4-Penten-2-ol, 3,3-dimethyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-, (4E)-

Endpoint summary

Description of key information

Repeated dose toxicity oral: NOAEL ≥ 1000 mg/kg bw/d (OECD 408, GLP, K, rel. 1)

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:

From March 26 to September 5, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Although not requested under REACH Annex VIII, the sub-chronic repeated dose toxicity study was performed to comply with worldwide regulations. See attached justification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD GLP (inspected from 05 to 07 February 2014/ signed on April 24, 2014)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 217-297 g; Females: 168-224 g
- Housing: Five animals/sex/cage (main study and recovery) in polycarbonate cages with a stainless steel mesh lid
- Diet: Rat and Mouse No. 1 Maintenance Diet, ad libitum; Non-restricted (except overnight for urine collection and blood sampling for haematology and blood chemistry).
- Water: Potable water from the public supply, ad libitum
- Acclimation period: Two weeks before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From March 26 to September 5, 2014

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was prepared for administration as a series of graded concentrations in the vehicle. Starting with the lowest concentration (20 mg/mL), the required amount of test material was weighed into a suitable container. Formulations were prepared by adding approximately 50 % of the final volume of vehicle to the test material. The suspension was then magnetically stirred until thoroughly mixed. The suspension was made up to the required volume with vehicle. The formulation was placed into a container and stirred using a magnetic stirrer until homogenous, forming a clear suspension; any samples that were required were taken at this point. During magnetic stirring, the suspension was transferred in daily aliquots to the final containers. Remaining concentrations were prepared in ascending group order using the same method. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- Storage of preparation: Refrigerated (nominally 2-8 °C)
- Frequency of preparation: Weekly

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: The stability of an homogenous suspension of the test substance in the vehicle was demonstrated over a period of up to eight days at ambient temperature and for 15 days following refrigerated storage (2-8 °C) in a previous study (Huntingdon Life Sciences Report No. HIK0005).
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 13 of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 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:

Main phase: 10
Recovery phase: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels employed in this study (0, 100, 300 and 1000 mg/kg bw/day) were selected in conjunction with the Sponsor, and were based on the results of a previously conducted OECD 421 reproductive screening study in CD rats (Huntingdon Life Sciences Report No. HIK0005). In that study, the high dose level of 1000 mg/kg bw/day was well tolerated throughout the treatment period (at least four weeks for males and at least six weeks for females) and was concluded to be the No Observed Adverse Effect Level. In this OECD 408 study, the high dose level was therefore set at 1000 mg/kg bw/day. The intermediate dose level of 300 mg/kg bw/day and the low dose level of 100 mg/kg bw/day were selected to provide an approximate 3-fold decrease in dose levels to allow evaluation of a possible dose relationship for any treatment-related changes.
- Rationale for animal assignment: Randomly allocated on arrival
- Group 1, 2, 3 and 4: 0, 100, 300 and 1000 mg/kg bw/day, respectively.

Positive control:

None

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: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation; One to two hours after completion of dosing all groups; As late as possible in the working day.

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), then weekly thereafter throughout the study and before necropsy.

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. On several occasions during the dosing period, animals in all treated groups (but predominantly those in Group 4) showed an increase in water consumption. This was anticipated based on the nature of the test formulations and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- 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 - All Main phase animals; Week 4 of Recovery - All Recovery phase animals
- Anaesthetic used for blood collection: Yes; animals were held under light general anaesthesia induced by isoflurane
- Animals fasted: Yes; overnight withdrawal of food and water
- Blood sampling was performed on the morning after overnight collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Parameters checked:
Haematology: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser:
Haematocrit (Hct)*, Haemoglobin concentration (Hb)*, Erythrocyte count (RBC)*, Absolute reticulocyte count (Retic)*, Mean cell haemoglobin (MCH)*, Mean cell haemoglobin concentration (MCHC)*, Mean cell volume (MCV)*, Red cell distribution width (RDW)*, Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC); Platelet count (Plt), Morphology: Anisocytosis, Microcytosis, Macrocytosis, Hypochromasia, Hyperchromasia
* Additional investigations for selected parameters only during Week 4 of recovery; analysed for female animals.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser: Prothrombin time (PT)**; 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: Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of:
Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Total bile acids (Bi Ac), Urea, Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), d’3 hydroxybutyrate (3DHY), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 - All Main phase animals; Week 4 of Recovery - All Recovery phase animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; animals were placed in an individual metabolism cage overnight, without food or water. Urine samples were collected over approximately 16 h.
- Parameters checked:
Using manual methods: Appearance (App) ^ - by visual assessment, Volume (Vol) ^- using a measuring cylinder, pH ^ - using a pH meter, Specific gravity (SG) ^ - by direct refractometry using a SG meter
Using Multistix reagent strips, interpreted using a Clinitek®500 instrument: Glucose (Gluc), Ketones (Keto), Bilirubin/bile pigments (Bili), Urobilinogen (Urob), Blood pigments (UBld)
Using a Roche P Modular analyser: Protein (T-Prot) ^, Creatinine (T-Creat), Sodium (T-Na) ***, Potassium (T-K) ***, Chloride (T-Cl) ***
*** Additional investigations for selected parameters only during Week 4 of recovery; samples only taken for female animals.
^ Additional investigations for selected parameters only during Week 4 of recovery; samples taken for male and female animals.
A microscopic examination of the urine sediment was performed: Epithelial cells (Epi), Leucocytes (WBC), Erythrocytes (RBC), Crystals (Cryst), Casts, Other abnormal components (A), Spermatozoa (Sperm)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during each week of treatment, 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 (before dosing during the treatment period), by an observer unaware of the experimental group identities.
- Battery of functions tested: sensory activity / grip strength / motor activity
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed (before dosing) on the first five Main phase animals and all Recovery phase animals in Groups 1 and 4, and all Main phase animals from Groups 2 and 3 during Week 12 of treatment.
Motor activity: During Week 12 of treatment (before dosing), the motor activity of the first five Main phase animals and all Recovery phase animals in Groups 1 and 4, and all Main phase animals from Groups 2 and 3.

Sacrifice and pathology:

SACRIFICE: Animals were killed by carbon dioxide asphyxiation with subsequent exsanguination. 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. All Main phase and Recovery phase animals were subject to a detailed necropsy.

GROSS PATHOLOGY: Yes; 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 interval.
Abnormalities only: All Main phase animals of Groups 2 and 3.
Routine staining: Sections were stained with haematoxylin and eosin.
Recovery phase animals: Tissues from Recovery phase animals were retained in fixative pending results from initial examinations of tissues from Main phase animals.
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 only.

Other examinations:

None

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. Please refer to the filed "Any other information on materials and methods incl. tables" for more details.

Clinical signs:

no effects observed

Description (incidence and severity):

- There were no test article-related signs observed during the detailed physical examination and arena observations and no signs observed in relation to dose administration.

Mortality:

no mortality observed

Description (incidence):

- There were no premature deaths

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males given 1000 mg/kg bw/day showed low body weight gain during Week 0-1 and Weeks 6 13 of dosing, such that overall mean weight gain during the 13-week dosing period was 86% of Control; the mean weight gain of males receiving 100 or 300 mg/kg bw/day was unaffected. Following the cessation of dosing, the mean body weight gain of males previously given 1000 mg/kg bw/day was slightly higher than Control over the 4-week recovery period. The body weight performance of all groups of treated females was slightly variable throughout the study, such that overall mean weight gain from Week 0 to 13 of treatment was slightly higher than Control, although an obvious dose response was absent (114, 110 and 104 % of Control at 100, 300 and 1000 mg/kg bw/day, respectively). During the 4 week recovery period, the body weight performance of females previously given 1000 mg/kg bw/day was similar to Control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Mean food consumption in Week 1 of treatment for all groups of males and females was similar to that recorded during the pre-treatment period. From Week 2 to Week 13, there was a non-adverse trend towards marginally higher food intake among all groups of treated females and for males receiving 300 or 1000 mg/kg bw/day when compared to Controls, which continued for the first 2 or 3 weeks of the recovery period for those animals which had previously been given 1000 mg/kg bw/day, with partial recovery evident by Recovery Week 3 (females) or 4 (males).

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- Ophthalmoscopy assessments for all groups of animals during Week 12 were unaffected by treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Analysis of haematological parameters during Week 13 of treatment revealed lower than Control erythrocyte and haemoglobin concentrations and haematocrit in all groups of treated females, associated with a slightly lower than Control mean cell haemoglobin concentrations and a concomitant higher than Control red cell distribution width in females given 1000 mg/kg bw/day. All groups of treated males showed longer than Control prothrombin times and activated partial thromboplastin times, with some evidence of a dose-related trend. Conversely, females receiving 300 or 1000 mg/kg bw/day showed shorter than Control prothrombin times. All of these differences were no longer evident 4 weeks after the cessation of dosing, demonstrating full recovery.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- During Week 13, biochemical analysis of plasma revealed a trend towards slightly low aspartate amino-transferase concentrations in all groups of treated males and females. Slightly low alkaline phosphatase concentrations were apparent in all groups of treated females, and slightly high plasma urea and d’3 hydroxybutyrate concentrations in all groups of treated males and females. Creatinine concentrations were also slightly high in all groups of treated males and in females given 100 or 1000 mg/kg bw/day. For males given 300 or 1000 mg/kg bw/day glucose concentrations were slightly low and gamma glutamyl transpeptidase levels were slightly high, with slightly higher bilirubin concentrations also apparent in males given 1000 mg/kg bw/day. In the 1000 mg/kg bw/day group, low triglyceride, slightly high cholesterol and an increase in total protein was apparent in both sexes; among females, the increase was associated with an increase in albumin levels, but in males the increase was attributable to globulin levels resulting in a slight decrease in albumin/globulin ratio. Males given 300 mg/kg bw/day and males and females given 1000 mg/kg bw/day showed slightly low chloride concentrations; calcium concentrations were also marginally high in females given 1000 mg/kg bw/day. All of these differences showed partial or full recovery after 4 weeks off-dose.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Analysis of urine collected during Week 13 of treatment revealed darker and/or orange urine in all groups of treated males and females, and was associated with slightly lower than Control urinary pH for males given 300 mg/kg/day and for males and females given 1000 mg/kg bw/day. Slightly higher than Control specific gravity was evident among males and females receiving 1000 mg/kg bw/day, and females given 300 or 1000 mg/kg bw/day showed lower than Control sodium and chloride concentrations. All of these differences were no longer evident 4 weeks after the cessation of dosing. Microscopic examination of the urine sediment did not reveal any abnormalities at any dose level investigated.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organ weights following 13 weeks of treatment indicated a dose-dependent increase in absolute liver weight for females at 300 mg/kg/day, and for males and females at 1000 mg/kg/day when compared to Control; statistical significance was attained for the differences in body weight-adjusted liver weight in females given 300 mg/kg/day (118% of Control) and in males and females given 1000 mg/kg/day (124% and 152% of Control, respectively). Body weight-adjusted kidney weights were statistically higher than Control in males given 1000 mg/kg/day (108% Control) and slightly low body weight-adjusted epididymal weights were apparent for males given 300 or 1000 mg/kg/day (91% and 89% of Control, respectively). In addition, body weight-adjusted ovary weights were slightly high in females given 1000 mg/kg/day (117% of Control).
After 4 weeks of recovery, body weight-adjusted liver weights in males and females previously given 1000 mg/kg/day remained slightly higher than Control, with statistical significance only in males, however the magnitude of the difference (117% and 113% Control for males and females, respectively) was lower than that recorded at the end of the treatment period suggestive of partial recovery. In males previously given 1000 mg/kg/day, body weight-adjusted kidney weights remained higher than Control although statistical significance was not attained, whereas epididymal weights were similar to Control indicating full recovery. In contrast to the end of the dosing period, the ovary weights of females in the 1000 mg/kg/day group were statistically significantly lower than Control at the end of the recovery period (75% of Control).
It was noted that the body weight-adjusted spleen weight of males and the body weight adjusted uterus and cervix weight of females in the 1000 mg/kg/day group were statistically significantly higher than Control at the end of the recovery period. In the absence of similar differences at the end of the 13-week treatment period, these changes in organ weights were considered to be attributable to natural variation, and unrelated to previous treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- 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 test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- Histopathological evaluation of the retained tissues of the 1000 mg/kg bw/day animals killed after 13 weeks of treatment did not reveal any test article-related micropathological changes.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

Formulation analysis: The mean concentrations of test item in test formulations analysed for the study were within 5 % of nominal concentrations, confirming accurate formulation. The precision of individual results from mean values was less than 1 %, confirming accurate analysis.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Critical effects observed:

no

Result tables are included in "Attached background documents".

Conclusions:

Based on results of the study, NOAEL was concluded to be 1000 mg/kg bw/day.

Executive summary:

A 28-day repeat dose gavage study was performed in the rat (Fraunhofer, 2013). The maximum dose level was limited to 300 mg/kg bw/day based on the observation of ketones in urine from an old study performed on the supporting substance (1989). However, the highest dose level did not induce toxic effects on the registered substance and therefore should have been higher.

In addition, this 28-day study is not in accordance with Japan requirements for CSCL notification since it was not inspected by quality assurance unit.

Therefore, to submit the notification in Japan, a new 90-day study was conducted (HLS, 2015).

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, test material solution in corn oil was administered by gavage to 10 male and 10 female CD rats at doses of 100, 300 and 1000 mg/kg bw/day. A similarly constituted control group received the vehicle, corn oil. A further five male and five female rats were assigned to each of the control and high dose group. 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, urinalysis, organ weights, macropathology and histopathology were undertaken.

Clinical signs and mortality

There were no premature deaths, no test article-related signs observed during the detailed physical examination and arena observations and no signs observed in relation to dose administration.

Body weight and weight gain

Males given 1000 mg/kg bw/day showed low body weight gain during Week 0-1 and Weeks 6 13 of dosing, such that overall mean weight gain during the 13-week dosing period was 86% of Control; the mean weight gain of males receiving 100 or 300 mg/kg bw/day was unaffected. Following the cessation of dosing, the mean body weight gain of males previously given 1000 mg/kg bw/day was slightly higher than Control over the 4-week recovery period. The body weight performance of all groups of treated females was slightly variable throughout the study, such that overall mean weight gain from Week 0 to 13 of treatment was slightly higher than Control, although an obvious dose response was absent (114, 110 and 104 % of Control at 100, 300 and 1000 mg/kg bw/day, respectively). During the 4 week recovery period, the body weight performance of females previously given 1000 mg/kg bw/day was similar to Control.

Food consumption

Mean food consumption in Week 1 of treatment for all groups of males and females was similar to that recorded during the pre-treatment period. From Week 2 to Week 13, there was a non-adverse trend towards marginally higher food intake among all groups of treated females and for males receiving 300 or 1000 mg/kg bw/day when compared to Controls, which continued for the first 2 or 3 weeks of the recovery period for those animals which had previously been given 1000 mg/kg bw/day, with partial recovery evident by Recovery Week 3 (females) or 4 (males).

Ophthalmoscopic examination

Ophthalmoscopy assessments for all groups of animals during Week 12 were unaffected by treatment.

 

Haematology

Analysis of haematological parameters during Week 13 of treatment revealed lower than Control erythrocyte and haemoglobin concentrations and haematocrit in all groups of treated females, associated with a slightly lower than Control mean cell haemoglobin concentrations and a concomitant higher than Control red cell distribution width in females given 1000 mg/kg bw/day. All groups of treated males showed longer than Control prothrombin times and activated partial thromboplastin times, with some evidence of a dose-related trend. Conversely, females receiving 300 or 1000 mg/kg bw/day showed shorter than Control prothrombin times. All of these differences were no longer evident 4 weeks after the cessation of dosing, demonstrating full recovery.

 

Clinical chemistry

During Week 13, biochemical analysis of plasma revealed a trend towards slightly low aspartate amino-transferase concentrations in all groups of treated males and females. Slightly low alkaline phosphatase concentrations were apparent in all groups of treated females, and slightly high plasma urea and d’3 hydroxybutyrate concentrations in all groups of treated males and females. Creatinine concentrations were also slightly high in all groups of treated males and in females given 100 or 1000 mg/kg bw/day. For males given 300 or 1000 mg/kg bw/day glucose concentrations were slightly low and gamma glutamyl transpeptide levels were slightly high, with slightly higher bilirubin concentrations also apparent in males given 1000 mg/kg bw/day. In the 1000 mg/kg bw/day group, low triglyceride, slightly high cholesterol and an increase in total protein was apparent in both sexes; among females, the increase was associated with an increase in albumin levels, but in males the increase was attributable to globulin levels resulting in a slight decrease in albumin/globulin ratio. Males given 300 mg/kg bw/day and males and females given 1000 mg/kg bw/day showed slightly low chloride concentrations; calcium concentrations were also marginally high in females given 1000 mg/kg bw/day. All of these differences showed partial or full recovery after 4 weeks off-dose.

 

Urinalysis

Analysis of urine collected during Week 13 of treatment revealed darker and/or orange urine in all groups of treated males and females, and was associated with slightly lower than Control urinary pH for males given 300 mg/kg bw/day and for males and females given 1000 mg/kg bw/day. Slightly higher than Control specific gravity was evident among males and females receiving 1000 mg/kg bw/day, and females given 300 or 1000 mg/kg bw/day showed lower than Control sodium and chloride concentrations. All of these differences were no longer evident 4 weeks after the cessation of dosing. Microscopic examination of the urine sediment did not reveal any abnormalities at any dose level investigated.

 

Neurobehaviour

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

 

Organ weights

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 females given 300 mg/kg/day and in males and females given 1000 mg/kg/day. Body weight-adjusted kidney weights were higher than Control in males given 1000 mg/kg/day and low body weight-adjusted epididymal weights were apparent for males given 300 or 1000 mg/kg/day. In addition, body weight-adjusted ovary weights were slightly high in females given 1000 mg/kg/day. Following 4 weeks of recovery, body weight-adjusted liver weights in males and females previously given 1000 mg/kg/day remained slightly higher than Control, although the magnitude of the difference was lower than that recorded at the end of the treatment period suggestive of partial recovery. In males previously given 1000 mg/kg/day, body weight-adjusted kidney weights remained higher than Control, whereas epididymal weights were similar to Control indicating full recovery. In contrast to the end of the dosing period, the ovary weights of females in the 1000 mg/kg/day group were statistically significantly lower than Control at the end of the recovery period.

 

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 test item.

 

Histopathology

Histopathological evaluation of the retained tissues of the 1000 mg/kg bw/day animals killed after 13 weeks of treatment did not reveal any test article-related micropathological changes.

Plasma biochemistry and analysis of urine 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 and urine parameters were considered not to be adverse. 

Based on results of the study, the NOAEL was concluded to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1).

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

A 28-day repeat dose gavage study was performed in the rat (Fraunhofer, 2013). The maximum dose level was limited to 300 mg/kg bw/day based on the observation of ketones in urine from an old study performed on the supporting substance (1989). However, the highest dose level did not induce toxic effects on the registered substance and therefore should have been higher. In addition, this 28-day study is not in accordance with Japan requirements for CSCL notification since it was not inspected by the quality assurance unit. Therefore, to submit the notification in Japan, a new 90-day study was conducted (HLS, 2015).

The 90-day study is considered as the key study for repeated dose toxicity endpoint.

The 90-day study was performed according to the OECD Test Guideline No. 408 and in compliance with GLP. Groups of male and female Crl:CD(SD) rats received daily oral (gavage) administration at 0, 100, 300 or 1000 mg/kg bw/day. Administration of the test material was well tolerated, no premature deaths occurred, there were no test article-related clinical signs observed or adverse effects on body weight, food consumption, sensory reactivity, grip strength, motor activity, ophthalmoscopy, haematology, blood chemistry, urinalysis, organ weights or macropathology. There were no target organs identified during the micropathological evaluations.

In males given 1000 mg/kg bw/day, low body weight gain was observed during Week 1 and from Week 6-13 of dosing when compared to Control. During the full 13-week dosing period, the weight gain of these males was only 14% lower than Controls, and as there was no impact on the clinical condition of these animals and the weight gain observed in the 4-week recovery period was slightly higher than Control, the lower weight gain observed during the dosing period was considered not to be adverse.

All groups of females given the test material and males given 300 or 1000 mg/kg bw/day showed slightly higher than Control food intake from Week 2-13 of dosing. These differences were considered to have occurred as a consequence of slightly low food intake by the Control animals due to the slightly greater nutritional benefit that the Control animals gained from the administration of a 100% corn oil. Anticipated food consumption was apparent in test and control animals following the cessation of treatment.

At the end of the dosing period several minor test article-related changes in blood plasma and urine composition were apparent that indicated effects on the liver and kidneys; these were supported by increases in absolute and body weight-adjusted liver weight in females given 300 mg/kg bw/day and both sexes at 1000 mg/kg bw/day and slightly increased body weight adjusted kidney weight in males given 1000 mg/kg bw/day. Plasma indicators of liver effects comprised changes in enzyme activity and concentrations of bilirubin, proteins, cholesterol and triglycerides. Markers of kidney effects comprised slight increases in urea, creatinine and d’3 hydroxybutyrate concentrations and slightly decreased chloride concentrations in blood plasma, and low pH, increased specific gravity and changes in protein, sodium and chloride concentrations in the urine. Despite these indicators of effects on the liver and kidney, there were no changes in the general clinical condition of the animals and no micropathological abnormalities in either of these organs. Assessments conducted following the 4-week off-dose period showed that full or partial recovery was evident for the blood chemistry and urinalysis changes. It was therefore concluded that these changes were a normal adaptive metabolism/excretion response in the liver and kidneys; such disturbances of biochemical and urine parameters following administration of a xenobiotic in the absence of corroborative pathology or functional change of the organs is considered not to be adverse.

Based on results of the study, the NOAEL was concluded to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to the Regulation (EC) No 1272/2008.