5,9-dimethyldec-4-enal

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-02-2016 to 06-06-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2016 ; signature: October 2016

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-5,9-dimethyldec-4-enal; (4Z)-5,9-dimethyldec-4-enal

EC Number:

943-495-7

Cas Number:

689-65-6

Molecular formula:

C12H22O

IUPAC Name:

(4E)-5,9-dimethyldec-4-enal; (4Z)-5,9-dimethyldec-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark
- Other: clear colourless

Test animals

Species:

rat

Strain:

other: Crl:CD (SD) IGS BR

Details on species / strain selection:

The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 6 to 8 weeks
- Weight at study initiation: males 211 - 315 g and females 176 - 230 g; individuals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Fasting period before study: None
- Housing: Polypropylene body with a stainless steel mesh lid with softwood flake bedding, changed at appropriate intervals; group housed (5 per group) by sex. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. Cage distribution within the holding rack was randomized.
- Diet (e.g. ad libitum): Rodent 2014C, Global Certified Diet, ad libitum
- Water (e.g. ad libitum): ad libitum (except during urine collection)
- Acclimation period: 5 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent 2014C, Global Certified Diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2016-03-02 To: 2016-04-13

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Arachis oil BP was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable for at least ten days when stored refrigerated. Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored at approximately 4 ºC in the dark.
- Concentration in vehicle: Samples of the test item formulations were taken on three occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within ± 5% of the nominal concentration. Arachis oil formulations was assessed for homogeneity and confirmed at nominal concentrations of 3.75 mg/mL and 250 mg/mL, during refrigerated storage. Furthermore the stability of the test item in Arachis Oil was assessed at nominal concentration of 7.5 mg/mL and 250 mg/mL during refrigerated storage. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 4 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group
- Other: Dose-formulations were analysed during the study and were reported as with ± 10% applied limits.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity and stability was confirmed in Arachis oil BP formulations at nominal concentrations of 3.75 mg/mL and 250 mg/mL , during refrigerated storage for at least ten (10) days.
- Refrigerated formulations were also analysed after refrigeration on receipt, then on day 10: the formulation was removed from storage and equilibrated to ambient temperature. The formulations were mixed according to mixing procedure (as used in the definitive test) and single samples were removed for analysis from the top, middle and bottom of the mixed formulation.
- The analysis consisted of GC FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). Samples of Arachis oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These were then subjected to analysis by GC FID and using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report).
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery control group

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low – Group

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High – Group

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Recovery High – Group

No. of animals per sex per dose:

5 per sex per dose (5 male / 5 female); 5 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 cited in the full study report). Dose levels were selected following 7-day sighting test as: Group 1: 0 mg/kg/day (Arachis oil BP) Group 2: 30 mg/kg/day, Group 3: 300 mg/kg/day Group 4: 1000 mg/kg/day (Treatment Group - High). In the 7-day range finder (administered consecutively, for 7-days) the following effects were determined was using a range finding dose range of 250, 500 and 1000 mg/kg/day: Increased salivation was evident in all females and one male treated with 1000 mg/kg bw/day on Day 7. No such effects were detected in animals of either sex treated with 500 or 250 mg/kg bw/day. Males from all treatment groups and females treated with 1000 and 500 mg/kg bw/day showed an increase in overall water consumption; however a true dose related response was not evident. No such effects were detected in females treated with 250 mg/kg bw/day. For organ weights: Males from all treatment groups and females treated with 1000 and 500 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weights. No such effects were detected in females treated with 250 mg/kg bw/day. It was considered that there were no adverse effects to exclude 1000 mg/kg bw/day as a dose level. Basis: other: nominal in vehicle (Arachis Oil BP).
- 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All individuals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment free period, animals were observed daily. All observations were recorded. Additional functional observations were made as ‘special evaluations’. This was prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to termination and, in the case of recovery group animals, on Days 36 and 43 prior to termination.

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: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

FOOD EFFICIENCY: Yes.
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily. Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically. A possible intergroup difference was detected during Week 3, therefore water consumption was continued to be measured and recorded for each cage group until the termination.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed. Additional histopathology was conducted on eyes after termination.
- Dose groups that were examined: All test animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all non-recovery test and control group individuals. End of recovery period (day 42) for all recovery group individuals.
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No.
- How many animals: All animals
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices – including: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count – including: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic). Additionally: Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all non-recovery test and control group individuals. End of recovery period (day 42) for all recovery group individuals.
- Animals fasted: No.
- How many animals: All animals
- Parameters checked: Urea, Aspartate aminotransferase (ASAT), Glucose, Alanine aminotransferase (ALAT), Total protein (Tot.Prot.), Alkaline phosphatase (AP), Albumin, Creatinine (Creat), Albumin/Globulin (A/G) ratio (by calculation), Total cholesterol (Chol), Sodium (Na+), Total bilirubin (Bili), Potassium (K+), Triglycerides (Tri), Chloride (Cl-), Bile acids, Calcium (Ca++), Gamma glutamyltranspeptidase, Inorganic phosphorus (P)

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld during time of urine collection; overnight)
- Parameters checked: urine volume, urine appearance, 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: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- organs weighed: Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles, Uterus with Cervix (with coagulating glands and fluids)

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in neutral buffered 10% formalin: Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and Rectum pons), Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides (Preserved in modified Davidson’s fluid), Skin, Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes (fixed in Davidson’s fluid), Gross lesions, Spleen, Heart, Stomach, Ileum ,Testes (Preserved in modified Davidson’s fluid), Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver,Trachea, Lungs (with bronchi) - inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina, Muscle (skeletal).
Microscopic analysis was conducted thereof. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
- Other: Since there were indications of treatment-related adrenal changes in both sexes, liver (both sexes) and thyroid (females), examination was subsequently extended to include similarly prepared sections of the adrenals (both sexes), liver (both sexes) and thyroid (females) in the low (30 mg/kg bw), intermediate (300 mg/kg bw) and recovery groups.

Other examinations:

- Thyroid Hormone Assessment: At termination, blood samples were taken from the exsanguination procedure and the plasma
from each animal was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Where appropriate, data transformations were performed using the most suitable method. Data were analysed using the decision tree from proprietary tables and statistics modules incorporating, homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day dose level: Animals of either sex treated with 1000 mg/kg bw/day showed increased salivation post dosing between Days 11 and 25. No such effects were detected in animals of either sex treated with 300 or 30 mg/kg bw/day.

Other clinical observations were considered by type and due to isolated nature and/or presence in control animals to be incidental findings.

There were no treatment-related changes in the behavioural parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance.

During the treatment-free period, no clinical observations were detected for any of the survivors in the recovery group..

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no adverse effects detected in body weight development at any dose level.

Recovery females that were previously given 1000 mg/kg bw/day showed a statistically significant (p<0.05) reduction in body weight gain during the first week of the treatment free period. No such effects were detected in any treated animal during the treatment period and body weight gain in these recovery females improved during the final week of the treatment free period. The intergroup difference was therefore considered not to be of toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related effects on food consumption or food conversion efficiency.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no adverse effects on food consumption or food conversion efficiency.

A reduction in food conversion efficiency was evident in recovery females that were previously given 1000 mg/kg bw/day during the first week of the treatment-free period however this was considered to be the result of the reduced body weight gain evident in these females during this period and was considered not to be of toxicological significance.

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

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg bw/day showed an increase in overall water consumption (40% for males and 24% for females) during the final two weeks of the treatment period. Recovery was evident in females previously treated with 1000 mg/kg bw/day during the treatment free period however males continued to show an increase, albeit to a lesser extent (21%), during the treatment free period.

No such effects were detected in animals of either sex treated with 300 or 30 mg/kg bw/day.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected in the hematological parameters examined at the end of the treatment or treatment-free periods.

Males treated with 1000 mg/kg bw/day showed statistically significant increases (p<0.05 - 0.01) in hemoglobin, erythrocyte count and hematocrit. Females from this treatment group showed statistically significant reductions in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration and a statistically significant increase in neutrophils. Whilst these findings may indicate some minor changes in red blood cells for animals of either sex treated with 1000 mg/kg bw/day, the affected parameters were generally not consistent between the two sexes. There were no corresponding intergroup differences for recovery animals of either sex at the end of the treatment-free period and in the absence of any associated histopathological findings in the bone marrow or spleen, these observations were considered to be of no toxicological relevance.

At the end of the treatment-free period, group mean lymphocyte values in females previously treated with 1000 mg/kg bw/day were statistically significantly (p<0.05) increased. In the absence of a similar effect in animals of either sex at the end of the treatment period, the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day: males showed a statistically significant increase in albumin and statistically significant reductions in glucose, triglycerides and bilirubin. Females treated also showed a statistically significant reduction in bilirubin.

No toxicologically significant effects were detected in animals of either sex treated with 300 or 30 mg/kg bw/day.

Urinalysis findings:

no effects observed

Description (incidence and severity):

At up to 1000 mg/kg bw/day: urinalysis evaluations at the end of the treatment or treatment-free periods did not identify any effects of toxicological importance in males or females receiving the test item.

The minor intergroup variations evident in the parameters measured did not follow a dose related response, were not consistent between sexes or did not have any associated renal microscopic changes evident, therefore these were considered to represent normal biological variation.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the behavioural parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day: males and females showed a statistically significant increase (p<0.05 - 0.01) in liver weight both absolute and relative to terminal body weight. No such effects were seen at 300 or 30 mg/kg bw/day. At 1000 mg/kg bw/day, similar differences were not evident following two weeks of recovery, indicating full reversibility of these changes.

The apparent slight increase in males receiving 30 mg/kg bw/day reflected one male with an incidentally large liver. This was not considered as related to treatment. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any degenerative or inflammatory changes is not classed as hepatotoxicity it was considered to be an adaptive change or considered to reflect a metabolic disturbance that is a result of hepatic enzyme induction.

All other observations (reduction in mean ovary weight, spleen weight, an isolated ovary weight increase and in the recovery males minimal significance increase in heart and reduction in brain weights) whilst having statistical significance did not demonstrate true dose related responses and due to similar observations in animals at the end of the treatment period plus absence of histopathological correlates and/or consideration of intergroup differences were not considered to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examination revealed no test substance related lesion.

One male treated with 1000 mg/kg bw/day had an enlarged liver and enlarged kidneys. Microscopic examination of this male did not reveal any associated histopathological changes, therefore the intergroup differences were considered to be incidental.

One male treated with 30 mg/kg bw/day had a mottled and enlarged liver, enlarged lymph nodes and an enlarged spleen. Microscopic examination only revealed midzonal vacuolation in the liver. In the absence of a similar effect at 1000 mg/kg bw/day, these findings were considered to be incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Adrenals: mild cortical vacuolation was evident in three males treated with 1000 mg/kg bw/day and in one male treated with 300 mg/kg bw/day. No such effects were detected in males treated with 30 mg/kg bw/day. Following fourteen days without treatment, one recovery male that was previously given 1000 mg/kg bw/day showed mild cortical vacuolation.

Hypertrophy of the zona glomerulosa was evident in three males and three females treated with 1000 mg/kg bw/day and in one male treated with 300 mg/kg bw/day. No such effects were detected in females treated with 300 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day. Following fourteen days without treatment, three recovery females that were previously given 1000 mg/kg bw/day showed hypertrophy of the zona glomerulosa.

Adrenal cortical vacuolation is seen occasionally at a low level as a background change, usually in males therefore the etiology is not clear. Hypertrophy of the zona glomerulosa of the adrenal gland is seen occasionally as a treatment related finding generally considered to be adaptive and linked to fluid and/or electrolyte balance. This may correlate with the increased water consumption noted in animals of either sex treated with 1000 mg/kg bw/day. One male at 300 mg/kg bw/day had both adrenal vacuolation and zona glomerulosa hypertrophy. As this finding is occasionally observed in control animals, an association with treatment could not be proven and was therefore considered incidental.


- Liver: minimal centrilobular hepatocyte hypertrophy was evident in three males and three females treated with 1000 mg/kg bw/day. No such effects were detected in animals of either sex treated with 300 or 30 mg/kg bw/day and this change was not present in recovery animals that were previously given 1000 mg/kg bw/day following fourteen days without treatment.

Similar differences in the minimal centribolular hepatocyte hypertrophy and increased absolute and relative liver weights were not evident following two weeks of recovery, indicating full reversibility of these changes. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any degenerative or inflammatory changes is not classed as hepatotoxicity but considered to be an adaptive change or considered to reflect a metabolic disturbance that is a result of hepatic enzyme induction.


- Thyroids: minimal follicular cell hypertrophy was evident in four females treated with 1000 mg/kg bw/day. No such effects were detected in treated males, or in females treated with 300 or 30 mg/kg bw/day and this change was not present in recovery females that were previously given 1000 mg/kg bw/day following fourteen days without treatment.

Microscopic examination of the thyroid revealed follicular epithelial hypertrophy in females treated with 1000 mg/kg bw/day. The morphological changes seen in the thyroid were considered to be an adaptive physiologic response of the thyroid gland to hepatic enzyme induction and represent a well understood and familiar response in rats, treated with high doses of xenobiotics (Capen, 2002, Hall, 2012, Zabka, 2011).

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected. There were no microscopic findings that were considered to be related to treatment with the test item.

Other effects:

no effects observed

Description (incidence and severity):

1. Thyroid Hormone Assessment: blood samples were taken at exsanguination and the plasma was stored frozen. No treatment-related effects on the pituitary-thyroid axis were identified and therefore further analysis was not conducted.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Remarks:

Applicant assessment: All findings were determined to be either of limited toxicological significance, adaptive changes and/or were reversible or have no relevance for human health.

Target system / organ toxicity

Key result

Critical effects observed:

no

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.

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 MOE 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) IGS BR 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. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination at termination. Histopathological examination of selected tissues was performed. There were no unscheduled deaths. Males and females treated at 1000 mg/kg bw/day showed increased salivation post dosing between Days 1 and 25. No effects were seen at 30 or 300 mg/kg bw/day. There were no treatment-related changes in the behavioural parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance. No adverse effects were detected in body weight development. There were no treatment-related effects on food consumption or food efficiency. Males and females treated with 1000 mg/kg bw/day showed an increase in overall water consumption during the final two weeks of the treatment period. Recovery was evident in females previously treated with 1000 mg/kg bw/day during the treatment free period however males continued to show an increase, albeit to a lesser extent, during the treatment free period. No such effects were detected at 300 or 30 mg/kg bw/day. No toxicologically significant effects were detected in the hematological parameters at the end of the treatment or treatment free periods. Males treated with 1000 mg/kg bw/day showed a statistically significant increase in albumin and statistically significant reductions in glucose, triglycerides and bilirubin. Females treated with 1000 mg/kg bw/day also showed a statistically significant reduction in bilirubin. No such effects were detected at 300 or 30 mg/kg bw/day. There were no treatment-related effects of toxicological importance detected in the urinalytical parameters. There were no treatment-related macroscopic abnormalities detected. Males and females treated at 1000 mg/kg bw/day showed statistically significant increase in liver weight both absolute and relative to terminal body weight. No such effect was seen at 300 or 30 mg/kg bw/day or in recovery groups following 14 days without treatment. In the histopathology assessment: in the adrenal glands: mild cortical vacuolation was evident in three males treated with 1000 mg/kg bw/day and in one male treated with 300 mg/kg bw/day. No such effects were detected in males treated with 30 mg/kg bw/day. Following fourteen days without treatment, one recovery male that was previously given 1000 mg/kg bw/day showed mild cortical vacuolation. Hypertrophy of the zona glomerulosa was evident in three males and three females treated with 1000 mg/kg bw/day and in one male treated with 300 mg/kg bw/day. No such effects were detected in females treated with 300 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day. Following fourteen days without treatment, three recovery females that were previously given 1000 mg/kg bw/day showed hypertrophy of the zona glomerulosa. In the liver: minimal centrilobular hepatocyte hypertrophy was evident in three males and three females treated with 1000 mg/kg bw/day. No such effects were detected in animals of either sex treated with 300 or 30 mg/kg bw/day. In the thyroids: minimal follicular cell hypertrophy was evident in four females treated with 1000 mg/kg bw/day. No such effects were detected in treated males, or in females treated with 300 or 30 mg/kg bw/day. All other histological changes were considered to be unrelated to treatment. The oral (gavage) administration of the test item to males/females at dose levels of 30, 300 or 1000 mg/kg bw/day resulted in treatment-related changes in animals of either sex treated with 1000 mg/kg bw/day. No treatment-related changes were evident at 300 or 30 mg/kg bw/day. The microscopic adrenal, liver and thyroid changes and associated blood chemistry changes identified in animals of either sex treated with 1000 mg/kg bw/day were all considered to represent adaptive changes and were considered not to represent an adverse toxicological finding. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg/day for males/females.