Allyl heptanoate

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Allyl heptanoate

EC Number:

205-527-1

EC Name:

Allyl heptanoate

Cas Number:

142-19-8

Molecular formula:

C10H18O2

IUPAC Name:

allyl heptanoate

Test animals

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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Remarks:

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

Doses / concentrations 2

Dose / conc.:

400 ppm

Remarks:

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

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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

3

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

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).