Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 28 November 2011 and 16 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study carried out according to internationally recognised guidelines and performed according to GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 28 November 2011 and 16 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: Approximately twelve weeks old
- Weight at study initiation: Males weighed 304 to 400g, the females weighed 193 to 222g
- Fasting period before study: No data
- Housing: All animals were housed in groups of five in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
During the mating phase, the animals were transferred to polypropylene grid floor cages
suspended over trays lined with absorbent paper on a one male: one female basis.
Following evidence of successful mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation, in solid floor
polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Rodent PMI 5002 (Certified) Ground Diet ad libitum
- Water (e.g. ad libitum): Mains drinking water, ad libitum
- Acclimation period: For five days during which time their health status was assessed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were therefore prepared three times during the study, as the results of stability and homogeneity tests showed the dietary admixtures to be stable for a period of at least eight days at room temperature and up to six weeks when stored at approximately -20°C.

- Mixing appropriate amounts with (Type of food):
The test item was incorporated into the diet at concentrations of 13000, 4000 and 1300 ppm as follows:
A known amount of test item was mixed with a small amount of basal laboratory diet using Robot Coupe Blixer 4 until homogenous. 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 QE200 mixer.

- Storage temperature of food:The diet was stored in labelled, double plastic bags and placed in a -20°C freezer when not in use.

Details on mating procedure:

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 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 ~ 8 to 12 mins

Duration of treatment / exposure:

Up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Daily

Details on study schedule:

Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
The male dose groups were killed and examined macroscopically on Day 43.
At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Dose / conc.:

1 300 ppm (nominal)

Remarks:

equivalent to a mean achieved dosage of 91.4mg/kg bw/d actually ingested

Dose / conc.:

4 000 ppm (nominal)

Remarks:

equivalent to a mean achieved dosage of 277mg/kg bw/d actually ingested

Dose / conc.:

13 000 ppm (nominal)

Remarks:

equivalent to a mean achieved dosage of 810mg/kg bw/d actually ingested

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, plain diet

Positive control:

None reported

Parental animals: Observations and examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change
daily. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for
males until termination and weekly for females until mating was evident. Body weights
were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and
4 post partum and at terminal kill.

Food Consumption
During the maturation period, weekly food consumption was recorded for each cage of
adults until pairing. This was continued for males after the mating phase. For females
showing evidence of mating, food consumption was recorded for the periods covering
post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption
was recorded during the lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for
males and for females during the pre-mating phase. Due to offspring growth and milk
production for lactation, food efficiency for females could not be accurately calculated
during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt
changes.

Reproduction Performance:
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of
up to fourteen days. Cage tray-liners were checked each morning for the presence of
ejected copulation plugs and each female was examined for the presence of a copulation
plug in the vagina. A vaginal smear was prepared for each female and the stage of
oestrus or the presence of sperm was recorded. The presence of sperm within the
vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day
0 of gestation) and the males were subsequently returned to their original holding cages
(unless required for additional pairing). Mated females were housed individually during
the period of gestation and lactation. Females failing to mate were also housed
individually after the mating phase.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and
around the period of expected parturition. Observations were carried out at
approximately 0830 and 1230 hours at weekends and public holidays. The following was
recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Litter observations:

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring
was recorded. Offspring were individually identified within each litter by tattoo on Day 1
post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post
partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were
calculated retrospectively from this data)
Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

Pathology
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by
exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium
pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring
were terminated via intracardiac overdose of sodium pentobarbitone. Any females which
failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.
For all females, the uterus was examined for signs of implantation and the number of
uterine implantations in each horn was recorded. This procedure was enhanced; as
necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski
1964).
All adult animals and offspring, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free
from fat and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group,
in buffered 10% formalin, except where stated:
Coagulating gland
Prostate
Epididymides♦
Seminal vesicles
Ovaries
Testes♦
Mammary gland (females only)
Uterus/Cervix
Pituitary
Vagina

♦ = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately
forty-eight hours later

The tissues from control and 13000 ppm dose group animals, any animals dying during
the study, and any animals which failed to mate or did not achieve a pregnancy were
prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with
Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections
of testes and epididymides from all control and 13000 ppm males were also stained with
Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment related mammary gland, prostate, seminal
vesicles and coagulating gland changes, examination was subsequently extended to
include similarly prepared sections of these organs from animals from the low and
intermediate groups.

Postmortem examinations (offspring):

Offspring, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight-relative organ weights (Males)
The following statistical procedures were used:
Data for males and females prior to pairing, 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 and ANCOVA and Bartletts’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
non-parametric data. If no dose response was found, but the data showed nonhomogeneity
of means, the data were analysed by a stepwise Dunnett (parametric) or
Steel (non-parametric) 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).
Data for females during gestation and lactation, and offspring data were assessed for
dose response relationships by linear regression analysis, followed by one way analysis
of variance (ANOVA) incorporating Levene’s ttest for homogeneity of variance. Where
variances were shown to be homogenous, pairwise comparisons were conducted using
Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed
using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.
Non-parametric methods were also used to analyse implantation loss, offspring sex ratio
and landmark developmental markers.

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating
period of the parental generation:
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and
parturition period of the parental generation:
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating
and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100

Offspring viability indices:

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were
first calculated for each litter and the group mean was calculated using their individual
litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:
% pre–implantation loss = ((Number of corpora lutea - Number of implantation sites)/Number of corpora lutea) x100

% post–implantation loss = ((Number of implantation sites - Total number of offspring born)/Number of implantation sites) x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100

Viability Index (%) = (Number of offspring alive on Day 4/ Number of offspring alive on Day 1)x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the
following formula: (Number of male offspring/Total number of offspring) x 100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 13000 ppm males showed statistically significant (p<0.05 and p<0.01) reductions in mean body weight gains during Weeks 2, 3 and 6 of treatment when compared with control values.
Males treated with 1300 or 4000 ppm also showed statistically significant (p<0.05) reductions in mean body weight gains during Week 3 of treatment.
Females treated with 13000 ppm also showed statistically significant (p<0.05 to p<0.01) reductions in mean body weight gains during Week 2 of treatment and during the gestation and lactation phases.
Females treated with 4000 ppm also showed a statistically significant (p<0.05) reduction in mean body weight gain during Week 2 of treatment.
Females treated with 1300 ppm showed a slight reduction in mean body weight gain during Week 2 of treatment, however statistical significance has not been reached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For animals treated with 13000 ppm treatment related reductions in food consumption and food efficiency was evident throughout the treatment period.
Whilst a relationship to treatment at 1300 and 4000 ppm cannot be excluded, reduction in food consumption and food efficiency was considered minimal and is therefore considered to be of no toxicological importance.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

For animals treated with 13000 ppm treatment related reductions in food consumption and food efficiency was evident throughout the treatment period.
Whilst a relationship to treatment at 1300 and 4000 ppm cannot be excluded, reduction in food consumption and food efficiency was considered minimal and is therefore considered to be of no toxicological importance.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual inspections of the water intake did not reveal any overt changes during the treatment period.

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

MATING
There were no treatment related effects on mating performance at any of the dose levels investigated. The majority of animals mated within the first five days of pairing (i.e. at the first oestrus opportunity) and the mating index of treated animals was comparable to controls.
One control female (No.13) and one female treated with 13000 ppm (No.74) did not mate following fourteen days of cohousing with their male partners.
The male partners for both of these non-mated pairings showed histopathological evidence of either diffuse tubular atrophy in the testes and increased cellular debris in the epididymides (control male No.3) or reduced secretory activity in the prostate (13000 ppm male No.64).
These findings may therefore be a contributory factor in the lack of reproductive performance.
The distribution of these findings shows no treatment related trend.

FERTILITY
There were no treatment related effects detected on fertility indices and pregnancy rate when compared with controls.
One female treated with 13000 ppm (No.79) showed evidence of pregnancy but no litter was observed.
Histopathological findings showed decidual contents in uterus, cervix and vagina indicative of pregnancy with possible prenatal and perinatal fetal deaths/losses.
The male partner (No.69) for this female showed a slight hypospermatogenesis in testes and slight oligospermia in epididymides during microscopic examinations.
There is no conclusive evidence to confirm the significance of male findings upon the observed total post-implantation litter loss.
One control female (No.19) did not achieve pregnancy following evidence of mating.
No histopathological correlates were evident in the female reproductive organs, however, the male partner (No.9) for this female showed minimal or slight reduction in secretory activity in prostate and seminal vesicles.
The significance of these findings in the infertility of this male/female pairing could not be established.

Mortality
There were no occurrences of mortality evident during the study period.

Clinical Observations
There were no clinically observable signs of toxicity or signs related to general
appearance throughout the study period for either male or female rats.

Body Weight
Lower mean body weights were evident in all treated groups at interim weighing points
and at termination.
At 13000 ppm males showed statistically significant (p<0.05 and p<0.01) reductions in
mean body weight gains during Weeks 2, 3 and 6 of treatment when compared with
control values. Males treated with 1300 or 4000 ppm also showed statistically significant
(p<0.05) reductions in mean body weight gains during Week 3 of treatment.
Females treated with 13000 ppm also showed statistically significant (p<0.05 to p<0.01)
reductions in mean body weight gains during Week 2 of treatment and during the
gestation and lactation phases. Females treated with 4000 ppm also showed a
statistically significant (p<0.05) reduction in mean body weight gain during Week 2 of
treatment. Females treated with 1300 ppm showed a slight reduction in mean body
weight gain during Week 2 of treatment, however statistical significance has not been
reached.

Food Consumption and Food Efficiency
For animals treated with 13000 ppm treatment related reductions in food consumption
and food efficiency was evident throughout the treatment period.
Whilst a relationship to treatment at 1300 and 4000 ppm cannot be excluded, reduction
in food consumption and food efficiency was considered minimal and is therefore
considered to be of no toxicological importance.

Water Consumption
Daily visual inspections of the water intake did not reveal any overt changes during the
treatment period.

Reproductive Performance:
Mating
There were no treatment related effects on mating performance at any of the dose levels
investigated. The majority of animals mated within the first five days of pairing (i.e. at the
first oestrus opportunity) and the mating index of treated animals was comparable to
controls.
One control female (No.13) and one female treated with 13000 ppm (No.74) did not mate
following fourteen days of cohousing with their male partners. The male partners for
both of these non-mated pairings showed histopathological evidence of either diffuse
tubular atrophy in the testes and increased cellular debris in the epididymides (control
male No.3) or reduced secretory activity in the prostate (13000 ppm male No.64). These
findings may therefore be a contributory factor in the lack of reproductive performance.
The distribution of these findings shows no treatment related trend.

Fertility
There were no treatment related effects detected on fertility indices and pregnancy rate
when compared with controls.
One female treated with 13000 ppm (No.79) showed evidence of pregnancy but no litter
was observed. Histopathological findings showed decidual contents in uterus, cervix and
vagina indicative of pregnancy with possible prenatal and perinatal fetal deaths/losses.
The male partner (No.69) for this female showed a slight hypospermatogenesis in testes
and slight oligospermia in epididymides during microscopic examinations. There is no
conclusive evidence to confirm the significance of male findings upon the observed total
post-implantation litter loss.
One control female (No.19) did not achieve pregnancy following evidence of mating. No
histopathological correlates were evident in the female reproductive organs, however,
the male partner (No.9) for this female showed minimal or slight reduction in secretory
activity in prostate and seminal vesicles. The significance of these findings in the
infertility of this male/female pairing could not be established.

Gestation Length
There were no treatment related effects detected in the length of gestation between
control and treated groups. All females showed a gestation length between 22 and 23½
days.

Pathology:
Organ weights
There were no treatment related effects in organ weights detected in all males when
compared to controls.

Necropsy
There were no toxicologically significant macroscopic abnormalities detected during the
post mortem procedure which were considered to be attributable to treatment with the
test item.
Four males treated with 13000 ppm had small seminal vesicles at necropsy, one of these
males also had pale kidneys with increased pelvic space of the right kidney and small
prostate whilst a further male had small epididymides, small prostate and also small and
flaccid testes. In the absence of any other supportive data on adverse effects correlated
to these findings, it was considered of no toxicological importance.
One control male had small testes whilst other control males had small seminal vesicles
and prostate at necropsy.
One female treated with 13000 ppm had brown mass in right horn of the uterus and
cervix at necropsy which corresponded to decidual contents and dilation of the right horn
recorded at histopathology examination. This finding was considered to represent normal background lesion which can be recorded in females of this strain and age. As
such, this finding was considered unrelated to treatment.
In the absence of treatment all findings detected in control animals are considered to be
fortuitous.

Histopathology
Histopathological examination of reproductive tissues from animals treated with
13000 ppm showed possible treatment related changes detected in prostate, seminal
vesicles, coagulating glands and mammary glands. Subsequent examinations of these
tissues from animals treated with 1300 or 4000 ppm were performed and no treatment
related abnormalities were detected.
The remaining microscopic findings were those commonly observed in laboratory
maintained rats of the age and strain employed and there were no differences in
incidence or severity between control and treatment groups that were considered to be of
toxicological significance

Key result

Dose descriptor:

NOAEL

Effect level:

>= 810 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

no effects observed

Litter Response
Eight females from both control and 13000 ppm dose groups and ten females from 1300
and 4000 ppm dose groups gave birth to a live litter and successfully reared young to
Day 5 of age. The following assessment of litter response is based on all litters reared to
termination on Day 5 of lactation/age.

Offspring Litter Size and Viability
At 13000 ppm a statistically significant reduction was detected in mean corpora lutea (p<0.01)
and a consequential implantation site (p<0.01) number. The mean corpora lutea value was slightly
below historical control numbers. Litter size at birth was also lower than controls showing statistically
significant reduction in total number of offspring born (p<0.05).
No such effects were detected at 1300 and 4000 ppm in mean numbers of corpora lutea,
implantation sites, litter size and implantation losses when compared to control numbers.
There were no treatment related effects detected in survival indices and sex ratio in all
treated litters when compared to controls.

Offspring Growth and Development
There were no clinically observable signs of toxicity detected for offspring from all treated
litters when compared to controls.
Mean values for total litter weight, offspring body weight, body weight changes and
surface righting reflex showed treatment related effects in litters treated with 13000 ppm.
Litter weight values for litters treated with 13000 ppm showed statistically significant
reduction (p<0.05 to p<0.01) during Day 1 and Day 4 evaluations. At this dose level
individual offspring weights were significantly lower than controls during Day 1 and Day 4
assessments whilst male offspring weights during Day 4 showed a statistically significant
effect (p<0.01). Offspring body weight change between Days 1 to 4 was subsequently
affected and a statistically significant reduction (p<0.01 and p<0.05) was evident. Mean
surface righting reflex values showed statistically significant reduction in 13000 ppm
offspring when compared to control values.
No clinically observable signs of toxicity were detected. Instances of swollen limb and
offspring found dead or missing from either control litters or litters from females treated
with 4000 ppm are commonly observed findings in studies of this type and considered to
be unrelated to test item toxicity.

Necropsy:
No treatment related macroscopic abnormalities were detected for interim death or
terminal kill offspring. The incidental findings observed were those occasionally
observed in reproductive studies of this type and were considered to be unrelated to
toxicity of the test item.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 810 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Reproductive effects observed:

no

DISCUSSION:

The oral administration of Patchouli Oil to rats for a period of up to eight weeks (including two weeks pre-mating, gestation and early lactation period for females) at dose levels of 1300, 4000 and 13000 ppm (equivalent to a mean achieved dosage of 91.4, 277 and 810 mg/kg bw/day respectively) resulted in treatment related effects detected only at 13000 ppm.

Adverse effects on body weight and food consumption values in adult animals were evident throughout the treatment period. The lower food consumption and food efficiency at 13000 ppm correlated with body weight gain loss in males and females.

At 13000 ppm there were differences in group mean corpora lutea counts compared with concurrent and historical control values. The result was considered equivocal because of the fact that there was no obvious effect upon reproductive performance of these females with no obvious impairment of oestrous cyclicity in the majority of females mating at the first opportunity. In addition it is of note that although mean values for corpora lutea are lower than historical controls the difference between the mean value recorded for this treatment group and the lower limit for control animals was very small and the variance within the treated group individual values was notable. The mean corpora lutea, implantation values and pre and post implantation loss values were skewed from the norm by one litter (No.79) whose values were different from others in the group. This resulted in a lower mean implantation number and live litter size at birth.

The differences in pre and post implantation losses were minimal and within background control values and therefore considered not to be a consequence of treatment. For subsequent live births there was no indication of increased neonatal mortality but there was an indication of reduced individual offspring body weight gain from Day 1 to Day 4 post partum. Offspring body weights were slightly lower than controls at Day 1 post partum which may suggest toxicity to the foetus/neonatal animal. The reduced litter size and weight therefore resulted in lower litter weights compared to control values.

Development and reflex markers assessed by percentage of offspring that passed surface righting reflex evaluation showed a reduction when compared to control values, however, the majority of individual values were within the normal range data for rats of the strain and age used.

Histopathological examinations of reproduction and accessory reproductive organs, particularly for males showed evidence of effects such as an increased incidence and severity of reductions of secretory activity. The conclusion was that these effects were directly correlated with inhibited body weight development amongst the animals at the highest dose level. Therefore these effects can be concluded as a general toxicity to the adult animals rather than a specific treatment effect upon organs of the reproduction system.

Conclusions:

The oral administration of the test substance to rats by dietary admixture, at dose levels of 1300, 4000 and 13000 ppm (equivalent to a mean achieved dosage of 91.4, 277 and 810 mg/kg bw/day respectively) resulted in treatment related systemic toxicity in adults treated with 13000 ppm only.
This dose level also resulted in a small number of treatment related effects on offspring pre and post partum with an equivocal effect on the nature of some in utero parameters. Those effects are attributed to the excess impact of the dietary dosing on normal nutrition found in the parents.
The following NOAELs can be set: NOAELparental toxicity ≥ 810 mg/kg bw/d and NOAELreproductive and developmental toxicity ≥ 810 mg/kg bw/d.

Executive summary:

Introduction. The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard

assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421

“Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). 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 dietary admixture to three groups, each of

ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for

females), at dose levels of 1300, 4000 and 13000 ppm (equivalent to a mean achieved

dosage of 91.4, 277 and 810 mg/kg bw/day respectively). A control group of ten males and ten females was dosed with basal laboratory diet.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently

being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving

offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy

examination and histopathological evaluation of reproductive tissues was performed.

Results.

Adult Responses:

Mortality. There were no unscheduled deaths during the study.

Clinical Observations. There were no clinically observable signs of toxicity detected.

Body Weight. Reductions in overall mean body weights were evident in all treated animals of either sex when compared to controls throughout the treatment period.

Food Consumption and Food Efficiency. Reductions in overall food consumption and food efficiency values were recorded in animals of either sex treated with 13000 ppm.

Water Consumption. No intergroup differences were detected.

Reproductive Screening:

Mating. There were no treatment related effects detected on mating performance in treated animals when compared to controls.

Fertility. There were no treatment related effects detected on fertility in treated animals when compared to controls.

Gestation Lengths. There were no treatment related effects detected on gestation length.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. No treatment related effects were evident in sex ratio or viability assessments. Litter size was reduced at 13000 ppm.

Offspring Growth and Development. Litter weight, offspring weights and surface righting reflex mean values were lower at 13000 ppm when compared to controls.

Offspring Observations. There were no clinically observable signs of toxicity detected for offspring from all treated litters when compared to controls.

Pathology:

Necropsy. There were no toxicologically significant macroscopic abnormalities detected.

Organ Weights. There were no treatment related effects detected in organ weights.

Histopathology. There were no treatment related abnormalities recorded in reproductive organs during microscopic examinations.

Conclusion. The oral administration of Patchouli Oil to rats by dietary admixture, at dose levels of 1300, 4000 and 13000 ppm (equivalent to a mean achieved dosage of 91.4, 277 and

810 mg/kg bw/day respectively) resulted in treatment related effects detected in adults treated with 13000 ppm only. This dose level also resulted in a small number of

treatment related effects on offspring pre and post partum with an equivocal effect on the nature of some in utero parameters. Those effects are attributed to the excess impact of the dietary dosing on normal nutrition found in the parents.

The following NOAELs can be set: NOAELparental toxicity ≥ 810 mg/kg bw/d and NOAELreproductive and developmental toxicity ≥ 810 mg/kg bw/d.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

EC Number:

939-227-3

Cas Number:

84238-39-1

Molecular formula:

Not applicable (a generic Molecular formula cannot be provided for this substance)

IUPAC Name:

Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): Patchouli Oil
- Physical state: Yellow liquid
- Analytical purity:
- Lot/batch No.: AS00068648
- Expiration date of the lot/batch: 29 June 2013
- Storage condition of test material: room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: Approximately twelve weeks old
- Weight at study initiation: Males weighed 304 to 400g, the females weighed 193 to 222g
- Fasting period before study: No data
- Housing: All animals were housed in groups of five in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
During the mating phase, the animals were transferred to polypropylene grid floor cages
suspended over trays lined with absorbent paper on a one male: one female basis.
Following evidence of successful mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation, in solid floor
polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Rodent PMI 5002 (Certified) Ground Diet ad libitum
- Water (e.g. ad libitum): Mains drinking water, ad libitum
- Acclimation period: For five days during which time their health status was assessed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were therefore prepared three times during the
study, as the results of stability and homogeneity tests showed the dietary admixtures to be stable for a
period of at least eight days at room temperature and up to six weeks when stored at
approximately -20°C.

- Mixing appropriate amounts with (Type of food): The test item was incorporated into the diet at concentrations of 13000, 4000 and
1300 ppm as follows:
A known amount of test item was mixed with a small amount of basal laboratory diet
using Robot Coupe Blixer 4 until homogenous. 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 QE200 mixer.

- Storage temperature of food: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:

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 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 ~ 8 to 12 mins

Details on mating procedure:

On Day 15, animals were paired on a 1 male: 1 female basis within each dose
group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were
returned to their original cages and females were transferred to individual cages.

Duration of treatment / exposure:

Up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females.

Frequency of treatment:

Daily

Duration of test:

55 days

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change
daily. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for
males until termination and weekly for females until mating was evident. Body weights
were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and
4 post partum and at terminal kill.

Food Consumption
During the maturation period, weekly food consumption was recorded for each cage of
adults until pairing. This was continued for males after the mating phase. For females
showing evidence of mating, food consumption was recorded for the periods covering
post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption
was recorded during the lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for
males and for females during the pre-mating phase. Due to offspring growth and milk
production for lactation, food efficiency for females could not be accurately calculated
during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt
changes.

Reproduction Performance:
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of
up to fourteen days. Cage tray-liners were checked each morning for the presence of
ejected copulation plugs and each female was examined for the presence of a copulation
plug in the vagina. A vaginal smear was prepared for each female and the stage of
oestrus or the presence of sperm was recorded. The presence of sperm within the
vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day
0 of gestation) and the males were subsequently returned to their original holding cages
(unless required for additional pairing). Mated females were housed individually during
the period of gestation and lactation. Females failing to mate were also housed
individually after the mating phase.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and
around the period of expected parturition. Observations were carried out at
approximately 0830 and 1230 hours at weekends and public holidays. The following was
recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Pathology
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by
exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium
pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring
were terminated via intracardiac overdose of sodium pentobarbitone. Any females which
failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.
For all females, the uterus was examined for signs of implantation and the number of
uterine implantations in each horn was recorded. This procedure was enhanced; as
necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski
1964).
All adult animals and offspring, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free
from fat and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group,
in buffered 10% formalin, except where stated:
Coagulating gland
Prostate
Epididymides♦
Seminal vesicles
Ovaries
Testes♦
Mammary gland (females only)
Uterus/Cervix
Pituitary
Vagina

♦ = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately
forty-eight hours later

The tissues from control and 13000 ppm dose group animals, any animals dying during
the study, and any animals which failed to mate or did not achieve a pregnancy were
prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with
Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections
of testes and epididymides from all control and 13000 ppm males were also stained with
Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment related mammary gland, prostate, seminal
vesicles and coagulating gland changes, examination was subsequently extended to
include similarly prepared sections of these organs from animals from the low and
intermediate groups.

Fetal examinations:

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring
was recorded. Offspring were individually identified within each litter by tattoo on Day 1
post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post
partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were
calculated retrospectively from this data)
Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Offspring, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight-relative organ weights (Males)

The following statistical procedures were used:
Data for males and females prior to pairing, 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 and ANCOVA and Bartletts’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
non-parametric data. If no dose response was found, but the data showed nonhomogeneity
of means, the data were analysed by a stepwise Dunnett (parametric) or
Steel (non-parametric) 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).
Data for females during gestation and lactation, and offspring data were assessed for
dose response relationships by linear regression analysis, followed by one way analysis
of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where
variances were shown to be homogenous, pairwise comparisons were conducted using
Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed
using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.
Non-parametric methods were also used to analyse implantation loss, offspring sex ratio
and landmark developmental markers.

Indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating
period of the parental generation:
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and
parturition period of the parental generation:
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating
and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were
first calculated for each litter and the group mean was calculated using their individual
litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:
% pre–implantation loss = ((Number of corpora lutea - Number of implantation sites)/Number of corpora lutea) x100

% post–implantation loss = ((Number of implantation sites - Total number of offspring born)/Number of implantation sites) x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100

Viability Index (%) = (Number of offspring alive on Day 4/ Number of offspring alive on Day 1)x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

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

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

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:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Reproductive Performance:
Mating
There were no treatment related effects on mating performance at any of the dose levels
investigated. The majority of animals mated within the first five days of pairing (i.e. at the
first oestrus opportunity) and the mating index of treated animals was comparable to
controls.
One control female (No.13) and one female treated with 13000 ppm (No.74) did not mate
following fourteen days of cohousing with their male partners. The male partners for
both of these non-mated pairings showed histopathological evidence of either diffuse
tubular atrophy in the testes and increased cellular debris in the epididymides (control
male No.3) or reduced secretory activity in the prostate (13000 ppm male No.64). These
findings may therefore be a contributory factor in the lack of reproductive performance.
The distribution of these findings shows no treatment related trend.

Fertility
There were no treatment related effects detected on fertility indices and pregnancy rate
when compared with controls.
One female treated with 13000 ppm (No.79) showed evidence of pregnancy but no litter
was observed. Histopathological findings showed decidual contents in uterus, cervix and
vagina indicative of pregnancy with possible prenatal and perinatal fetal deaths/losses.
The male partner (No.69) for this female showed a slight hypospermatogenesis in testes
and slight oligospermia in epididymides during microscopic examinations. There is no
conclusive evidence to confirm the significance of male findings upon the observed total
post-implantation litter loss.
One control female (No.19) did not achieve pregnancy following evidence of mating. No
histopathological correlates were evident in the female reproductive organs, however, the male partner (No.9) for this female showed minimal or slight reduction in secretory
activity in prostate and seminal vesicles. The significance of these findings in the
infertility of this male/female pairing could not be established.

Gestation Length
There were no treatment related effects detected in the length of gestation between
control and treated groups. All females showed a gestation length between 22 and 23½
days.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 810 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Response
Eight females from both control and 13000 ppm dose groups and ten females from 1300
and 4000 ppm dose groups gave birth to a live litter and successfully reared young to
Day 5 of age. The following assessment of litter response is based on all litters reared to
termination on Day 5 of lactation/age.

Offspring Litter Size and Viability
At 13000 ppm a statistically significant reduction was detected in mean corpora lutea
(p<0.01) and a consequential implantation site (p<0.01) number. The mean corpora lutea value was slightly below historical control numbers. Litter size at birth was also
lower than controls showing statistically significant reduction in total number of offspring
born (p<0.05).
No such effects were detected at 1300 and 4000 ppm in mean numbers of corpora lutea,
implantation sites, litter size and implantation losses when compared to control numbers.
There were no treatment related effects detected in survival indices and sex ratio in all
treated litters when compared to controls.

Offspring Growth and Development
There were no clinically observable signs of toxicity detected for offspring from all treated
litters when compared to controls.
Mean values for total litter weight, offspring body weight, body weight changes and
surface righting reflex showed treatment related effects in litters treated with 13000 ppm.
Litter weight values for litters treated with 13000 ppm showed statistically significant
reduction (p<0.05 to p<0.01) during Day 1 and Day 4 evaluations. At this dose level
individual offspring weights were significantly lower than controls during Day 1 and Day 4
assessments whilst male offspring weights during Day 4 showed a statistically significant
effect (p<0.01). Offspring body weight change between Days 1 to 4 was subsequently
affected and a statistically significant reduction (p<0.01 and p<0.05) was evident. Mean
surface righting reflex values showed statistically significant reduction in 13000 ppm
offspring when compared to control values.
No clinically observable signs of toxicity were detected. Instances of swollen limb and
offspring found dead or missing from either control litters or litters from females treated
with 4000 ppm are commonly observed findings in studies of this type and considered to
be unrelated to test item toxicity.

Necropsy:
No treatment related macroscopic abnormalities were detected for interim death or
terminal kill offspring. The incidental findings observed were those occasionally
observed in reproductive studies of this type and were considered to be unrelated to
toxicity of the test item.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 810 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Mortality

There were no occurrences of mortality evident during the study period.

Clinical Observations

There were no clinically observable signs of toxicity or signs related to general

appearance throughout the study period for either male or female rats.

Body Weight

Lower mean body weights were evident in all treated groups at interim weighing points

and at termination.

At 13000 ppm males showed statistically significant (p<0.05 and p<0.01) reductions in

mean body weight gains during Weeks 2, 3 and 6 of treatment when compared with

control values. Males treated with 1300 or 4000 ppm also showed statistically significant

(p<0.05) reductions in mean body weight gains during Week 3 of treatment.

Females treated with 13000 ppm also showed statistically significant (p<0.05 to p<0.01)

reductions in mean body weight gains during Week 2 of treatment and during the

gestation and lactation phases. Females treated with 4000 ppm also showed a

statistically significant (p<0.05) reduction in mean body weight gain during Week 2 of

treatment. Females treated with 1300 ppm showed a slight reduction in mean body

weight gain during Week 2 of treatment, however statistical significance has not been

reached.

Food Consumption and Food Efficiency

For animals treated with 13000 ppm treatment related reductions in food consumption

and food efficiency was evident throughout the treatment period.

Whilst a relationship to treatment at 1300 and 4000 ppm cannot be excluded, reduction

in food consumption and food efficiency was considered minimal and is therefore

considered to be of no toxicological importance.

Water Consumption

Daily visual inspections of the water intake did not reveal any overt changes during the

treatment period.

Necropsy

Adults

There were no toxicologically significant macroscopic abnormalities detected during the

post mortem procedure which were considered to be attributable to treatment with the

test item.

Four males treated with 13000 ppm had small seminal vesicles at necropsy, one of these

males also had pale kidneys with increased pelvic space of the right kidney and small

prostate whilst a further male had small epididymides, small prostate and also small and

flaccid testes. In the absence of any other supportive data on adverse effects correlated

to these findings, it was considered of no toxicological importance.

One control male had small testes whilst other control males had small seminal vesicles

and prostate at necropsy.

One female treated with 13000 ppm had brown mass in right horn of the uterus and

cervix at necropsy which corresponded to decidual contents and dilation of the right horn

recorded at histopathology examination. This finding was considered to represent normal background lesion which can be recorded in females of this strain and age. As

such, this finding was considered unrelated to treatment.

In the absence of treatment all findings detected in control animals are considered to be

fortuitous.

Histopathology

Histopathological examination of reproductive tissues from animals treated with

13000 ppm showed possible treatment related changes detected in prostate, seminal

vesicles, coagulating glands and mammary glands. Subsequent examinations of these

tissues from animals treated with 1300 or 4000 ppm were performed and no treatment

related abnormalities were detected.

The remaining microscopic findings were those commonly observed in laboratory

maintained rats of the age and strain employed and there were no differences in

incidence or severity between control and treatment groups that were considered to be of

toxicological significance.

Applicant's summary and conclusion

Conclusions:

The NOAELdevelopmental toxicity is found to be ≥ 810 mg/kg bw/d.

Executive summary:

Introduction. The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard

assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421

“Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). 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 dietary admixture to three groups, each of

ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for

females), at dose levels of 1300, 4000 and 13000 ppm (equivalent to a mean achieved

dosage of 91.4, 277 and 810 mg/kg bw/day respectively). A control group of ten males and ten females was dosed with basal laboratory diet.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently

being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving

offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy

examination and histopathological evaluation of reproductive tissues was performed.

Results.

Adult Responses:

Mortality. There were no unscheduled deaths during the study.

Clinical Observations. There were no clinically observable signs of toxicity detected.

Body Weight. Reductions in overall mean body weights were evident in all treated animals of either sex when compared to controls throughout the treatment period.

Food Consumption and Food Efficiency. Reductions in overall food consumption and food efficiency values were recorded in animals of either sex treated with 13000 ppm.

Water Consumption. No intergroup differences were detected.

Reproductive Screening:

Mating. There were no treatment related effects detected on mating performance in treated animals when compared to controls.

Fertility. There were no treatment related effects detected on fertility in treated animals when compared to controls.

Gestation Lengths. There were no treatment related effects detected on gestation length.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. No treatment related effects were evident in sex ratio or viability assessments. Litter size was reduced at 13000 ppm.

Offspring Growth and Development. Litter weight, offspring weights and surface righting reflex mean values were lower at 13000 ppm when compared to controls.

Offspring Observations. There were no clinically observable signs of toxicity detected for offspring from all treated litters when compared to controls.

Pathology:

Necropsy. There were no toxicologically significant macroscopic abnormalities detected.

Organ Weights. There were no treatment related effects detected in organ weights.

Histopathology. There were no treatment related abnormalities recorded in reproductive organs during microscopic examinations.

Conclusion. The oral administration of Patchouli Oil to rats by dietary admixture, at dose levels of 1300, 4000 and 13000 ppm (equivalent to a mean achieved dosage of 91.4, 277 and

810 mg/kg bw/day respectively) resulted in treatment related effects detected in adults treated with 13000 ppm only. This dose level also resulted in a small number of

treatment related effects on offspring pre and post partum with an equivocal effect on the nature of some in utero parameters. Those effects are attributed to the excess impact of the dietary dosing on normal nutrition found in the parents.

The following NOAELs can be set: NOAELparental toxicity ≥ 810 mg/kg bw/d and NOAELreproductive and developmental toxicity ≥ 810 mg/kg bw/d.