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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 December 2016 - 24 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyl propionate
EC Number:
204-559-3
EC Name:
Benzyl propionate
Cas Number:
122-63-4
Molecular formula:
C10H12O2
IUPAC Name:
benzyl propanoate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes (females were determined to have a normal estrous cycle)
- Age at study initiation (at start of administration): 10 weeks (males), 12 weeks (females)
- Weight at study initiation (at start of administration): 360.2 - 419.9 g (males), 223.3 - 287.5 g (females)
- Fasting period before study: no
- Housing: 1 (during the quarantine-acclimation period), 1 (during the dosing period), 1 male and 1 female (during the mating period), 1 female and neonates (during the lactation period); Stainless wire mesh cages, 260W×350D×210H (mm), Polycarbonate cage 260W×420D×180H (mm)
- Diet: ad libitum (pelleted rodent chow)
- Water: ad libitum (public tap water was filtered and irriadiated by UV light)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the supplier and the results of feed analysis met the allowable standard of the test facility.
Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year according to the Regulation of Quality Criteria for Potable Water and Test. The results of water analysis met the allowable standard of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8–24.9
- Humidity (%): 41.4–65.8
- Air changes (per hr): 10–15 clean, fresh, filtered air
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was chosen to assess the toxicity by oral exposure of the test substance.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations.
The dosing formulations were prepared every day before dosing until confirmed stability for 7 days.
After the confirmation, the dosing formulations were stored in a refrigerator (4.4–6.1°C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was well dissolved in it.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBW9504V, MKBV2080V, MKBZ9899V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using a gas chromatography (GC-2010 series, Shimadzu Corp., Japan). Samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analyses were determined to be in the range of 96.90–99.67%. These results were within the acceptable limits (±15% of nominal values).
Duration of treatment / exposure:
- Males of the main groups were dosed for a total of 50 days (for 2 weeks prior to mating, during 2 weeks of mating and 22 days post-mating).

- Also, males and females of the recovery groups were dosed for 50 days.

- Females of the main groups were dosed for 2 weeks prior to mating until Postpartum Day 13. Also, females showing no evidence of copulation or parturition signs were dosed until Gestation Day 25.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 (test and control group)
6 (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In previously conducted 2-week repeated oral dose range finding study (Biotoxtech Study No.: B16325), salivation was observed in one male at 300 mg/kg/day, and in males and females at 1,000 mg/kg/day. A decrease in the body weight and an increasing tendency in AST were noted in males at 1,000 mg/kg/day. The absolute and relative weights of the spleen were increased in females at 1,000 mg/kg/day. Therefore, the high dose level was selected at 500 mg/kg/day. Then, the mid and low dose levels were selected at 150 and 50 mg/kg/day, respectively.
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
NA

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: general condition and clinical signs at least once daily; morbidity and mortality twice daily
- Cage side observations: general condition, clinical signs, morbidity, mortality and females also signs of abortion and premature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing and once weekly for the dosing and recovery periods

BODY WEIGHT: Yes
- Time schedule for examinations: males of main groups and males/females of recovery groups: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period and recovery period, the day prior to necropsy and on the day of necropsy (fasted body weights); females of main groups: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (approximately 18 hours prior to necropsy)
- How many animals: Six females and six males from the main groups and all animals from the recovery groups.
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes (approximately 18 hours prior to necropsy)
- How many animals: Six females and six males from the main groups and all animals from the recovery groups.
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: two days before necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, but free access to drinking water
- Parameters checked in table No. 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: few days before necropsy
- Dose groups that were examined: all main groups and the recovery groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: on PND 4 and 13; adults: at termination
- How many animals: at least two pups per litter (if available above culling target), all adult males and dams of the main group
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (T4) [ug/dL] Method: Chemiluminescent competitive immunoassay

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (Complete gross postmortem examinations were conducted on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.)

ORGAN WEIGHTS: The testis and epididymis of all adult males were weighed. Six males and six females were randomly selected from the main group animals in addition to all recovery animals for necropsy. The organ weights from organs listed in Table No. 4 were determined.

HISTOPATHOLOGY: Yes. The testis, epididymis and eyes with optic nerves were fixed in Davidson’s fixative. All other tissues were preserved in 10 % neutral buffered formalin. The thyroid from one male and one female pups per litter were preserved in 10% neutral buffered formalin on PND 13. Organs listed in Table No. 5 were prepared for histopathology. Examinations were conducted in six males and six females from the control, low, mid and high groups, for all gross, macroscopic lesions in all animals.

Statistics:
For the data including body weights, food consumption, estrous cycle, mating period, gestation period, post-implantation loss, body weights and birth and survival rates of pups, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneous data, then if significant (significance level: 0.05), Dunnett’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data, then if significant (significance level: 0.05), Steel’s test was be performed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The data of mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01).
For the data of recovery groups, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed for homogeneity, but if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the main groups, salivation was observed in seven males from Day 11 to the end of the dosing. Salivation was also observed in eight females from Gestation Day (GD) 8 to Post-partum Day (PPD) 13.
In the recovery groups, salivation was observed in three males from Day 12 to the end of the dosing.
However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics.

No detailed clinical signs were observed in males and females of the main and recovery groups in the detailed examinations once a week.
Mortality:
no mortality observed
Description (incidence):
All animals of the main and recovery groups survived the duration of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight changes were noted in males and females of the main and recovery groups compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant differences in food consumption were noted in males and females of the main and recovery groups compared to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No effects were observed in any animal in the main and recovery groups.
Other statistical significance was considered not to be a test substance-related change because of small difference and the value was within the range of historical reference data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effects were observed in any animal in the main and recovery groups.
Other statistical significance was considered not to be a test substance-related change because of small difference and the value was within the range of historical reference data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the main and recovery groups, no effects were noted in urinalysis in males of any dosing group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main and recovery groups when compared to the control group.

In the main groups, there was no test substance-related effect on the grip strength in both sexes when compared to the control group. In spontaneous motor activity, statistically significant increases in 10–20 min ambulatory and vertical counts were noted in males at 50 mg/kg/day. However, these statistical significances had little toxicological significance because they were temporary changes and had no dose-dependency.

In the recovery groups, a statistically significant increase in hindlimb grip strength was noted in males at 500 mg/kg/day. However, this statistical significance had little toxicological significance because it was small difference. There was no test substance-related effect on the spontaneous motor activity in both sexes when compared to the control group.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse effects in total thyroxine (T4) level in F0 males and F1 pups of PND 13 at 50, 150 and 500 mg/kg/day.
Other statistical significance was considered not to be a test substance-related change because of small difference and the value was within the range of historical reference data.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related adverse effects on organ weights were observed in any animal in the main and recovery groups.
Other statistical significances in the absolute and/or relative organ weights were considered not to be test substance-related effects because of small difference and/or the values were within the range of historical reference data.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination at necropsy did not reveal treatment-related changes. In addition, the two dams, whose pups were all dead, commonly showed stress-related gross findings such as small thymus.
The other macroscopic findings were considered to be incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment-related changes were not observed in this study.
No test substance-related histopathological findings were noted in the reproductive organs of either sex.
In addition, severe cortical atrophy of the thymus was commonly observed in the two dams, whose pups were all dead. It was not considered to be related to the test substance since the dose-dependent response was not clear, and this non-specific finding was frequently noted in poor condition/stress.
All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test-item related adverse effects were observed.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL for systemic toxicity was considered to be 500 mg/kg bw/day for animals of both sexes.
Executive summary:

The purpose of this study was to determine the No Observed Adverse Effect Level (NOAEL) of the test item for both the general systemic when administered orally to male and female rats at dose levels of 0 (control), 50, 150 and 500 mg/kg/day.

All animals of the main and recovery groups survived the duration of the study. Salivation was observed in seven males and eight females at 500 mg/kg/day in the main groups and in three males at 500 mg/kg/day in the recovery group during the dosing period, but it was not considered to have toxicological significance.

No test substance-related adverse effects were noted in the results of body weights, food consumption, estrous cycle, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights, necropsy, histopathology and thyroid hormone analysis in adult animals of the test substance-dosed groups.

In conclusion, the NOAEL of the test item for systemic toxicity was considered to be 500 mg/kg/day for animals of both sexes.