Allyl heptanoate

Endpoint summary

Description of key information

Oral (OECD 408), rat: NOAEL = 84.25 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Nov 2015 - 13 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21 Sep 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Advinus Therapeutics Ltd, Bengaluru, India
- Age at study initiation: 5 - 7 weeks
- Weight at study initiation: 149.3 - 218.3 g (males), 131.9 - 172.5 g (females)
- Housing: one/two rats per sex per cage in standard polysulfone cages (L 425 x B 266 x H 185 mm) on steam sterilized clean corn cob, polycarbonate rat huts were used as cage enrichment
- Diet: Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet (Harlan Laboratories (Envigo), Madison, USA), ad libitum
- Water: deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier (Eureka Forbes Ltd., Mumbai, India), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 56 - 68
- Air changes (per hr):12 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: The test substance fortified diet was prepared at an interval of 15 days.
- Mixing appropriate amounts with Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet
The required quantities of the test substance (1.5, 6 and 22.5 g for the low, mid and high dose) were weighed and mixed with 10 mL of acetone. This was mixed manually with approximately 1.0 kg of powder food in a stainless steel drum for 2 minutes. Afterwards, the mixture was added in portions to the remaining portion of the food (approximately 14 kg) and mixed in a ribbon blender for 20 minutes.
- Storage temperature of food: The experimental feed was stored in the polyethylene bags within labeled stainless steel drums in the experimental room.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and test substance concentration analysis, the prepared test substance mixed experimental diet was sampled (one replicate each from top, middle and bottom layer) from each dose for analysis on Days 1, 57 and 85 of the treatment period. One composite sample was sampled from vehicle control group. The analysis were done by gas chromatography (G.C) equipped with FID and PC based data system.
The homogeneity of the samples was considered acceptable as the overall mean (calculated using all the 3 replicate values) of all the layers and mean of each layer was within ± 20% of the claimed concentration and the relative standard deviation (calculated using all the 3 replicate values) of assay of top, middle and bottom layers was less than 15%.

Stability and homogeneity of the test substance in the experimental fortified diet was carried out at the dose levels of 100 and 15000 ppm. The results of the analysis showed that the test item in the fortified diet was stable for 15 days when stored at room temperature.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously (via diet)

Dose / conc.:

100 ppm

Remarks:

equivalent to 6.37 and 6.85 mg/kg bw/day in males and females, respectively

Dose / conc.:

400 ppm

Remarks:

equivalent to 24.43 and 27.05 mg/kg bw/day in males and females, respectively

Dose / conc.:

1 500 ppm

Remarks:

equivalent to 84.25 and 93.08 mg/kg bw/day in males and females of the main group, respectively, and equivalent to 81.73 and 91.58 mg/kg bw/day in males and females of recovery group, respectively

No. of animals per sex per dose:

10 (main groups)
5 (recovery groups; for control and high dose groups)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The three dose levels of 100, 400 and 1500 ppm in diet were selected based on the results of a previously conducted 28-days oral feeding dose range-finding study in Wistar rats, in which, the dose levels of 150, 500, 1500, and 5000 ppm were used. The results of the 28-day study revealed that the body weights, body weight gain and food consumption were reduced at 1500 and 5000 ppm. However, the percent decrease was more at 5000 ppm. The terminal fasting body weights were lower in both sexes at 5000 ppm. In addition, decreased heart, liver (absolute and relative to brain), adrenals, kidneys and prostate (absolute) weights were observed at 5000 ppm and were considered as secondary changes associated with reduced body weight. However, no test substance related alterations were observed in clinical pathology (hematology, coagulation and clinical chemistry) parameters, organ weights and their ratio and gross pathology. The histopathology of stomach, liver and kidney did not reveal any treatment-related changes at 5000 ppm.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on holidays)
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to the test substance administration on Day 1 and at weekly intervals during treatment and recovery period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membrane, occurrence of secretions and excretions, autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies and bizarre behaviour like self-mutilation, walking backwards.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to test substance administration on Day 1 and at weekly intervals thereafter (± 1 day) during treatment and recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment (Day -1) and on Days 91 and 116 for the main and recovery group, respectively
- Dose groups that were examined: 100, 400 and 1500 ppm

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (Day 92) and at the end of the recovery period (Day 120) for the main and recovery group, respectively
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haematocrit, haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, mean platelet volume, platelets, red blood cells, white blood cells, differential leukocyte counts (neutrophils, lymphocytes, monocytes, eosinophils and basophils), reticulocytes, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (Day 92) and at the end of the recovery period (Day 120) for the main and recovery group, respectively
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alanine aminotransferase, albumin, alkaline phosphatase, albumin / globulin ratio, aspartate amino transferase, blood urea nitrogen, creatinine, calcium, chloride, gamma glutamyl transpeptidase, globulin, glucose, inorganic phosphorous, potassium, sodium, total cholesterol, total plasma protein, total bilirubin, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment and recovery period, respectively
- Metabolism cages used for collection of urine: specially fabricated cages were used
- Animals fasted: Not specified
- Parameters examined: specific gravity, nitrite, pH, proteins, glucose, ketone bodies, urobilinogen, bilirubin, creatinine, urea, appearance (colour and clarity), volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Week 12 (males) and 13 (females) of the treatment period and towards the end of the recovery period
- Dose groups that were examined: 100, 400 and 1500 ppm
- Battery of functions tested: motor activity, sensory evaluation, landing hindlimbs, footsplay and measurement of grip performance
- Home cage observations: rats were observed in the home cage for posture and presence or absence of abnormal vocalizations, tremors and convulsions
- Observations during removal from home cage and handling: ease of removal from home cage, handling reactivity, palpebral closure, eye examination, piloerection, lacrimation, salivation, skin/fur examination, perineum wetness, respiration, muscle tone and extensor thrust response
- Open field observations: gait, posture, tremors, mobility score, arousal level, clonic or tonic movements, stereotypic behaviour, bizarre behaviour, urination, defecation, rearing, abnormal vocalizations

IMMUNOLOGY: No

OTHER: The body temperature (rectal temperature) was measured.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following tissues and organs were collected and weighed: adrenal glands, aorta, axillary lymph nodes, bone marrow smear, brain (cerebrum, cerebellum, medulla/pons), cecum, colon, duodenum, epididymides, esophagus, eyes (with optic nerve), gross lesions, femur bone with distal joint, femoral muscle (skeletal muscle), heart, harderian glands, ileum with Peyer’s Patch, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, mesenteric lymph node, mandibular lymph node, mammary gland, nerves sciatic, ovaries, oviduct, pancreas, pharynx, pituitary, prostate, rectum, salivary glands (submandibular, sublingual and parotid), seminal vesicles and coagulating glands, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with marrow, stomach, testes, thymus, thyroid with parathyroids, tongue, trachea, urinary bladder, ureters, uterus with cervix, vagina

10% neutral buffered formalin (NBF) was used for fixation except eyes and testes which were preserved in Davidson’s fluid and modified Davidson’s fluid, respectively.

HISTOPATHOLOGY: Yes
Histopathological examination was carried out on all the preserved organs and tissues (including all gross lesions) of control and high dose group.

Tissues were processed for routine paraffin embedding and 4 - 5 micron sections were stained with Mayer’s Haematoxylin and Eosin stain.

Statistics:

Parameters such as body weight, terminal fasting body weight and organ weights, haematology and clinical chemistry data was analysed using Provantis™. Derived data like net body weight change, food consumption and organ weight ratios were also analyzed using Provantis™.
The statistical analysis of the experimental data captured other than Provantis™ was carried out using SYSTAT Statistical package Ver.12.0. All quantitative variables like functional observation tests (neuromuscular observations, motor activity, body weight and body temperature) were tested for normality and homogeneity of variances (Levene’s test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. The non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. ANOVA was done using log transformation. Comparison of means between treatment groups and control group was done using Dunnett’s t-test when the overall treatment, ‘F’ test was found significant.
In case of recovery groups, the data was analysed using two-sample Dennett’s t-test. Comparison of means between treatment recovery and control recovery group was performed.
All analyses and comparisons were evaluated at p < 0.05.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

An incidence of sparse hair loss each in control and 400 ppm dose group females was observed. This finding is common in this species and considered incidental and not related to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected by the treatment at 100 ppm when compared to the vehicle control.
At 400 ppm, the mean body weights were significantly lower in males during Weeks 4-13 and in females during Weeks 7-10. At the end of the treatment period, body weights were reduced up to 14% and 6% in males and females, respectively.
The mean body weights were significantly reduced up to 23% and 12% in males and females, respectively, at 1500 ppm.
The mean body weights were apparently lower without statistical significance in the males and females of the high dose recovery group when compared to control recovery group.

The body weight gain was significantly lower in males during Week 7 at 100 ppm, during Weeks 1, 2, 5 and 7 at 400 ppm and 1-3, 5-7, 11 and 12 at 1500 ppm, whereas the same was significantly higher during Week 13 at 1500 ppm. In females, the body weight gains were significantly lower during Weeks 3 and 7 at 100 ppm, Week 7 at 400 ppm and during Weeks 1, 3, 6 and 7 at 1500 ppm, whereas the same was significantly higher during weeks 5 and 13 at 100 ppm and Week 13 at 1500 ppm. When compared to the control recovery group, the body weight gain was apparently lower (comparable in few instances) without statistical significance in the males and females of the high dose recovery group.

The decrease in the mean body weights and body weight gains at 400 and 1500 ppm are correlated with the reduced food consumption at the respective dose levels and considered as treatment-related effect. During the recovery period, the weight change in the high dose group was higher than that observed in the concurrent control group, indicating the reversal of the effect of the test substance on body weights.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption was significantly reduced up to 6% and 14% in males throughout the treatment period at 400 and 15000 ppm, respectively. In addition, an incidence of significantly lower food consumption was observed at 100 ppm during Week 11. In females, the mean daily food consumption was significantly reduced during Weeks 1, 7, 10, 12 and 13 at 400 ppm and during Weeks 1 to 13 at 1500 ppm when compared to the vehicle control.
The mean daily food consumption was significantly reduced (except on Week 3) in males of the recovery group at 1500 ppm throughout the treatment period. In females, the mean daily food consumption was significantly reduced during Weeks 1, 3, 4, 7, 8 and 10-12 of the treatment period and Weeks 3 and 4 of the recovery period.
The decrease in the food consumption at 400 and 1500 ppm correlated with the reduced body weights at the respective dose levels and was considered as treatment-related effect.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examination carried out prior to initiation of the treatment did not reveal any eye abnormalities.
The ophthalmological examination did not reveal any test item-related eye abnormalities at the end of treatment or recovery periods.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related changes were observed in haematological and coagulation parameters of both sexes across the treated groups. All statistically significant haematological alterations observed were not related to test substance administration as the changes were minimal in magnitude and also lacked the dose progression. Decreased prothrombin time at 400 ppm and decreased activated partial thromboplastin time at 100 ppm and above was observed in males. The findings were considered incidental as the changes were minimal in magnitude and also lacked the dose progression.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related changes were observed in clinical chemistry parameters of both sexes across the treated groups. All the statistically significant biochemical changes observed were not attributed to test substance administration as the alterations were of minimal magnitude and also lacked the dose progression/microscopic correlation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test substance-related changes were observed in the urinalysis parameters evaluated in both the sexes across the treated groups.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Neurological examinations including functional tests, conducted on male and female rats did not reveal any treatment-related significant changes at any of the tested dose levels. Statistically significant differences were considered incidental as either the changes were of minimal magnitude or not dose-related and all the observed values were within the laboratory historical control data range.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, test substance-related decrease in weights (absolute and relative to brain weight) of liver, heart, kidneys, lungs, prostate, spleen and thymus was observed at 400 ppm and above. In addition, decreased pituitary and thyroid with parathyroid weights were also noted at 1500 ppm. The changes observed in liver, heart, lungs, thymus and thyroid with parathyroid were partially reversible while that of kidneys, prostate, spleen and pituitary were completely reversible at the end of recovery period.
In females, decreased liver, heart and thymus weights (absolute and relative to brain weight) were observed at 400 ppm and above and decreased kidneys and lungs weights (absolute and relative to brain weight) were noted at 1500 ppm. All the changes were completely reversible except liver and heart at the end of recovery period.
The organ weight changes observed in both the sexes were correlated to decreased body weight and considered as secondary changes to test substance administration.
All other differences observed in organ weight and their ratios were considered incidental as the changes were minimal in magnitude and/or lacked the dose progression/microscopic correlation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross changes were observed in both sexes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All microscopic findings observed in males and females at 1500 ppm were considered incidental/spontaneous and not attributed to test substance administration as they were normally present in rats of this age group. In addition, observed microscopic findings were comparable to vehicle control group.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: the observed effects on body weight were considered as a secondary effect due to reduced food consumption

Key result

Dose descriptor:

NOAEL

Effect level:

>= 84.25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

other: the observed effects on body weight were considered as a secondary effect due to reduced food consumption

Key result

Dose descriptor:

NOAEL

Effect level:

>= 93.08 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

other: the observed effects on body weight were considered as a secondary effect due to reduced food consumption

Key result

Critical effects observed:

no

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was set at ≥ 1500 ppm (equivalent to 84.25 and 93.08 mg/kg bw/day in males and females, respectively).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-10 to 2009-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, adopted 2008-10-03

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

440/2008/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Main groups: males 36 days and females 37 days; Recovery groups: males 34 days and females 35 days
- Weight at study initiation: Main groups: males 144.3 - 170.2 g and females 126.2 - 146.4 g; Recovery period groups: males 128.7 - 144.7 g and females 113.9 - 130.3 g
- Fasting period before study:
- Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (ad libitum): Commercial ssniff®-R/M-H V1530 (ssniff® Spezialdiäten GmbH, 59494 Soest, Germany); Food residue was removed and weighed.
- Water (ad libitum): Drinking water
- Acclimation period: 6 days (recovery animals) or 8 days (main study animals)

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C +/- 3°C (maximum range)
- Relative humidity: 55% +/- 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Sesame oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item-vehicle mixture was freshly prepared every day.
The test item was diluted in Sesame oil to the appropriate concentrations and administered at a constant volume of 5 mL/kg b.w./day. The control animals received the vehicle only at a constant volume of 5 mL/kg b.w./day. The amount of the test item was adjusted to the animal's current body weight daily.

VEHICLE
- Batch no.: 4683301

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each test item that was mixed with the vehicle, tests by appropriate analytical methods were conducted to determine the concentration, stability and homogeneity of the test item in the solutions.
For the analysis of the test-vehicle mixtures, samples of approx. 20 mL were taken at the following times and stored at -20°C or colder until analysis.
At study initiation (Test day 1):
- Analysis of stability and concentration: Immediately after preparation of the solution as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples/dose group). Total number of samples: 9
- Additionally, analysis of concentration: Immediately after preparation of the solution (3 samples/dose group). Total number of samples: 9
- Homogeneity: At the start of administration, during (middle) ad-ministration and before administration to the last animal of each dose group (3 samples/dose group). Total number of samples: 9
At study termination:
- Analysis of stability: During treatment with the test item always before administration to the last animal/dose group (1 sample/dose group). Total number of samples: 3
- Additionally: Analysis of concentration: Immediately after preparation of the solution (3 samples/dose group). Total number of samples: 9

The results indicate that the content of Allyl heptanoate (Sym09/611041) in the samples from the 28-day sub-chronic oral toxicity and reproduction/development toxicity screening study corresponded well with the nominal concentrations in the concentration range of 2 to 20 mg/mL, so that maintenance of the dosing regimen can be concluded.
Moreover, the dosing suspensions were shown to be stable and homogeneous during the application period.
In addition, the stability of frozen samples over a period of 113 days was confirmed.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 10, 30, and 100 mg/kg b.w./day (males/females)
Basis:
actual ingested

No. of animals per sex per dose:

Main study:
0 mg/kg b.w./day: 5 males/ 5 females
10 mg/kg b.w./day: 5 males/ 5 females
30 mg/kg b.w./day: 5 males/ 5 females
100 mg/kg b.w./day: 5 males/ 5 females
Recovery groups:
0 mg/kg b.w./day: 5 males/ 5 females
100 mg/kg b.w./day: 5 males/ 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the 7-day dose-range-finding study in rats. The animals were treated orally for 7 days with daily dose levels of 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w. and for 4 days with a daily dose level of 300 mg Allyl heptanoate (Sym09/611041)/kg b.w.
The rats treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w. did not reveal any influence on behaviour or external appearance, body weight and food and drinking water consumption. Necropsy revealed changes in the liver in form of foci-like indurations accompanied by white-yellow stained discolourations. None of the animals died prematurely.
All five male animals and two of five female animals treated with 300 mg Allyl heptanoate (Sym09/611041)/kg b.w. were found dead in the morning of test day 3 or 4. The treatment with 300 mg Allyl heptanoate (Sym09/611041)/kg b.w. caused slightly to severely reduced motility, pilo-erection and/or apathy. Necropsy revealed changes in the liver in form of generalised white spots, dark-red discoloured right lobe, red-yellowish discoloured/marbled.
The following dose levels were suggested for the 28-day repeated dose study:
- 10, 30, and 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day plus one control group.
- Post-exposure recovery period in satellite groups: 2 weeks.

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed individually at least once daily for any signs of behavioural changes, reaction to treatment or illness. In addition, animals were checked regularly throughout the working day.
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was followed.
- Cage side observations checked: Any signs of illness or reaction to treatment were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first treatment (to allow for within-subject comparisons) and once a week thereafter (1, 2, 4, 8 and 24 hours after administration), detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of group allocation, on the day of commencement of treatment and once a week thereafter always on the same day of the week throughout the experimental period.

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
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week. From these data the food consumption (in g/kg b.w./day) was determined.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed prior to the start of administration, at the end of test week 4 and at the end of the recovery period.
- Dose groups that were examined: All animals
- Ocular structures examined: adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from the retrobulbar venous plexus at the following times: From main study animals at main study termination (on the day of dissection) and from recovery animals at the end of the recovery period (on the day of dissection).
- Anaesthetic used for blood collection: Yes; light ether anaesthesia
- Animals fasted: Yes; Overnight
- How many animals: All main study animals and all recovery animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative) (neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes, monocytes and large unstained cells), differential blood count (absolute) (neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes, monocytes and large unstained cells), reticulocytes, platelets, haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the retrobulbar venous plexus at the following times: From main study animals at main study termination (on the day of dissection) and from recovery animals at the end of the recovery period (on the day of dissection).
- Animals fasted: Yes; Overnight
- How many animals: All main study animals and all recovery animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acid, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (in blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, and aspartate aminotransferase

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In test week 4 (TD 27) approx. 1 - 2 hours after dosing and before any blood sampling for laboratory examinations, screening of sensory reactivity to stimuli of different types as well as the assessment of grip strength and motor activity assessment were conducted in all animals.
- Dose groups that were examined: all dose groups
- Battery of functions tested:
Observational screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function, grip strength, and locomotor activity (stereotype, static movement and active locomotion).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On test day 29 (one day after the last administration) the animals of the main study were dissected following a randomisation scheme. Dissection of all animals allocated to the recovery period has been carried out on test day 43.
The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
HISTOPATHOLOGY: Yes
The following organs or parts of organs of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution and the testes in Bouin’s solution for optimum fixation.
Organs: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla / pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: left and right ventricle, septum), intestine, large (colon, rectum), intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches; Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles; preserved by inflation with fixa-tive and then immersion), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 levels: cervical, midthoracic, lumbar), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts) and vagina.
The afore-listed organs of all animals of groups 1 and 4 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made and stained with scarlet R.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.
In addition the following target organs of the animals of the low and intermediate dose level groups (groups 2 and 3) were examined histologically: liver (all animasl) and kidney stained with scarlet R (all females).

Other examinations:

Organ weights:
The weights of the following organs of all animals were determined before fixation: adrenal gland (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), spleen, testicle (2), thymus, and as a whole prostate, seminal vesicles with coagulating glands.
Adrenal glands, gonads and kidneys were weighed individually and identified as left or right.

Statistics:

The test item groups 2 - 4 were compared with the control group 1.
The following statistical methods were used:
- STUDENT's t-test: all numerical functional tests
- Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control Biometrics, 482 - 491 (September 1964): Body weight / food consumption / haematology / clinical biochemistry / relative and absolute organ weights.
- Exact test of R. A. FISHER: Histopathology
These statistical procedures were used for all data.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Treatment and recovery period: None of the animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day or previously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day revealed any changes of behaviour or external appearance. No mortality was noted at 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day. The faeces of all animals were of normal consistency throughout the experimental period.

BODY WEIGHT AND WEIGHT GAIN
Treatment and recovery period: Body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day or previously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day compared to the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Treatment and recovery period: No test item-related influence was noted on the food consumption of the male and female animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day or previously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day compared to the control group.
The slight increases of food consumption observed in the high dosed (100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day) male animals in test weeks 2 and 4 and in the high dosed female animals in test week 2 (statistically significant at p ≤ 0.01) are considered to be within the normal range of biological variability and are regarded to be a spontaneous change and not test item-related.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Treatment and recovery period: The visual appraisal of the drinking water consumption did not reveal any test item-related changes.

OPHTHALMOSCOPIC EXAMINATION
Treatment and recovery period: Ophthalmological examination revealed no test item-related changes of the eyes and the optic region in the male and female rats treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day or previously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day. No changes were noted for the control animals, either.

HAEMATOLOGY
Treatment and recovery period: No test item-related influence on the haematological parameters was observed for the animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day. No test item-related changes were observed at end of the recovery period (test day 43).
No test item-related influence was observed for the haemoglobin content, the number of erythrocytes, leucocytes, reticulocytes and platelets, the relative and absolute differential blood count, the haematocrit value, the thromboplastin time, the activated partial thromboplastin time, the mean corpuscular volume (MCV), the mean corpuscular hae-moglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC).
The slight decreases of the mean corpuscular volume (MCV) and the mean corpuscular haemoglobin (MCH) values observed in the high dosed (100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day) males on test day 29 (statistically significant at p ≤ 0.01) are considered to be within the normal range of biological variability and are regarded to be a spontaneous change and not test item-related.

CLINICAL CHEMISTRY
Treatment and recovery period: No test item-related influence was noted for the biochemical parameters of the animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day or previ-ously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day compared to the control group.
No test item-related influence was noted for the plasma levels of albumin, globulin, bile acid, the plasma levels of bilirubin, cholesterol, creatinine, glucose, protein (total), urea, calcium, chloride, potassium and sodium. The plasma activities of ALAT, aP and ASAT were not increased.
The slight decrease of the albumin/globulin ratio and the slight increase of the calcium value observed in the high dosed (100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day) female animals on test day 29 (statistically significant at p ≤ 0.01) are considered to be within the normal range of biological variability and are regarded to be a spontaneous change and not test item-related.

NEUROBEHAVIOUR
Treatment and recovery period: The observational screening performed in test week 4 approximately 1 - 2 hours after dosing did not reveal any test item-related influence on the main study animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day or on the recovery animals previously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day.
The slight decrease of the hindlimp grip strength observed in the low and intermediate dosed (10 or 30 mg Allyl heptanoate (Sym09/611041)/kg b.w./day) female animals in test week 4 (statistically significant at p ≤ 0.01) is considered to be within the normal range of biological variability and is regarded to be a spontaneous change and not test item-related.
The slight increase of the spontaneous motility (high sensitivity) observed in the high dosed (100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day) male animals in test week 4 (statistically significant at p ≤ 0.01) is considered to be within the normal range of biological variability and is regarded to be a spontaneous change and not test item-related.

ORGAN WEIGHTS
Treatment and recovery period: The absolute and relative organ weights were not influenced in the male and female animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day or previously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day compared to the control group.

GROSS PATHOLOGY
Treatment period: At necropsy, no test item-related systemic changes were noted in the male and female animals treated with 10 or 30 mg Allyl heptanoate (Sym09/611041)/kg b.w./day for 28 days.
Macroscopic changes were noted in livers of the high dosed animals. One male and two female animals treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day showed a yellow-stained discoloured liver. These findings are considered to be test item-related.
Further macroscopic changes were noted in the lungs (red-stained discoloured) and in the thymus (red discoloured or red-stained discoloured) of individual male and female animals of the control and high dosed item-treated groups. These changes are considered to be incidental findings and not test item-related.
Recovery period: Macroscopic inspection on test day 43 did not reveal any test item-related changes in female and male animals previously treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day for 4 weeks.
Macroscopic changes were noted in the thymus (dark-red discoloured, reddish disco-loured or reddened) of three male animals of the control and high dose groups. These changes are considered to be incidental findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment period: Treatment-related effects were observed in both sexes of the high dose group (100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day) in livers in form of chronic inflammation (portal triangle), oval cell hyperplasia (portal triangle) and pigment deposition (multifocal).
No test item-related changes were observed in livers of lower dose groups for both sexes.
No test item-related changes were noted in kidneys of female animals at all dose levels.
All other macroscopic and microscopic changes observed were not considered to be related to the test compound.
Recovery period: For the recovery animals of both sexes, only one of five male animals (no. 49) showed treatment-related liver changes in form of chronic inflammation (portal triangle), oval cell hyperplasia (portal triangle), pigment deposition and porto-portal fibrosis. Female recovery animals did not show any hepatotoxicity anymore.
All other macroscopic and microscopic changes observed were not considered to be related to the test compound.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no data

Critical effects observed:

not specified

- Dose verification (content of Allyl heptanoate (Sym09/611041) in samples in the concentration range of 20 to 200 mg/mL was successful.

- Homogeneity of the samples during the application period was given.

- Stability of the test item Allyl heptanoate (Sym09/611041) over a period of 113 days at -20 ± 2 °C was confirmed (sampling at LPT Laboratory of Pharmacology and Toxicology until further sample dilution and analysis at the test facility).

For further information please refer to the appendix of this robust study summary.

 

 

Conclusions:

Under the present test conditions, the NOEL was 30 mg Allyl heptanoate/kg bw/day.

Executive summary:

Analytical results indicate that the content of Allyl heptanoate in the dosing suspensions corresponded reasonably well with the nominal concentrations in the concentration range of 6 to 20 g/L, and the dosing suspensions were shown to be stable and homogeneous during the application period.

The subacute toxicity (28-days) of Allyl heptanoate, given daily by oral administration via gavage, was investigated in CD® rats. Animals were treated with dose levels of 10, 30 or 100 mg Allyl heptanoate/kg bw/day. On the basis of treatment-related macroscopic changes in livers (yellow-stained discolouration) of three main study animals at the high dose level (100 mg/kg bw/day), and treatment-related histopathological changes in livers of male and female high dose animals (chronic inflammation (portal triangle), oval cell hyperplasia (portal triangle) and pigment deposition (multifocal)), the NOEL was established at the mid dose level of 30 mg Allyl heptanoate (Sym09/611041)/kg b.w./day.

 

At the end of the recovery period, all changes had nearly subsided. Only in one of five high dose recovery males, histopathology revealed liver changes (chronic inflammation (portal triangle), oval cell hyperplasia (portal triangle), pigment deposition and porto-portal fibrosis) indicating reversibility of the findings.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

84.25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The repeated dose toxicity of the test substance was assessed in a 28-day oral gavage and in a 90-day oral feeding toxicity study.

A short-term repeated dose toxicity study was performed according to OECD Guideline 407 and in compliance with GLP (Leuschner, 2010). CD rats were treated via oral gavage with the test substance at dose levels of 10, 30 and 100 mg/kg bw/day. Treatment-related macroscopic chances in livers, yellow-stained discolouration, were observed in 1 male and 2 females at 100 mg/kg bw/day. In addition, histopathological examination revealed treatment-related changes, chronic inflammation (portal triangle), oval cell hyperplasia (portal triangle) and pigment deposition (multifocal), in the livers of both sexes at 100 mg/kg bw/day. At the end of the recovery period, almost all changes were reversible. Histopathological liver changes were present only in 1/5 high dose recovery males, indicating reversibility of the findings. Based on the results of the study the NOEL was set at 30 mg/kg bw/day.

In a second repeated dose toxicity study, the subchronic toxicity of the test substance was assessed in a 90-day oral feeding toxicity study with a 28-day recovery period according to OECD Guideline 408 and in compliance with GLP (Sathish, 2016). The dose levels for the main study were selected based on a previous 28-day oral feeding dose range-finding study in Wistar rats using dose levels of 150, 500, 1500 and 5000 ppm (equivalent to 10.06, 32.54 and 249.24 mg/kg bw/day in males and 10.76, 33.18, 98.12 and 273.05 mg/kg bw/day in females). Four treatment groups and one control group consisting of 6 males and 6 females were treated continuously via diet over a period of 29 days. Food consumption was significantly reduced in males (-13%) at 1500 ppm and in both sexes (-27% and -20% in males and females, respectively) at 5000 ppm. Due to reduced food consumption mean body weights were reduced at the end of the treatment period up to -11% in males at 1500 ppm and up to -24% and -11% in males and females, respectively, at 5000 ppm. At necropsy, decreased heart, liver (absolute and relative to brain), adrenals, kidneys and prostate (absolute) weights were observed at 5000 ppm. These findings were considered as secondary changes associated due to reduced body weight. No treatment-related effects on hematology and clinical chemistry were observed. Histopathological examination of the stomach, liver and kidney did not reveal any treatment-related changes at 5000 ppm. Based on the results of the dose range-finding study, dose levels of 100, 400 and 1500 ppm were selected for the main study.

In the main study, 10 Wistar rats per sex and dose were treated via diet with the test substance over a period of 90 days. The control group received plain diet. Additionally, a recovery group of 5 rats per sex was allocated to the control and high dose group. No mortality and treatment-related clinical signs occurred during the study period. The mean body weights were significantly reduced in males (-14%) at 400 ppm and in both sexes (-23% and -12% in males and females, respectively) at 1500 ppm. The mean body weights were reduced without statistical significance in males and females of the high dose recovery group when compared to the control recovery group. The mean daily food consumption was significantly reduced in both sexes (-14% and -11% in males and females, respectively) at 1500 ppm. In the absence of treatment-related findings in the clinical pathology parameters and histopathology, the decreased body weights and food consumption at 400 and 1500 ppm were considered as treatment-related non adverse effect. Gross pathological examination revealed decreased weights of liver, heart, kidneys, lungs and thymus in both sexes at 1500 ppm and prostate, spleen, pituitary and thyroid with parathyroid in males at 1500 ppm. The decrease in the organ weights were considered as secondary changes associated with reduced body weight and no microscopic changes were observed in any of the organs evaluated. No test substance-related findings were observed on hematology, clinical chemistry, urinalysis and in functional observations.

In conclusion, no adverse effects on systemic toxicity after repeated exposure were observed up to and including 1500 ppm, the highest dose tested. Therefore, a NOAEL of ≥ 1500 ppm (equivalent to 84.25 and 93.08 mg/kg bw/day in males and females, respectively) was derived in the OECD 408 Guideline study.

Justification for classification or non-classification

The available data on repeated oral dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.