Registration Dossier

Administrative data

Description of key information

NOAEL for males and females (OECD guideline 408): 891 mg/kg bw/day and 1109 mg/kg bw/day, respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 January - 17 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 408 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
15 September 2014
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 6-7 weeks
- Weight at study initiation: Males: 212-290 g; Females: 155-208 g
- Housing: Animals were housed 5/sex/cage in polycarbonate cages with a stainless steel mesh lid
- Diet: Rat and Mouse No. 1 Maintenance Diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: feed
Vehicle:
other: Stabiliser: Corn oil (test material to corn oil ratio 5:1).
Details on oral exposure:
DIET PREPARATION
- Vehicle: The test material was dissolved in corn oil to minimise evaporation and then mixed with the carrier diet Rat and Mouse No. 1 Maintenance Diet.
- Carrier diet: Rat and Mouse No. 1 Maintenance Diet (RMI).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of the test substance and corn oil required for the premix was added to an equal amount of sieved diet and stirred. An amount of sieved diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added or the mix appeared dry. At this stage, the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a Turbula mixer for 100 cycles.
This premix was diluted with further quantities of plain diet to prepare the two highest test concentration mixes (5000 ppm and 15000 ppm) and a second premix. The second premix was used to prepare the lowest concentration test mix (1500 ppm). Each formulation was mixed using a Turbula mixer for 100 cycles.
For Control diet, the corn oil (equal to the amount used for the 15000 ppm concentration) was added directly to the diet and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of preparation: Frozen (nominally -20 °C) until required for feeding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 100 and 24000 ppm were analysed to assess the stability and homogeneity of the test substance in the diet matrix. A further diet preparation at 500 ppm was analysed for stability only. Stability was confirmed as follows:
100 ppm: 1 day ambient; 16 days frozen.
500 ppm: 1 day ambient; 22 days frozen.
24000 ppm: 8 days ambient; 22 days frozen.

Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment were analysed for achieved concentration of the test substance.
Duration of treatment / exposure:
Main phase: Minimum of 90 days (13 weeks)
Recovery phase: 4 weeks
Frequency of treatment:
Continuously
Remarks:
Doses / Concentrations:
1500, 5000 and 15000 ppm (100, 330 and 981 mg/kg bw/day for males and 109, 362 and 1109 mg/kg bw/day for females, respectively.)
Basis:
nominal in diet
No. of animals per sex per dose:
Main phase: 10 animals/sex/dose
Recovery phase: 5 animals/sex/dose (2 groups only)
Control animals:
other: Untreated diet of the same batch (with corn oil)
Details on study design:
- Dose selection rationale: The dietary levels for investigation in this study (0, 1500, 5000 and 15000 ppm) were selected in conjunction with the Sponsor based on the results of a 3-week palatability/ preliminary study (Study Number OAD0018). In that study, dietary levels of 5000, 10000 and 15000 ppm were investigated. For this 13-week toxicity study, the high dose level of 15000 ppm was selected as there was no evidence to suggest that a dietary level of 15000 ppm would be unsuitable for use and this level was expected to result in an achieved dosage of 1000 mg/kg bw/day (the highest dosage required by the test guidelines to which this study was designed to meet) in both sexes when averaged over the full 13-week treatment period. The lowest dietary concentration (1500 ppm) was expected to provide an achieved dosage of 100 mg/kg bw/day and to be the No Observed Adverse Effect Level and the intermediate dietary concentration (5000 ppm) was selected as it represented the approximate geometric mean of the low and high dietary concentrations.
- Rationale for animal assignment: Randomly allocated on arrival.
- Group 1, 2, 3 and 4: Control, 1500, 5000 and 15000 ppm, respectively
- Post-exposure recovery period in satellite groups (exposure 0 and 15000 ppm): 4 weeks
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced (Week P1), on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily for the week before treatment started (reported as Week P1) and for each day throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No treatment-related effect was observed and, consequently, quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of the animals were examined by means of a binocular indirect ophthalmoscope and a slit-lamp biomicroscope as follows:
- Time schedule for examinations: Pre-treatment: all animals; Week 12: All animals of Groups 1 and 4
- Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (at termination): All main phase animals; Week 4 of Recovery (at termination): All recovery phase animals
- Anaesthetic used for blood collection: Yes; animals were held under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes; blood samples were collected after overnight withdrawal of food.
- Parameters checked:
HAEMATOLOGY: Haematocrit (Hct)*, Haemoglobin concentration (Hb)*, Erythrocyte count (RBC)*, Absolute reticulocyte count (Retic)*, Mean cell haemoglobin (MCH)*, Mean cell volume (MCV)*, Mean cell haemoglobin concentration (MCHC)*, Red cell distribution width (RDW)* Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) & Large unstained cells (LUC), Platelet count (Plt), Morphology: Anisocytosis, Microcytosis, Macrocytosis, Hypochromasia & Hyperchromasia, Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
* Additional investigations for selected parameters only during Week 4 of recovery; samples taken for male and female animals.
CLINICAL CHEMISTRY: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot) and Albumin (Alb)
Samples obtained from animals during Recovery Week 4 (at termination) were examined for the following characteristics:
Alkaline phosphatase (ALP) – males only; Aspartate aminotransferase (AST) – females only; Urea– males and females; Creatinine (Creat) – males and females; Glucose (Gluc) – males and females; Total cholesterol (Chol) – males and females; Total protein (Total Prot) – males only; Albumin (Alb) – males only; Albumin/globulin ratio (A/G Ratio) – males only


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity
Time schedule for examinations:
- Sensory reactivity and grip strength assessments were performed on the first five Main phase animals and all Recovery phase animals in Group 1 and 4, and all Main phase animals from Group 2 and 3 (10 animals from each group) during Week 12 of treatment.
- During Week 12 of treatment, the motor activity of the first five Main phase animals and all Recovery phase animals in Group 1 and 4, and all Main phase animals from Group 2 and 3 (10 animals from each group) was measured.
Sacrifice and pathology:
SACRIFICE: Main phase animals were killed following 13 weeks of treatment. Recovery phase animals were killed following 13 weeks of treatment and 4 weeks of recovery. Animals were killed by carbon dioxide asphyxiation with subsequent exsanguination.

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

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed together, unless specified in Table 7.5.1/1. The requisite organs were weighed for Main phase and Recovery phase animals killed at scheduled termination.

HISTOPATHOLOGY: Yes
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid; Eyes: In Davidson’s fluid.
Histology:
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: Main phase animals of Groups 1 and 4 killed at a scheduled termination.
Processing - abnormalities, adrenals and thymus: All Main phase animals of Group 2 and 3 killed at a scheduled termination.
Processing - adrenal and thymus: All Recovery phase animals of Group 1 and 4 killed at a scheduled termination.
Routine staining: Sections were stained with haematoxylin and eosin.
Light microscopy: Tissues preserved for examination were examined as follows:
All Main phase animals of Groups 1 and 4: All specified in Table 7.5.1/1
All Main phase animals of Groups 2 and 3: Abnormalities, adrenals and thymus only.
All recovery animals of Groups 1 and 4: Adrenals and thymus
Other examinations:
None
Statistics:
See "Any other information on materials and methods incl. tables"
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Due to the lack of patability of the preparation at the highest dose
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Reversible and non-adverse changes in adrenal glands and thymus
Details on results:
CLINICAL SIGNS AND MORTALITY
- During the 13-week treatment period and 4-week recovery period, there were no premature deaths and no test article-related signs observed during the detailed physical examination and arena observations.

BODY WEIGHT AND WEIGHT GAIN
- Mean body weight gain of males and females given 15000 ppm was generally lower than Control throughout the treatment period, with statistical significance attained for differences in the majority of weekly recording periods. Effects were most pronounced during Week 1, where 2/15 males and 8/15 females showed body weight stasis or weight loss. During the remainder of the treatment period, a clear improvement in mean body weight gain was apparent, such that overall mean weight gain for males and females during Weeks 0-13 was 64 % and 61 % of Controls for males and females, respectively.
- Following the cessation of treatment, males in the 15000 ppm group showed immediate marked increase in mean body weight gain during Week 1 of recovery, which continued throughout the 4-week recovery period, such that overall mean weight gain during recovery was 246 % of Controls. Females in the 15000 ppm group also showed a marked increase in mean body weight gain during Week 1 of recovery (26 grams compared to 4 grams among Control females). In Week 2 of recovery these females showed mean body weight loss; there was no apparent reason for this weight loss and thereafter to the end of the recovery period weekly body weight change remained highly variable; despite this variation overall mean body weight gain in the 4-week recovery period was 209 % of Controls.
- Males given 5000 ppm showed statistically significantly low mean body weight gain during Weeks 1-2 of treatment; thereafter mean weight gain was essentially similar to Control, such that overall mean weight gain during Weeks 0-13 was 85% of Controls.
- The mean body weight gain of females given 5000 ppm and of males and females given 1500 ppm was unaffected by treatment.

FOOD CONSUMPTION
- During Week 1 of treatment, the mean food intake of males and females given 15000 ppm was markedly lower than Control (68 % for males and 73 % for females). Thereafter, a clear increase in food consumption was apparent although weekly food intake remained lower than Control throughout the treatment period; overall mean food consumption from Week 1-13 was 83 % and 88 % of Control for males and females, respectively.
- After replacement of the treated diet by untreated diet (with vehicle), males and females in the 15000 ppm group showed a marked increase in food consumption in Week 1 of recovery; this increase persisted throughout the recovery period for the males, whereas the food intake of the females was similar to Controls during Weeks 2-4 of recovery. Overall food consumption during recovery period was 113 % and 109 % of Control for males and females, respectively.
- The mean food intake of males and females given 1500 or 5000 ppm was considered to be unaffected by treatment.

WATER CONSUMPTION
- The visual assessment of water intake did not reveal any clear or consistent treatment-related effect and, consequently, quantitative measurements were not performed.
- A visual assessment of water intake indicated an apparently higher water consumption than the controls, on Days 19, 20 (males only), 22, 26 (females only), 34 (a single cage of each sex only) and 90 for animals receiving 15000 ppm, on Day 22 for animals receiving 5000 ppm and on Day 40 for males receiving 1500 ppm. Since no further differences were seen throughout the study, these isolated findings were considered to have occurred by chance. In addition, an apparently higher water consumption than controls was recorded on a number of occasions during the Recovery period for previously treated females and conversely, lower water consumption was recorded on two occasions for previously treated males. These differences were considered to have occurred by chance.

OPHTHALMOSCOPIC EXAMINATION
- There were no test article-related ophthalmoscopy findings apparent during Week 12 of treatment.

HAEMATOLOGY
- Analysis of haematological parameters, including morphology, following 13 weeks of treatment revealed a slight decrease in erythrocyte counts and haemoglobin concentration and haematocrit in males and females given 15000 ppm, associated with a slight decrease in mean cell haemoglobin concentrations in females and a concomitant minor increase in red cell distribution width in males and females; statistical significance was attained for the majority of these differences. The absence of any effect on reticulocyte counts would indicate that there was no change in red blood cell turnover. In addition, all groups of treated males showed a dose-dependent shortening of prothrombin times and all groups of treated females showed a non-dose-dependent shortening of activated partial thromboplastin times. For all of these minor differences from Control, values were within 95% confidence limits of the Historical Control Data (HCD) range.
- There was no clear effect of treatment on red cell parameters for males and females given 1500 or 5000 ppm, or on white cell parameters, platelet counts or blood morphology at any dietary inclusion level investigated.
- Four weeks after the withdrawal of treatment, erythrocyte counts and haemoglobin concentration, haematocrit, mean cell haemoglobin concentrations and clotting times for males and females in the 15000 ppm group were similar to Control, demonstrating full recovery. It was noted that statistically significant slight reductions in red cell distribution width was evident in males and females in the 15000 ppm group, along with reduced reticulocyte concentrations in these females. Such differences were either not apparent at the end of treatment or had a converse direction of change; therefore these minor differences at the end of the recovery period were considered to be attributable to natural variation, and unrelated to previous treatment.

CLINICAL CHEMISTRY
- Biochemical analysis of plasma after 13 weeks of treatment revealed a non-dose-dependent decrease in urea, blood urea nitrogen and creatinine concentrations in all groups of treated males and females when compared to Controls, though the inter-group differences from controls for urea and blood urea nitrogen in females did not attain statistical significance. In addition, cholesterol concentrations were slightly high in males and females given 15000 ppm, changes in glucose concentrations were apparent in 15000 ppm group (slightly low in males and slightly high in females), alkaline phosphatase, albumin concentrations and albumin/globulin ratio were slightly high in males given 15000 ppm and aspartate amino transferase concentrations were slightly low in females given 15000 ppm; these differences from Control attained statistical significance. For all of these minor differences from Control, values were within 95 % confidence limits of the Historical Control Data (HCD) range.
- All of the above differences were no longer apparent four weeks after the withdrawal of treatment, demonstrating full recovery.

NEUROBEHAVIOUR
- Sensory reactivity observations and grip strength values for all groups of animals during Week 12 were considered unaffected by treatment.
- Females given 5000 or 15000 ppm showed a slight reduction in mean forelimb grip strength when compared to Control, with differences attaining statistical significance. Mean values were within the Historical Control Data range (0.84-1.15 kg), and in the absence of a dose response relationship or similar effects on hindlimb grip strength in these groups of females, these minor differences in forelimb grip strength were considered incidental and unrelated to treatment.
- Motor activity scores for males and females during Week 12 showed some intra and inter group variation, with differences in occasional 6-minute recording periods attaining statistical significance; however there was no consistent evidence of an adverse effect of treatment.

ORGAN WEIGHTS
- At scheduled termination after 13 weeks of treatment statistically significantly low mean terminal body weights were apparent for males and females given 15000 ppm, indicative of the lower weight gain recorded throughout the treatment period.
- The analysis of organ weights for the Main phase animals indicated a dose-dependent increase in body weight-adjusted liver weight in all groups of treated males (110, 117 and 128 % of Control) and in females given 5000 or 15000 ppm (109 and 136 % of Control). Increased body weight-adjusted kidney weights were evident in females given 15000 ppm (109 % Control) and low body weight-adjusted uterus and cervix weights were apparent for females given 5000 or 15000 ppm (70% of Control). All values were within the 5-95% confidence limits of the Historical Control Data (HCD) range.
- After 4 weeks of recovery, terminal body weights remained slightly lower than Control. Body weight-adjusted liver weights in males previously given 15000 ppm remained slightly higher than Control, although the magnitude of the difference (110 % Control) was lower than that recorded at the end of the treatment period; liver weights for females were essentially similar to Control. In females previously given 15000 ppm, body weight-adjusted kidney weights remained higher than Control; the magnitude of the differences were greater than that observed at the end of treatment (121 % Control).

GROSS PATHOLOGY
- There were no macroscopic abnormalities detected at scheduled termination after 13 weeks of treatment or after 4 weeks of recovery that were attributable to treatment with the test substance. All findings were similar to the background of changes commonly seen at these laboratories in this species and strain.

HISTOPATHOLOGY
- Histopathological evaluation of the retained tissues revealed changes in the adrenal glands and thymus. Treatment-related hypertrophy of the zona glomerulosa in the adrenal glands was seen in females given 5000 or 15000 ppm, and in one male given 15000 ppm. Full recovery was apparent after the four week recovery period. In addition, an increased incidence of tingible body macrophages was seen in the thymic cortex of animals in all treated groups. This change was considered to be secondary and stress-related and was no longer apparent after the 4-week recovery period.
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Equivalent to 981 mg/kg bw/day for males and 1109 mg/kg bw/day for females; no evidence of adverse toxic effects up to the highest dose tested
Critical effects observed:
not specified

Formulation analysis:

Homogeneity was confirmed for the test substance in Rat and Mouse No. 1 maintenance diet formulations at nominal concentrations of 100 ppm and 24000 ppm. Stability was confirmed at ambient storage for up to one day and frozen storage for 16 days for 100 ppm, at ambient storage for up to one day and frozen storage for 22 days for 500 ppm and at ambient storage for up to 8 days and frozen storage for 22 days for 24000 ppm.

The mean concentrations of the test substance in test formulations analysed for the study was within +10 %/-15 % of nominal concentrations, confirming accurate formulation. The percent difference from mean remained within 3 % confirming the precision of the analysis.

Achieved dose:

The overall achieved doses during Weeks 1-13 at 1500, 5000 and 15000 ppm were 100, 330 and 981 mg/kg bw/day for males and 109, 362 and 1109 mg/kg bw/day for females, respectively.
Conclusions:
The No Observed Adverse Effect Level (NOAEL) was concluded to be 15000 ppm, equivalent to 981 mg/kg bw/day for males and 1109 mg/kg bw/day for females.
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, test substance 2-ETHYL-4 -(2,2,3- TRIMETHYLCYCLOPENT- 3-EN-1-YL) BUT-(2E)-EN-1-OL was administered orally via the diet to groups of Crl:CD(SD) rats (10/sex/dose) at doses of 1500, 5000 or 15000 ppm for 13 weeks. A similarly constituted Control group received untreated diet, with vehicle (corn oil) only, throughout the treatment period. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 13 weeks, followed by a four week period without treatment to assess the potential for any treatment-related change to recover. During the study, assessment of clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (visual observations), ophthalmoscopy, haematology, coagulation, blood chemistry, organ weights, macropathology and histopathology investigations were undertaken.

The overall achieved doses during Weeks 1-13 at 1500, 5000 and 15000 ppm were 100, 330 and 981 mg/kg bw/day for males and 109, 362 and 1109 mg/kg bw/day for females, respectively.

 

There were no premature deaths and no test article-related signs observed during the detailed physical examination and arena observations.

 

Sensory reactivity observations, grip strength, motor activity scores and ophthalmoscopy assessments for all groups of animals during Week 12 were unaffected by treatment.

 

Mean body weight gain of males and females given 15000 ppm was generally lower than Control throughout the treatment period (64% and 61% of Controls for males and females, respectively), with the most pronounced effects occurring in Week 1. Males given 5000 ppm showed statistically significant low mean body weight gain during Weeks 1-2 of treatment but thereafter were essentially similar to Control. Following the replacement of treated diet by untreated diet (with vehicle), the mean body weight gain of males and females previously given 15000 ppm was statistically significantly higher than Control (at least 200% of Control) over the 4-week recovery period. The mean body weight gain of females given 5000 ppm and of males and females given 1500 ppm was unaffected by treatment.

 

The mean food intake of animals given 15000 ppm was markedly lower than Control in Week 1 of treatment; thereafter, a clear increase in food consumption was apparent although weekly food intake remained lower than Control throughout the treatment period (overall food intake was 68 % and 73 % of Control for males and females, respectively). After replacement of the treated diet by untreated diet (with vehicle), animals in the 15000 ppm group showed a marked increase in food consumption.

Reduced food consumption, with associated reductions in body weight gain was thus evident in the 15000 ppm group, however this was deemed attributable to the treated diet being slightly unpalatable due to the class and high concentration of the test article, but not adverse as there was no evidence of primary toxicity.

 

Analysis of haematological parameters during Week 13 of treatment revealed lower than Control erythrocyte and haemoglobin concentrations and haematocrit in males and females given 15000 ppm, associated with a minor increase in red cell distribution width, and a slight decrease in mean cell haemoglobin concentration in females. All groups of treated males showed shorter than Control prothrombin times, with some evidence of a dose-related trend. Conversely, all groups of treated females showed shorter than Control activated partial thromboplastin times. All these differences were within 95 % confidence limits of the Historical Control Data range and were no longer evident 4 weeks after the cessation of dosing.

 

During Week 13, biochemical analysis of plasma revealed a non-dose-dependent decrease in urea, blood urea nitrogen and creatinine concentrations in all groups of treated males and females when compared to Controls. In addition, cholesterol concentrations were slightly high in males and females given 15000 ppm, changes in glucose concentrations were apparent in 15000 ppm group (slightly low in males and slightly high in females), alkaline phosphatase, albumin concentrations and albumin/globulin ratio were slightly high in males given 15000 ppm and aspartate amino-transferase concentrations were slightly low in females given 15000 ppm. All these differences were within 95% confidence limits of the Historical Control Data range and were no longer evident after 4 weeks off-dose.

 

The analysis of organ weights following 13 weeks of treatment indicated dose-dependent and statistically significantly higher than Control body weight-adjusted liver weight in all groups of treated males and in females given 5000 or 15000 ppm. Body weight-adjusted kidney weights were higher than Control in females given 15000 ppm, and body weight-adjusted uterus and cervix weights were slightly low in females given 5000 or 15000 ppm. Following 4 weeks of recovery, body weight-adjusted liver weights in males previously given 15000 ppm remained slightly higher than Control, although the magnitude of the difference was lower than that recorded at the end of the treatment period. In females previously given 15000 ppm, body weight-adjusted kidney weights remained higher than Control.

 

Plasma biochemistry revealed several slight changes in composition which were indicative of adaptations of metabolism/excretion in the liver and kidneys, and were accompanied by increases in liver and kidney weight. In the absence of any evidence of degenerative or functional change in the liver and kidneys during histopathological evaluation, the slight disturbances of biochemical parameters were considered not to be adverse.

There were no macroscopic abnormalities detected at scheduled termination after 13 weeks of treatment or after 4 weeks of recovery that were attributable to treatment.

Histopathological evaluation of the retained tissues revealed limited changes in the adrenal glands and thymus. Non-adverse minimal diffuse hypertrophy of the zona glomerulosa in the adrenal glands was seen in females given 5000 or 15000 ppm, and in one male given 15000 ppm. Full recovery was apparent after the four week recovery period. In addition, a minimal to slight increase in the incidence of tingible body macrophages was seen in the thymic cortex of animals in all treated groups. This change was considered to be secondary and stress-related and was no longer apparent after the 4-week recovery period.

 

 It was concluded that dietary administration of the test item to Crl:CD(SD) rats at dietary inclusion levels of 1500, 5000 or 15000 ppm for 13 weeks provied clearevidence of systemic exposure but no effects which were deeme to be adverse.

Therefore, the No Observed Adverse Effect Level (NOAEL) was concluded to be 15000 ppm, equivalent to 981 mg/kg bw/day for males and 1109 mg/kg bw/day for females.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
981 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well conducted and well described studies in accordance with GLP and OECD Guidelines 422 (1996) and 408 without any deviation.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an oral repeated dose toxicity range-finding experiment, male and female rats exposed for 14 days showed increased liver weight in both sexes and a slight increase in kidney weight in females.

In a recent 28-day repeated dose toxicity study conducted according to GLP and OECD guideline 422, biochemical changes in females at 1000 mg/kg/day, such as increased alanine aminophosphatase, alanine aminotransferase, cholesterol and bilirubin levels also confirmed that the metabolic function of the liver was affected by administration of 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-(2E)-buten-1-ol.

Also, in a 13-week repeated dose toxicity study conducted according to GLP and OECD guideline 408, males and/or females exposed by diet to the registered substance showed slight non-adverse and reversible changes in haematological parameters (erythrocyte and haemoglobin concentrations, prothrombin time and haematocrit), biochemical parameters (urea, blood urea nitrogen and creatinine concentrations, glucose,alkaline phosphatase, albumin concentrations and albumin/globulin ratio), associated with increases in liver and/or kidney weights. These observations are thought to be indicative of adaptations of metabolism/excretion in the liver and kidneys, with absence of degenerative or functional change in liver or kidneys. Therefore, the NOAEL was concluded to be 15000 ppm, equivalent to 981 mg/kg bw/day for males and 1109 mg/kg bw/day for females.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Well conducted and well described study in accordance with GLP and OECD Guideline 408 without any deviation. This study describes effects in rats exposed to the registered substance during the longest period of treatment.

Justification for classification or non-classification

No adverse effects were observed below the limit of classification of 300 mg/kg bw/day for subacute studies or 100 mg/kg bw/day for subchronic studies, therefore the registered substance is not classified for repeated dose toxicity according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.