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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

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
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Official notice of Japan MHLW, METI and ME (31 March 2011), YAKUSHOKUHATSU No. 0331. 7, SEIKYOKU No. 5, KANPOKIHATSU No. 110331009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2014; signature: September 2014
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one
EC Number:
700-956-5
Molecular formula:
not applicable (reaction mass of constitutional isomers)
IUPAC Name:
Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: liquid
- Storage condition of test material: Refrigerator (ca. 4°C) in the dark under nitrogen.
- Other: Pale yellow

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Age at study initiation: approximately 44 - 57 days
- Weight at study initiation: males 233 293 g (groups 1, 2 and 3) and 285 - 367 g (groups 2 and 5); females 187 - 233 g (groups 1, 2 and 3) and 201 - 242 g (groups 2 and 5). All were within +/- 20 % of the mean.
- Fasting period before study: None
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals; group housed by sex but dispersed in batteries so possible environmental influences were equilibrated. Aspen chew block and plastic shelter provided to each cage and replaced when necessary, for enrichment.
- Diet (e.g. ad libitum): Rat and Mouse No. 1 Maintenance Diet ad libitum
- Water (e.g. ad libitum): ad libitum (except during urine collection)
- Acclimation period: 15 days before commencement of treatment for Groups 1, 3 and 4 and 22 days for Groups 2 and 5.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 (monitored between: 19-23 ºC; highest temperature attained 24 °C; with no significant impact on the study)
- Humidity (%): 55 +/- 10 (monitored between: 40 - 70%)
- Air changes (per hr): Not reported. Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2014-03-20 To: 2014-04-17

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Approximately 50% of the final volume of vehicle was added to the required amount of test substance and magnetically stirred to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance. Prepared weekly and referigerated (nominally 4°C). Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. Corn oil formulations was assessed and confirmed at nominal concentrations of 1 mg/mL and 200 mg/mL, during ambient and refrigerated storage.
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles,during magnetic stirring for 2 hours, ambient temperature storage for up to 24 hours and refrigerated storage for up to 17 days.
- The mean concentrations in test formulations analysed for the study were within +10/-15% of nominal concentrations, with the exception of Week 1 Group 2, confirming accurate formulation; subsequent analyses for Group 2 showed acceptable achieved concentration. The precision of individual results from mean values was less than 2%, confirming the precision of analysis.
- The analysis consisted of GC FID analysis with internal calibration (within a dedicated formulation analysis report attached to the full study report). A resentative sample of test formulation (1 mL, accurately weighed) and dissolved using ultrasonic vibration in a suitable volume of acetone. The extract was diluted using acetone, to provide a solution containing test item at an expected concentration of 40 μg/mL. Solutions also contained internal standard solution at 20 μg/mL, by adding the appropriate amount of internal standard to each sample before making to volume. The internal standard was 1 μL/mL tetradecane (> 99%) in acetone.
Duration of treatment / exposure:
Minimum period 28 days followed by a 14 day recovery period. The last dose was administered on Day 28.
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control Group (corn oil)
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low Group - 30 mg/kg/day (GHS Category 2 threshold dose)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate Group - 300 mg/kg/day (GHS Category 1 threshold dose)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Group - 1000 mg/kg/day (regulatory limit dose)
No. of animals per sex per dose:
10 per sex per dose (5 male / 5 female); 10 per sex per dose for recovery phase groups
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 7-day sighting study (Report number attached to and citedi n the full study report).
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: 14 days.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (a.m. and p.m. on working days, a.m. on weekends and holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Examination was carried out daily, and observations were recorded at least weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- 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: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily. No effect was observed and consequently quantitative measurements were not performed. Minor deviation in that was not assessed on one day. However, has no impact on the study oucome.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examined prior to dosing, towards the end of the treatment period and at the end of the recovery period.
- Dose groups that were examined: All test animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period / recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (food was witheld overnight prior to blood removal)
- How many animals: All animals
- Parameters checked: Haematocrit, Haemoglobin concentration, Erythrocyte count, Absolute reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count; 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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment period / recovery period
- Animals fasted: Yes (food was witheld overnight prior to blood removal)
- How many animals: All animals
- Parameters checked: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyl transferase, Total bilirubin, Bile acid, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Total protein, Albumin. A/G ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: End of treatment period / recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food and water witheld during time of urine collection)
- Parameters checked: urine volume, urine colour, urine density, pH, ketones, bilirubin, urobilnogen, blood pigments, protein, sodium, potassium, chloride, creatinine, glucose. Microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Casts, Spermatozoa, Other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: body, brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, spleen, thyroid.
- Organs and tissues preserved in neutral buffered 4% formalin: skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland (both), liver, pancreas, oesauphagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), kidney (both), urinary bladder, prostrate, seminal vesicles, testes, epididymis (both), uterus, vagina, ovaries, pituitary gland, adrenal glands, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), zymbal gland (both), muzzle, tongue, any tissue with gross lesions.

HISTOPATHOLOGY: Yes
- microscopic analysis of: spleen, mesenteric lymph node, axillary lymph node, femur with joint (including bone marrow), trachea, lung, heart, aorta, submandibular salivary gland (both), liver, pancreas, esophagus, stomach, small intestine (duodenum, ileum, jejunum), large intestine (cecum, colon, rectum), kidney (both), urinary bladder, testis (both), epididymis (both), uterus, vagina, ovary (both) ,pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve (sciatic nerve), brain (including medulla, pons, cerebral cortex, cerebellar cortex), any organ with gross lesions.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analysed at each timepoint separately:
Grip strength and motor activity
Body weight, using gains over appropriate study periods
Haematology
Blood chemistry
Urinalysis
Organ weights, absolute and adjusted for terminal body weight
The following comparisons were performed:
Group 1 vs Groups 3 and 4 for Main study animals
Group 5 vs Group 2 for Main study animals
Group 1 vs Group 4 for Recovery phase animals
A sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, organ weight and clinical pathology data was performed. Not limited to A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. Departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. Specific statistical analyses were completed for grip strength, motor activity and clinical pathology data e.g. Fisher’s Exact tests and/or organ weights - analysis of covariance was performed using terminal body weight as covariate.
Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No death and no clinical sign of reaction to treatment.
Mortality:
no mortality observed
Description (incidence):
No death and no clinical sign of reaction to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related lower than control bodyweight gain was apparent in males treated at 1000 mg/kg/day during the first two weeks of treatment; these animals showed slightly higher than control gain during the last two weeks of treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination after 28 days of treatment revealed treatment-related increased platelet numbers and prothrombin times in males treated at 1000 mg/kg/day; there was evidence of recovery.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Examination of the plasma after 28 days of treatment revealed treatment-related electrolyte changes (low chloride and high calcium and phosphorous concentrations) in males receiving 1000 mg/kg/day; there was evidence of recovery.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis revealed a treatment-related increase in acidity amongst males treated at 300 or 1000 mg/kg/day and a treatment-related decreased potassium and creatinine concentrations in females treated at 1000 mg/kday; there was evidence of recovery.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related higher than Control liver weights were evident amongst males and females treated at 300 or 1000 mg/kg/day; there was evidence of recovery.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination revealed no test substance related lesion
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology changes related to treatment were seen in the liver (centrilobular hypertrophy in males and females treated at 1000 mg/kg/day) and kidneys (hyaline droplets in males treated at 300 or 1000 mg/kg/day) but recovered completely.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Due to the reversible nature of the histopathology findings, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg/day and, since these findings are either adaptive or have no relevance to human health

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

All findings were determined to be either adaptive and/or reversible or have no relevance for human health.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is defined as 1000 mg/kg body weight per day in males and females; findings were either adaptive or have no relevance for human health.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and ME (2011) guidelines under GLP conditions. Following a previously conducted 7-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 30, 300 or 1000 mg/kg/day. Two similarly constituted control groups received the vehicle, corn oil, at the same dose volume. The animals were treated for 28 days to assess the oral toxic potential of the test substance. A further five male and five female rats were assigned to one of the control and the high dose groups (Groups 1 and 4). These animals were treated for 28 days, followed by a 14 day period without treatment to assess the potential for any treatment-related change to recover. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

There was no death and no clinical sign of reaction to treatment. There was no effect of treatment on sensory reactivity observations, grip strength or motor activity. Treatment-related lower than control bodyweight gain was apparent in males treated at 1000 mg/kg/day during the first two weeks of treatment; these animals showed slightly higher than control gain during the last two weeks of treatment. Bodyweight gain of males treated at 30 or 300 and of females was considered to have been unaffected by treatment. Treatment-related higher than control food consumption, associated with the increased body weight gain, was apparent in males treated at 1000 mg/kg/day during the last two weeks of treatment. Food consumption of males treated at 30 or 300 and of females was considered to have been unaffected by treatment. Daily visual assessment of the water bottles did not reveal any effect of treatment. Haematological examination after 28 days of treatment revealed treatment-related increased platelet numbers and prothrombin times in males treated at 1000 mg/kg/day; there was evidence of recovery. Examination of the plasma after 28 days of treatment revealed treatment-related electrolyte changes (low chloride and high calcium and phosphorous concentrations) in males receiving 1000 mg/kg/day; there was evidence of recovery. Analysis of the urine after 28 days of administration revealed a treatment-related increase in acidity amongst males treated at 300 or 1000 mg/kg/day and a treatment-related decreased potassium and creatinine concentrations in females treated at 1000 mg/kg/day; there was evidence of recovery. Treatment-related higher than Control liver weights were evident amongst males and females treated at 300 or 1000 mg/kg/day; there was evidence of recovery. The macroscopic examination revealed no test substance related lesion. Histopathology changes related to treatment with the test item were seen in the liver (centrilobular hypertrophy in males and females treated at 1000 mg/kg/day) and kidneys (hyaline droplets in males treated at 300 or 1000 mg/kg/day) but recovered completely following the off-dose period.

Under the conditions of this study, due to the reversible nature of the histopathology findings, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg/day and, since these findings are either adaptive or have no relevance to human health, they can be excluded when consideration of the NOEL is made.