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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June 2012- 18 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (z)-hex-3-enyl acetate (leaf acetate)
- Physical state: liquid
- Analytical purity: 98.45 %
- Lot/batch No.: 1Z00151
- Expiration date of the lot/batch: 13 June 2015
- Stability under test conditions: stable at room temperature (15- 25 °C) for at least 8 d
- Storage condition of test material: at room temperature (15- 25 °C) away from direct sunlight, N2 into the container

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst/ Netherlands
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 310 to 360 g; Females: 190 to 234 g
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. During the pairing period females were housed with sexually mature males (1:1) until evidence of copulation was observed.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were not corrected for purity and were prepared as supplied. The dose formulations were prepared weekly as indicated by the results of stability analyses in the dose range-finding study (Harlan Laboratories Ltd, 2012). The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added (w/v). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Lot/batch no. (if required): BCBG8285V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed using HPLC. Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start.
- Transport of dose formualtions to Analytical Laboratory: At ambient temperature (20 ± 5 °C)
- Storage of dose formulations in Analytical Laboratory: Frozen (ca. -20 ± 5 °C)
Duration of treatment / exposure:
Males: minimum 4 weeks
Females: approximately 7 weeks
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11/sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected bsed on the results of a non-GLP dose range-finding study (Harlan Laboratories Ltd, 2012)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item in males and females. It was further performed in males and females twice during pre-pairing and 3 times during pairing. In females, it was performed additionally, on days 0, 6, 13 and 20 of the gestation period as well as on 3 days of the lactation period.
- Females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing
- Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and 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 or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded daily from treatment start to day of necropsy

FOOD CONSUMPTION:
- Time schedule for males: weekly during pre-pairing and after pairing periods
- Time schedule for females: pre-pairing period days 1-8 and 8- 14; gestation days 0- 7, 7- 14 and 14- 21 post coitum and days 1- 4 post partum
- No food consumption was recorded during the pairing period

HAEMATOLOGY: Yes
The following hematology parameters were determined:
- Complete Blood Cell Count: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count, total, Differential leukocyte count, Platelet count
- Coagulation: Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL BIOCHEMISTRY
- The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin, total, Cholesterol, total, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein, total, Albumin, Globulin, Albumin/Globulin ratio

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained at the end of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms
- Animals fasted: Yes. The animals were fasted for approximately 18 h before blood sampling but allowed access to water ad libitum
- How many animals: Blood samples were drawn sublingually from all animals under light isoflurane anesthesia

OTHER:
- Pup data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

- Termination of the Study: Males were sacrificed after treatment of at least 28 d, when no longer needed for the assessment of reproductive effects. Dams and pups were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

- Necropsy: All animals sacrificed or found dead were weighed and subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital or CO2-asphyxia. All P generation animals were exsanguinated. Dead pups, except those excessively cannibalized, were examined macroscopically. All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

- Organ Weights: At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Liver, Thymus, Spleen

-Tissue Preservation: The following tissues from all parental males were preserved in neutral phosphate buffered 4%: formaldehyde solution: Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative). The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: Ovaries. In addition, from the 5 males and females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: Gross lesions, Brain, Spinal chord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus, Thyroids, and parathyroids if possible, Trachea and lungs (preserved by inflation, with fixative and then immersion), Uterus (with vagina), Urinary bladder, Lymph nodes (mesenterial, mandibular), Peripheral nerve (sciatic), Bone marrow

-Histotechnique: All organ and tissue samples to be examined by the prinicipal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 µm and stained with hematoxylin and eosin. Additionally, the testes were stained by PAS-hematoxylin.

-Histopathology: Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the prinicipal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. If test item-related morphologic changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible. Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Other examinations:
Functional Observation Battery (FOB)
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum), relevant parameters from a modified Irwin screen test were performed with 5 P generation males and 5 P generation females from each group in place of the usual weekly behavioral observation. This FOB assessment was conducted following the daily dose administration.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 10-min intervals over a period of 60 min. These data and the total activity over 60 min were reported.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
One male (no. 39) treated with 1000 mg/kg/day of the test item was found dead on day 4 of the pre-pairing period. One female (no. 80) of this dose group was sacrificed in extremis on day 20 post coitum. One male (no. 30) was found dead on day 4 of the pairing period and one female (no. 67) was found dead on day 11 post coitum, both were treated with 300 mg/kg/day of the test item. Two females ( (nos. 57, 63) treated with 100 mg/kg/day of the test item were found dead on day 22 and on day 3 post coitum, respectively. These deaths were considered to be caused by aspiration during gavage procedures. The deaths were not related to systemic toxicity of the test item. All other animals survived until the scheduled necropsy.

CLINICAL SIGNS OR OBSERVATIONS
Almost all males and females of all dose groups including controls showed slightly soft feces from treatment day 6 (pre-pairing period) onwards. This could be considered to be an effect of the vehicle.

No other clinical signs were noted in males and females treated with 100 mg/kg/day of the test item during the course of the study except an isolated breathing noise, noted in one female on one day during the gestation period.

One male treated with 300 mg/kg/day had slight salivation on one day of the pairing period and 1 female of this dose group had slight dyspnea on two days of the pre-pairing period. Another female of this group had slight breathing noises on four days of the pairing period and on two days of the gestation period as well as a third female had this on one day of the gestation period.

Moderate or slight dyspnea was noted in 3 males treated with 1000 mg/kg/day of the test item on up to 3 days of the pre-pairing period, slight dyspnea was noted in 1 male of this group on one day of the pairing period and marked to slight breathing noises were noted in 1 male on one day and in 2 males of this dose group on four or six days of the pairing period.

Slight breathing noises were noted also in 4 females of this dose group on up to six days at the end of the gestation period. Slight reddish nasal secretion and slight ptosis were noted in 1 female on one day of the gestation period.

All these findings could be observed by aspiration during gavage procedures and are therefore considered to be no adverse findings.

No clinical findings were noted in males and females during weekly observations in any period.

FUNCTIONAL OBSERVATIONAL BATTERY
No statistically significant differences in mean values of grip strength (fore- and hind paws) were noted in test item treated males and females when compared with the control animals. The mean body temperature of test item treated males and females showed no statistically significant differences when compared with the control animals.

LOCOMOTOR ACTIVITY
The mean locomotor activity, observed during 1 h, measured in 10 min intervals, of males treated with 100 mg/kg/day of the test item was significantly (p<0.01) increased (20 to 30 min) when compared with the controls. This was considered to be incidental because it was noted in the low dose group and during one interval only. No other changes in mean locomotor activity were noted in test item treated males and females when compared with the control animals.

FOOD CONSUMPTION
Males
- Pre-Pairing Period: The mean food consumption of test item treated males showed no statistically significant differences when compared with the controls.
Females
- Pre-Pairing, Gestation and Lactation Periods: No effects on mean food consumption of females treated with 100, 300 or 1000 mg/kg/day were noted when compared with the controls.

BODYWEIGHT
Males
- Pre-Pairing and Pairing Periods: The mean body weights of test item treated males showed no statistically significant differences to the control males. The mean body weight gain was increased (p<0.05) in males treated with 1000 mg/kg/day on one single day during the pairing period, this is without any toxicological relevance.
Females
- Pre-Pairing, Pairing, Gestation and Lactation Periods: No statistically significant differences in mean body weight of test item treated females were noted when compared with the control females. The mean body weight gain was decreased (p<0.05) in females of all test item treated groups on several days of the paring period. The mean body weight gain of females treated with 1000 mg/kg/day was increased (p<0.05) on one day of the pre-pairing period. These differences were considered to be of no toxicological relevance.

CLINICAL LABORATORY INVESTIGATIONS
Haematology
- Males: No statistically significant differences in parameters of hematology were noted in test item treated males when compared with control males.
- Females: The mean value of total leucocytes was slightly decreased (p<0.05) in females treated with 100 mg/kg/day of the test item when compared with controls. The mean value of haemoglobin concentration distribution width and the mean value of large unstained cells were slightly decreased (p<0.05) in females treated with 300 mg/kg/day. No dose response relationship could be observed in these cases. Therefore the slightly decreased values were considered to be incidental.

Clinical biochemistry
- Males: The mean value of creatinine was slightly increased (p<0.05) in males treated with 100 mg/kg/ day when compared with controls. This was considered to be incidental because no dose response relationship could be observed. The mean total bilirubin was slightly decreased (p<0.05) in males treated with 1000 mg/kg/day when compared with controls. This was considered to be incidental because the value lies within the range of the historical reference data. The mean activity of alanine aminotransferase was decreased (p<0.01) in males of this dose group when compared with controls. It was considered to be not test item related because no related findings in the liver were observed.
- Females: No statistically significant differences in parameters of clinical biochemistry were noted in test item treated females when compared with control males.

ORGAN WEIGHTS
No differences in mean organ weights, mean organ to body weight ratios and mean organ to brain weight ratios were noted in test item treated males when compared with the control animals at the end of the 28 d treatment period. In females treated with 100 mg/kg/day the mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio were decreased when compared with the controls (p<0.05, p<0.01, p<0.05; respectively). The mean thymus to body weight ratio was also decreased (p<0.05) in females treated with 1000 mg/kg/day of the test item. This was considered to be not test item related because no dose response relationship could be observed. The mean heart to body weight ratio was increased (p<0.05) in females treated with 100 mg/kg/day what was also considered to be not test item related.

MACROSCOPICAL FINDINGS
In animals found spontaneously dead or which were sacrificed in extremis in dose groups, reddish discoloration and incomplete collapse of the lung, and red discoloration and red focus/foci of the thymus were recorded. There were no gross lesions that could be attributed to treatment with the test item in sacrificed animals. All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
Findings in Animals Decedents
The respiratory disorder consisted of necrosis and inflammatory cell infiltration of the trachea, congestion, alveolar edema, interstitial edema, alveolar hemorrhage and alveolar macrophages of the lung were recorded. Major microscopic findings with macroscopic findings in each animal were described as follow:
No.30
- Trachea: minimal inflammatory cell infiltration was recorded.
- Lung: reactive change consisting of slight congestion, moderate alveolar edema, slight alveolar hemorrhage and slight alveolar macrophages was recorded. (Macroscopic findings: Incompletely collapse and Discoloration, dark red)
- Thymus: Moderate hemorrhage as lesion during agonal period. (Macroscopic findings: Discoloration, dark red)

No.39
- Trachea: marked mucosal necrosis and moderate inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
- Lung: reactive change consisting of moderate congestion, slight alveolar edema, minimal interstitial edema, slight alveolar hemorrhage and slight alveolar macrophages was recorded. (Macroscopic findings: Discoloration, dark red)
- Thymus: Multifocal minimal hemorrhage as lesion during agonal period. (Macroscopic findings: Focus/Foci, dark red)

No.57
- Trachea: minimal inflammatory cell infiltration was recorded.
- Lung: reactive change consisting of slight congestion, slight alveolar edema, minimal alveolar hemorrhage and minimal alveolar macrophages was recorded. (Macroscopic findings: Incompletely collapse and Discoloration, dark red)
- Thymus: Multifocal slight hemorrhage as lesion during agonal period. (Macroscopic findings: Discoloration, dark red)

No.63
- Trachea: moderate mucosal necrosis and slight inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
- Lung: reactive change consisting of slight congestion, slight alveolar edema, slight interstitial edema, minimal alveolar hemorrhage and slight alveolar macrophages was recorded. (Macroscopic findings: Discoloration, dark red)
- Thymus: Multi focal slight hemorrhage as lesion during agonal period. (Macroscopic findings: Focus/Foci, dark red)

No.67
- Trachea: marked mucosal necrosis and moderate inflammatory cell infiltration was recorded and acute reactive and inflammatory changes were considered.
- Lung: reactive change consisting of moderate congestion, slight alveolar edema, slight interstitial edema, slight alveolar hemorrhage and minimal alveolar macrophages was recorded. (Macroscopic findings: Discoloration, dark red)
- Thymus: Multi focal slight hemorrhage as lesion during agonal period. (Macroscopic findings: Focus/Foci, dark red)

No.80
- Trachea: moderate mucosal necrosis and moderate inflammatory cell infiltration was recorded and acute reactive and inflammatory changes were considered.
- Lung: minimal inflammatory exudate in Bronchi was recorded.

Findings in Schedule Sacrificed Animals
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age


Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Litter Data - F1 Pups

External Examination at First Litter Check and during Lactation

No abnormal findings were noted in pups of dose groups 100 mg/kg bw/day and 1000 mg/kg bw/day at first litter check or during the first 4 days post partum. One male control pup had a blue nose. One female pup of dose group 300 mg/kg bw/day had a missing tail and another female pup of this dose group had a blue discolored abdomen. These isolated findings were considered to be not test item-related.

 

Sex ratios

Sex ratios at first litter check were unaffected by exposure to the test item. The proportion of males was 44, 45, 50 and 55%, in order of ascending dose level.

 

Body Weights to Day 4 Post Partum

No statistically significant differences in mean pup weights of groups two to four were noted when compared with control pups on day 0, day 1 and day 4 post partum.

 

Macroscopical Findings

No test item-related findings were noted at macroscopic examination of F1 pups.

Applicant's summary and conclusion

Conclusions:
The repeat dose toxicity of the test material was assessed according to OECD guideline 422. Based on the results of this study, the NOAEL of the test material administered orally by gavage to Wistar rats, could be established at 1000 mg/kg bw/day.