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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 October 2005 to 31 January 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 407 and in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on 2005-08-30)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(3S)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Molecular formula:
C25 H46 O S
IUPAC Name:
(3S)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Constituent 2
Chemical structure
Reference substance name:
(3R)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Molecular formula:
C25 H46 O S
IUPAC Name:
(3R)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Constituent 3
Chemical structure
Reference substance name:
(3S)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Molecular formula:
C25 H46 O S
IUPAC Name:
(3S)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Constituent 4
Chemical structure
Reference substance name:
(3R)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Molecular formula:
C25 H46 O S
IUPAC Name:
(3R)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone
Test material form:
liquid
Details on test material:
- Physical state: colourless to pale yellow slightly viscous liquid
Specific details on test material used for the study:
- Storage condition of test material: Approximately 4 °C under nitrogen in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent - UK
- Age at study initiation: ca. 6 to 8 weeks old.
- Weight at study initiation: males 140 -171g , females 124-153g
- Fasting period before study: animal were not fasted before blood sampling
- Housing: Groups of five by sex in solid floor polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum (pelleted diet Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK), routinely analysed
- Water (e.g. ad libitum): ad libitum (tap water, routinely analysed)
- Acclimation period: 6 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 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.
Formulations (showed to be stable for at least 14 days) were prepared weekly and stored at +4°C in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not soluble in water (water solubility < 0.1mg/L @20°C)
- Concentration in vehicle: 0, 3.75, 37.5 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of ST 15 C 05 in the test material formulations was determined by gas chromatography (GC) using an external standard technique. The test material formulations in Arachis oil BP were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/mL. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/mL. Actual concentration measured in test material formulations were greater than 92% (between 93-102%) of nominal dosage, in consequence, variance between actual and nominal concentration is acceptable.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily, 7 days each week
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (See Table 7.5.1/1 in any other information on material and methods incl. tables)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Following single dose sighting work treating one male and one female with 1000 mg/kg, two groups, each of six rats (three males and three females) were dosed with 0 or 1000 mg/kg bw of the test substance. The test material was administered daily, for fourteen consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg bw/day of Arachis oil BP. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at Days 4, 8 and 11.
The observations performed were the following:
1. No death during the 14 days
2. no clinical signs of toxicity or ill health or behavioural change
3. bodyweight: no adverse effect on bodyweight development was detected for animals of either sex treated with 1000 mg/kg/day
4. Necropsy: no macroscopic findings were observed at post-mortem examination

In function of these observations, the following doses were chosen for the 28 day study: 15, 150 & 1000 mg/kg/day (plus a control group treated with vehicle alone)

- Rationale for animal assignment (if not random): Not Applicable
- Rationale for selecting satellite groups: Not performed
- Post-exposure recovery period in satellite groups: Not performed
- Section schedule rationale (if not random): Not Applicable
Positive control:
Not Applicable/Not required by OECD Guideline 407

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing
at weekends. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on Days 5, 12, 19 and 26 all animals were observed for signs of functional/behavioural toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to start of dosing) and at weekly intervals thereafter. Individual bodyweights were also recorded prior to terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): NOT A FEEDING STUDY
(Food consumption was recorded for each cage group at weekly intervals throughout the study.)

FOOD EFFICIENCY: Not required in OECD Guideline 407
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes (Dietary intake were recorded weekly for each cage group. Weekly food efficiency (bodyweight gain/food intake) were calculated

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes (NOT A DRINKING WATER STUDY)
Water consumption was recorded daily for each cage group throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters checked in table 7.5.1/2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Animals fasted: No
- How many animals: all
- Parameters checked in table 7.5.1/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 5, 12, 19 and 26, 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 from approximately two hours after dosing on each occasion.
- Dose groups that were examined: all
- Battery of functions tested: sensory reactivity (auditory, visual, proprioceptive stimuli), forelimb/hindlimb grip strength, motor activity
1.) Behavioural Assessments: Detailed individual clinical observations were performed for each animal using a purpose built arena (The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation).
2.) 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 percentage of time 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) and 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.
3.) Sensory Reactivity (Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals) see table 7.5.1/3
HISTOPATHOLOGY: Yes (all animals) see table 7.5.1/3
Other examinations:
none
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
All data was summarised in tabular form. 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 non-parametric 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 (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).
Probability values (p) are presented as follows:
p<0.0l **
p < 0.05 *
P ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically significant clinical signs were detected:
1000 mg/kg bw/day: Increased salivation was detected immediately after dosing for animals of either sex throughout the treatment period. One male showed noisy respiration immediately after and up to 1 hour after dosing. This is likely to be attributable of slight mis-administration of the test material and is considered not to be treatment-related.
150 mg/kg bw/day: one incident of increased salivation immediately after dosing
15/mg/kg bw/day: no clinically observable signs of toxicity were detected for animals of either sex
Mortality:
no mortality observed
Description (incidence):
There were no mortalities during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effect on bodyweight change
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related effect on dietary intake
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection revealed no intergroup differences
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes in haematological parameters measured.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes in blood chemistry parameters measured.
A significant decrease (p<0.05) in plasma potassium was observed for females treated with 1000 and 150 mg/kg bw/day. In the absence of a dose related response this was considered to be incidental and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the behavioural parameters measured.
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Functional Performance Tests: There were no treatment-related changes in the functional performance parameters measured.
A statistically significant (p<0.05) decrease in activity and mobility in the last 20% of the period for males treated with 1000 and 150 mg/kg bw/day was observed. In isolation and in the absence of similar findings in females at these dose levels, this was considered to be incidental and unrelated
to treatment.
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 age used, and was of no toxicological importance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
A significant increase in both absolute and relative kidney (p<0.01) and liver (p<0.05) weights were observed in males treated with 1000 mg/kg bw/day. The effect on liver weight extended to males treated with 150 mg/kg bw/day. In the absence of any histological correlates these differences were considered not to be treatment related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic abnormalities were detected.
One male treated with 1000 mg/kg bw/day showed pale kidneys. In isolation, this was considered to be unrelated to treatment.
One male treated with 150 mg/kg bw/day showed small testes and epididymides. In the absence of similar findings in animals treated with 1000 mg/kg bw/day this was considered not to be treatment related.
One control female showed reddened lungs. As this was observed in a control animal it was considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
All remaining morphological changes 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.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material to rats, by gavage, at dose levels of 15, 150 and 1000 mg/kg/day for twenty-eight consecutive days resulted in no treatment-related changes. The No Observed Effect Level (NOEL) was therefore considered to be 1000 mg/kg/day.
Executive summary:

In a subacute oral toxicity study performed similarly to OECD test guideline No. 407 and in compliance with GLP, the test material diluted in Arachis oil BP was administered to 5 Sprague- DawleyCrl:CD® (SD) IGS BR rats/sex/dose by gavage at dose levels of 0, 15, 150 and 1000 mg/kg bw/day.

The dose levels were selected based on the absence of effects seen in the fourteen-day preliminary study performed at the dose of 0 and 1000 mg/kg bw/d.

Clinical signs, functional observations, bodyweight development and food 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 from high dose and control animals was performed.

Administration of the test material did not produce any toxic effects in this study. It was concluded that the NOEL was 1000 mg/kg bw/d. The test material is therefore not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.

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