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Toxicological information

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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Beige waxy solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age/weight at study initiation: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 388 g (range: 335 g to 438 g) and the females were approximately 9 weeks old had a mean body weight of 236 g (range: 203 g to 270 g)
- Housing: the animals were individually housed, except during pairing and lactation, in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 d before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per h): approximately 12 cycles/h of filtered, non-recycled air
- Photoperiod (h dark / h light): 12 h/12 h.

IN-LIFE DATES: 13 February 2013 to 09 April 2013.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Preparation of dosing solutions:
The test substance was administered as a suspension in the vehicle. The dose formulations were prepared according to the following process, as described in an homogeneity/stability study:
- a small amount of test substance was melt using water bath at a temperature of +50°C,
- the vehicle was warmed at +50°C using water bath,
- the appropriate amount of melt test item was weighed in the preparation beaker and the corresponding amount of vehicle was added,
- the test item and the vehicle were mixed using magnetic stirrer in the water bath at +50°C during 10 minutes,
- the obtained suspension was stirred at ambient temperature at least 30 minutes.
No correction factor was applied. The test substance dose formulations were prepared on a weekly basis, stored at room temperature protected from light prior to use and delivered to the study room at room temperature and protected from light. When not on the day of formulation preparation, test substance formulations were warmed under magnetic stirring at +50°C using water bath during at least 30 minutes and then kept under magnetically stirring at ambient temperature for at least 30 minutes before daily delivery to the study room.

Vehicle
- Choice of vehicle: good suspension in corn oil
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test substance concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: assessed in homogeneity study (satisfactory results)
Stability: assessed in stability study (stable after 9 d at room temperature and protected from light)
Duration of treatment / exposure:
In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (at least 5 weeks in total),

In the females:
− 2 weeks before mating,
− during the mating period (1 week),
− during pregnancy,
− during lactation until day 5 post-partum inclusive,
− until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations: 100, 300 and 1000 mg/kg bw/day
Basis: actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2-week toxicity study. In this study, three groups of three male and three female Sprague-Dawley rats received the test substance as a suspension in corn oil at 100, 300 or 1000 mg/kg bw/day for 2 weeks by gavage. There were no unscheduled deaths. Reduced mean body weight gain and mean food consumption were noted in males during the first week of treatment. Clinical signs were ptyalism in all test substance treated animals and hunched posture in two males and one female at the high-dose during the second week of treatment. There were no test substance-related macroscopic findings.
- Animal assignment: computerized stratification procedure.
Positive control:
Not required

Examinations

Observations and examinations performed and frequency:
The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post coital. and lactation on Days 1 and 5 post partem. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 post partem. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females.
Sacrifice and pathology:
The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 post partem. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control and high dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low and intermediate dose groups and on all macroscopic lesions.
Statistics:
Body weight, food consumption and reproductive data :Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Hematology and blood biochemistry: A specific sequence was used for the statistical analyses of hematology and blood biochemistry data.
Organ weight: PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) according to a specific sequence

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg bw/day from the first or second week of treatment and in most of the animals at 100 mg/kg bw/day but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg bw/day but more at 1000 mg/kg bw/day were considered to be of limited toxicological significance. Incidental findings consisted of half-closed eyes, reflux at dosing, cutaneous lesion and abnormal growth of teeth.
Mortality:
mortality observed, treatment-related
Description (incidence):
Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg bw/day from the first or second week of treatment and in most of the animals at 100 mg/kg bw/day but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg bw/day but more at 1000 mg/kg bw/day were considered to be of limited toxicological significance. Incidental findings consisted of half-closed eyes, reflux at dosing, cutaneous lesion and abnormal growth of teeth.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See details below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See details below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details below
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Food consumption: In males, mean food consumption at 1000 mg/kg bw/d was statistically significantly reduced in the first week of treatment when compared with controls (which correlated with a non-statistically significantly lower mean body weight gain at that time). It became similar to controls in the second week of dosing. This slight and transient effect was considered to be of limited toxicological significance. Mean food consumption at 100 and 300 mg/kg bw/d was not affected. Mean food consumption in females was considered to be unaffected by the test substance treatment.

Hematology: APPT values were not considered to be affected (one control data in males was higher than the others hence the shortened mean values in the test substance treated groups; there was no dose-relationship in females). For the slightly higher mean white blood cell counts when compared with controls, a relationship with the test substance treatment was considered to be doubtful (no clear dose-relationship, individual values generally included in historical control data, and no statistical level).

Blood biochemistry: Mean cholesterol level was higher in test substance treated female groups in a dose-related manner, reaching statistical and toxicological significance at 300 and 1000 mg/kg bw/d. All individual values in both groups were included in historical control data and in absence of adverse correlates in the study, this was considered to be non-adverse. There was no dose-relationship in males, but an effect of the test substance may be suspected in two males treated at 1000 mg/kg bw/d which had a high cholesterol level (2.3 and 2.4 mmol/L). Mean sodium concentration was also statistically significant higher in females treated at 1000 mg/kg bw/d when compared with controls. Males or other electrolytes in females were not affected and the variation was not severe. Therefore, this finding was not considered to be of toxicological significance. An effect of the test substance treatment on mean albumin concentration at 300 mg/kg bw/d in both sexes was considered unlikely as there was no dose-relationship. The increase observed in triglyceride levels at 300 and 1000 mg/kg bw/d in both sexes was considered to be incidental as there was no statistical level reached for the group means and no dose-relationship seen in individual data. Moreover, the increase at 1000 mg/kg bw/d was due to isolated animals per sex only.

Functional Observation Battery (FOB) and motor activity: An effect of the test substance treatment in males and females at 300 and/or 1000 mg/kg bw/d on mean motor activity data was considered to be equivocal but of limited toxicological significance. The vast majority of the individual data were similar to what can usually be seen in this type of study.

Organ weights: When compared with controls, the mean absolute and relative liver weights were higher in males and females given 1000 mg/kg bw/d and in males given 300 mg/kg bw/d, reaching statistically significant values in both sexes at 1000 mg/kg bw/d (p<0.01 except for the absolute weight in females where it was p<0.05). This correlated histologically with minimal hepatocellular hypertrophy and was considered to be test substance related. The mean absolute and relative thymus weights were lower in females given 1000 mg/kg bw/d, without reaching a statistically significant value. This correlated with minimal to slight atrophy in two females and was considered to be probably test substance related. The mean absolute heart weight was higher in males given 100 mg/kg bw/d. In the absence of a dose-related trend, any relationship with the test substance was considered to be excluded. Other changes in the mean organ weights were considered to be part of the normal variation in rats and without relationship with the test substance.

Macroscopic post-mortem examination: The thymus was reduced in one female given 1000 mg/kg bw/d correlating in this animal with a small weight (0.1200 g) and histologically with slight atrophy. A relationship with the test substance was considered to be likely. Irregular color was seen in the lungs of 3/5 males given 1000 mg/kg bw/d. This correlated histologically with presence of mucus and inflammation (chronic) and/or alveolar macrophages. These changes were consistent with aspiration or regurgitation of the oily vehicle or test item during the technical gavage procedures.

Microscopic examination: Test substance related changes were observed in the liver and the thymus.

Liver: Minimal hepatocellular hypertrophy was seen in 1/5 males given the test item at 300 mg/kg bw/d and 4/6 males and 2/5 females given 1000 mg/kg bw/d. This non-adverse change was considered to be test substance related and correlated with the higher weight noted at necropsy. Other changes seen in the liver, including the minimal foci of subcapsular necrosis seen in 1/5 control females, 1/5 males at 300 mg/kg bw/d and 2/5 females at 1000 mg/kg bw/d were considered to be fortuitous and without any relationship with the test substance.

Thymus: Minimal or slight lymphoid atrophy was seen in 2/5 females given 1000 mg/kg bw/d, correlating with the lower weight at necropsy. Any relationship with the test substance was considered to be likely but non-adverse (low number of animals affected and low grades). No test substance related changes were seen at 100 or 300 mg/kg bw/d.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Parental or systemic toxicity (non adverse)

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under study conditions, the NOAEL for parental or systemic toxicity was determined to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance, Oleamide MIPA, according to OECD Guideline 422, in compliance with GLP. Three groups of ten male and ten female Sprague-Dawley rats received the test substance daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5 post-partum. The test substance was administered as a suspension in the vehicle, corn oil, at dose-levels of 0,100, 300 or 1000 mg/kg bw/day. Another group of ten males and ten females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg bw/day was used. The concentration of the dose formulation was checked in study weeks 1, 3 and 6. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post coital and lactation on Days 1 and 5 post-partum. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 post-partum. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (haematology and blood biochemistry) were carried out on five males and five females. The males were sacrificed after at least 5 weeks of treatment and the females on day 6 post-partum. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control and high dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low and intermediate dose groups and on all macroscopic lesions. The test substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no test substance in control formulations. There were no test substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/d and in most of the animals at 100 mg/kg bw/d (minor toxicological significance), as well as hypoactivity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg bw/day for a few days (limited toxicological significance). There were no relevant effects on mean body weight, mean Functional Observation Battery (FOB), as well as on mean haematology parameters in any group and sex. An effect of the test substance treatment on mean motor activity data at 300 and/or 1000 mg/kg bw/day was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg bw/day was reduced in the first week of treatment only (23 g/male/d, vs. 27, p<0.001). This effect was considered to be of limited toxicological significance. Mean food consumption in males at 100 and 300 mg/kg bw/d and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg bw/day (up to 1.9 mmol/L, vs. 1.2, p<0.01) and considered to be non-adverse in absence of adverse correlates in the study. There were no relevant blood biochemistry findings in females at 100 mg/kg bw/d or in males. At histopathology at 1000 mg/kg bw/day, minimal hepatocellular hypertrophy correlating with higher mean liver weight was seen in the liver of both sexes (about +28% in males and +20% in females compared to controls, p<0.01 generally). In females, mild lymphoid atrophy was seen in the thymus of 2/5 females, correlating with lower mean weight at necropsy (about -22% from controls). At 300 mg/kg bw/day, only minimal hepatocellular hypertrophy was seen in the liver of a single male correlating with minor higher mean absolute weight in this group. All these microscopic findings were considered to be non-adverse (low number of animals affected and/or minimal to slight grades). There were no histopathological effects at 100 mg/kg bw/day. Based on the results of the study, the study author determined the NOAEL for parental or systemic toxicity to be at 1000 mg/kg bw/day (Bentz, 2013).