Registration Dossier

Administrative data

Description of key information

Oral (dietary) administration of Patchouli Oil to rats for a period of twenty-eight consecutive days at dose levels of up to 13000 ppm resulted in toxicologically significant effects at 13000 ppm (equivalent to a mean achieved dosage of 977 mg/kg bw/day). The changes seen were liver changes indentified as centrilobular hepatocellular hypertrophy, thyroid changes identified as increased incidence and/or severity of follicular hypertrophy and kidney changes identified as exacerbation of hyaline droplets and an increased incidence of tubular degeneration/regeneration. These changes are considered to be adaptive in nature and not to represent “serious damage” to health. Therefore the “No Observable Adverse Effect Level” (NOAEL) for males and females is considered to be 13000 ppm (977 mg/kg bw/day). No such effects were detected for females at 500 ppm therefore, the “No Observed Effect Level” (NOEL) is considered to be 500 ppm for females only.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
NA
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK.
- Age at study initiation: Seven to eight weeks old
- Weight at study initiation: Males weighed 191 to 221g, the females weighed 163 to 189g
- Housing: In groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): Rat and Mouse RM1 SQC Ground Diet No. 1 ad libitum
- Water (e.g. ad libitum): Mains drinking water ad libitum
- Acclimation period: Six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled
to give twelve hours continuous light and twelve hours darkness
Route of administration:
oral: feed
Details on route of administration:
NA
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The test item was incorporated into the diet at concentrations of 500, 4000 and
13000 ppm as follows:
A known amount of test item was mixed with a small amount of basal laboratory diet for
nineteen minutes at a constant speed, setting 1 in a Hobart QE200 mixer. This pre-mix
was then added to a larger amount of basal laboratory diet and mixed for a further thirty
minutes at a constant speed, setting 1 in a Hobart H800 mixer.
The stability and uniformity of distribution of the test item in the diet were determined. The results showed the
dietary admixtures to be stable for a period of up to six weeks when stored at
approximately -20°C. Dietary admixtures were therefore prepared twice during the
study. The diet was stored in labelled, double plastic bags and placed in a -20°C freezer
when not in use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each dietary admixture and analysed for uniformity of distribution
and concentration.

The concentration of Patchouli Oil in the dietary admixtures was determined by gas
chromatography (GC) using an external standard technique.

Samples: The dietary admixtures were extracted with acetonitrile to give a final, theoretical test
item concentration of approximately 100 ppm.

Standards
Standard solutions of test item were prepared in acetonitrile at a nominal concentration
of 100 ppm. The standard solutions relevant to the dose levels of 200 and 500 ppm
contained the equivalent amount of diet to that of the samples.

The standard and sample solutions were analysed by GC using the following conditions:
GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Column : DB-5 (30 m x 0.25 mm id x 0.25 μm film)
Oven temperature program : initial 100 ºC for 0 mins; rate 10 ºC/min; final 325 ºC for 2 mins
Injection temperature : 250 ºC
Flame ionisation detector temperature : 250 ºC
Injection volume : 1 μl
Retention time : Profile of peaks from ~ 7 to 10 mins

The results indicate that the mean prepared dietary admixture concentrations
were within acceptable limits for the purpose of this study.
Duration of treatment / exposure:
Twenty-eight consecutive days
Frequency of treatment:
Continuously by dietary admixture
Dose / conc.:
500 ppm
Remarks:
Doses / Concentrations: 500 ppm Basis: nominal in diet
Dose / conc.:
4 000 ppm
Remarks:
Doses / Concentrations: 4000 ppm Basis: nominal in diet
Dose / conc.:
13 000 ppm
Remarks:
Doses / Concentrations: 13000 ppm Basis: nominal in diet
No. of animals per sex per dose:
Five/sex/dose
Control animals:
yes, plain diet
Details on study design:
As per guideline
Positive control:
No
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change
daily from the start of treatment. All observations were recorded.

Functional Observations
Prior to the start of treatment and on Days 6, 13, 20 and 24, all animals were observed
for signs of functional/behavioural toxicity. Functional performance tests were also
performed on all animals during Week 4, together with an assessment of sensory
reactivity to different stimuli. Observations were carried out at a similar time on each
occasion, whenever possible.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose
built arena. The following parameters were observed:
Gait and co-ordination
Hyper/Hypothermia
Tremors
Skin colour
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

Functional Performance Tests
Motor Activity.
Twenty purpose built 44 infra-red beam automated activity monitors
were used to assess motor activity. Animals of one sex were tested at each occasion
and were randomly allocated to the activity monitors. The evaluation period was one
hour for each animal. The time in seconds each animal was active and mobile was
recorded for the overall one hour period and also during the final 20% of the period
(considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength.
An automated grip strength meter was used. Each
animal was allowed to grip the proximal metal bar of the meter with its forepaws. The
animal was pulled by the base of the tail until its grip was broken. The animal was drawn
along the trough of the meter by the tail until its hind paws gripped the distal metal bar.
The animal was pulled by the base of the tail until its grip was broken. A record of the
force required to break the grip for each animal was made. Three consecutive trials
were performed for each animal. The assessment was developed from the method
employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and
proprioceptive stimuli. This assessment was developed from the methods employed by
Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response
Touch escape
Vocalisation
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to the start of treatment) and at
weekly intervals thereafter. Body weights were also performed prior to terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the
study.

Water Consumption
Water intake was measured and recorded daily for each cage group.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from
each test and control group at the end of the study (Day 28). Blood samples were
obtained from the lateral tail vein. Where necessary repeat samples were obtained by
cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Haematology
The following parameters were measured on blood collected into tubes containing
potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices:
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated Partial Thromboplastin
Time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate
solution (0.11 mol/l).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes
containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)
Sacrifice and pathology:
Pathology
On completion of the dosing period all animals were killed by intravenous overdose of
sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any
macroscopic abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the
serum from each animal was stored frozen at -20°C. No treatment-related effects on the
pituitary-thyroid axis were identified, therefore these samples were discarded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study,
were dissected free from fat and weighed before fixation:
Adrenals
Liver
Brain
Ovaries
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Pituitary (post fixation)
Thyroid/Parathyroid (post-fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Uterus with Cervix

Histopathology
Samples of the following tissues were removed from all animals and preserved in
buffered 10% formalin except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Salivary glands (submaxillary)
Caecum
Sciatic nerve
Colon
Seminal vesicles (with coagulating glands and fluids)
Duodenum
Epididymides ♦
Skin (hind limb)
Eyes *
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer's patches)
Stomach
Jejunum
Testes ♦
Kidneys
Thymus
Liver
Thyroid/parathyroid
Lungs (with bronchi)#
Trachea
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus & Cervix
Muscle (skeletal)
Vagina
Oesophagus

♦ = preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately
48 hours later
* = eyes fixed in Davidson’s fluid
# = Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

Any macroscopically
observed lesions were also processed together with the liver and spleen from all 500 and
4000 ppm dose group animals. In addition, sections of testes and epididymides from all
Control and 13000 ppm males were stained with Periodic Acid-Schiff (PAS) stain and
examined.
Since there were indications of treatment related liver, kidney and thyroid changes
examination was subsequently extended to include similarly prepared sections of kidney
and thyroid from animals from the low and intermediate groups.
Other examinations:
None
Statistics:
Data were processed to give group mean values and standard deviations where
appropriate.
Where appropriate, quantitative data were
analysed by the Provantis™ Tables and Statistics Module. For each variable, the most
suitable transformation of the data was found, the use of possible covariates checked
and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test.
The transformed data were analysed to find the lowest treatment level that showed a
significant effect, using the Williams Test for parametric data or the Shirley Test for nonparametric
data. If no dose response was found, but the data showed non-homogeneity
of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric)
test to determine significant differences from the control group. Finally, if
required, pair-wise tests were performed using the Student t-test (parametric) or the
Mann-Whitney U test (non-parametric).
Probability values (P) are presented as follows:
P<0.01 **
P<0.05 *
p≥0.05 (not significant)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 13000 ppm showed an overall reduction in dietary intake during the treatment period. Food efficiency was reduced in the high dose males during Weeks 2 to 4 and high dose females during Weeks 1 to 3, in comparison to controls.
No such effect was detected in males or females treated with 500 and 4000 ppm.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see Food consumption above
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increase in liver weights was evident in animals of either sex treated with 4000 and 13000 ppm.
An increase in kidney weight was seen in male rats at 4000 and 13000 ppm in comparison to controls.
No such effect was detected in males treated with 500 ppm.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: Hepatic centrilobular-diffuse hepatocellular hypertrophy of minimal to moderate severity degree was evident in the liver of treated animals of either sex treated with 4000 and 13000 ppm. Vacuolation (fatty change) of the liver was observed for animals of either sex treated with 500 and 4000 ppm and males treated with 13000 ppm.

Thyroid: Increased incidence and/or severity of follicular hypertrophy in the thyroid were detected in animals of either sex treated with 4000 and 13000 ppm and males treated with 500 ppm.

Kidney: Hyaline droplets nephropathy in the kidneys was recorded throughout the male treatment groups. In these animals tubular degeneration/regeneration along with increased mean severity of hyaline droplets was recorded in a dose dependent manner.
This finding is consistent with the presence of hydrocarbon nephropathy which results from the excessive accumulation of alpha 2-microglobulin in renal proximal tubular epithelial cells. This is found only in the proximal tubular epithelium of adult male rats and is considered to be of no toxicological significance.

All other findings are those which are commonly found in the untreated laboratory rat of this strain and age and were considered to be of no toxicological significance
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
Mortality
There were no deaths during the study.

Clinical Observations
No treatment related findings were observed throughout the study.
Generalised fur loss was seen in two control males. This finding is considered incidental
and not related to treatment.

Behavioural Assessments
There were no treatment related changes in the behavioral assessment measurements.

Functional Performance Tests
There were no adverse changes in the functional performance test measurements for
treated animals in comparison to controls.
A statistically significant increase in overall activity (P<0.05) was evident in males treated
with 13000 ppm. In the absence of any supporting clinical observations this finding is
considered to be of no toxicological significance. Females from this treatment group
showed a statistically significant increase in hindlimb grip strength (P<0.01). A slight but
statistically significant reduction in forelimb grip strength (P<0.05) was seen throughout
the male treatment groups in comparison to controls. These findings were only observed
in one out of the three tests performed on each parameter and as such these are
considered to be of no toxicological importance.

Sensory Reactivity Assessments
There were no treatment related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a
result of normal variation for rats of the strain and ages used, and were of no
toxicological importance.

Functional Observations
There were no adverse changes in the functional
performance test measurements for treated animals in comparison to controls.

Body Weight
Animals of either sex treated with 13000 ppm showed statistically significant reductions
in body weight gain during the treatment period. The males showed reductions during
Week 2 and 4 of treatment (P<0.05 and P<0.01 respectively) whilst the females showed
a reduction during Week 1 (P<0.05), in comparison to controls. The overall absolute and
percentage gain was also markedly lower than the controls.
No such effect was detected in females treated with 500 or 4000 ppm.
During Weeks 1 and 3 of treatment a statistically significant increase (P<0.05) in body
weight gain was noted in the 500 ppm males. The 4000 ppm males showed a statistically
significant reduction in body weight gain (P<0.01) during Week 4 of treatment in
comparison to controls. In the absence of an overall effect on body weight gain noted in
these treatment groups, such findings are considered to be incidental and of no
toxicological significance.

Food Consumption
Animals of either sex treated with 13000 ppm showed reductions in dietary intake and
food efficiency during the treatment period, in comparison to controls. The males showed
reductions in dietary intake throughout the treatment period whilst reductions in food
efficiency were seen during Weeks 2 to 4 of treatment. The females showed reductions
in dietary intake throughout the treatment period whilst reductions in food efficiency were
seen during Weeks 1 to 3 of treatment.
No such effect was detected in males or females treated with 4000 or 500 ppm.

Water Consumption
No treatment related effect on water intake was detected for treated animals in
comparison to controls.

Haematology
There was no adverse effect on the haematological parameters measured for treated
animals in comparison to controls.
A statistically significant reduction in total leucocyte counts (P<0.05), activated partial
thromboplastin time (P<0.05) and reticulocyte counts (P<0.01) was evident in the
13000 ppm males in comparison to controls. A statistically significant reduction in
lymphocyte counts was seen throughout the male treatment groups and females treated
with 500 ppm in comparison to controls. The majority of the individual values were within
normal ranges for the strain and aged of the rat used and in the absence of any
histopathological correlated these findings are considered to be of no toxicological
significance.

Blood Chemistry
There was no adverse effect on the blood chemical parameters measured for treated
animals in comparison to controls.
A statistically significant reduction in bilirubin levels was seen throughout the male
treatment groups (P<0.01), with a statistically significant reduction in alkaline
phosphatase (P<0.05) evident in the 13000 ppm males. All the individual values were within normal ranges for rats of the strain used therefore these findings are considered to
be of no toxicological significance.
A statistically significant increase in creatinine levels (P<0.01) was seen in the 4000 and
13000 ppm males. All the individual values were within normal ranges for rats of the
strain and age used and this affect is attributed to lower than expected control values. As
such this is not considered to be of any toxicological importance.
A slight but statistically significant increase in glucose levels (P<0.05) was noted in the
4000 ppm males. The majority of the individual values were within normal ranges for rats
of the strain and age used and in the absence of a dose-related response this is
considered to be of no toxicological importance.
A statistically significant reduction in urea levels (P<0.05) was evident throughout the
female treatment groups. This effect can be attributed to higher than expected control
values and as such is considered to be of no toxicological significance.
Females treated with 4000 and 13000 ppm showed statistically significant reductions in
aspartate and alanine aminotransferase levels (P<0.05). In the absence of a dose related
response and as these findings are in contradiction to the histopathological
affects seen in the liver, these are considered to be of no toxicological importance.

Organ Weights
A statistically significant increase in liver weights both absolute and relative to terminal
body weight was evident in animals of either sex treated with 4000 and 13000 ppm
(P<0.01 in intermediate males and high dose animals of either sex; P<0.05 in
intermediate females) in comparison to controls.
A statistically significant increase in kidneys weights both absolute and relative to
terminal body weight was seen in the 4000 and 13000 ppm males (P<0.05 and P<0.01
respectively).
No such effect was detected in males treated with 500 ppm.
A statistically significant reduction in brain and spleen weights (P<0.05), both absolute
and relative to terminal body weight was evident in males treated with 13000 ppm.
Females from this dose group showed a statistically significant reduction in heart weight
(P<0.05), both absolute and relative to terminal body weight. In the absence of any
histopathological correlates this finding is considered to be of no toxicological
significance.
A statistically significant reduction in thyroid weights (P<0.05), both absolute and relative
to terminal body weight was seen throughout the female treatment groups, in comparison
to controls. The reduction did not show a dose-related response and is in contradiction to
the histopathological findings. As such this finding is considered to be of no toxicological
importance.

Necropsy
No macroscopic abnormalities were detected in treatment groups at terminal kill.

Histopathology
The following treatment related changes were observed:
Liver: Hepatic centrilobular-diffuse hepatocellular hypertrophy of minimal to moderate
severity degree was evident in the liver of treated animals of either sex treated with 4000
and 13000 ppm. Vacuolation (fatty change) of the liver was observed for animals of
either sex treated with 500 and 4000 ppm and males treated with 13000ppm.
Hepatocyte hypertrophy is commonly observed in the rodent liver following the
administration of xenobiotics and in the absence of associated inflammatory or
degenerative changes is considered to be adaptive in nature.
Thyroid: Increased incidence and/or severity of follicular hypertrophy in the thyroid were
detected in animals of either sex treated with 4000 and 13000 ppm and males treated
with 500 ppm.
Follicular hypertrophy of the thyroids is deemed to be associated with the increased
hepatic metabolization of thyroid hormones (T3/T4) due to hepatocellular hypertrophy in
the 4000 and 13000 ppm dose groups. Therefore, these findings are deemed to
represent a secondary effect of treatment.
Follicular hypertrophy was present in males treated with 500 ppm, but no hepatocellular
hypertrophy was identified at this treatment level. However, it is deemed that the altered
hepatic enzymatic pattern, which is eventually reflected by hypertrophy seen in the 4000
and 13000 ppm animals is equally present in the 500 ppm males and affects the
thyroideal morphology in these animals.
Kidney: Hyaline droplets nephropathy in the kidneys was recorded throughout the male
treatment groups. In these animals tubular degeneration/regeneration along with
increased mean severity of hyaline droplets was recorded in a dose dependent manner.
This finding is consistent with the presence of hydrocarbon nephropathy which results
from the excessive accumulation of alpha 2-Microglobulin in renal proximal tubular
epithelial cells. This is found only in the proximal tubular epithelium of adult male rats and
is considered to be of no toxicological significance.
All other findings are those which are commonly found in the untreated laboratory rat of
this strain and age and were considered to be of no toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
13 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no effects observed in females at this dose
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
13 000 ppm
System:
other: whole system
Organ:
other:
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
4 000 ppm
System:
other: whole system
Organ:
other:
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
500 ppm
System:
other: whole system
Organ:
other:
Conclusions:
Oral (dietary) administration of Patchouli Oil to rats for a period of twenty-eight consecutive days at dose levels of up to 13000 ppm resulted in toxicologically significant effects at 13000 ppm (equivalent to a mean achieved dosage of 977 mg/kg bw/day). The
changes seen were liver changes indentified as centrilobular hepatocellular hypertrophy, thyroid changes identified as increased incidence and/or severity of follicular hypertrophy and kidney changes identified as exacerbation of hyaline droplets and an increased incidence of tubular degeneration/regeneration. These changes are considered to be adaptive in nature and not to represent “serious damage” to health. Therefore the “No Observable Adverse Effect Level” (NOAEL) is considered to be 13000 ppm. No such effects were detected for females at 500 ppm therefore, the “No Observed Effect Level” (NOEL) is considered to be 500 ppm for females only.
Executive summary:

Introduction. The study was designed to investigate the systemic toxicity of the test item. It is compatible with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Methods. The test item was administered by continuous dietary admixture to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dietary concentrations of 500, 4000 and 13000 ppm (equivalent to a mean achieved dosage of 41, 323 and 977 mg/kg bw/day respectively). A control group of five males and five females were treated with basal laboratory diet. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed. Results. Mortality. There were no deaths during the study. Clinical Observations. No treatment related findings were observed throughout the study. Behavioural Assessment. There were no treatment related changes in the behavioral assessment measurements for treated animals in comparison to controls.

Functional Performance Tests. There were no adverse changes in the functional performance test measurements for treated animals in comparison to controls. Sensory Reactivity Assessments. There were no treatment related changes in sensory reactivity. Body Weight. Animals of either sex treated with 13000 ppm showed a reduction in overall body weight gain during the treatment period. The males showed reductions during Week 2 to 4 of treatment whilst the females showed reductions during Weeks 1 to 3, in comparison to controls. No such effect was detected in animals of either sex treated with 500 or 4000 ppm. Food Consumption. Animals of either sex treated with 13000 ppm showed an overall reduction in dietary intake during the treatment period. Food efficiency was reduced in the high dose males during Week 2 to 4 and high dose females during Weeks 1 to 3, in comparison to controls. No such effect was detected in males or females treated with 500 and 4000 ppm. Water Consumption. No treatment related effect on water intake was detected for treated animals in comparison to controls. Haematology. There was no adverse effect on the haematological parameters measured for treated animals in comparison to controls. Blood Chemistry. There was no adverse effect on the blood chemical parameters measured for treated animals in comparison to controls. Organ Weights. An increase in liver weights was evident in animals of either sex treated with 4000 and 13000 ppm. An increase in kidney weight was seen in male rats at 4000 and 13000 ppm in comparison to controls. No such effect was detected in males treated with 500 ppm. Necropsy. No macroscopic abnormalities were detected at terminal kill. Histopathology. The following treatment related changes were observed: Liver: Hepatic centrilobular-diffuse hepatocellular hypertrophy of minimal to moderate severity degree was evident in the liver of treated animals of either sex treated with 4000 and 13000 ppm. Vacuolation (fatty change) of the liver was observed for animals of either sex treated with 500 and 4000 ppm and males treated with 13000 ppm. Thyroid: Increased incidence and/or severity of follicular hypertrophy in the thyroid were detected in animals of either sex treated with 4000 and 13000 ppm and males treated with 500 ppm. Kidney: Hyaline droplets nephropathy in the kidneys was recorded throughout the male treatment groups. In these animals tubular degeneration/regeneration along with increased mean severity of hyaline droplets was recorded in a dose dependent manner. This finding is consistent with the presence of hydrocarbon nephropathy which results from the excessive accumulation of alpha 2-microglobulin in renal proximal tubular epithelial cells. This is found only in the proximal tubular epithelium of adult male rats and is considered to be of no toxicological significance. All other findings are those which are commonly found in the untreated laboratory rat of this strain and age and were considered to be of no toxicological significance. Conclusion. Oral (dietary) administration of Patchouli Oil to rats for a period of twentyeight consecutive days at dose levels of up to 13000 ppm resulted in toxicologically significant effects at 13000 ppm (equivalent to a mean achieved dosage of 977 mg/kg bw/day). The changes seen were liver changes indentified as centrilobular hepatocellular hypertrophy, thyroid changes identified as increased incidence and/or severity of follicular hypertrophy and kidney changes identified as exacerbation of hyaline droplets and an increased incidence of tubular degeneration/regeneration. These changes are considered to be adaptive in nature and not to represent “serious damage” to health. Therefore the “No Observable Adverse Effect Level” (NOAEL) is considered to be 13000 ppm (977 mg/kg/day) for male and female animals. No such effects were detected for females at 500 ppm therefore, the “No Observed Effect Level” (NOEL) is considered to be 500 ppm for females only.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
977 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1
System:
other: Whole system exposure
Organ:
other: Based on clinical signs.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Oral (dietary) administration of Patchouli Oil to rats for a period of twenty-eight consecutive days at dose levels of up to 13000 ppm resulted in toxicologically significant effects at 13000 ppm (equivalent to a mean achieved dosage of 977 mg/kg bw/day). The changes seen were liver changes indentified as centrilobular hepatocellular hypertrophy, thyroid changes identified as increased incidence and/or severity of follicular hypertrophy and kidney changes identified as exacerbation of hyaline droplets and an increased incidence of tubular degeneration/regeneration. These changes are considered to be adaptive in nature and not to represent “serious damage” to health. Therefore the “No Observable Adverse Effect Level” (NOAEL) for males and females is considered to be 13000 ppm (977 mg/kg bw/day). No such effects were detected for females at 500 ppm therefore, the “No Observed Effect Level” (NOEL) is considered to be 500 ppm for females only.

Justification for classification or non-classification

Treatment related effects observed at the highest dose of (equivalent to) 977 mg/kg/bw were considered to be adaptive and not relevant to humans. In the absence of significant treatment related adverse effects at the highest dose the substance is not considered to be classified.