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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 31, 2013 to June 17, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
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 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs 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 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 substance was weighed in the preparation beaker and the corresponding amount of vehicle was added,
- the test substance 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/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred as Day 0 post-coitum
- After successful mating each pregnant female was caged individually
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 days 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
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/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 item as a suspension in corn oil at 100, 300 or 1000 mg/kg/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 item-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 item-related macroscopic findings.

- Animal assignment: computerized stratification procedure.
Positive control:
Not required

Examinations

Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

DETAILED CLINICAL SIGNS:
- Time schedule: once a week during the treatment period.

BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: Males: once a week until sacrifice. Females: once a week until mating, on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.

- NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: at the end of the treatment period.

HAEMATOLOGY:
- Time schedule: on the day of sacrifice.

CLINICAL CHEMISTRY:
- Time schedule: on the day of sacrifice.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
Fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below
- special emphasis to the spermatogenic cycle and testicular interstitial cells (groups 1 and 4).
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after the end of the mating period
- Female animals: all surviving animals = Day 6 post-partum or, for females which had not delivered yet, Day 25 post-coitum.

ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination.

HISTOPATHOLOGY:
- on all tissues listed in the table below for the first five control and high dose animals (groups 1 and 4) sacrificed as scheduled,
- on all macroscopic lesions,
- all females sacrificed because of no delivery to investigate possible causes,
- liver (both sexes), thymus (females only), seminal vesicles (males only) and bone marrow (females only) from the first five sacrificed as scheduled males and the first five females sacrificed on day 6 p.p. of the low- and intermediate-dose groups (groups 2 and 3).
Postmortem examinations (offspring):
SACRIFICE: on Day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on Day 4 p.p. = 100 * (Number of surviving pups on Day 4 p.p. / Number of live born pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For details see section 7.5.1
Mortality:
mortality observed, treatment-related
Description (incidence):
For details see section 7.5.1
Body weight and weight changes:
no effects observed
Description (incidence and severity):
For details see section 7.5.1
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For details see section 7.5.1
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):
For details see section 7.5.1
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For details see section 7.5.1
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY:
There were no deaths considered as test substance related. One female given 1000 mg/kg/day was prematurely sacrificed on Day 23 p.c. due to difficulty to deliver. Piloerection, round back, cold to the touch, abdominal breathing, reddish vaginal discharge, chromodacryorrhea and chromorhinorrhea were observed on Day 22 and/or 23 p.c. Blood in the bedding, fetuses (mostly dead) and a few placentae were found in the bedding on Day 23 p.c. This death was considered to be incidental. There were no other unscheduled deaths during the study.

CLINICAL SIGNS (see table 1):
Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg/day from the first or second week of treatment and in most of the animals at 100 mg/kg/day but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day but more at 1000 mg/kg/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 (GAIN):
There were no relevant effects on mean body weights and mean body weight gains. The low mean body weight gain in high-dose males for the interval days 1 to 8 when compared to controls was considered to be of no toxicological significance (no statistical level and transient decrease).

FOOD CONSUMPTION (see table 2):
In males, mean food consumption at 1000 mg/kg/day 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/day was not affected. Mean food consumption in females was considered to be unaffected by the test item treatment.

NEUROBEHAVIOURAL EXAMINATION (see table 3):
There were no relevant findings on FOB in test substance-treated groups when compared to the control group. An effect of the test substance treatment in males and females at 300 and/or 1000 mg/kg/day 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.

HAEMATOLOGY:
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).

CLINICAL CHEMISTRY (see table 4):
Mean cholesterol level was higher in test item-treated female groups in a dose-related manner, reaching statistical and toxicological significance at 300 and 1000 mg/kg/day. 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/day 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/day 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/day in both sexes was considered unlikely as there was no dose-relationship. The increase observed in triglyceride levels at 300 and 1000 mg/kg/day 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/day was due to isolated animals per sex only.

REPRODUCTIVE PERFORMANCE:
Mating and fertility data
There were no test substance-related effects on mean mating and fertility data. At 1000 mg/kg/day, one female was blocked in metestrous/diestrous for 12 d and mated with its male after 13 d, and one female was prematurely sacrificed on Day 23 p.c. because of difficulty to deliver as previously mentioned. Both events were considered to be incidental as isolated. A total of five females (three controls and two females treated at 1000 mg/kg/day) were sacrificed on Day 25 p.c. because of no delivery. They were non-pregnant.

Delivery data
There were no test substance-related effects on mean delivery data. Mean pre- and post-implantation loss data were lower than the highest percentages seen in historical control data, thus there were no effects of the test substance treatment.

ORGAN WEIGHTS (see table 5):
When compared with controls, the mean absolute and relative liver weights were higher in males and females given 1000 mg/kg/day and in males given 300 mg/kg/day, reaching statistically significant values in both sexes at 1000 mg/kg/day (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/day, 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/day. 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.

GROSS PATHOLOGY:
The thymus was reduced in one female given 1000 mg/kg/day 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/day. 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. Other changes were considered to be part of the normal background commonly seen in the rats and were considered to be without relationship with the test substance.

HISTOPATHOLOGY:
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 substance at 300 mg/kg/day and 4/6 males and 2/5 females given 1000 mg/kg/day. 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/day and 2/5 females at 1000 mg/kg/day 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/day, 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/day.

Miscellaneous changes
- Seminal vesicles
Minimal apoptosis was seen in 1/5 males given 100 mg/kg/day and 3/6 animals given 1000 mg/kg/day. As this change was minimal, and in the absence of a dose-related trend and other test substance-related changes in testes or epididymides, any relationship with the test substance was considered to be unlikely.

- Bone marrow
There was a trend towards higher presence of adipose tissue in the bone marrow of females at all dose levels when compared with controls. As this was not seen in males, poorly dose-related and as this was not correlated with any changes in the hematological parameters, any relationship with the test substance was excluded.

- Lungs
Mucus was seen in bronchi and/or bronchioli of 3/6 males given 1000 mg/kg/day and eosinophilic infiltrate in 3/6 males at this dose-level. Increased alveolar macrophages were seen at a similar incidence and severity between controls and treated animals in both sexes. These changes were consistent with aspiration or regurgitation of the oily vehicle or test substance during the technical gavage procedures. Any direct relationship with the test substance was excluded.

A careful examination of testes, epididymides, and female genital tract did not show any test substance-related effects on these organs.

Other histopathological changes were considered to be part of the normal background of changes commonly seen in rats and without any relationship with the test substance.

Effect levels (P0)

open allclose all
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 toxicity (non adverse)
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: Reproductive performance (mating and fertility)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
See below for details
Mortality / viability:
no mortality observed
Description (incidence and severity):
See below for details
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See below for details
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See below for details

Details on results (F1)

MORTALITY/VIABILITY:
There were no effects on pup viability.

CLINICAL SIGNS:
None of the clinical signs were considered to be test substance related (comparable incidences in control pups).

BODY WEIGHT (GAIN):
There were no effects of the test substance treatment on mean body weight and mean body weight changes in pups. The trend toward higher mean body weights and mean body weight gain at 100 mg/kg/day was considered to be incidental (not dose-related).

SEX RATIO (see table 6)
Despite the fact that the pup sex ratio at 1000 mg/kg/day was slightly above the historical control range, it was considered to be of no toxicological significance in view of its low amplitude.

GROSS PATHOLOGY:
Both findings are not very often recorded in controls of such studies, although they can appear spontaneously. An effect of the test substance treatment was suspected since they were observed with a dose relationship at both the highest dose-levels. However, the effect was considered as of minor toxicological significance as these findings are generally considered as morphological variations (not malformations) and as their incidence was not severe.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Toxic effects on progeny (minor toxicological significance)

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under study conditions, NOAEL for parental toxicity, reproductive performance (mating and fertility), and toxic effects on progeny was determined to be at 1000 mg/kg bw/day.
Executive summary:

A study was conducted to determine the reproductive toxicity potential of the test substance, Oleamide MIPA, according to OECD Guideline 422, in compliance with GLP. The test substance in corn oil was administered daily by oral gavage to three groups of ten male and ten female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until up to Day 5 post partem, at dose-levels of 100, 300 or 1000 mg/kg/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/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 coitus 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. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 post partem. 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 partem. Final body weights and selected organs weights (adrenals, brain, epididymis, 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 pups were sacrificed on Day 5 post partem and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. 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/day and in most of the animals at 100 mg/kg/day (minor toxicological significance), as well as hypo activity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg/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/day was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg/day was reduced in the first week of treatment only (23 g/male/day, 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/day and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg/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/day or in males. At histopathology at 1000 mg/kg/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/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/day. There were no effects on mean mating, fertility and delivery data in any group. There were no test substance related effects on pup viability, clinical signs, body weight and sex ratio. Dilated ureter and renal pelvis were recorded with dose-relationship in a few litters at necropsy at 300 (1 or 2 litters affected out of 10) and 1000 (2/7 litters) mg/kg/day Vs none in the controls. An effect of the test substance treatment was considered to be of minor toxicological significance. Under the study conditions, NOAEL for parental toxicity, reproductive performance (mating and fertility), and toxic effects on progeny was determined to be 1000 mg/kg bw/day (ECHA, 2013).