/dose
Control animals:
no
Details on study design:
- Dose selection rationale: The dose level selection of 250 mg/kg bw/day for Group 1 animals was selected in conjunction with the sponsor based on the results of a previous OECD422 combined toxicity and reproductive screening study (Huntingdon Life Sciences Report No. OAD0004) in the CD rat and OECD414 study (Huntingdon Life Sciences Report No. OAD0011) in the CD rat in which the NOAEL was 250 mg/kg bw/day for unmated females and 600 mg/kg bw/day for maternal toxicity and embryo-fetal survival. In addition, in a previous pilot study in the non-pregnant female rabbit (Envigo Study No. QS48JF), there was no discernible difference in the response of females given 200 mg/kg bw/day in corn oil to that seen in vehicle controls.
- Rationale for animal assignment: Random, avoiding allocation of more than one sibling per group; on the day of dosing.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration:
Pre-dose observation; 10 to 20 minutes after the end of dosing each group; One to two hours after completion of dosing; As late as possible in the working day
A detailed physical examination was performed on each animal to monitor general health. This was performed weekly during acclimatization and then daily from three days prior to the start of treatment (Day -3), on the day that treatment commenced and daily throughout the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded weekly during acclimatization and then daily for three days prior to the start of treatment, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily throughout the study from three days before treatment started until scheduled termination.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
NECROPSY
- All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded. Any abnormal tissues were sampled as appropriate and preserved in appropriate fixative.
Schedule: Animals were killed once a clear reaction to treatment was established or maximum practical or maximum desired dose was attained;
Method of kill: Intravenous injection of sodium pentobarbitone
Any preserved tissues were not processed but were held pending any possible future requirement for further examination.
Other examinations:
None
Statistics:
No statistical analysis was performed on the study.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- At 600 mg/kg bw/day, following administration on Day 1, all females showed signs of constricted pupils, abnormal/unsteady gait and reddened ears. A similar spectrum of signs were also evident after the second dose administration, however all three females showed additional signs including underactive behavior and flat posture, and one female (No. 5) was also observed to be unresponsive, with reduced body temperature (cold to touch), shallow respiration, partially closed eyelids and prostrate posture. It was concluded that the 600 mg/kg/day level exceeded the maximum tolerated dose and all females were killed for welfare reasons; no macroscopic abnormalities were evident.
- There were no test item-related signs observed at routine physical examination or following dose administration at 250 or 400 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
- Administration of Terpineol multiconstituent at 600 mg/kg bw/day was not tolerated, with all females killed for reasons of animal welfare after the second dose administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- The body weight performance of females receiving 250 or 400 mg/kg bw/day was in line with expectation and no effect of Terpineol multiconstituent administration was considered to be inferred.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- The food consumption of females receiving 250 or 400 mg/kg bw/day was in line with expectation and no effect of Terpineol multiconstituent administration was considered to be inferred.
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
Description (incidence and severity):
There were no test item-related macroscopic abnormalities apparent at any dose level investigated.
Neuropathological findings:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
Key result
Critical effects observed:
not specified
Conclusions:
It is concluded that, due to the steep dose response between 400 and 600 mg/kg bw/day in this pilot study, and the potential for the toxicity profile to be different between the non-pregnant and pregnant female rabbit, a cautious approach should be taken in the preliminary embryo fetal development study, with the study conducted in two phases, in order to establish a high dose level suitable for repeated administration for the required 23-day dosing period (Day 6 to Day 28 after mating). The vehicle used will be methylcellulose.
Executive summary:

In a repeated dose toxicity study (pilot study), groups of New Zealand White rabbits (3 females/dose) received Terpineol multiconstituent, via oral (gavage) at the dose levels of 250, 400 and 600 mg/kg bw/day (in vehicle methylcellulose). During the study, clinical condition, body weight, food consumption and macropathology investigations were undertaken. Initially three non-pregnant rabbits received Terpineol multiconstituent at a dose of 250 mg/kg bw/day for a period of 14 days. Based on the results obtained, a further three non‑pregnant rabbits were assigned to the study receiving a dose level of 600 mg/kg bw/day; overt toxicity was observed after dosing on Day 2 of treatment and this group of animals was killed for reasons of animal welfare. A further three non-pregnant rabbits were subsequently assigned to the study receiving a dose level of 400 mg/kg bw/day for a period of 14 days.

The dose level of 600 mg/kg bw/day Terpineol multiconstituent exceeded the maximum tolerated dose to non-pregnant female rabbits, with a broad spectrum of marked signs apparent after the second dose administration requiring premature termination of the group. In all three females in the group, these signs comprised underactive behavior, flat posture, constricted pupils and abnormal/unsteady gait; one of the females was also found to be unresponsive, with reduced body temperature (cold to touch), shallow respiration, partially closed eyelids and prostrate posture. The performance of females receiving 250 or 400 mg/kg bw/day was in line with expectation and did not indicate any evidence of treatment-related effects.

The body weight performance and food consumption of females receiving 250 or 400 mg/kg bw/day was in line with expectation and no effect of Terpineol multiconstituent administration was considered to be inferred. There were no test item-related macroscopic abnormalities apparent at any dose level investigated.

It is concluded that, due to the steep dose response between 400 and 600 mg/kg bw/day in this pilot study, and the potential for the toxicity profile to be different between the non-pregnant and pregnant female rabbit, a cautious approach should be taken in the preliminary embryo fetal development study, with the study conducted in two phases, in order to establish a high dose level suitable for repeated administration for the required 23-day dosing period (Day 6 to Day 28 after mating).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From March 24 to April 21, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Preliminary study used as range-finding experiment for a OECD 422 screening test, performed in GLP laboratory. Only 4 animals/dose/sex and no statisctical analysis. Non GLP study.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Range-finding experiment for an OECD guideline 422 study, where clinical condition, bodyweight, food consumption, water consumption, oestrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken in male and female rats exposed to test substance by oral gavage for 2 weeks.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 70 days (groups 1-3) or 79 days (group 4)
- Weight at study initiation: Males: 343-382 g (groups 1-3) and 397-439 g (group 4); females: 224-253 g (groups 1-3) and 252-270 g (group 4)
- Housing: Housed in groups of 4/sex in polycarbonate cages
- Diet: Standard rodent diet (SDS VRF1 Certified Diet); ad libitum except overnight before blood sampling
- Water (e.g. ad libitum): Potable water from public supply; ad libitum
- Acclimation period: 5 days (groups 1-3) or 14 days (group 4)

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70%
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours artificial light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: All formulations were prepared daily with corn oil and used within two hours of completion of preparation.

VEHICLE
- Concentration in vehicle: 30, 120 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
Two weeks
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Four
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 150 and 600 mg/kg bw/day were selected based on the results of an acute oral toxicity study in which LD50 was identified as > 2000 mg/kg bw. No mortalities were observed but signs of piloerection and reduced levels of activity were observed 6 hours post-dosing. Dose level of 1000 mg/kg bw/day was selected since there were no significant effects on bodyweight or food consumption after one week of treatment at 150 and 600 mg/kg bw /day.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily; immediately before and after dosing of each animal, on completion of dosing of each group, between 1-2 hours after completion of dosing of all groups and as late as possible in the working day

BODY WEIGHT:
- Time schedule for examinations: Daily throughout the treatment period and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: Visually assessed daily from day of commencement of study; measured daily in control and 1000 mg/kg bw/day treatment groups from Days 17-23 and Days 8-14, respectively.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At the end of the second week of treatment blood samples were obtained from the sublingual vein of each animal.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes; overnight
- How many animals: All animals (4/sex/dose)
- Parameters examined: Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and creatinine levels

OESTROUS CYCLE:
- Time schedule for examinations: Daily vaginal smears were taken from all female animals to establish the duration and regularity of the oestrous cycle.
Sacrifice and pathology:
GROSS PATHOLOGY: surviving animals were killed by carbon dioxide asphyxiation followed by subsequent exsanguination and subjected to detailed necropsy.
ORGAN WEIGHTS: organ weights of adrenals, epididymides, heart, kidneys, liver, ovaries, prostate, spleen and testes were recorded for each animal killed after completion of the study.
HISTOPATHOLOGY: No but tissues with macroscopic abnormalities were preserved for microscopic examinations in 10% neutral buffered formalin except the testes which were retained in modified Davidson’s fluid, pending any future requirement for microscopic examination.
Other examinations:
None
Statistics:
No statistical analysis was performed as the small sample size precluded meaningful statistical evaluation.
Clinical signs:
effects observed, treatment-related
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):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
- No mortality occurred during the study.
- Chin rubbing and salivation were observed at 600 and 1000 mg/kg bw/day.
- Isolated occasions of reduced activity, reduced body tone and unsteady gait were observed in few females at 1000 mg/kg bw/day

BODY WEIGHT AND WEIGHT GAIN: Slight bodyweight loss was recorded at the start of treatment in males at 600 and 1000 mg/kg bw/day and in females at 150, 600 and 1000 mg/kg bw/day, however overall bodyweight change was unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE : No effect

WATER CONSUMPTION AND COMPOUND INTAKE: Increased water consumption was reported in animals at 1000 mg/kg bw/day during the second week of treatment. Incidences of increased visual water consumption were observed at 600 mg/kg bw/day.

CLINICAL CHEMISTRY: Plasma urea and creatinine levels were elevated in males at 600 and 1000 mg/kg bw/day and in females at 1000 mg/kg/day. Plasma activity levels of alanine phosphotase were lower in males and females receiving 1000 mg/kg bw/day but a reduction in this parameter is not considered to be adverse.

OESTROUS CYCLE: No effect

ORGAN WEIGHT: Dose-related increased relative liver weight was observed in males (120, 139, and 142% of control at 150, 600 and 1000 mg/kg bw /day, respectively) and females (132 and 137% of control at 600 and 1000 mg/kg bw/day, respectively). Reduced testis weights (67% of control) and epididymal weights (77% of control) and increased kidney weights (115% of control) were observed in males at 1000 mg/kg bw/day. Testes were observed to be flaccid in 2/4 males.

GROSS PATHOLOGY: Small testes and epididymides were recorded for all males receiving 1000 mg/kg bw/day, the testes of two of these males were also described as flaccid.
Key result
Dose descriptor:
dose level:
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased liver weight at 600 and 1000 mg/kg bw/day. Small testes and epididymides at 1000 mg/kg/day.
Critical effects observed:
not specified

None

Conclusions:
Terpineol multiconstituent was considered to increase liver weight at 600 and 1000 mg/kg bw/day and to increase urea and creatinine levels at 1000 mg/kg bw/day in female rats. In males, reduced testis and epididymal weights with flaccid testis and increased kidney weights were observed at 1000 mg/kg bw/day and a dose-related increased liver weight was reported at all doses. The liver and the testes were considered to be the primary targets of terpineol multiconstituent.
Executive summary:

In a 14-day repeated dose toxicity range-finding study performed, groups of Crl:CD(SD) rats (four/dose/sex) were administered daily with terpineol multiconstituent at dose levels of 0 (vehicle control), 150, 600 and 1000 mg/kg bw/day in corn oil by oral gavage. Animals were then observed for mortality, clinical condition, bodyweight, food consumption, water consumption, oestrous cycles, blood chemistry and organ weight and were all macroscopically necropsied after sacrifice.

 

No mortality occurred during the study. Chin rubbing and salivation were observed at 600 and 1000 mg/kg bw/day. Isolated occasions of reduced activity, reduced body tone and unsteady gait were observed in few females at 1000 mg/kg bw/day. Overall bodyweight gain and food consumption were not affected during the treatment but water consumption was high at 600 and 1000 mg/kg bw/day. Plasma urea and creatinine levels were elevated in males at 600 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day. Dose-related increased liver weight was observed in males at all doses and in females at 600 and 1000 mg/kg bw/day. Reduced testis and epididymal weights with flaccid testis and increased kidney weights were observed in males at 1000 mg/kg bw/day. Oestrous cycles were not affected by treatment.

 

Under the test conditions, terpineol was considered to increase liver weight at 600 and 1000 mg/kg bw/day and to increase urea and creatinine levels at 1000 mg/kg bw/day in female rats. In males, reduced testis and epididymal weights with flaccid testis and increased kidney weights were observed at 1000 mg/kg bw/day and a dose-related increased liver weight was reported at all doses. The Liver and the testes were considered to be the primary targets of terpineol multiconstituent when administered by gavage.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Recent GLP study conducted according to OECD Guideline 422 without any deviation (Klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February - 01 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD 413 Guideline without deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP compliance monitoring programme (inspected on 01 July 2014 / signed on 07 October 2015)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 60-67 days
- Weight at study initiation: 264-338 g (males); 173-219 g (females)
- Housing: 5 rats/cage/sex in polycarbonate cages with a stainless steel mesh lid.
- Diet: Harlan Teklad 2014C pelleted diet, ad libitum (except overnight before blood sampling for haematology or blood chemistry and during exposure)
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 ºC
- Humidity: 40-70 %
- Air supply: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h light / 12 h dark
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
other: snout-only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD: <0.52, 0.7 and 1.6 µm for achieved concentrations of 0.202, 0.572 and 2.23 mg/L, respectively.
GSD: 2.99 and 1.75 for achieved concentrations of 0.572 and 2.23 mg/L, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through snout only chamber
Aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre chamber.
Groups 3 and 4 exposure chamber contained a chamber liner to reduce the internal volume.
- Method of holding animals in test chamber: during exposure, the rats were held in restraining tubes with their snouts protruding from the ends of the tubes into the exposure chambers.
- Training for dosing: the animals were acclimated to the method of restraint, over a 5 day period immediately preceding the first test substance exposure
- Aerosol Generation: a stainless steel concentric jet atomiser (manufactured in house by Inhalation Engineering Services), designed to produce and maintain an atmosphere containing a high proportion of respirable droplets. The test substance was supplied to the generator, via a Polyethylene feed line, from a plastic syringe driven at a constant rate by a syringe pump (Harvard).
- Inlet Airflow:
From in-house compressed air system – breathing quality
Generator flow: 9 L/minute per system
- Extract Airflow:
Drawn by in-house vacuum system; filtered locally; Flow: 25 L/minute per system
- Airflow Monitoring:
High quality tapered tube flowmeters – calibrated daily
In-line flowmeters monitored continuously
- Temperature in air chamber: Measured using an electronic thermometer and recorded at 60 minute intervals during exposure. The chamber temperatures were similar for each group on each day of the study. The observed temperatures for all groups remained within the acceptable range for inhalation exposure of rats.
- Air flow rate: 2.0 L/minute
- Method of particle size determination: determined by cascade impaction.
Samples collected as follows:
Impactor type: Marple 298
Collection media: stainless steel substrates and glass fibre (GF10) final stage filter
Sample flow: 2.0 L/minute
Sample volume: measured by Apex Pro pump
Sample frequency: minimum of 1 sample/week/group
Sample location: animal exposure port
Sample analysis: gravimetric

ATMOSPHERE ANALYSIS
Aerosol samples collected as follows:
Filter type: glass fibre filter (GF10) held in open faced filter holder
Sample flow: 2.0 L/minute
Sample volume: measured by Apex Pro pump
Sample frequency: minimum of 3 sample/group/day (test groups); 1 sample/day (Group 1)
Sample location: animal exposure port
Sample analysis: gravimetric
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Total amount of test material on the collection substrates were determined by gravimetric analysis and test article concentration by chemical analysis (GC method of analysis)
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
0.2, 0.6 and 2 mg/L
Basis:
other: target concentration
No. of animals per sex per dose:
10
Control animals:
other: air
Details on study design:
- Dose selection rationale: test concentrations were selected on the basis of the results of a preliminary toxicity study by inhalation administration to rats for 2 Weeks (Study Code: OAD0029), where terpineol multiconstituent was administered to Crl:CD(SD) rats for six hours per day, for five days, using a snout-only exposure system at achieved exposure levels of 0.194, 0.637 and 2.15 mg/L. The only treatment related findings observed in life consisted of reduced body weight gain for all males exposed to terpineol multiconstituent and for females exposed to 2.15 mg/L. Histopathological changes related to treatment were evident for both sexes at an exposure level of 2.15 mg/L. These consisted of minimal to slight hyperplasia/inflammation of the nasal epithelium. The No Observed Adverse Effect Level (NOAEL) for the 2 week study was 2.15 mg/L.
Consequently, the same exposure levels were used for this 13 week study. An exposure level of 2.15 mg/L was expected to be tolerated for 13 weeks, with minor body weight effects described above. The lowest exposure level of 0.2 mg/L is a tenth of the highest level and is expected to provide no adverse effects. The intermediate exposure level of 0.6 mg/L is the geometric mean of the high and low exposure target levels and provided information on the dose relationship of any effects seen.
- Rationale for animal assignment: randomly allocated on arrival; using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Post-exposure recovery period in satellite groups: control and high dose groups (10/sex/dose)
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: yes
Time schedule:
Detailed observations were recorded daily at the following times in relation to dose administration:
- pre-exposure observation and during exposure. However, observation is severely restricted due to tube restraint.
- as each animal is returned to its home cage,
- as late as possible in the working day.
In addition, observations were made in the treatment period, on days without exposures and at the following times during the day:
- early in the working day (equivalent to pre-exposure observations),
- as late as possible in the working day.
A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: yes
Time schedule for examinations: the weight of each animal was recorded twice weekly from Week 1 to Week 4 , weekly from Week 5 to Week 13 and before necropsy.

FOOD CONSUMPTION: yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment and for each week throughout the study.

WATER CONSUMPTION: no

OPHTHALMOSCOPIC EXAMINATION: yes
Time schedule for examinations
- Pretreatment: all animals (Main, Recovery and spares)
- Week 13: all Main and Recovery animals of Groups 1 and 4
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide, ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: yes
Time schedule for collection of blood: during Week 13, all animals (Main and recovery); during Week 4 (recovery), all recovery animals.
Anaesthetic used for blood collection: yes;
Animals were held under light general anaesthesia induced by isoflurane.
Animals fasted: yes;
Blood samples were collected after overnight withdrawal of food and prior to dosing.
Parameters checked:
Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) & Large unstained cells (LUC), Platelet count (Plt), Morphology: Anisocytosis, Microcytosis, Macrocytosis, Hypochromasia & Hyperchromasia
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. In the presence of platelet clumping a manual count of the differential white blood cell parameters was performed.

Using citrate as anticoagulant - Prothrombin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: yes
Time schedule for collection of blood: Week 13, all animals (Main and recovery).
Anaesthetic used for blood collection: yes;
Animals were held under light general anaesthesia induced by isoflurane.
Animals fasted: yes;
Blood samples were collected after overnight withdrawal of food and prior to dosing.
Parameters checked: blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and albumin concentration was analysed.

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: no
Sacrifice and pathology:
Necropsy: animals were killed by overdose of intraperitoneal pentobarbitone sodium followed by exsanguination.
Schedule:
Main study animals were killed following 13 weeks of treatment.
Recovery animals were killed following 13 weeks of treatment and 4 weeks of recovery.

All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals (Table 7.5.2/1).

HISTOPATHOLOGY: yes
Fixation: tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below.
Testes were preserved in modified Davidson’s fluid.
Eyes were preserved in Davidson’s fluid.

Histology:
Processing: tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required (Table 7.5.2/1).
Full List: main study and recovery animals of Groups 1 and 4 were killed at a scheduled interval.
Nasal turbinates and nasal pharynx: main study animals of Groups 2 and 3, and Recovery Phase animals of Groups 1 and 4 were killed at a scheduled interval.
Routine staining: sections were stained with haematoxylin and eosin.

Light microscopy:
Tissues preserved for examination were examined as follows.
Main study
- All animals of Groups 1 and 4: all specified tissues are in Table 7.5.2/1.
- All animals of Groups 2 and 3 and recovery animals: abnormalities only.
The following tissues, which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study animals of Groups 2 and 3 and recovery animals of Groups 1 and 4: nasal turbinates and nasal pharynx.
Other examinations:
Haematology, bone marrow: bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods.
Fixation: smears were air dried and subsequently fixed in methanol.
Analysis: no examinations were performed, however, the smears were retained for possible future examination.
Statistics:
See section "Any other information on materials and methods incl. tables
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
group mean body weight gains were lower than controls for males (all dose levels) and at 2.23 and 0.572 mg/L for females
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with terpineol multiconstituent were seen in the nasal turbinates and nasal pharynx
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- There were no treatment related deaths in this study.
- Salivation and chin rubbing were observed from Day 23 onwards in all animals exposed to 2.23 mg/L, in several animals exposed to 0.572 mg/L and one female exposed to 0.202 mg/L. These signs were observed on consecutive days for the majority of animals.
- Unsteady gait was observed between Day 30 and 64 of treatment in 7/20 males and 14/20 females exposed to 2.23 mg/L and in 1/10 females exposed to 0.572 mg/L. Partially closed or closed eyelids were observed between Day 26 and 60 of treatment in 2/20 males and 7/20 females exposed to 2.23 mg/L and in 1/10 females exposed to 0.572 mg/L. Elevated posture was observed between Day 30 and 33 in 8/20 males and 7/20 females exposed to 2.23 mg/L. With the exception of a few isolated incidences, the aforementioned signs resolved prior to the final check at the end of the working day. These observations were intermittent for all effected animals, usually presenting on non-consecutive days.
- Signs associated with the method of restraint included wet fur and red/brown staining of the head, muzzle and nose which were observed in some animals from all groups on return to the home cage.
- There were no test article related clinical signs during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
- Males exposed to 2.23 mg/L showed a group mean body weight loss of 6 g between Day 1 and 4, compared with a group mean gain of 5 g in Control males. Body weight changes for females exposed to 2.23 mg/L showed a group mean body weight loss of 2 g over the same period, compared with a group mean gain of 1 g in Control females.
- Over the 13 weeks of exposures, group mean body weight gains for males exposed to 0.202, 0.572 and 2.23 mg/L were lower than controls (0.87X, 0.87X and 0.81X respectively), with changes for males exposed to 2.23 mg/L achieving statistical significance. For females exposed to 0.572 and 2.23 mg/L, group mean body weights were statistically significantly lower than controls (0.81X and 0.84X respectively). Females exposed to 0.202 mg/L had a similar weight gain to controls.
- Over the 4 week recovery period, body weight gains for males and females previously exposed to 2.23 mg/L showed complete recovery to control values.

FOOD CONSUMPTION
There were no treatment related effects on food consumption.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment related effects in ophthalmoscopic examination.

HAEMATOLOGY
- The group mean reticulocyte percentage and the absolute reticulocyte count were lower than control values for males exposed to 0.572 (0.90X and 0.92X respectively) and males exposed to 2.23 mg/L (0.82X and 0.82X respectively), with males exposed to 2.23 mg/L achieving statistical significance. A similar effect was seen for females exposed to 2.23 mg/L (0.92X and 0.87X control values for reticulocyte percentage and absolute reticulocyte count, respectively), but this did not attain statistical significance. During Recovery Week 4, values for both sexes previously exposed to 2.23 mg/L were similar to controls.
- Other changes from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra group variation.

CLINICAL CHEMISTRY
- There were no treatment related effects.
- Differences from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra group variation.

ORGAN WEIGHTS
- There were no treatment related effects.
- Differences from control, some of which attained statistical significance, were generally small, inconsistent between groups and sexes and are considered due to intra group variation.

GROSS PATHOLOGY
Animals killed after 13 weeks of treatment
- The macroscopic examination performed after 13 weeks of treatment revealed no test substance related lesions.
- The incidence and distribution of all findings were consistent with the common background changes seen in Sprague-Dawley rats at these laboratories.

Animals killed after 4 weeks of recovery
- The macroscopic examination performed after 4 weeks of recovery revealed no test substance related lesions.
The incidence and distribution of all findings were consistent with the common background changes seen in Sprague-Dawley rats at these laboratories.

HISTOPATHOLOGY: NON-NEOPLASTIC
Animals killed after 13 weeks of treatment:
- Treatment related findings: Changes related to treatment with terpineol multiconstituent were seen in the nasal turbinates and nasal pharynx.
- Nasal turbinates: hyperplasia of the mucous cells (minimal to slight severity) was present in almost all males treated with terpineol multiconstituent, the majority of females receiving 0.572 mg/L and 2.23 mg/L and several females receiving 0.202 mg/L. The respiratory epithelium associated with the ventral nasal septum in the anterior portion of the nasal cavity was predominantly affected. Minimal inflammation was present in the respiratory epithelium of two animals exposed to 2.23 mg/L and was typified by the presence of a predominantly neutrophilic cellular infiltrate. Minimal degeneration of the olfactory and/or respiratory epithelium lining the anterior portion of the dorsal meatus was present, predominantly in females treated with 2.23 mg/L terpineol multiconstituent.
- Nasal pharynx: hyperplasia of the mucous cells was present in the respiratory epithelium lining the nasal pharynx of predominantly females treated with 0.572 mg/L or 2.23 mg/L of terpineol multiconstituent.
- Incidental findings: all other changes were considered to be incidental and consistent with the common background lesions seen in Sprague-Dawley rats at these laboratories.

Animals killed after 4 weeks of recovery:
Treatment related findings: changes related to treatment with terpineol multiconstituent were seen in the nasal turbinates following the 4 week recovery period.
Nasal cavity: hyperplasia of the mucous cells (minimal to slight severity) was present in the majority of males and females treated with 2.23 mg/L and in one control male. The respiratory epithelium associated with the ventral nasal septum in the anterior portion of the nasal cavity was predominantly affected. Minimal degeneration of the olfactory epithelium lining the anterior portion of the dorsal meatus was present in one male and two females treated with 2.23 mg/L of terpineol multiconstituent.
Incidental findings: all other changes were considered to be incidental and consistent with the common background lesions seen in Sprague-Dawley rats at these laboratories.
Key result
Dose descriptor:
NOAEC
Effect level:
2.23 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEC is the highest dose tested
Critical effects observed:
not specified

ATMOSPHERE ANALYSIS AND ESTIMATION OF ACHIEVED DOSE

 

Table 7.5.2/2: Summary data

 

Group

Achieved concentration (mg/L)

Particle size

MMAD (µm)

GSD

1

-

-

-

2

0.202

<0.52

-

3

0.572

0.7

2.99

4

2.23

1.6

1.75

MMAD: Mass median aerodynamic diameter

GSD: Geometric standard deviation

 

The achieved levels were 101, 95 and 112% of the target concentrations for Groups 2, 3 and 4 respectively.

The MMAD values showed a general increase with increasing aerosol concentration. The general trend to larger MMAD with increasing aerosol concentration is normal and is attributed to increased particle interaction and subsequent aggregation.

The MMAD for Group 4 was within the ideal range of 1 to 3 µm indicating that the terpineol multiconstituent aerosol was respirable to the rats.

The Group 3 particle size distribution values show a bi-modal distribution across the impactor stages with an average of 49% of the captured droplet having an MMAD below 0.52 µm.

The MMAD for Group 2 was not calculable as virtually all the measurable test material was captured on the final filter stage. The value presented is based on the cut point of the penultimate stage of the impactor (Stage 8) which has a cut point of 0.52 µm. This value is below the ideal range of 1 to 3 µm. The concern with how respirable particles with an MMAD below 1 µm are that they may not impact on airway surfaces and be exhaled, thus reducing exposure. In this study, due to the delivered aerosol being a liquid, it is likely that inhaled droplets would impact on airway surfaces and not be exhaled.

 

HISTOPATHOLOGY

 

Table 7.5.2/3: Summary of treatment related findings in the nasal cavity for animals killed after 13 weeks of treatment

 

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Achieved Exposure Level (mg/L)

0

0.202

0.572

2.23

0

0.202

0.572

2.23

Hyperplasia, Mucous Cell, Respiratory Epithelium

Minimal

0

5

5

5

0

3

6

5

Slight

0

4

4

5

0

1

2

3

Total

0

9

9

10

0

4

8

8

Degeneration, Respiratory Epithelium

Minimal

0

0

0

0

0

0

0

2

Total

0

0

0

0

0

0

0

2

Degeneration, Olfactory Epithelium

Minimal

0

0

0

2

0

0

0

4

Total

0

0

0

2

0

0

0

4

Inflammation, Respiratory Epithelium

Minimal

0

0

0

1

0

0

0

1

Total

0

0

0

1

0

0

0

1

Number of tissues examined

10

10

10

10

10

10

10

10

Table 7.5.2/4: Summary of treatment related findings in the nasal pharynx for animals killed after 13 weeks of treatment

 

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Achieved Exposure Level (mg/L)

0

0.202

0.572

2.23

0

0.202

0.572

2.23

Hyperplasia, Mucous Cell

Minimal

0

0

1

1

0

0

2

4

Total

0

0

1

1

0

0

2

4

Number of tissues examined

10

10

10

10

10

10

10

10

 

Table 7.5.2/5: Summary of treatment-related changes in the nasal turbinates for animals killed after 4 weeks of recovery

 

Group/sex

1M

4M

1F

4F

Achieved Exposure Level (mg/L)

0

2.23

0

2.23

Hyperplasia, Mucous Cell, Respiratory Epithelium

Minimal

1

5

0

7

Slight

0

4

0

2

Total

1

9

0

9

Degeneration, Olfactory Epithelium

Minimal

0

1

0

2

Total

0

1

0

2

Number of tissues examined

10

10

10

10

Conclusions:
The No Observed Adverse Effect Concentration (NOAEC) was considered to be 2.23 mg/L.
Executive summary:

In a repeated dose toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, terpineol multiconstituent was administered by inhalation-aerosol to groups of Crl:CD(SD) rats (10 rats/sex/ group) by snout-only inhalation exposure at target exposure levels of 0.2, 0.6 and 2 mg/L for 6 hours per day, 5 days per week for 13 weeks. Control animals received air only. Recovery animals were similarly treated for 13 weeks followed by a 4 week off dose period. Control and high dose recovery groups were included (10/sex/group). During the study, clinical condition, body weight, food consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

The achieved levels were 101, 95 and 112% of the target concentrations of 0.2, 0.6 and 2 mg/L, respectively (achieved concentrations 0.202, 0.572 and 2.23 mg/L).

MMAD: <0.52, 0.7 and 1.6 µm for achieved concentrations of 0.202, 0.572 and 2.23 mg/L, respectively.

GSD: 2.99 and 1.75 for achieved concentrations of 0.572 and 2.23 mg/L, respectively.

MMAD showed a general increase with increasing aerosol concentration. The MMAD for Group 2 could not be calculated, as virtually all the measurable test material was captured on the final filter stage, and the value presented is based on the cut point of the penultimate impactor stage. The Group 3 particle size distribution values showed a bi-modal distribution with an average of 49% of the captured droplet having a MMAD below 0.52 µm. The MMAD value for Group 4 was within the ideal range (1 to 3 µm), indicating that the terpineol multiconstituent aerosol was respirable to the rats. The MMADs for Groups 2 and 3 were below the ideal range of 1 to 3 µm. However, since the delivered aerosol was a liquid, it is likely that those inhaled droplets with an aerodynamic diameter below 1 µm would still have impacted on airway surfaces and not been exhaled.

There were no treatment related deaths or effects on food consumption, blood chemistry, ophthalmoscopy, organ weights or macropathology findings.

Group mean body weight gains were lower than control for males exposed to 0.202 mg/L and for both sexes exposed to 0.572 and 2.23 mg/L. In both sexes, no relationship between exposure concentration and body weight gain was observed but the decrease in mean body weight gain was statistically significant for males exposed to 2.23 mg/L. Body weights showed full recovery for animals previously exposed to 2.23 mg/L.

Clinical pathology measurements following 13 weeks of exposure revealed statistically significantly lower group mean reticulocyte percentages and absolute counts for males exposed to 0.572 or 2.23 mg/L, compared to control (as low as 0.82X control). A similar effect was observed for females exposed to 2.23 mg/L (as low as 0.87X control) but this did not attain statistical significance. During Recovery Week 4, values for both sexes previously exposed to 2.23 mg/L were similar to controls.

Histopathological changes related to treatment were observed in the nasal turbinates for the majority of animals given terpineol multiconstituent and nasal pharynx for a limited number of animals given 0.572 or 2.23 mg/L.

The nasal cavity was identified as a target organ for local effects. Changes related to treatment with terpineol multiconstituent were reported in the nasal turbinates and nasal pharynx in both males and females. In the nasal pharynx, minimal hyperplasia of the mucous cells was also seen in the respiratory epithelium in a small number of animals exposed to 0.572 mg/L or 2.23 mg/L. The changes in the nasal pharynx were also not associated with an inflammatory cell infiltrate or degenerative changes. Examination of recovery phase animals showed no changes in the nasal pharynx respiratory epithelium, suggesting complete recovery after 4 weeks which is therefore not considered adverse.

In the nasal turbinate, mucous cell hyperplasia was present at all exposure levels and did not exhibit a clear dose response in terms of incidence or severity in males, although there were slightly higher incidences in females at 0.572 mg/L or 2.23 mg/L compared with females exposed to 0.202 mg/L. Following a 4 week recovery period, similar incidences of mucous cell hyperplasia in the respiratory epithelium were observed in animals exposed to 2.23 mg/L compared to those at the end of the main study phase, although most were of minimal severity. Mucous cell hyperplasia is considered to be an adaptive change in the epithelium in response to chronic irritation (Renne et al 2009). This may correlate with clinical signs of salivation and chin rubbing that were observed on consecutive days (from Day 23 onwards) and displayed a dose related response in terms of the number of animals affected. Given that the histopathology changes were of minimal or slight severity and were generally not associated with inflammatory or degenerative changes in the respiratory epithelium, particularly at lower exposure levels, it would be expected that these changes would reverse following a suitable recovery period. Therefore, mucous cell hyperplasia in the nasal turbinates is also not considered adverse.

Therefore, the No Observed Adverse Effect Concentration (NOAEC) was considered to be 2.23 mg/L.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29 August - 18 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Preliminary study designed to select the appropriate dose levels for the upcoming 14-day inhalation preliminary study.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
Preliminary study designed to select the appropriate dose levels for the upcoming 14-day inhalation preliminary study.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: Charles River Laboratories, Sulzfeld, Germany
Age at study initiation: < 9 Weeks
Weight at study initiation: males: 179-215 g; females: 125-161 g
Housing: group caging (in groups of 3 rats/cage, by sex) in polycarbonate solid floor cages (type II or III) with stainless steel mesh lids.
Diet: animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance", ssniff Spezialdiäten GmbH, Germany, ad libitum
Water: tap water, ad libitum
Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
Temperature: 22 ± 3 °C
Humidity: 30-70 %
Air changes: at least 15 air exchanges per hour
Photoperiod: 12 h dark / 12 h light
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: An aerosol atmosphere was generated to contain particles with a mass median aerodynamic diameter (MMAD) between 1 to 3 μm with a geometric standard deviation (δg) in the range of 1.5 to 3. Measurements of aerodynamic particle size were performed from the animal’s breathing zone.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: for the test atmosphere generation, compressed air jet nebulizer (TSE Systems GmbH, Bad Homburg, Germany) was used connected to the infusion pump (TSE) and to pressurized air supply.
- Method of holding animals in test chamber: animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was approximately 1.5 L/min.
- System of generating aerosols: atmosphere generation was dynamic. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, the aerosol was distributed to the individual exposure ports.
- Exposure conditions: temperature of the test atmosphere and air used for the control group exposure in some cases were increased over the limit of 25ºC (up to 25.2ºC) due to temporary malfunctioning of the cooling system. The relative humidity was lower than the optimal range of 30-70% due to the use of filtered, dry air for the dispersion of the test item.
- Air flow rate: for dispersion of the test item in air, the air flow rate was 30 L/min (in the nebulizer). The test item was supplied by a constant rate (approximately 6, 13 and 23 mL/h, for low, mid and high concentrations, respectively).
- Method of particle size determination: particle size analysis of generated atmospheres was performed using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Samples were collected once a week at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage < 4 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: after passing through the animal’s breathing zone, spent aerosol entered the outer cylinder from where it was exhausted through a suitable filter system [GF10 glass fibre filters (Whatman, Germany)].

TEST ATMOSPHERE
- Brief description of analytical method used: several testing was performed in order to select a proper sampling method of the test atmosphere, which would be suitable both by gravimetry and analytical analysis. A charcoal filled tubes Anasorb CSC were tested but better results were achieved using glass fibre filters F10.
The stability of particle size distribution was determined by performing multiple particle size analyses during characterisation using a cascade impactor.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test atmosphere concentrations were monitored based on the gravimetric analysis and confirmed by validated GC method.
Duration of treatment / exposure:
5 days
Frequency of treatment:
6 h/day for 5 consecutive days
Remarks:
Doses / Concentrations:
1, 2 and 3 mg/L
Basis:
other: target concentrations
No. of animals per sex per dose:
3 rats/sex/group in 3 groups, one additional female in high concentration group
Control animals:
no
Details on study design:
- Dose selection rationale: test concentrations were selected on the basis of the results of a preliminary acute study in the rat (Safepharm Laboratories Ltd. study project No.: 2227/0001), where test item atmosphere at a mean concentration level of 4.8 mg/L was tested during the single four-hour exposure in Sprague Dawley rats followed by 14-day recovery period.
- Preparation of the test item before dispersion: the test atmosphere was generated from the test item as supplied.
- Rationale for animal assignment: animals were randomly allocated to exposure groups before the animal exposure.
Positive control:
not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: yes
Mortality / morbidity: Mortality / morbidity checks were made twice daily, early and late during the normal working day.
Clinical signs: individual clinical observations were performed prior to exposure and twice during exposure whilst the animals are still restrained. Following exposure, clinical observations were performed at least twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure).

BODY WEIGHT: yes
Time schedule for examinations: body weight of each animal was recorded with precision of 1 g at randomization, then on Day 0 (before the exposure), and on Days 1, 3 and 5.

FOOD CONSUMPTION: no

WATER CONSUMPTION: no

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: no

CLINICAL CHEMISTRY: no

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: yes
Gross necropsy was performed on each animal terminally one day following the last exposure. All animals were euthanised under pentobarbital anaesthesia by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. All abnormalities were recorded.
ORGAN WEIGHTS:
The following organs were trimmed of fat and weighed in all animals: lungs, liver, kidney, brain, testis, epididymis and thymus and adrenals
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.
HISTOPATHOLOGY: yes
Weighed organs larynx, trachea, nasal cavity and all organs showing macroscopic lesions of all animals were preserved. Lungs after weighing were infused with formalin. Unilateral testis with epididymis were retained in modified Davidson’s fixative and all other organs in 10 % buffered formalin solution.
Lungs of all animals were evaluated microscopically to assess irritative effect on the respiratory tract at the first instance. Testes (unilateral) of all animals were also evaluated. The tissues were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
Other examinations:
Spermiology assessment:
The potential effect on the male reproductive system was investigated in all males using sperm analysis.
One of testis with epididymis was subjected to the sperm analysis.
The motility and morphology were evaluated from the sperm samples collected from the distal part of ductus epididymis. At least 200 cells per sample were counted twice in each animal. For additional morphology evaluation sperm smears were made and the slides retained.
Sperm count was evaluated from testis (from a frozen sample) after the appropriate homogenization and dilution.
Sperm analysis (motility, count and morphology) was made manually.
Statistics:
Data were collected using the software PROVANTIS v.7 or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the Microsoft Office Word and/or Excel, as appropriate.
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.
Data from additional female (No. 254) were excluded from mean for body weight and body weight gain calculation, due to considerable difference from group mean at the initiation of the treatment. All individual data are presented in the report.
No statistical analysis was performed on the data.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
There was no mortality at any of the exposure level.

CLINICAL SIGNS
- In animals exposed to 3 mg/L, laboured breathing, ataxia and decreased activity were observed. In males these signs were generally slight and were observed by the end of the exposure (following removal from the restrain), with ceasing tendency. In addition, slight sneezing was observed from Day 2 and was noted on the following days also before the treatment. In females, similar clinical signs were observed but were more pronounced, i.e. ataxia and decreased activity were of severe grade, laboured breathing was slight to moderate. Ceasing tendency was also noted for females. Permanent slight sneezing was also observable from Day 2-3. In three females, noisy respiration was noted toward the end of the treatment period (Days 2/3-4/5). The most pronounced clinical signs were observed in one female (No. 252) following the first exposure, i.e. severely decreased activity was followed by prone position. Prone position was also noted on Day 1 following the exposure. Ruffled fur was additionally observed in this female from Day 3 together with hunched posture. In this female, significant body weight loss (approximately 20%) was noted between Days 0-3. Bilateral white area of cornea was also noted in this animal from Day 4.
- In animals exposed to 2 mg/L, slightly laboured breathing was the most important clinical sign. It was transient and was observed toward the end of the exposure and following removal from the restrain in both males and females. One hour after the exposure, the animals were symptom-free.
- Animals exposed to 1 mg/L, were symptom free during the 5-day exposure period.
- Additionally, red-brown staining of the fur on the nose and on the head was occasionally observed in both sexes. This clinical sign, related to porphyrin discharge was considered to be a common observation in animals treated by the inhalation route. Wet fur was commonly recorded in both sexes and was considered to be related to the restraint and exposure procedures and not to be toxicologically significant.

BODY WEIGHT AND WEIGHT GAIN
- The terminal mean body weight of males and females at 3 mg/L was lower than initial mean values by approximately 3-4 %, due to transient body weight losses observed in both males and females between Days 0-3. Thereafter (Days 3-5) the body weight gain normalized. The most pronounced effect was observed in one female (No. 252), with a body weight loss of approximately 20% compared to initial body weight between Days 0-3.
- At 2 mg/L, slight body weight loss (no more than 3% of the individual initial values) or body weight gain suppression was noted in 2/3 males and 1/3 females. In one male (No. 303) and two females (No. 351 and 353) the body weight gain was within the range commonly recorded for this range and strain and was comparable to values of animals exposed to 1 mg/L. In one female (No. 352) a body weight loss of 3 % was evident over the 5 days of exposure. However, this occurred only between Day 0 and Day 1.
- No effects on body weight were observed at 1 mg/L.

ORGAN WEIGHTS
- Thymus weights were decreased at 3 mg/L in both sexes. When compared to the values of animals exposed to 1 mg/L, mean thymus weights in females were lower by approximately 42 % (absolute values) and 47 % (body weight relative values). The lowest value was recorded in female No. 252, however, without this value, the difference was still considerable (approximately 30-40 %). In males thymus weights were lower by approximately 20 % (both values). It should be noted that age differences between groups were negligible in spite of the staggered initiation of the treatment, approximately one week differences between groups and the last group animals were exposed to 2 mg/L.
- Slightly higher adrenal weights were recorded at 3 mg/L in both males (by approximately 20-25 %) for absolute and brain relative values, respectively and in females (by approximately 45 %, both values). Also in this group higher liver weights were noted for 1/3 male and one female (No. 252).

GROSS PATHOLOGY
No test item-related findings were noted during the gross pathological investigation, however small thymus was observed in one female (No. 252) at 3 mg/L. In this female marked body weight loss and clinical signs were noted. Bilateral, diffuse opacity of cornea was noted in this single female and was regarded as individual finding.

HISTOPATHOLOGY
No signs of irritation or other test item related changes were observed in the lungs of the animals exposed to 1, 2 or 3 mg/L during histopathological evaluation. No microscopic changes were observed in the testes.

OTHER FINDINGS
Spermiology: no significant effects were noted during sperm analysis in the sperm count and morphology, but the number of non-motile sperm was slightly higher in animals exposed to 2 and 3 mg/L. However due to the small number of animals and the absence of clear dose response, these changes were considered to be of no toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
1 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 1 mg/L exposed groups no effects were observed.
Critical effects observed:
not specified

TEST ATMOSPHERE DATA

Actual and nominal concentrations

The exposure concentrations were monitored intermittently by gravimetrical analysis of the test item deposited on a sampling filter. Results of the confirmatory specific analysis of the test atmosphere were in agreement with the gravimetrical results. 

The nominal concentration (mass of the test item dispersed into the exposure system in total air flow used for exposure) was 3.7, 7.2 and 12.5 mg/L, for low, mid and high concentration, respectively.

 

Table 7.5.2/1: Mean achieved actual aerosol concentrations

 

Group

No.

Group

Designation

Target

Concentration

(mg/L)

Achieved Concentration (mg/L)

Gravimetry

(mean)

Specific analysis

(mean)

1

Low

1.0

1.02

(SD: 0.04)

1.03

(SD: 0.06)

3

Mid

2.0

1.99

(SD: 0.12)

1.81

(SD: 0.12)

2

High

3.0

2.97

(SD: 0.08)

2.80

(SD: 0.14)

 

Particle size analysis

According to particle size analysis of samples taken from the animal’s breathing zone the Mass Median Aerodynamic Diameter in the test atmospheres of all groups was in the range of 2.4-2.9 μm with Geometric Standard Deviation of 1.7-1.9.

 

Table 7.5.2/2: Particle size distribution data (MMAD and GSD)

 

Group

Mean Mass Median

Aerodynamic Diameter (MMAD) (μm)

Geometric

Standard Deviation

(GSD)

Inhalable Fraction

(% < 4μm)

1

2.41

1.92

78.0

2

2.94

1.72

71.5

3

2.71

1.72

76.3

 

 

 

Table 7.5.2/3: Mean body weight (g) on Day 5 and body weight gain (g) values between days 0-5 of males and females

Males / Females

Groups/Exposure level (mg/L)

1.0

2.0

3.0

Males

Body weight (g)

197

209

195

Body weight gain (g)

13.7

0.3

-7.3

Females

Body weight (g)

141

158

143

Body weight gain (g)

8.0

4.0

-4.7

 

Table 7.5.2/4: Mean selected organ weights of males and females (Day 5)

Males / Females

Groups/Exposure level (mg/L)

1.0

2.0

3.0

Males

Thymus weight absolute (g)

0.503

0.497

0.400

Thymus weight bw relative (%)

0.256

0.231

0.205

Adrenals weight absolute (g)

0.0453

0.0510

0.0563

Adrenals weight bw relative (%)

0.0230

0.0236

0.0289

Liver weight absolute (g)

7.98

8.83

8.74

Liver weight bw relative (%)

4.054

4.101

4.486

Females

Thymus weight absolute (g)

0.363

0.390

0.210

Thymus weight bw relative (%)

0.257

0.246

0.136

Adrenals weight absolute (g)

0.0513

0.0590

0.0740

Adrenals weight bw relative (%)

0.0364

0.0373

0.0489

Liver weight absolute (g)

5.69

6.87

6.79

Liver weight bw relative (%)

4.048

4.343

4.481

Conclusions:
The No Observed Adverse Effect Level (NOAEL) of Terpineol multiconstituent was considered to be 1 mg/L in rats.
Executive summary:

This 5-day inhalation study was designed to select the appropriate dose levels for the upcoming 14-day inhalation study. In this pilot study, terpineol multiconstituent was administered in the form of aerosol to Hannover Wistar rats Crl:WI (Han) (3 rats/sex/ group in 3 groups, one additional female was exposed 6 h/day for 4 consecutive days at 3 mg/L concentration) for 6 h per day, for five consecutive days, using a nose-only exposure system at levels of 1, 2 and 3 mg/L. No control groups were used. Parameters monitored during the study included mortality, clinical observations and body weight. Gross macroscopic examination was performed at necropsy and selected organs were weighed. Lungs of all animals and testes of all males were subjected to histopathological examination. The test atmosphere concentrations were monitored based on the gravimetric analysis and confirmed by validated GC method.

 

There were no unscheduled deaths during the treatment period. Clinical signs (laboured and noisy respiration, ataxia and decreased activity) were noted in animals exposed to 3 mg/L. These signs were more severe in females and decreasing tendency was observed in both sexes, i.e. ataxia and decreased activity were noted up to Day 2, while sneezing and slightly laboured and noisy respiration were evident throughout the study. At 2 mg/L, slight laboured breathing was the only clinical sign of toxicological importance and one hour after the end of exposure, animals were symptom-free. At 1 mg/L, all animals were symptom free, no clinical signs of toxicological importance were noted. At 3 mg/L, on Day 5, body weight of males and females was lower than initial mean values by approximately 3-4 %, due to transient body weight loss observed in both sexes between Days 0-3. At 2 mg/L, slight body weight losses or body weight gain suppression were noted in 2/3 males and 1/3 females. No effects on body weight were observed in animals exposed to 1 mg/L.

 

No test item-related findings were noted during gross pathological investigation. Decreased mean thymus weights were observed in animals exposed to 3 mg/L in both sexes (20 and 42 % decreases for absolute values in males and females, respectively) when compared to the values of animals exposed to 1 mg/L. At this concentration, increased mean adrenal weights were recorded in both males and females (by approximately 25 and 45%, respectively). Increased liver weights were also noted in 1/3 male and 1/4 female. No differences were noted in organ weights in animals exposed to 1 or 2 mg/L. No microscopic changes were observed in the lungs and testes. The number of non-motile sperm was higher in animals exposed to 2 and 3 mg/L. However due to the small number of animals and the absence of clear dose response, these changes were considered to be of no toxicological significance. Also, no significant effects were noted during sperm analysis in the sperm count and morphology.

 

Therefore, the No Observed Adverse Effect Level (NOAEL) of Terpineol multiconstituent was considered to be 1 mg/L in rats.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 2-week dose range finding inhalation study performed without deviation that would have an impact on the scientific reliability of the results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
GLP 2-week dose range finding inhalation study designed to select the appropriate dose levels for the upcoming 90-day inhalation study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 60-67 days old
- Weight at study initiation: males: 320 to 354 g; females: 223 to 257 g
- Housing: 3 rats/cage/sex in polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: Harlan Teklad 2014 rodent diet, ad libitum (except at scheduled necropsy and during dosing)
- Water, ad libitum (except during dosing)
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 ºC
- Humidity: 40-70 %
- Photoperiod: 12 h light / 12 h dark
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD: <0.52, 1.1 and 1.4 µm for achieved concentrations of 0.194, 0.637 and 2.15 mg/L, respectively
GSD: 2.05 and 1.81 for achieved concentrations of 0.637 and 2.15 mg/L, respectively
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ADG exposure chamber
- Method of holding animals in test chamber: during exposure, the rats were held in restraining tubes with their snouts protruding from the ends of the tubes into the exposure chambers.
- Training for dosing: animals were acclimated to the method of restraint, normally over a 5-day period preceding the first test substance exposure.
- Source and rate of air: from in-house compressed air system
– Breathing quality: 9 L/minute per system
- System of generating particulates/aerosols: a stainless steel concentric jet atomiser (manufactured in house by Inhalation Engineering Services), designed to produce and maintain an atmosphere containing a high proportion of respirable droplets.
- Method of particle size determination: by cascade impaction (impactor type: Marple 298)
- Treatment of exhaust air: filtered locally
- Temperature: between 18.4 and 20.2 °C.

TEST ATMOSPHERE
- Brief description of analytical method used: total amount of test material on the collection substrates were determined by gravimetric analysis and test article concentration by chemical analysis (GC method of analysis).
- Samples taken from breathing zone: yes (aerosol samples were collected with glass fibre filter held in open faced filter holder at minimum of 3 samples/group/day)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Total amount of test material on the collection substrates were determined by gravimetric analysis and test article concentration by chemical analysis (GC method of analysis: test samples were extracted with acetone then injected by split injection onto GC column (SPB-5) with flame ionisation detection)
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
0.2, 0.6 and 2 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
3
Control animals:
other: air only
Details on study design:
Dose selection rationale: test concentrations were selected on the basis of the results of the 5-day dose range finding inhalation toxicity study (Study Code: 14/260-212PEA), where terpineol multiconstituent was administered to Crl:WI (Han) rats for six hours per day, for five days, using a snout-only exposure system at achieved exposure levels of 1.03, 1.81 or 2.80 mg/L.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: yes
Time schedule: detailed observations were recorded daily, on exposure days, at the following times in relation to dose administration (pre-exposure observation); as each animal is returned to its home cage; as late as possible in the working day
Observation during exposure was severely restricted due to tube restraints.
In addition observations were recorded daily, on days without exposures and at the following times during the day: early in the working day (equivalent to pre-exposure observation); as late as possible in the working day
A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: yes
Time schedule for examinations: the weight of each animal was recorded twice weekly during the week before treatment, on the day that treatment commenced (Day 1), twice weekly throughout the study and before necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatmentand for each week throughout the study.

WATER CONSUMPTION: no

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: no

CLINICAL CHEMISTRY: no

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: no
Sacrifice and pathology:
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule: animals were killed following 2 weeks of treatment.
Method of kill: overdose of intraperitoneal pentobarbitone sodium followed by exsanguination.
Sequence: to allow satisfactory inter-group comparison.

Organ weights
Brain, heart, kidneys, liver, lungs and spleen were weighed. Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

HISTOPATHOLOGY
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of testes (stained with periodic Acid Schiffs (PAS)) and eyes (Davidson’s fluid).
Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with haematoxylin and eosin.
Tissues examined:
- All specified organs in table 7.5.2/1 were examined in all animals of control and high dose groups.
- Nasal turbinates were examined in all animals of low and mid dose groups.
Other examinations:
None
Statistics:
See "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no treatment-related clinical signs. Signs associated with dosing included red staining of the head and wet fur in some animals from all groups on return to the home cage. These were considered to be due to the method of restraint used.

BODY WEIGHT AND WEIGHT GAIN
A reduction in group mean body weight gain was evident for males exposed to terpineol multiconstituent (0.62, 0.45 and 0.56X control values, respectively) and for females exposed to 2.15 mg/L (0.55X control values), although there was no relationship to dose levels. Body weight gains for females exposed to 0.194 or 0.637 mg/L were similar to control values. Between Days 1 to 4, no body weight gain was observed for males given 0.637 or 2.15 mg/L. However, from Day 4, body weights for the majority of these males increased steadily for the remainder of the study, although at a slightly lower rate than that of Control males.

FOOD CONSUMPTION
A reduction in food consumption was evident for males exposed to terpineol multiconstituent and for females exposed to 0.637 and 2.15 mg/L compared with pretreatment or concurrent control values (as low as 0.79 and 0.87 X control values for males and females respectively).

ORGAN WEIGHTS
- Group mean, body weight adjusted, liver weights were lower than control values for females given 2.15 mg/L (0.89 X control values), achieving statistical significance. A similar effect was not evident for males.
- Remaining intergroup differences seen were small and largely inconsistent between groups and sexes. Therefore, these were considered to be due to individual variation in results and not treatment related.

GROSS PATHOLOGY
- The macroscopic examination performed after 2 weeks of treatment revealed no test substance related lesions.
- The incidence and distribution of all findings were consistent with the common background seen in Sprague-Dawley rats at these laboratories.

HISTOPATHOLOGY
- Treatment related findings: change related to treatment with terpineol multiconstituent was seen in the nasal turbinates with hyperplasia/inflammation in the respiratory epithelium of the nasal turbinates of animals exposed to 2.15 mg/L.
- All other histological changes were considered to be unrelated to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
2.15 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Atmosphere analysis and estimation of achieved dose

 

Table 7.5.2/2: Summary data

 

Group

Aerosol Concentration (mg/L)

Particle size

Target

Achieved

MMAD (µm)

GSD

1

0

-

-

-

2

0.2

0.194

<0.52

-

3

0.6

0.637

1.1

2.05

4

2

2.15

1.4

1.81

MMAD: Mass median aerodynamic diameter; GSD: Geometric standard deviation

 

The achieved levels were 97, 106 and 108% of the target concentrations for Groups 2, 3 and 4 respectively. The mass median aerodynamic diameter (MMAD) for Groups 3 and 4 were within the ideal range (1 to 3 µm) for a repeat dose inhalation study.

The MMAD values showed a general increase with increasing aerosol concentration. The general trend to larger MMAD with increasing aerosol concentration is normal and is attributed to increased particle interaction and subsequent aggregation.

The MMAD value for Group 2 was not calculable as all the measurable test material was captured on the final filter stage. The value presented is based on the cut point of the penultimate stage of the impactor (Stage 8) which has a cut point of 0.52 μm. This value is below the ideal range of 1 to 3 μm. The concern with the respirability of particles with an MMAD below 1 μm is that they may not impact on airway surfaces and be exhaled, thus reducing exposure. In this study, due to the delivered aerosol being a liquid, it is likely that inhaled droplets would impact on airway surfaces and not be exhaled. Therefore, it is considered that the Group 2 animals have also been exposed.

The Group 3 particle size distribution values show a bi-modal distribution across the impactor stages with an average of 34% of the captured droplet having an MMAD below 0.52 μm.

 

Table 7.5.2/3: Body weight - Group mean values (g) - Males

 

Group/Sex

Day

Day

Change

P1

P4

1

4

8

11

15

1-15

Statistics test

Wi

1M

Mean

299

318

342

352

373

385

406

64

SD

10.6

10.5

8.1

8.0

11.9

11.2

13.9

11.2

N

3

3

3

3

3

3

3

3

2M

Mean

304

319

341

350

364

372

381

40

SD

15.3

9.6

14.5

9.6

13.1

10.2

9.9

6.3

N

3

3

3

3

3

3

3

3

% of 1M

62

3M

Mean

302

318

327

327

339

346

357

29

SD

8.6

9.3

4.8

16.5

17.0

21.9

26.3

29.7

N

3

3

3

3

3

3

3

3

% of 1M

45

4M

Mean

296

310

328

327

345

345

363

36

SD

6.7

6.5

11.1

5.6

2.2

5.1

9.9

15.1

N

3

3

3

3

3

3

3

3

% of 1M

56

 

 

Table 7.5.2/4: Body weight - Group mean values (g) – Females

Group

/Sex

Day

Day

Change

P1

P4

1

4

8

11

15

1-15

Statistics test

Wi

1F

Mean

231

230

241

239

246

246

256

15

SD

12.4

14.6

15.4

22.4

23.7

20.3

24.2

8.8

N

3

3

3

3

3

3

3

3

2F

Mean

224

227

235

234

242

241

250

15

SD

8.0

9.9

8.9

4.5

3.2

5.4

4.5

6.1

N

3

3

3

3

3

3

3

3

% of 1F

97

3F

Mean

222

228

229

231

238

239

244

15

SD

4.3

6.3

5.8

6.8

5.4

4.6

2.5

7.3

N

3

3

3

3

3

3

3

3

% of 1F

99

4F

Mean

225

235

241

237

248

243

250

8

SD

6.7

5.9

9.5

8.4

9.8

7.7

8.3

12.6

N

3

3

3

3

3

3

3

3

% of 1F

55

 

Table 7.5.2/5: Histopathology - group distribution of findings

 

Tissue/Organ and Findings

Group/Sex

Number of animals affected

1M

2M

3M

4M

1F

2F

3F

4F

No. of animals

3

3

3

3

3

3

3

3

Larynx

No. examined

3

0

0

3

3

0

0

3

Infiltrate, Inflammatory Cell

Minimal

2

0

0

1

1

0

0

0

 

Total

2

0

0

1

1

0

0

0

Lungs and Bronchi

No. examined

3

0

0

3

3

0

0

3

Alveolar Macrophages, Foamy

Minimal

3

0

0

2

1

0

0

0

Total

3

0

0

2

1

0

0

0

Inflammation, Alveoli

Minimal

3

0

0

2

2

0

0

1

Total

3

0

0

2

2

0

0

1

Metaplasia, Osseous, Alveolar

Minimal

1

0

0

1

0

0

0

0

Total

1

0

0

1

0

0

0

0

Infiltrate, Inflammatory Cell, Perivascular

Minimal

3

0

0

2

3

0

0

1

Total

3

0

0

2

3

0

0

1

Lymph Node, Tracheobronchial

No. examined

3

0

0

3

3

0

0

3

Erythrocytosis/Erythrophagocytosis, Sinuses

Minimal

0

0

0

0

0

0

0

1

Total

0

0

0

0

0

0

0

1

Germinal Center Development Increased

Minimal

1

0

0

0

0

0

0

1

Total

1

0

0

0

0

0

0

1

Nose/Turbinates

No. examined

3

3

3

3

3

3

3

3

Hyperplasia/Inflammation, Respiratory Epithelium

Minimal

0

0

0

2

0

0

0

3

Slight

0

0

0

1

0

0

0

0

Total

0

0

0

3

0

0

0

3

Trachea

No. examined

3

0

0

3

3

0

0

3

Infiltrate, Inflammatory Cell

Minimal

1

0

0

0

0

0

0

0

Total

1

0

0

0

0

0

0

0

Tracheal Bifurcation

No. examined

3

0

0

3

3

0

0

3

Conclusions:
The No Observed Adverse Effect Level (NOAEL) is considered to be 2.15 mg/L. These exposure levels are expected to be suitable for use during a 13-week study in the same species.
Executive summary:

In a 2-week dose range finding inhalation study, terpineol multiconstituent was administered in the form of aerosol to Crl:CD(SD) rats (3 rats/sex/ group) by snout-only inhalation exposure at nominal concentration levels of 0.2, 0.6 and 2 mg/L for 6 hours per day, 5 days per week for two weeks. Control animals received air only. During the study, clinical condition, body weight, food consumption, organ weight, macropathology and histopathology investigations were undertaken.

 

The achieved levels were 97, 106 and 108% (achieved aerosol concentrations of 0.194, 0.637 and 2.15 mg/L) of the target concentrations for Groups 2, 3 and 4, respectively. The mass median aerodynamic diameter (MMAD) for Groups 3 and 4 were within the ideal range (1 to 3 µm) for a repeated dose inhalation study. The MMAD for Group 2 could not be determined because all the test material was captured on the final filter stage. The value presented is based on the cut point of the penultimate stage of the impactor (Stage 8) which has a cut point of 0.52 µm. This value is below the ideal range of 1 to 3 µm. The concern with the ability to respire particles with an MMAD below 1 µm is that they may not impact on airway surfaces and be exhaled, thus reducing exposure. In this study, due to the delivered aerosol being a liquid, it is likely that inhaled droplets would impact on airway surfaces and not be exhaled. Therefore, it is considered that the Group 2 animals have also been exposed.

There were no treatment-related clinical signs. A reduction in group mean body weight gain was evident for all males exposed to terpineol multiconstituent and for females exposed to 2.15 mg/L. A reduction in food consumption was evident for males exposed to terpineol multiconstituent and for females exposed to 0.637 mg/L or 2.15 mg/L compared with pretreatment or concurrent control values. Group mean, body weight adjusted, liver weights were statistically significantly lower than control values for females given 2.15 mg/L. A similar effect was not evident for males. Hyperplasia/inflammation was seen in the respiratory epithelium of the nasal turbinates of animals exposed to 2.15 mg/L. Due to the low severity of these findings, they are considered not to be adverse.

Therefore, the No Observed Adverse Effect Level (NOAEL) is considered to be 2.15 mg/L. These exposure levels are expected to be suitable for use during a 13-week study in the same species.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 230 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Recent GLP study conducted according to OECD Guideline 413 without any deviation (Klimisch score = 1).

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 230 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Recent GLP study conducted according to OECD Guideline 413 without any deviation (Klimisch score = 1).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Daily administration of terpineol multiconstituent by gavage for 5 weeks to males and unmated females was generally well tolerated at dose levels of 60, 250 and 750 mg/kg/day.

The liver was identified as a target organ in this study, to a greater extent in females than males. Minimal centrilobular hepatocyte hypertrophy was seen in the liver of three toxicity phase females dosed with terpineol multiconstituent at 750 mg/kg/day for 5 weeks and accounts for the increases in liver weight at necropsy. Other biochemical findings in this study, such as bile acids and cholesterol levels in females at 750 mg/kg/day may also indicate an alteration of the metabolic function of the liver following administration of terpineol multiconstituent. However, the changes in liver weight and histopathology findings showed complete recovery after 2 weeks.

The relative weight of the kidneys were higher than control in males receiving 750 mg/kg/day. Histopathological changes associated with hyaline droplets were observed in the kidneys of male rats receiving 250 or 750 mg/kg/day but such changes are commonly associated with administration of volatile hydrocarbons and are of no consequence to human risk assessment.

The major effects considered to be related to the systemic exposure to terpineol multiconstituent were related to the testes and epididymides of males receiving 750 mg/kg/day. Testis weight was markedly low in males receiving 750 mg/kg/day and there was also an indication of low epididymal weights at this dose. Reduced numbers or complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) were observed in the epididymides and were still present following the 2week recovery period. Moderate to severe seminiferous tubular atrophy/degeneration was seen in the testes of all animals dosed with terpineol multiconstituent at 750 mg/kg/day, accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation. Similar findings were still evident following the 2week recovery period but at a lower incidence and severity suggesting a degree of recovery.

Therefore, the No Observed Adverse Effect Level (NOAEL) for males and unmated females was 250 mg/kg bw/day when considering oral route.

A repeated dose oral dietary toxicity study was performed with terpineol multiconstituent. The test item was dissolved in corn oil, mixed in Ssniff powder feed at the dose level of 12000 ppm and fed to male Sprague-Dawley rats (10/dose) daily ad libitum for 13 weeks. Rats in the control group were fed basal diet only without any test item admixtures. All rats were observed for clinical signs, mortality, and changes in the body weights and food intake. Sperm evaluations were conducted at termination for all the males from each group. Sperm motility, count and morphology were evaluated for all the groups. All rats were subjected to detailed necropsy at termination and organs were weighed. Histopathological examination of the testes and the epididymides were carried out.

No treatment related mortality or signs of toxicity were noted. The body weights were significantly reduced in rats receiving test item at 12000 ppm. This decrease was associated with a decrease in the food intake throughout the treatment period. The Food consumption was significantly reduced in males receiving test item at 12000 ppm during the treatment period. The calculated mean daily test item consumption was 0 and 622.65 mg/kg bw/day corresponding to 0 and 12000 ppm, respectively.

A slight significant increase in the percentage of abnormal (4.8 %) sperms was noted at 12000 ppm as compared to the control group. However, the change was considered incidental as it was well within the range of normal biological variation noted among male rats [the range of the in-house historical control data for mean percentage of abnormal sperms: 0.1- 7.4%]. The sperm motility remained unaffected by dietary administration of test item. There were no test item-related changes observed in cauda epididymal weight/sperm count and testicular weight/spermatid count.

There were no test item-related changes in the terminal fasting body weights. Increased liver weights (absolute-13 % and relative-20 %) were noted in the treatment group. Increased relative weights (paired and unpaired) of testes and epididymides were observed in the treatment group. There were no test item-related histological changes observed in the testis and the epididymis.

No testicular and epididymal toxicity was evidenced in animals receiving terpineol multiconstituent at 12000 ppm, corresponding to 623 mg/kg bw/day, for 90 days to Sprague-Dawley rats.

By inhalation administration, terpineol multiconstituent was well tolerated at dose levels of 0.202, 0.572 or 2.23 mg/L, for 6 hours a day, 5 days a week, for 13 weeks.

The nasal cavity was identified as a target organ for local effects. Changes related to treatment with terpineol multiconstituent were reported in the nasal turbinates and nasal pharynx in both males and females. In the nasal pharynx, minimal hyperplasia of the mucous cells was also seen in the respiratory epithelium in a small number of animals exposed to 0.572 mg/L or 2.23 mg/L. The changes in the nasal pharynx were also not associated with an inflammatory cell infiltrate or degenerative changes. Examination of recovery phase animals showed no changes in the nasal pharynx respiratory epithelium, suggesting complete recovery after 4 weeks which is therefore not considered adverse.

In the nasal turbinate, mucous cell hyperplasia was present at all exposure levels and did not exhibit a clear dose response in terms of incidence or severity in males, although there were slightly higher incidences in females at 0.572 mg/L or 2.23 mg/L compared with females exposed to 0.202 mg/L. Following a 4 week recovery period, similar incidences of mucous cell hyperplasia in the respiratory epithelium were observed in animals exposed to 2.23 mg/L compared to those at the end of the main study phase, although most were of minimal severity.  Mucous cell hyperplasia is considered to be an adaptive change in the epithelium in response to chronic irritation (Renne et al 2009). This may correlate with clinical signs of salivation and chin rubbing that were observed on consecutive days (from Day 23 onwards) and displayed a dose related response in terms of the number of animals affected. Given that the histopathology changes were of minimal or slight severity and were generally not associated with inflammatory or degenerative changes in the respiratory epithelium, particularly at lower exposure levels, it would be expected that these changes would reverse following a suitable recovery period. Therefore, mucous cell hyperplasia in the nasal turbinates is also not considered adverse.

The only effects considered to be related to the systemic exposure to terpineol multiconstituent were reduced body weight gains for all males exposed to terpineol multiconstituent and females exposed to 0.572 and 2.23 mg/L although clearly related to treatment, this did not exhibit a clear dose response.

The No Observed Adverse Effect Concentration (NOAEC) for this study was considered to be 2.23 mg/L when considering the inhalation route.


Justification for classification or non-classification

The toxic effects reported in the repeated dose toxicity study were seen to occur at 750 mg/kg bw/day in males (liver and testes) and in a lesser extend in females (liver) (i.e. at least at dose level > 250 mg/kg/day).

No adverse effects were reported in the 90-day repeated dose toxicity study by inhalation up to 2.23 mg/L.

Terpineol multiconstituent is therefore not classified according to CLP Regulation (EC) No 1272/2008.