Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to relevant guidelines 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'.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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'.
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: 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
Principles of method if other than guideline:
The micronucleus assessment phase of the study was compatible with the following regulatory guidelines:
1. OECD Guidelines for Testing of Chemicals No. 474 “Mammalian Erythrocyte Micronucleus Test” (adopted 26 September 2014)
2. Commission Regulation (EC) 440/2008 Method B12 “Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test”, dated May 30, 2008
3. US EPA (TSCA) OPPTS 870.5395 “Mammalian Erythrocyte Micronucleus Test” EPA 712-C-98-226, August 1998
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2016 ; signature: October 2016
Limit test:
yes
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 205 - 311 g and females 139 - 215 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: 2016-03-04 To: 2016-04-15
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 two 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 ± 3% of the nominal concentration. Arachis oil formulations was assessed and confirmed at nominal concentrations of 3.75 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; positive control was 10 mL/kg (The positive control for Micronucleus Assessment was shared with a parallel study: full details available in the full study report). For further information see 'Doses / concentrations'.
- 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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Recovery control group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low – Group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Intermediate – Group
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
High – Group
Dose / conc.:
400 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: 150 mg/kg/day Group 4: 400 mg/kg/day (Treatment Group - High). In the 7-day range finder (administered consecutively, for 8-days) the following effects were determined was using a range finding dose range of 100, 300 and 500 mg/kg/day: increased salivation post dosing at 500 mg/kg bw/day and an effect on body weight performance and food consumption in males at 500 and 300 mg/kg bw/day. An increase in water consumption was also evident in females treated with 500 mg/kg bw/day. The plasma analysis indicated exposure of the bone marrow to the test item, and therefore the suitability of incorporating a micronucleus assessment as part of a 28 day toxicity study. 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.
Positive control:
Positive control males: five (5) males - single dose were used for micronucleus test, treated with cyclophosphamide at 25 mg/kg bw/day - dosed on Day 28 only. No concurrent positive control was used for other effects. The PC group was shared with another study. Full details provided in the full study report.
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 400 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 males, examination was subsequently extended to include similarly prepared sections of the adrenals from males in the low (30 mg/kg bw), intermediate (150 mg/kg bw) and recovery groups.
Other examinations:
- Thyroid Hormone Assessment: blood samples were taken at exsanguination and the plasma was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

- Micronucleus assessment (non-recovery groups only):
- Following termination (i.e. 24 hours following dosing), from the left femur: fetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald / Giemsa, allowed to air-dry and a cover slip applied using mounting medium. Separate positive control slides were prepared. Stained bone marrow smears were coded and examined blind using light microscopy.
- The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
- An additional group of five males were given a single oral dose of cyclophosphamide (25 mg/kg) , to serve as a positive control. This was a positive control group for micronucleus assessment, shared with another study. Full details are available in the full study report.
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, Micronucleus results, 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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg bw/day dose level: Animals of either sex treated with 400 and 150 mg/kg bw/day showed increased salivation post dosing between Days 6 and 28 and Days 17 and 26 (respectively). Four males and four females treated with 400 mg/kg bw/day also showed incidences of body tremors between Days 18 and 24. No such effects were detected in animals of either sex treated with 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. Therefore there was evidence of recovery.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 400 mg/kg bw/day dose level: One female treated with 400 mg/kg bw/day that was designated as a recovery animal was found dead on Day 2. This animal did not show any clinical signs prior to being found dead nor were there any macroscopic or microscopic findings that could account for the death. There were no further unscheduled deaths. In view of the nature of the clinical signs (body tremors) evident later in the study in surviving animals at this dose level, an association to treatment for this death at 400 mg/kg bw/day cannot be excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg bw/day: males showed a reduction in body weight gain during Weeks 1, 3 and 4. Statistical significance however was not achieved. Females from this treatment group showed a reduction in body weight gain during the final week of treatment with five females showing an actual body weight loss. No adverse effects were evident in food consumption for these animals, although a reduction in food efficiency was evident in females during Week 4 and increased water consumption was evident in animals of either sex. No effects were observed at 150 or 30 mg/kg bw/day.

Following the cessation of treatment, body weight gain in animals of either sex that had previously been given 400 mg/kg bw/day was generally greater than controls, with statistical significance achieved during the final week of the treatment free period.

Following the cessation of treatment recovery in body weight gain and water consumption was evident in animals of either sex that were previously given 400 mg/kg bw/day. Therefore, there was limited effects seen on bodyweight post-exposure and evidence of recovery at all dose levels in males and females.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no adverse effects on food consumption or food conversion efficiency.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg bw/day: males and females showed an increase in overall water consumption during the final two weeks of the treatment period. Males and females at 150 mg/kg bw/day and females treated with 30 mg/kg bw/day also showed a slight increase in overall water consumption, however a true dose related response was not evident in females from these groups. No such effects were detected in males treated with 30 mg/kg bw/day.

Following the cessation of treatment, males/females that had previously been given 400 mg/kg bw/day showed water consumption that was comparable to controls. There was evidence of recovery.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined at the end of the treatment or treatment-free periods.

Males from all treatment groups showed a statistically significant increase (p<0.01) in platelet counts whilst females from all treatment groups showed a statistically significant reduction (p<0.05) in prothrombin time. A true dose related response was not evident for either parameter nor were these findings observed in the other sex, therefore in the absence of any associated histopathological findings, these observations were considered to be of no toxicological relevance.

Although there were some statistically significant differences in treated animals from controls for hematological parameters measured, these differences were seen in the absence of any associated histopathological correlates and were considered not to be of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the blood chemical parameters examined at the end of the treatment or treatment-free periods.

For example: Males treated with 400 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase (p<0.05) and bile acids (p<0.01) whilst females from this treatment group showed a statistically significant increase in bilirubin (p<0.05). Bile acids is a variable parameter and whilst this and the other intergroup differences may indicate minor perturbations in metabolism, in the absence of any histopathological hepatic changes these were considered unlikely to be of any toxicological relevance.

Other effects (calcium concentration, urea, potassium, creatinine, albumin and/or gamma glutamyltranspeptidase) either did not show effects in the corresponding treatment/treatment free period or correlation with histopathological findings.

Therefore, although there were some statistically significant differences in treated animals from controls for blood chemical parameters measured, these differences were seen in the absence of any associated histopathological correlates and were considered not to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
At up to 400 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:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured at the end of treatment or treatment-free periods.

Males treated with 400 and 150 mg/kg bw/day showed a statistically significant reduction (p<0.01) in spleen weight both absolute and relative to terminal body weight. In the absence of a true dose related response, an effect on the overall hematological profile or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

At the end of the treatment free period, males that were previously given 400 mg/kg bw/day showed a statistically significant increase (p<0.05) in heart and liver weights both absolute and relative to terminal body weight. The level of significance achieved was minimal and in the absence of a similar effect observed in animals at the end of the treatment period or any histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Females treated with 30 mg/kg bw/day showed a statistically significant increase in absolute and relative kidney weight. In the absence of a similar effect seen in females at 150 or 400 mg/kg bw/day or any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination revealed no test substance related lesion
Neuropathological findings:
not examined
Histopathological findings: non-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.

Microscopic examinations performed after 28-day treatment and 14-days of recovery, respectively revealed no test item related lesions or effects.

Adrenal Glands: Cortical vacuolation was seen in the adrenals of three out of five males treated with 400 mg/kg bw/day following the termination of treatment. On examination of the adrenals from males treated with 150 and 30 mg/kg bw/day and recovery males this finding was considered not to be treatment related due to it also being seen at a similar level in two recovery control males and two recovery males previously treated with 400 mg/kg bw/day. All other histological changes were considered to be unrelated to treatment.

Thyroids: No treatment-related effects on the pituitary-thyroid axis were identified.
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.

One female treated with 150 mg/kg bw/day had a mass (approximately 2mm x 3mm) present in the left horn of the uterus. Microscopic examination of the uterus for this female revealed the presence of a cyst and thrombus together with moderate focal inflammation which correlated with the macroscopic finding observed. In the absence of a similar effect at 400 mg/kg bw/day, this finding was considered to be incidental and unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
1. Micronucleus Test:
- There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item groups when compared with the vehicle control group.
- There were no statistically significant decreases in the PCE/NCE ratio in any of the test item groups when compared with the vehicle control group.
- The vehicle control groups were within the normal range for the frequency of micronucleated PCEs and for PCE/NCE ratio.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes. Confirming sensitivity of the test system to the known mutagenicity of the cyclophosphamide under the conditions of the test.
Note: one PC male slide, was disregarded due to excessive bone marrow toxicity. This was considered to have no impact on the results. (Full details on the shared positive controls and other results are available in the full study report).

2. 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.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
mortality
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is defined as 150 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. Additional parameters covering an in vivo, micronucleus assessment was included under the following guidelines OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 as part of the study. 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, 150 or 400 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 (400 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Furthermore, as part of micronucleus assessment a concurrent (shared with another study) positive control group of five males was exposed to 25 mg/kg bw/day cyclophosphamide on day 28 only. 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 and at termination, a bone marrow sample was taken from all non-recovery animals for micronucleus assessment. Histopathological examination of selected tissues was performed. There was an unscheduled mortality in one female at 400 mg/kg bw/day in a recovery group on day 2. The female did not show any clinical signs prior to mortality and/or there were no macro or microscopic findings. Treatment related mortality could not be excluded. Males and females treated at 400 and 150 mg/kg bw/day showed increased salivation post dosing between Days 6 and 28 and Days 17 and 26 (respectively). At 400 mg/kg bw/day Four males and four females treated with 400 mg/kg bw/day also showed incidences of body tremors between Days 18 and 24. No effects were seen at 30 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. Males treated with 400 mg/kg bw/day showed a reduction in body weight gain during Weeks 1, 3 and 4. Females from this treatment group showed a reduction in body weight gain during the final week of treatment with five females showing an actual body weight loss. No effects were detected in males or females at 150 or 30 mg/kg bw/day. There were no adverse effects related to treatment on food consumption or food conversion efficiency. Males and females treated with 400 mg/kg bw/day showed an increase in overall water consumption during the final two weeks of the treatment period. Males and females at 150 mg/kg bw/day and females at 30 mg/kg bw/day showed a slight increase in overall water consumption, however a true dose related response was not evident in females from these groups and these effects insolation were considered not to reflect an adverse effect of treatment. Following the cessation of treatment, males/females that had previously been given 400 mg/kg bw/day showed water consumption that was comparable to controls. There was evidence of recovery. No toxicologically significant effects were detected in the hematological parameters or blood chemical parameters examined at the end of the treatment or treatment free periods. There were no treatment-related effects of toxicological importance detected in the urinalytical parameters. There were no treatment-related macroscopic abnormalities detected. There were no toxicologically significant effects detected in the organ weights measured at the end of the treatment or treatment-free periods. In the histopathology assessment: in the adrenal glands: cortical vacuolation was seen in the adrenals of three out of five males treated with 400 mg/kg bw/day following the termination of treatment. On examination of the adrenals from males treated with 150 and 30 mg/kg bw/day and recovery males this finding was considered not to be treatment related due to it also being seen at a similar level in two recovery control males and two recovery males previously treated with 400 mg/kg bw/day. All other histological changes were considered to be unrelated to treatment. Within the micronucleus assessment, there was no indications of genotoxicity. The oral (gavage) administration of the test item to males/females at dose levels of 30, 150 or 400 mg/kg bw/day resulted in one 400 mg/kg bw/day female being found dead, although it was not possible to determine whether or not this was related to treatment. Clinical signs of toxicity (body tremors) and an effect on body weight development were also evident at 400 mg/kg bw/day. No adverse effects were evident at 150 or 30 mg/kg bw/day. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 150 mg/kg/day for males/females.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
The study was conducted as part of a sub-chronic, 28-day repeated-dose toxicity study under OECD TG 407 and EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP. The study was modified according to the relevant listed regulatory guidelines in order to incorporate micronucleus assessment following exposure to the test item in a manner compatible with OECD TG 474 (2014) and listed guidelines.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Chemical structure
Reference substance name:
phenyl pent-4-enoate
EC Number:
812-668-8
Cas Number:
51231-09-5
Molecular formula:
C11H12O2
IUPAC Name:
phenyl pent-4-enoate
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 guideline and consistent with the OECD TG 474 guideline.
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 222 - 356 g and females 154 - 223 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: Polycarbonate 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: 2016-03-04 To: 2016-04-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: 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 of test material in vehicle: Samples of the test item formulations were taken on two 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 ± 3% of the nominal concentration. Arachis oil formulations was assessed and confirmed at nominal concentrations of 3.75 mg/mL and 250 mg/mL, during refrigerated storage.
- Amount of vehicle (if gavage or dermal): Treatment volume was 4 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group; positive control was 10 mL/kg. For further information see 'Doses / concentrations'.
- Type and concentration of dispersant aid (if powder): Not applicable.
- Other: Dose-formulations were analysed during the study and were reported as within ±10% applied limits.
Details on 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.
Duration of treatment / exposure:
Minimum period 28 days followed by a 14 day recovery period (treatment free) for the OECD 407 part of the study. The last dose was administered on Day 28. Micronucleus assessment proceeded after in non-recovery groups thereafter.
Frequency of treatment:
Once daily at approximately the same time each day.
Post exposure period:
24-hours following final dosing in relevant treatment groups only
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control - Group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
High - Group
No. of animals per sex per dose:
10 per sex per dose (5 male / 5 female)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): The choice of positive control was made in accordance with the OECD TG 474 guideline.
- Route of administration: Oral gavage
- Doses / concentrations: 1 dose; 25 mg/kg bw
- Other: five (5) males shared with another study (study number reported in full study report) - single dose were used for micronucleus test, treated with cyclophosphamide at 25 mg/kg bw/day - dosed on Day 28 only.

Examinations

Tissues and cell types examined:
Bone marrow was extracted and smear preparations were made and stained Polychromatic (PCE) and nomtochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCEINCE ratio was calculated as an indicator for toxicity.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The test item dose levels used in the main study were 30, 150 and 400 mg/kg bw/day. The maximum dose level was considered to be adequate to investigate the genotoxic endpoint in this study (confirmed as part of investigations in 7-d range finder and 28-day repeated dose toxicity testing).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Males/females were treated daily for 28-days at the specified dose levels. 24-hours following the last treatment (day 28) the treatment groups were terminated and then bone marrow was extracted from the left femur as part of sampling. Smear preparations were subsequently made and stained.

DETAILS OF SLIDE PREPARATION:
24-hours following last treatment, left femurs were extracted, aspirated with foetal bovine serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald / Giemsa. Then allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS:
Stained smears were coded and examined blind using microscopy (x1000) magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with the appropriate group mean values and standard deviations.

OTHER: Not applicable.
Evaluation criteria:
1. Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group:
- A positive response would be demonstrated when: there is a statistically significant dose response, toxicologically relevant increases in the number of micronucleated polychromatic erythrocytes observed at the 24-hour termination time compared with the vehicle control group
- A negative response would be fulfilled if the positive response criteria were not fulfile (no statistically significant dose responses, no toxicologically relevant increases in micronucleated polychromatic erythrocytes.

2. A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically lower than the vehicle control group.

Data is subject to statistical analysis, where appropriate.
Statistics:
Where appropriate, data is subject to statistical analysis using appropriate methods as recommended in UKEMS Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No statistically significant PCE/NCE decreases at up to 400 mg/kg bw/day observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: positive control male (number 65; PC group shared with this study) slide was disregarded due to excessive bone marrow toxicity. The loss of one individual from the PC scoring data was considered to have no impact on the outcome of the study.
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not applicable.
- Induction of micronuclei (for Micronucleus assay):
Vehicle Control, 0 mg/kg bw/day: Group mean (SD) = 5.8 (3.2) PCE with micronuclei per 4000 PCE
Low dose, 30 mg/kg bw/day: Group mean (SD) = 7.4 (3.6) PCE with micronuclei per 4000 PCE
Intermediate dose, 150 mg/kg bw/day: Group mean (SD) = 11.7 (12.4) PCE with micronuclei per 4000 PCE
High dose, 400 mg/kg bw/day: Group mean (SD) = 8.1 (6.5) PCE with micronuclei per 4000 PCE
Positive Control, 25 mg/kg bw/day: Group mean (SD) = 83.8 * (71.7) PCE with micronuclei per 4000 PCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; * P < 0.05 ; ** P < 0.01
- Ratio of PCE/NCE (for Micronucleus assay):
Vehicle Control, 0 mg/kg bw/day: Group mean (SD) = 1.29 (0.39) PCE/NCE
Low dose, 30 mg/kg bw/day: Group mean (SD) = 1.22 (0.62) PCE/NCE
Intermediate dose, 150 mg/kg bw/day: Group mean (SD) = 0.90 (0.52) PCE/NCE
High dose, 400 mg/kg bw/day: Group mean (SD) = 1.01 (0.39) PCE/NCE
Positive Control, 25 mg/kg bw/day: Group mean (SD) = 0.50 ** (0.06) PCE/NCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; ** P < 0.01
- Appropriateness of dose levels and route: The highest dose was set in accordance with the findings of the 7-day Range Finding study and the OECD TG 407 study. The appropriateness of the dose and route was confirmed during the study (recorded in the full study report). Applicant assessment: The maximum dose 400 mg/kg bw/day is in accordance with the OECD TG 474 guideline for a period of administration greater than 14-days and considering toxicity. Where three dose levels with a separation factor less than 4.0 were utilised.
- Statistical evaluation: No statistically significant responses were observed in the test item treatment groups up to 400 mg/kg bw/day relative to the concurrent vehicle control.
- Other: positive control male (shared with study number – reported in full study report) number 65 slide was disregarded due to excessive bone marrow toxicity. The loss of one individual from the PC scoring data was considered to have no impact on the outcome of the study. A statistically significant (P < 0.05) response was observed in PCE with micronuclei per 4000 PCE therefore confirming the sensitivity of the test system under the conditions of the test.

Any other information on results incl. tables

Table 1.0 – Table of results

Treatment Group

Number PCE with micronuclei per 4000 PCE

PCE/NCE Ratio

 

Group Mean

SD

Group Mean

SD

Vehicle Control, 0 mg/kg bw/day

5.8

3.2

1.16

0.34

Low dose, 30 mg/kg bw/day

7.4

3.6

1.17

0.44

Intermediate dose, 150 mg/kg bw/day

11.7

12.4

1.06

0.40

High dose, 400 mg/kg bw/day

8.1

6.5

1.00

0.25

 

 

 

 

 

Positive Control, 25 mg/kg bw/day

83.8 *

71.7

0.50 **

0.06

 

 

 

 

 

Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; * P < 0.05 and ** P < 0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-genotoxic.
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. Additional parameters covering an in vivo, micronucleus assessment was included under the following guidelines OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 as part of the study. 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, 150 or 400 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 (400 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Furthermore, as part of micronucleus assessment a positive control group (PC group; shared with another study) of five males was exposed to 25 mg/kg bw/day cyclophosphamide on day 28 only. Within the micronucleus assessment, there was no indications of genotoxicity. The vehicle control group frequency of micronucleated PCE and PCE/NCE was within the normal range. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item groups when compared with the vehicle control group. There were no statistically significant decreases in the PCE/NCE ratio in any of the test item groups when compared with the vehicle control group. Additionally, the vehicle control groups were within the normal range for the frequency of micronucleated PCEs and for PCE/NCE ratio. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes. Confirming sensitivity of the test system to the known mutagenicity of the cyclophosphamide under the conditions of the test. Under the conditions of this study, the test item would not be considered as genotoxic.

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