Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Well conducted and comparable to guideline study, no information on GLP status
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Mus-Rattus, Brunntal, Germany
- Strain: Wistar rat, MuRa Han 67 SPF
- Age at study initiation: between 6-7 weeks
- Weight at study initiation: 109 (f) - 114 (m) g
- Housing: plastic cages, 3 males and 5 females/cage
- Diet (e.g. ad libitum): ad libitum (Altromin Ratdiet No. 1424 DK, Altromin GmbH, Lage, Germany)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 60-80
- Air changes (per hr): 11
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Doses were adapted weekly to the body weight; application volume - 5 mL/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 d
Frequency of treatment:
Daily once, 5 times/wk
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Remarks:
Days 1-28
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Days 1-28
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Days 1-14
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
Days 15-28
No. of animals per sex per dose:
10/sex/dose for main study; 5/sex/dose for 4 months recovery period


Control animals:
yes, concurrent no treatment
Details on study design:
According to standard procedure
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: end of study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per dose and sex
- Parameters checked: Hematocrit, erythrocytes, leukocytes, hemoglobin, thrombocytes, mean corpuscular volume, white blood cell differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- How many animals: 10 per sex and dose
- Parameters checked: GPT, GOT, AP, glucose, urea, total protein, calcium, phosphate, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: end of study
- Metabolism cages used for collection of urine: No
- Parameters checked: urea, creatinine, sodium, potassium, glucose, calcium, AP

NEUROBEHAVIOURAL EXAMINATION: No

Other: Groups of 5 male and 5 female rats kept for an additional 4 month recovery period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
the following tissues/organs were examined:
adrenal gland
aorta thoracica
brain (cornu ammonis)
coagulating gland with seminal vesicle
epididymis
eye with optic nerve
heart
intestine, large
intestine, small
kidney
liver
lungs
lymph node (cervical)
lymph node (mesenteric)
mucles
oesophagus
ovary
pancreas
prostate
salivary glands (mandibular,
parotid and sublingual gland)
skin
spleen
stomach
testicle
thymus
thyroid (incl. parathyroids)
tongue
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)


HISTOPATHOLOGY: Yes
see gross pathology
Statistics:
't' test used for statistical analysis of all parameters except organ weight (U-test)


Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
The doses up to 1500 mg/kg bw/day were tolerated by all animals without lethality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight gain and total increase of body weights did not differ from the control group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterine, GGT, GOT, GPT and LDH did not show any critical signs. Only slight shifts which were not dose-dependent could be observed. These signs were considered as not substance depending.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative organ weights showed no significant changes in the substance groups compared to the control group, except the organ weight of the liver which is slightly increased for the males of group 4 (750/1500 mg/kg bw) and increased adrenal glands weight in high dose females. This result is considered of no relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No biologically relevant treatment-related effects observed on any of the parameters recorded at any dose, also test animals treated with 1500 mg/kg bw (Days 15-28) showed no adverse effect
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C12-18 and C18-unsatd. DEA, according to design based on OECD guideline 407. Groups of 10 male and 10 female Wistar rats were orally gavaged with the substance diluted in olive oil, 5 d/week for 28 d at doses of 0, 70, 250, 750 (Days 1-14) and 1500 (Days 15-28) mg/kg bw/d. Clinical signs, bodyweight, haematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. No treatment-related adverse effects were observed at any of the doses. Changes in the forestomach at some doses including controls were attributed to the use of olive oil and found to be reversible after end of exposure. Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day (Potokar, 1983).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2016-January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Remarks:
No major deviation during the course of the study
GLP compliance:
yes (incl. QA statement)
Remarks:
23 march 2017
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
SPF (Specific Pathogen Free) Sprague-Dawley - Crl: OFA (SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 L'ARBRESLE Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks on the day of the first administration.
- Weight at study initiation: Between 184.2 and 231.9 g in males and 125.5 g and 171.4 g in females. The weight variation of animals used did not exceed +/- 20% of the mean weight of each sex.
- Housing: Animals were housed (separated by sex) in cages of standard dimensions with sawdust bedding (or equivalent).
- Diet: RM1 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilised) was available ad libitum except during the fasting experimental period. A certificate of analysis concerning this feed product is included to the study report. The criteria for acceptable levels of contaminants in the feed supply were within the limits of the analytical specifications established by the diet manufacturer.
- Water: Drinking water was available ad libitum in polycarbonate bottles with a stainless steel nipple. A specimen of water is obtained approximately every 6 months and sent to Laboratoire de la Touraine (ZA n°1 du Papillon - Rue de l'Aviation - 37210 Parçay-Meslay - France), for analysis. The certificates of analysis are included in the report. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications
- Acclimation period: A minimum of five days in the laboratory animal house where the experiment took place. Only animals without any visible sign of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The room temperature was between 19°C and 24°C and was recorded at the beginning and at the end of all observations.
- Humidity (%) and air changes (per hr): The animals were placed in an air-conditioned (20-24°C) animal house kept at relative humidity between 45% and 65% (except during the cleaning slot) in which nonrecycled filtered air was changed approximately 10 times per hour. Between 28 Nov at 08.00 p.m. and 29 Nov 2016 at 11.00 a.m. (for about 15 hours) and between 16 Jan at 10.00 p.m and 17 Jan 2017 at 12.30 a.m, an abnormal decrease in hygrometry was noted in the animal room.
- Photoperiod (hrs dark / hrs light): The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 a.m. except on 30 Oct 2016.

IN-LIFE DATES: From: 18 october 2016 to: 20 January 2017
Route of administration:
oral: gavage
Details on route of administration:
N-(2-hydroxypropyl) Oleamide or its vehicle was administered once a day at approximately the same time (a maximum range of 4 hours between the start and the end of the daily treatment) at each chosen dose level, by the oral route for 13 weeks. A constant administration volume of 5 mL/kg body weight (except some animals) was used in accordance with the previous study (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route (gavage) in rats - OECD 422 - No.39644 RSR).
Vehicle:
corn oil
Details on oral exposure:
- DIET PREPARATION: no information available
- VEHICLE : Corn oil was used (Reference C8267)
- Lot/batch no.: Batch Nos. MKBS6944V and MKBW9504V, Expiry dates: 08 Oct 2020 and 06 Oct 2021 respectively
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of test item in formulations were checked during the first and last week of the study. Each concentration level and the vehicle was checked. The analytical method is validated within the range 85-115% of the theoretical concentration for formulations between 19.765 mg/mL and 198.120 mg/mL, with a precision better than 10%. The samples in corn oil must be analysed within the stability period (i.e. 30 days at room temperature) and must be within the range 85-115% to meet the acceptance criteria. Formulation analysis was performed on one formulation prepared during the first and the last week of the study. The concentrations tested were 20 mg/mL, 60 mg/mL and 200 mg/mL. The concentrations found were within the range of acceptance (+/-15% of the intended concentration). The absence of test item was also confirmed in the vehicle samples. These results show that the formulations were properly prepared.
Duration of treatment / exposure:
Main phase: Minimum of 90 days (13 weeks)
Frequency of treatment:
Once a day
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
The study was conducted on 4 groups of 10 males and 10 females including a control group (80 animals in the main group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Proposed doses are selected in agreement with the Sponsor. The choice is based on previous studies (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route (gavage) in rats - OECD 422 - Study No. 39644 RSR). Moreover, the highest dose should reveal signs of toxicity and the lowest dose should represent a no-observed-adverse effects level.
- Allocation of each animal to treatment : randomly determined before the start of the study. Homogeneity of groups was validated on the criterion of body weight measured on the day of randomisation, separately for males and females.
- Justification of the number of animals per group: The number of animals per group is the minimum number enabling an accurate assessment of the studied toxicological effect and comparisons using the appropriate statistical tests.
- Dose adjustement: Doses of test item were expressed as mg/kg bw. Doses were adjusted on the basis of body weight measured at the most recent weighing.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed in the cage before the first dosing and at least once a day except for some animals. The time of observation was between 21 minutes and 1h45 post dose. The general disposition, behaviour and activity were observed. Once a week except for one female on week 3, animals were submitted to a full clinical examination in a cage without sawdust according to a standardised observation battery for general clinical signs, neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. The time of observation was at 1 hour (+/- 30 min) or 3h35 on d7 for Female No. 1602396 post dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the following days:
• on the day of randomisation
• at predose
• then weekly during the study
• on the day of necropsy (not exsanguinated), this is the reference weight used in the calculation of relative organ weights

FOOD CONSUMPTION : Yes
Food consumption was measured for each treatment cage except on the day of fasting. Animals were fasted during the night before scheduled blood sampling for haematology/coagulation parameters, clinical chemistry and during urine collection for urinalysis.

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination (Retinography) was performed under the responsability of a person who has followed a veterinary ophthalmological training on all animals before the first dosing and during the last week.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken from all animals during the last week. Samples were taken just before killing for moribund animals when possible.
- Anaesthetic used for blood collection: Yes (isoflurane inhalation)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken from all animals during the last week. Samples were taken just before killing for moribund animals when possible.
- Animals fasted: Yes

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Urine was collected from all animals, during the last week. Urine collection was performed individually in metabolism cages for a period of about 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (Food and water were withheld during collection)

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: Before the first dosing and on the last week, animals were observed according to a standardised observation battery for neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. The method is based on an Irwin screen modified by suppressing the graduation of intensity of clinical signs. Animals were observed individually in a cage without sawdust in a quiet room.The time of observation was 1 hour (+/- 36 min) post dose. The rectal temperature was measured at the end of each observation.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Hyperalertness, Decrease in visual placing, Passive response to finger approach, Passivity when touched, Absence of reactivity, Stereotyped movements, Aggressiveness/irritability, Increase/decrease in fear, Reduced/increased spontaneous locomotor activity, Increasing grooming, Staggering gait, Abnormal gait, Loss of righting reflex, Absence/increase of startle response, Insensitivity/Hyperreactivity to pinching of tail, Body sag, Decrease/Increase in limb tone , Decrease in grip strength, Decrease in body tone, Decrease in abdominal tone, Sitting up, Rearing, Virtually permanent reared position, Loss of pinna reflex, Loss of corneal reflex, Hind limb reflex, Pallor, Tachycardia, Bradycardia, Myosis, Mydriasis, Ptosis, Exophthalmos, Lacrimation, Liquid/soft faeces
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals dying during the course of the study and animals killed in a moribund condition were necropsied as quickly as possible and specimens as required by the study plan obtained whenever practically possible. All animals (including any found dead or moribund) were submitted to full necropsy procedures including an examination of:
• the external surface
• all orifices
• the cranial cavity
• the external surface of the brain and samples of the spinal cord
• the thoracic and abdominal cavities and organs
• the cervical tissues and organs
• the carcass
Paired organs were weighed together. From all animals, excluding those found dead, the organs were dissected free of fat and other contiguous tissues at the discretion of the prosector and weighed.

HISTOPATHOLOGY: Yes
Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically
The organs and tissues sampled were fixed and preserved in 4% buffered formalin with the following exceptions:
• testes and epididymides were fixed in alcoholic Bouin0s fluid (about 5 days), then transferred to ethanol 95%
• eyes and optic nerves were fixed and preserved in Davidson0s fluid (about 3 days), then transferred to ethanol 70%

Organs for organ weight determination and for histopathological examination are presented in table below.

SACRIFICE: Main phase animals will be killed following 13 weeks of treatment.
Other examinations:
None
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In decedent animals, the day before or the day of death, there were the following clinical signs:
• At 100 mg/kg bw, in Male No.1602323, there was no clinical sign.
• At 300 mg/kg bw, there was an increased salivation and absence of spontaneous locomotor activity in Male No.1602335 on d87 (the day before death). In male No.1602340, there was blood around and in the mouth on d58 (the day before death).
• At 1000 mg/kg bw, there were increased salivation, chromodacryorrhoea, dyspnoea, bradypnoea, decrease then absence of locomotor activity and it was lying on its sides on d59, in Male No. 1602344. In Male No. 1602345, there was increased salivation. In Female No. 1602388, there was increased salivation.

In surviving males and females treated with test item whatever the dose, there was a dose dependent:
• increased salivation (8.8%, 19.8% and 41.7% in males and 0.8%, 3.2% and 22.6% in females respectively) from d26 up to the end of the study
• chromodacryorrhoea (2.1%, 6.1% and 8.8% in males and 0.1%, 3.4% and 9.1% in females respectively) from d28 up to the end of the study
There were also isolated clinical signs such as a decrease in spontaneous locomotor activity from d57 up to d60 in at most 3/10 males treated with test item at 300 and 1000 mg/kg bw and also in 1/8 males treated with test item at 1000 mg/kg bw on d91, a decrease in fear in 1/8 males treated at 1000 mg/kg bw or 1/10 females treated at 100 mg/kg bw, an increase in fear or insensitivity to pinching of the tail in 1/10 females at 100 mg/kg bw.
Mortality:
mortality observed, treatment-related
Description (incidence):
6 animals treated with test item were found dead between d59 and d91:
• at 100 mg/kg bw, Male No. 1602323 was killed early on d77
• at 300 mg/kg bw, Male No. 1602335 on d88 and Male No. 1602340 on d59 were found dead
• at 1000 mg/kg bw, Male No. 1602344 was moribund and was found dead during the isoflurane anaesthesia, just before exsanguination on d59, Male No. 1602345 was found dead on d80, Female No. 1602388 was found dead on d91.
There was no mortality in the control group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no change in body gain in animals found dead except for male No. 1602345 treated at 1000 mg/kg bw which had a body weight loss (-9.6%) between d63 and d70. There was a statistically significant lower body weight gain in males treated with test item at 1000 mg/kg bw from d14 up to the end of the study (on d91, body weight gain was +71% vs +107% in the control group) and at 100 mg/kg bw from d70 up to the end of the study (on d91, body weight gain was +87% vs +107% in the control group). In males treated at 300 mg/kg bw, based on the mean values, there was no difference when compared to the control group due to Male No. 1602331 which had very high body weight (649 g vs 510 g the mean values of the group). However, if considering the individual values, there was also a lower body weight gain in the other males treated at 300 mg/kg bw. When the values of this animal were excluded, the body weight gain was +97% vs +107% in the control group. One male treated with test item at 100 mg/kg bw (No. 1602324) had a body weight loss between d84 and d91 (-5.4%). There was no change in body weight gain in females.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slightly lower food consumption in all males treated with test item whatever the dose (consumption was approximately 17-18 g/animal/day vs 20 g/animal/day in the control group. In cage 6 (with Male Nos. 1602324 and 1602325) of group 2, the food consumption was lower during the last week of the treatment period. There was no marked change in food consumption in females whatever the dose.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Not necessary
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There was no abnormality at the ophthalmological examination on d91 when compared to the records before the start of the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the decedent animals only the animal killed on d77 yielded data. A slight reduction of the white cell series was seen (total white cell count, neutrophil count, eosinophil count, basophil count, lymphocyte count). At 1000 mg/kg bw, there was a higher total white blood cell count in males (+20%) and in females (+30%) on d92. In females, there was also a statistically significant higher lymphocyte count (+33%) and monocyte count (+58%) on d92. There was also a statistically significant higher reticulocyte count in males treated with test item at 100 and 1000 mg/kg bw mainly due to two lower values in the control group (in male Nos. 1602312 and 1602320). Therefore this changes was not attributed to the test item. At 300 and 100 mg/kg bw, there was no change in haematology parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the decedent animals only the animal killed on d77 yielded data. A slight higher ALT, AST and ALP activities and slightly lower total bilirubin and albumin.
There was dose related statistically significant higher plasma ALT, AST and ALP activities in the males treated with test item at 300 and 1000 mg/kg bw and a statistically significant higher ALT activity (+45%) in females treated at 1000 mg/kg bw. There was also a statistically significant higher A/G ratio in male treated at 1000 mg/kg bw due to lower globulins. This was considered to be of minor degree (+14%) and was not considered as toxicologically relevant. At 100 mg/kg bw in surviving animals and at 300 mg/kg bw in females, there was no change in blood chemistry parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no change in urinary volume and semi-quantitative parameters. There was a higher creatinine level in urine in Male No. 1602324 treated with test item at 100 mg/kg bw.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was statistically significant changes at 1000 mg/kg bw (in absolute and/or relative values body or brain weight):
• higher liver weight in males and females
• higher adrenals weight in males
• lower thymus weight in males
• higher testes and epididymides weight in males
There was a statistically significant higher adrenals weight at 100 and 300 mg/kg bw in males (in relative values % body weight). This was low in amplitude (+18% when compared to the control relative values % body weight) and was not considered as toxicologically relevant. There were also statistically significant lower (in absolute values) spleen, thymus, heart and brain weights related to the lower body weight in males treated at 1000 mg/kg bw and lower thymus weight in males treated at 100 mg/kg bw due to the low individual value in Male No. 1602324. There was no other change in organ weight in animals treated at 300 or at 100 mg/kg bw.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In decedent animals, there were mainly dark organs (mucosa, liver, spleen, lung or heart) or mottled lung and soft brain. These observations were usually observed in animals found dead or in moribund state. In male No. 1602323 treated at 100 mg/kg bw and male No. 1602335 treated at 300 mg/kg bw, there was blood in the mouth. In males 1602335 and 1602340 treated at 300 mg/kg bw, there was liquid in the thoracic cavity and a white film in the thoracic cavity in Male No. 1602340. For male No. 1602344 treated at 1000 mg/kg bw, there was a subcutaneous hole under the left forelimb and a subcutaneous mass on the neck. In surviving animals, there were isolated macroscopic findings found at a low incidence such as point change in the heart (1/8 in males treated at 300 mg/kg bw) or dark mesenteric lymph nodes (1/8 in males treated at 1000 mg/kg bw) or observations found at the same incidence in the control and test item dose groups, such as point changes in thymus or liver (1/10 control males vs 1/8 treated males at 300 mg/kg bw), dark or point changes in sub maxillary lymph node. In Male No. 1602350 treated at 1000 mg/kg bw, there was pale and enlarged lung with dark area.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In decedent animals, there were no lesions suggesting the cause of death in any of these animals. In the male treated at 100 mg/kg bw and killed early on d77, there was only mild periacinar hepatocytic hypertrophy and minimal sternal lipid hyperplasia. In Male No. 1602345 and in Female No. 1602388 treated at 1000 mg/kg bw, there was minimal femoral lipid hyperplasia and minimal periacinar hepatocytic hypertrophy respectively. In other decedent animals treated at 300 or 1000 mg/kg bw, there were no relevant changes. In surviving animals, there were minor lesions in the thymus, liver and bone marrow related to the treatment.
Thymic atrophy was present in the majority of animals whatever the treatment, the degree was greatest in animals treated at 1000 mg/kg bw but the incidence and degree in other groups including the control group was sufficiently inconsistent to deny any true link to the test item. Hepatic changes in males treated at 1000 mg/kg bw and females treated at 300 and 1000 mg/kg bw were biliary prominence and periacinar hypertrophy. These findings were present in the control group, at the lower incidence and degree. The changes were adaptative in nature and very slight in degree, different following the gender and were not deemed significant. The partial replacement of bone marrow by lipid vacuolation in the femur was present in males treated at 1000 mg/kg bw and in the sternum in males treated at 300 and 1000 mg/kg bw. There were slight histopathological changes in the liver and the bone marrow in animals treated with test item at 1000 mg/kg bw.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There was statistically changes in mobile spermatozoa at 100 and 1000 mg/kg bw, midpiece anomaly at 100 mg/kg bw, cytoplasmie droplet anomaly at 1000 mg/kg bw. since there were no changes in % mobile spermatozoa or the variation was very low, these changes were not attributed to the test item. There was no change in sperm analysis.
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
clinical signs
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Conclusions:
Under the study conditions, a NOAEL could not be determined in males, whereas in females, the NOAEL was considered as 300 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C18-unsatd. MIPA, according to OECD Guideline 408, in compliance with GLP. The substance diluted in corn oil was administered by gavage to groups of male and female Sprague-Dawley rats (10/sex/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/day for 13 weeks (at constant administration volume of 5 mL/kg bw). Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. A full clinical examination was performed once a week. Functional and neurobehavioral tests were performed before the first dosing and in the last week. Body weight was recorded at pre-dose and once a week. Food consumption was measured weekly. Ophthalmological examination was performed before the first dosing and during the last week. Blood samples for haematology parameters and clinical chemistry analysis and urine were collected on d92. All animals were killed on d92 (or d91 for one female). Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically. Epidydimides were collected on d92 for sperm analysis. 5 males treated with test substance (2 males at 300 mg/kg bw/day and 2 males at 1000 mg/kg bw/day) were found dead between d59 and d88 and one male treated with test substance at 100 mg/kg bw/day was killed early on d77. One female treated with test substance at 1000 mg/kg bw/day was found dead on d91. On the days before death, there were no particular clinical signs but on the day of the death, there was mainly bradypnoea or polypnoea or absence or decrease in spontaneous locomotor activity. There was dose related statistically significant higher ALT, AST and ALP activities in males treated with test substance at 300 and 1000 mg/kg bw and statistically significant higher ALT activity in females treated at 1000 mg/kg bw/day. There were statistically significant changes at 1000 mg/kg bw/day, including higher liver weight in males and females, higher adrenals weight in males, lower thymus weight in males, and higher testes and epididymides weight in males. There were also bone marrow microscopic changes. No lesions at histopathology examination suggested the cause of death; however similar effects as those seen at the highest dose was noted such as higher hepatic enzymes activities or mild periacinar hepatocytic hypertrophy. In other animals treated at 100 mg/kg bw/day, there were no changes in hepatic plasma enzyme and no microscopic changes. Whatever the tested doses, the substance induced mortality, a lower food consumption and lower body weight gain in males. The male gender seems to more sensitive than female. Under the study conditions, a NOAEL could not be determined in males, whereas in females, the NOAEL was considered as 300 mg/kg bw/day (Mortier, 2018).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
31 January 2013 - 17 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item 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 item 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 item 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 item 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test item 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
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Controls
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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 bw 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:
no (not required)
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 p.c. and lactation on days 1 and 5 p.p.. 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 p.p.. 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 p.p.. 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
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/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw 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/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw 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):
In males, mean food consumption at 1000 mg/kg/day bw 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 bw was not affected. Mean food consumption in females was considered to be unaffected by the test item treatment.
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):
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 item-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 item treatment was considered to be doubtful (no clear dose-relationship, individual values generally included in historical control data, and no statistical level).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 bw. All individual values in both groups were included in historical control data (see Appendix 38) 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 item may be suspected in two males treated at 1000 mg/kg/day bw 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 bw 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 item treatment on mean albumin concentration at 300 mg/kg/day bw 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 bw 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 bw was due to isolated animals per sex only.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
An effect of the test item treatment in males and females at 300 and/or 1000 mg/kg/day bw 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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, the mean absolute and relative liver weights were higher in males and females given 1000 mg/kg/day bw and in males given 300 mg/kg/day bw, reaching statistically significant values in both sexes at 1000 mg/kg/day bw (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 item-related. The mean absolute and relative thymus weights were lower in females given 1000 mg/kg/day bw, without reaching a statistically significant value. This correlated with minimal to slight atrophy in two females and was considered to be probably test item-related. The mean absolute heart weight was higher in males given 100 mg/kg/day bw. In the absence of a dose-related trend, any relationship with the test item 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 item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The thymus was reduced in one female given 1000 mg/kg/day bw correlating in this animal with a small weight (0.1200 g) and histologically with slight atrophy. A relationship with the test item was considered to be likely. Irregular color was seen in the lungs of 3/5 males given 1000 mg/kg/day bw. 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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-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/day bw and 4/6 males and 2/5 females given
1000 mg/kg/day bw. This non-adverse change was considered to be test item-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 bw and
2/5 females at 1000 mg/kg/day bw were considered to be fortuitous and without any relationship with the test item.

Thymus
Minimal or slight lymphoid atrophy was seen in 2/5 females given 1000 mg/kg/day bw, correlating with the lower weight at necropsy. Any relationship with the test item was considered to be likely but non-adverse (low number of animals affected and low grades). No test item-related changes were seen at 100 or 300 mg/kg/day bw.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
See details below.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
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
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C18-unsatd. MIPA, according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. 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 p.c. and lactation on Days 1 and 5 p.p. 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 p.p. 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 p.p. 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. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. 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 pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/day and in most of the animals at 100 mg/kg bw/day (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 hematology parameters in any group and sex. An effect of the read across 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/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 bw/day 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/day 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. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013). 

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2020 - 2021 (planned study)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Species:
rat
Sex:
male/female
Route of administration:
oral: gavage
Remarks on result:
other: Testing planned
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Well conducted and comparable to guideline study, no information on GLP status
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Mus-Rattus, Brunntal, Germany
- Strain: Wistar rat, MuRa Han 67 SPF
- Age at study initiation: between 6-7 weeks
- Weight at study initiation: 109 (f) - 114 (m) g
- Housing: plastic cages, 3 males and 5 females/cage
- Diet (e.g. ad libitum): ad libitum (Altromin Ratdiet No. 1424 DK, Altromin GmbH, Lage, Germany)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 60-80
- Air changes (per hr): 11
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Doses were adapted weekly to the body weight; application volume - 5 mL/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 d
Frequency of treatment:
Daily once, 5 times/wk
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Remarks:
Days 1-28
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Days 1-28
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Days 1-14
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
Days 15-28
No. of animals per sex per dose:
10/sex/dose for main study; 5/sex/dose for 4 month recovery period


Control animals:
yes, concurrent no treatment
Details on study design:
According to standard procedure
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: end of study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per dose and sex
- Parameters checked: Hematocrit, erythrocytes, leukocytes, hemoglobin, thrombocytes, mean corpuscular volume, white blood cell differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- How many animals: 10 per sex and dose
- Parameters checked: GPT, GOT, AP, glucose, urea, total protein, calcium, phosphate, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: end of study
- Metabolism cages used for collection of urine: No
- Parameters checked: urea, creatinine, sodium, potassium, glucose, calcium, AP

NEUROBEHAVIOURAL EXAMINATION: No

Other: Groups of 5 male and 5 female rats kept for an additional 4 month recovery period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
the following tissues/organs were examined:
adrenal gland
aorta thoracica
brain (cornu ammonis)
coagulating gland with seminal vesicle
epididymis
eye with optic nerve
heart
intestine, large
intestine, small
kidney
liver
lungs
lymph node (cervical)
lymph node (mesenteric)
mucles
oesophagus
ovary
pancreas
prostate
salivary glands (mandibular,
parotid and sublingual gland)
skin
spleen
stomach
testicle
thymus
thyroid (incl. parathyroids)
tongue
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)


HISTOPATHOLOGY: Yes
see gross pathology
Statistics:
't' test used for statistical analysis of all parameters except organ weight (U-test)


Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
The doses up to 1500 mg/kg body weight/day were tolerated by all animals without lethality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight gain and total increase of body weights did not differ from the control group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterine, GGT, GOT, GPT and LDH did not show any critical signs. Only slight shifts which were not dose-dependent could be observed. These signs were considered as not substance depending.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative organ weights showed no significant changes in the substance groups compared to the control group, except the organ weight of the liver which is slightly increased for the males of group 4 (750/1500 mg/kg bw) and increased adrenal glands weight in high dose females. This result is considered of no relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No biologically relevant treatment-related effects observed on any of the parameters recorded at any dose, also test animals treated with 1500 mg/kg bw (Days 15-28) showed no adverse effect
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to design based on OECD Guideline 407. Groups of 10 male and 10 female Wistar rats were orally gavaged with the substance diluted in olive oil, 5 d/week for 28 d at doses of 0, 70, 250, 750 (Days 1-14) and 1500 (Days 15-28) mg/kg bw/d. Clinical signs, bodyweight, haematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. No treatment-related adverse effects were observed at any of the doses. Changes in the forestomach at some doses including controls were attributed to the use of olive oil and found to be reversible after end of exposure. Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day (Potokar, 1983). Based on the results of the read across study, a similar NOAEL value is expected for the test substance.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
October 2016-January 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Remarks:
No major deviation during the course of the study
GLP compliance:
yes (incl. QA statement)
Remarks:
23 march 2017
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
SPF (Specific Pathogen Free) Sprague-Dawley - Crl: OFA (SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 L'ARBRESLE Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks on the day of the first administration.
- Weight at study initiation: Between 184.2 and 231.9 g in males and 125.5 g and 171.4 g in females. The weight variation of animals used did not exceed +/- 20% of the mean weight of each sex.
- Housing: Animals were housed (separated by sex) in cages of standard dimensions with sawdust bedding (or equivalent).
- Diet: RM1 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilised) was available ad libitum except during the fasting experimental period. A certificate of analysis concerning this feed product is included to the study report. The criteria for acceptable levels of contaminants in the feed supply were within the limits of the analytical specifications established by the diet manufacturer.
- Water: Drinking water was available ad libitum in polycarbonate bottles with a stainless steel nipple. A specimen of water is obtained approximately every 6 months and sent to Laboratoire de la Touraine (ZA n°1 du Papillon - Rue de l'Aviation - 37210 Parçay-Meslay - France), for analysis. The certificates of analysis are included in the report. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications
- Acclimation period: A minimum of five days in the laboratory animal house where the experiment took place. Only animals without any visible sign of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The room temperature was between 19°C and 24°C and was recorded at the beginning and at the end of all observations.
- Humidity (%) and air changes (per hr): The animals were placed in an air-conditioned (20-24°C) animal house kept at relative humidity between 45% and 65% (except during the cleaning slot) in which nonrecycled filtered air was changed approximately 10 times per hour. Between 28 Nov at 08.00 p.m. and 29 Nov 2016 at 11.00 a.m. (for about 15 hours) and between 16 Jan at 10.00 p.m and 17 Jan 2017 at 12.30 a.m, an abnormal decrease in hygrometry was noted in the animal room.
- Photoperiod (hrs dark / hrs light): The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 a.m. except on 30 Oct 2016.

IN-LIFE DATES: From: 18 october 2016 to: 20 January 2017
Route of administration:
oral: gavage
Details on route of administration:
N-(2-hydroxypropyl) Oleamide or its vehicle was administered once a day at approximately the same time (a maximum range of 4 hours between the start and the end of the daily treatment) at each chosen dose level, by the oral route for 13 weeks. A constant administration volume of 5 mL/kg body weight (except some animals) was used in accordance with the previous study (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route (gavage) in rats - OECD 422 - No.39644 RSR).
Vehicle:
corn oil
Details on oral exposure:
- DIET PREPARATION: no information available
- VEHICLE : Corn oil was used (Reference C8267)
- Lot/batch no.: Batch Nos. MKBS6944V and MKBW9504V, Expiry dates: 08 Oct 2020 and 06 Oct 2021 respectively
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of test item in formulations were checked during the first and last week of the study. Each concentration level and the vehicle was checked. The analytical method is validated within the range 85-115% of the theoretical concentration for formulations between 19.765 mg/mL and 198.120 mg/mL, with a precision better than 10%. The samples in corn oil must be analysed within the stability period (i.e. 30 days at room temperature) and must be within the range 85-115% to meet the acceptance criteria. Formulation analysis was performed on one formulation prepared during the first and the last week of the study. The concentrations tested were 20 mg/mL, 60 mg/mL and 200 mg/mL. The concentrations found were within the range of acceptance (+/-15% of the intended concentration). The absence of test item was also confirmed in the vehicle samples. These results show that the formulations were properly prepared.
Duration of treatment / exposure:
Main phase: Minimum of 90 days (13 weeks)
Frequency of treatment:
Once a day
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
The study was conducted on 4 groups of 10 males and 10 females including a control group (80 animals in the main group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Proposed doses are selected in agreement with the Sponsor. The choice is based on previous studies (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route (gavage) in rats - OECD 422 - Study No. 39644 RSR). Moreover, the highest dose should reveal signs of toxicity and the lowest dose should represent a no-observed-adverse effects level.
- Allocation of each animal to treatment : randomly determined before the start of the study. Homogeneity of groups was validated on the criterion of body weight measured on the day of randomisation, separately for males and females.
- Justification of the number of animals per group: The number of animals per group is the minimum number enabling an accurate assessment of the studied toxicological effect and comparisons using the appropriate statistical tests.
- dose adjustement: Doses of test item were expressed as mg/kg. Doses were adjusted on the basis of body weight measured at the most recent weighing.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed in the cage before the first dosing and at least once a day except for some animals. The time of observation was between 21 minutes and 1h45 post dose. The general disposition, behaviour and activity were observed. Once a week except for one female on week 3, animals were submitted to a full clinical examination in a cage without sawdust according to a standardised observation battery for general clinical signs, neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. The time of observation was at 1 hour (+/- 30 min) or 3h35 on d7 for Female No. 1602396 post dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the following days:
• on the day of randomisation
• at predose
• then weekly during the study
• on the day of necropsy (not exsanguinated), this is the reference weight used in the calculation of relative organ weights

FOOD CONSUMPTION : Yes
Food consumption was measured for each treatment cage except on the day of fasting. Animals were fasted during the night before scheduled blood sampling for haematology/coagulation parameters, clinical chemistry and during urine collection for urinalysis.

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination (Retinography) was performed under the responsability of a person who has followed a veterinary ophthalmological training on all animals before the first dosing and during the last week.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken from all animals during the last week. Samples were taken just before killing for moribund animals when possible.
- Anaesthetic used for blood collection: Yes (isoflurane inhalation)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken from all animals during the last week. Samples were taken just before killing for moribund animals when possible.
- Animals fasted: Yes

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Urine was collected from all animals, during the last week. Urine collection was performed individually in metabolism cages for a period of about 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (Food and water were withheld during collection)

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: Before the first dosing and on the last week, animals were observed according to a standardised observation battery for neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. The method is based on an Irwin screen modified by suppressing the graduation of intensity of clinical signs. Animals were observed individually in a cage without sawdust in a quiet room.The time of observation was 1 hour (+/- 36 min) post dose. The rectal temperature was measured at the end of each observation.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Hyperalertness, Decrease in visual placing, Passive response to finger approach, Passivity when touched, Absence of reactivity, Stereotyped movements, Aggressiveness/irritability, Increase/decrease in fear, Reduced/increased spontaneous locomotor activity, Increasing grooming, Staggering gait, Abnormal gait, Loss of righting reflex, Absence/increase of startle response, Insensitivity/Hyperreactivity to pinching of tail, Body sag, Decrease/Increase in limb tone , Decrease in grip strength, Decrease in body tone, Decrease in abdominal tone, Sitting up, Rearing, Virtually permanent reared position, Loss of pinna reflex, Loss of corneal reflex, Hind limb reflex, Pallor, Tachycardia, Bradycardia, Myosis, Mydriasis, Ptosis, Exophthalmos, Lacrimation, Liquid/soft faeces
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals dying during the course of the study and animals killed in a moribund condition were necropsied as quickly as possible and specimens as required by the study plan obtained whenever practically possible. All animals (including any found dead or moribund) were submitted to full necropsy procedures including an examination of:
• the external surface
• all orifices
• the cranial cavity
• the external surface of the brain and samples of the spinal cord
• the thoracic and abdominal cavities and organs
• the cervical tissues and organs
• the carcass
Paired organs were weighed together. From all animals, excluding those found dead, the organs were dissected free of fat and other contiguous tissues at the discretion of the prosector and weighed.

HISTOPATHOLOGY: Yes
Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically
The organs and tissues sampled were fixed and preserved in 4% buffered formalin with the following exceptions:
• testes and epididymides were fixed in alcoholic Bouin0s fluid (about 5 days), then transferred to ethanol 95%
• eyes and optic nerves were fixed and preserved in Davidson0s fluid (about 3 days), then transferred to ethanol 70%

Organs for organ weight determination and for histopathological examination are presented in table below.

SACRIFICE: Main phase animals will be killed following 13 weeks of treatment.
Other examinations:
None
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In decedent animals, the day before or the day of death, there were the following clinical signs:
• At 100 mg/kg bw, in Male No.1602323, there was no clinical sign.
• At 300 mg/kg bw, there was an increased salivation and absence of spontaneous locomotor activity in Male No.1602335 on d87 (the day before death). In male No.1602340, there was blood around and in the mouth on d58 (the day before death).
• At 1000 mg/kg bw, there were increased salivation, chromodacryorrhoea, dyspnoea, bradypnoea, decrease then absence of locomotor activity and it was lying on its sides on d59, in Male No. 1602344. In Male No. 1602345, there was increased salivation. In Female No. 1602388, there was increased salivation.

In surviving males and females treated with test item whatever the dose, there was a dose dependent:
• increased salivation (8.8%, 19.8% and 41.7% in males and 0.8%, 3.2% and 22.6% in females respectively) from d26 up to the end of the study
• chromodacryorrhoea (2.1%, 6.1% and 8.8% in males and 0.1%, 3.4% and 9.1% in females respectively) from d28 up to the end of the study
There were also isolated clinical signs such as a decrease in spontaneous locomotor activity from d57 up to d60 in at most 3/10 males treated with test item at 300 and 1000 mg/kg bw and also in 1/8 males treated with test item at 1000 mg/kg bw on d91, a decrease in fear in 1/8 males treated at 1000 mg/kg bw or 1/10 females treated at 100 mg/kg bw, an increase in fear or insensitivity to pinching of the tail in 1/10 females at 100 mg/kg bw.
Mortality:
mortality observed, treatment-related
Description (incidence):
6 animals treated with test item were found dead between d59 and d91:
• at 100 mg/kg bw, Male No. 1602323 was killed early on d77
• at 300 mg/kg bw, Male No. 1602335 on d88 and Male No. 1602340 on d59 were found dead
• at 1000 mg/kg bw, Male No. 1602344 was moribund and was found dead during the isoflurane anaesthesia, just before exsanguination on d59, Male No. 1602345 was found dead on d80, Female No. 1602388 was found dead on d91.
There was no mortality in the control group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no change in body gain in animals found dead except for male No. 1602345 treated at 1000 mg/kg bw which had a body weight loss (-9.6%) between d63 and d70. There was a statistically significant lower body weight gain in males treated with test item at 1000 mg/kg bw from d14 up to the end of the study (on d91, body weight gain was +71% vs +107% in the control group) and at 100 mg/kg bw from d70 up to the end of the study (on d91, body weight gain was +87% vs +107% in the control group). In males treated at 300 mg/kg bw, based on the mean values, there was no difference when compared to the control group due to Male No. 1602331 which had very high body weight (649 g vs 510 g the mean values of the group). However, if considering the individual values, there was also a lower body weight gain in the other males treated at 300 mg/kg bw. When the values of this animal were excluded, the body weight gain was +97% vs +107% in the control group. One male treated with test item at 100 mg/kg bw (No. 1602324) had a body weight loss between d84 and d91 (-5.4%). There was no change in body weight gain in females.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slightly lower food consumption in all males treated with test item whatever the dose (consumption was approximately 17-18 g/animal/day vs 20 g/animal/day in the control group). In cage 6 (with Male Nos. 1602324 and 1602325) of group 2, the food consumption was lower during the last week of the treatment period. There was no marked change in food consumption in females whatever the dose.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Not necessary
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There was no abnormality at the ophthalmological examination on d91 when compared to the records before the start of the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the decedent animals only the animal killed on d77 yielded data. A slight reduction of the white cell series was seen (total white cell count, neutrophil count, eosinophil count, basophil count, lymphocyte count). At 1000 mg/kg bw, there was a higher total white blood cell count in males (+20%) and in females (+30%) on d92. In females, there was also a statistically significant higher lymphocyte count (+33%) and monocyte count (+58%) on d92. There was also a statistically significant higher reticulocyte count in males treated with test item at 100 and 1000 mg/kg bw mainly due to two lower values in the control group (in male Nos. 1602312 and 1602320). Therefore this changes was not attributed to the test item. At 300 and 100 mg/kg bw, there was no change in haematology parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the decedent animals only the animal killed on d77 yielded data. A slight higher ALT, AST and ALP activities and slightly lower total bilirubin and albumin.
There was dose related statistically significant higher plasma ALT, AST and ALP activities in the males treated with test item at 300 and 1000 mg/kg bw and a statistically significant higher ALT activity (+45%) in females treated at 1000 mg/kg bw. There was also a statistically significant higher A/G ratio in male treated at 1000 mg/kg bw due to lower globulins. This was considered to be of minor degree (+14%) and was not considered as toxicologically relevant. At 100 mg/kg bw in surviving animals and at 300 mg/kg bw in females, there was no change in blood chemistry parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no change in urinary volume and semi-quantitative parameters. There was a higher creatinine level in urine in Male No. 1602324 treated with test item at 100 mg/kg bw.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was statistically significant changes at 1000 mg/kg bw (in absolute and/or relative values body or brain weight):
• higher liver weight in males and females
• higher adrenals weight in males
• lower thymus weight in males
• higher testes and epididymides weight in males
There was a statistically significant higher adrenals weight at 100 and 300 mg/kg bw in males (in relative values % body weight). This was low in amplitude (+18% when compared to the control relative values % body weight) and was not considered as toxicologically relevant. There were also statistically significant lower (in absolute values) spleen, thymus, heart and brain weights related to the lower body weight in males treated at 1000 mg/kg bw and lower thymus weight in males treated at 100 mg/kg bw due to the low individual value in Male No. 1602324. There was no other change in organ weight in animals treated at 300 or at 100 mg/kg bw.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In decedent animals, there were mainly dark organs (mucosa, liver, spleen, lung or heart) or mottled lung and soft brain. These observations were usually observed in animals found dead or in moribund state. In male No. 1602323 treated at 100 mg/kg bw and male No. 1602335 treated at 300 mg/kg bw, there was blood in the mouth. In males 1602335 and 1602340 treated at 300 mg/kg bw, there was liquid in the thoracic cavity and a white film in the thoracic cavity in Male No. 1602340. For male No. 1602344 treated at 1000 mg/kg bw, there was a subcutaneous hole under the left forelimb and a subcutaneous mass on the neck. In surviving animals, there were isolated macroscopic findings found at a low incidence such as point change in the heart (1/8 in males treated at 300 mg/kg bw) or dark mesenteric lymph nodes (1/8 in males treated at 1000 mg/kg bw) or observations found at the same incidence in the control and test item dose groups, such as point changes in thymus or liver (1/10 control males vs 1/8 treated males at 300 mg/kg bw), dark or point changes in sub maxillary lymph node. In Male No. 1602350 treated at 1000 mg/kg bw, there was pale and enlarged lung with dark area.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In decedent animals, there were no lesions suggesting the cause of death in any of these animals. In the male treated at 100 mg/kg bw and killed early on d77, there was only mild periacinar hepatocytic hypertrophy and minimal sternal lipid hyperplasia. In Male No. 1602345 and in Female No. 1602388 treated at 1000 mg/kg bw, there was minimal femoral lipid hyperplasia and minimal periacinar hepatocytic hypertrophy respectively. In other decedent animals treated at 300 or 1000 mg/kg bw, there were no relevant changes. In surviving animals, there were minor lesions in the thymus, liver and bone marrow related to the treatment.
Thymic atrophy was present in the majority of animals whatever the treatment, the degree was greatest in animals treated at 1000 mg/kg bw but the incidence and degree in other groups including the control group was sufficiently inconsistent to deny any true link to the test item. Hepatic changes in males treated at 1000 mg/kg bw and females treated at 300 and 1000 mg/kg bw were biliary prominence and periacinar hypertrophy. These findings were present in the control group, at the lower incidence and degree. The changes were adaptative in nature and very slight in degree, different following the gender and were not deemed significant. The partial replacement of bone marrow by lipid vacuolation in the femur was present in males treated at 1000 mg/kg bw and in the sternum in males treated at 300 and 1000 mg/kg bw. There were slight histopathological changes in the liver and the bone marrow in animals treated with test item at 1000 mg/kg bw.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There was statistically changes in mobile spermatozoa at 100 and 1000 mg/kg bw, midpiece anomaly at 100 mg/kg bw, cytoplasmie droplet anomaly at 1000 mg/kg bw. Since there were no changes in % mobile spermatozoa or the variation was very low, these changes were not attributed to the test item. There was no change in sperm analysis.
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
clinical signs
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Conclusions:
Under the study conditions, a NOAEL could not be determined in males, whereas in females, the NOAEL was considered as 300 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 408, in compliance with GLP. The substance diluted in corn oil was administered by gavage to groups of male and female Sprague-Dawley rats (10/sex/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/day for 13 weeks (at constant administration volume of 5 mL/kg bw). Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. A full clinical examination was performed once a week. Functional and neurobehavioral tests were performed before the first dosing and in the last week. Body weight was recorded at pre-dose and once a week. Food consumption was measured weekly. Ophthalmological examination was performed before the first dosing and during the last week. Blood samples for haematology parameters and clinical chemistry analysis and urine were collected on d92. All animals were killed on d92 (or d91 for one female). Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically. Epidydimides were collected on d92 for sperm analysis. 5 males treated with read across substance (2 males at 300 mg/kg bw/day and 2 males at 1000 mg/kg bw/day) were found dead between d59 and d88 and one male treated with read across substance at 100 mg/kg bw/day was killed early on d77. One female treated with read across substance at 1000 mg/kg bw/day was found dead on d91. On the days before death, there were no particular clinical signs but on the day of the death, there was mainly bradypnoea or polypnoea or absence or decrease in spontaneous locomotor activity. There was dose related statistically significant higher ALT, AST and ALP activities in males treated with read across substance at 300 and 1000 mg/kg bw and statistically significant higher ALT activity in females treated at 1000 mg/kg bw/day. There were statistically significant changes at 1000 mg/kg bw/day, including higher liver weight in males and females, higher adrenals weight in males, lower thymus weight in males, and higher testes and epididymides weight in males. There were also bone marrow microscopic changes. No lesions at histopathology examination suggested the cause of death; however similar effects as those seen at the highest dose was noted such as higher hepatic enzymes activities or mild periacinar hepatocytic hypertrophy. In other animals treated at 100 mg/kg bw/day, there were no changes in hepatic plasma enzyme and no microscopic changes. Whatever the tested doses, the substance induced mortality, a lower food consumption and lower body weight gain in males. The male gender seems to more sensitive than female. Under the study conditions, a NOAEL could not be determined in males, whereas in females, the NOAEL was considered as 300 mg/kg bw/day (Mortier, 2018). Based on the results of the read across study, a similar NOAEL value is expected for the test substance.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
31 January 2013 - 17 June 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item 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 item 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 item 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 item 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test item 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
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Controls
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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 bw 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:
no (not required)
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 p.c. and lactation on days 1 and 5 p.p.. 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 p.p.. 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 p.p.. 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
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/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw 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/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw 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):
In males, mean food consumption at 1000 mg/kg/day bw 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 bw was not affected. Mean food consumption in females was considered to be unaffected by the test item treatment.
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):
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 item-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 item treatment was considered to be doubtful (no clear dose-relationship, individual values generally included in historical control data, and no statistical level).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 bw. 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 item may be suspected in two males treated at 1000 mg/kg/day bw 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 bw 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 item treatment on mean albumin concentration at 300 mg/kg/day bw 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 bw 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 bw was due to isolated animals per sex only.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
An effect of the test item treatment in males and females at 300 and/or 1000 mg/kg/day bw 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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, the mean absolute and relative liver weights were higher in males and females given 1000 mg/kg/day bw and in males given 300 mg/kg/day bw, reaching statistically significant values in both sexes at 1000 mg/kg/day bw (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 item-related. The mean absolute and relative thymus weights were lower in females given 1000 mg/kg/day bw, without reaching a statistically significant value. This correlated with minimal to slight atrophy in two females and was considered to be probably test item-related. The mean absolute heart weight was higher in males given 100 mg/kg/day bw. In the absence of a dose-related trend, any relationship with the test item 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 item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The thymus was reduced in one female given 1000 mg/kg/day bw correlating in this animal with a small weight (0.1200 g) and histologically with slight atrophy. A relationship with the test item was considered to be likely. Irregular color was seen in the lungs of 3/5 males given 1000 mg/kg/day bw. 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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-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/day bw and 4/6 males and 2/5 females given
1000 mg/kg/day bw. This non-adverse change was considered to be test item-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 bw and
2/5 females at 1000 mg/kg/day bw were considered to be fortuitous and without any relationship with the test item.

Thymus
Minimal or slight lymphoid atrophy was seen in 2/5 females given 1000 mg/kg/day bw, correlating with the lower weight at necropsy. Any relationship with the test item was considered to be likely but non-adverse (low number of animals affected and low grades). No test item-related changes were seen at 100 or 300 mg/kg/day bw.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
See details below.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
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
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. 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 p.c. and lactation on Days 1 and 5 p.p. 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 p.p. 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 p.p. 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. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. 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 pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/day and in most of the animals at 100 mg/kg bw/day (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 hematology parameters in any group and sex. An effect of the read across 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/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 bw/day 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/day 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. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013). 

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Mus-Rattus, Brunntal, Germany
- Age at study initiation: between 6-7 wk
- Weight at study initiation: 111 (f) - 125 (m) g
- Housing: plastic cages, 3 males and 5 females/cage
- Diet (e.g. ad libitum): ad libitum (Altromin Ratdiet No. 1424 DK, Altromin GmbH, Lage, Germany)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-20
- Humidity (%): 60-75
- Air changes (per hr): 11
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
doses were adpated weekly to the body weight
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no information
Duration of treatment / exposure:
28 d
Frequency of treatment:
daily once, 5 times/wk
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Controls
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Day 1 - 14
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
Days 15-28
No. of animals per sex per dose:
10/sex/dose for main study; 5/sex/dose for 4 month recovery period
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: end of study


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per dose and sex
- Parameters checked: Hematocrit, erythrocytes, leukocytes, hemoglobin, thrombocytes, mean corpuscular volume, white blood cell differntial


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- How many animals: 10 per sex and dose
- Parameters checked: GPT, GOT, AP, glucose, urea, total protein, calcium, phosphate, cholestrol


URINALYSIS: Yes
- Time schedule for collection of urine: end of study
- Metabolism cages used for collection of urine: No
- Parameters checked: urea, creatinin, sodium, potassium, glucose, calcium, ap


NEUROBEHAVIOURAL EXAMINATION: No


Other: Groups of 5 male and 5 female rats kept for an additional 4 month recovery period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
the following tissues/organs were examined:
adrenal gland (2)
aorta thoracica
brain (cornu ammonis)
coagulating gland with seminal vesicle
epididymis (2)
eye with optic nerve (2)
heart
intestine, large
intestine, small
kidney (2)
liver
lungs
lymph node (cervical) (1)
lymph node (mesenteric) (1)
mucles
oesophagus
ovary (2)
pancreas
prostate
salivary glands (mandibular,
parotid and sublingual gland)
skin
spleen
stomach
testicle (2)
thymus
thyroid (2) (incl. parathyroids)
tongue
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)



HISTOPATHOLOGY: Yes
see gross pathology
Statistics:
't' test used for statistical analysis of all parameters except organ weight (U-test)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
The doses up to 1500 mg/kg bw/day were tolerated by all animals without lethality. The body weight gain and total increase of body weights did not differ from the control group. Absolute and relative organ weights showed no significant changes in the substance groups compared to the control group, accept the organ weight of the adrenal gland which is increased for the females of group 3 (250 mg/kg) and 4 (750/1500 mg/kg bw). This result is considered of no relevance. The pathological, histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose independent reversible local findings were restricted to the fore stomach mucose of the highest group (hyperplastic and cellular changes in the forestomach but not dose-related and also found in controls. Attributed to the use of olive oil as carrier. Effects disappeared during 4 month recovery period). The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterine, GGT, GOT, GPT and LDH did not show any irregular signs. Slight alterations of phosphate in the highest group were noted and regarded as dose/compound related but not critical effect.
Key result
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: biologically relevant effects observed on any of the parameters recorded at any dose , also test animals treated with 1500 mg/kg bw /d (Days 15-28) showed no adverse effect
Key result
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the 28 d NOAEL for systemic toxicity was considered to be > 750 mg/kg bw.
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance to rat according to OECD Guideline 407. Groups of 10 male and 10 female rats were orally gavaged with the substance diluted in olive oil 5 days per week for 28 d at doses of 0, 70, 250, 750 (Days 1 – 14) and 1,500 (Days 15-28) mg/kg bw. Clinical signs, bodyweight, hematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. The doses up to 1500 mg/kg body weight/day were tolerated by all animals without lethality. The body weight gain and total increase of body weights did not differ from the control group. The pathological, histological evaluation did not show significant compound related gross or microscopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose independent reversible local findings were restricted to the fore stomach mucosa of the highest group. The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterol, GGT, GOT, GPT and LDH did not show any irregular signs. Slight alterations of phosphate in the highest group were noted and regarded as dose/compound related but not critical effect. Under the study conditions, the 28 d NOAEL for systemic toxicity was therefore considered to be >750 mg/kg bw (Gloxhuber and Potokar, 1983).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Mus-Rattus, Brunntal, Germany
- Age at study initiation: between 6-7 wk
- Weight at study initiation: 111 (f) - 125 (m) g
- Housing: plastic cages, 3 males and 5 females/cage
- Diet (e.g. ad libitum): ad libitum (Altromin Ratdiet No. 1424 DK, Altromin GmbH, Lage, Germany)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-20
- Humidity (%): 60-75
- Air changes (per hr): 11
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
doses were adpated weekly to the body weight
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 d
Frequency of treatment:
daily once, 5 times/wk
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Controls
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Day 1 - 14
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
Days 15-28
No. of animals per sex per dose:
10/sex/dose for main study; 5/sex/dose for 4 month recovery period
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: end of study


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per dose and sex
- Parameters checked: Hematocrit, erythrocytes, leukocytes, hemoglobin, thrombocytes, mean corpuscular volume, white blood cell differntial


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- How many animals: 10 per sex and dose
- Parameters checked: GPT, GOT, AP, glucose, urea, total protein, calcium, phosphate, cholestrol


URINALYSIS: Yes
- Time schedule for collection of urine: end of study
- Metabolism cages used for collection of urine: No
- Parameters checked: urea, creatinin, sodium, potassium, glucose, calcium, ap


NEUROBEHAVIOURAL EXAMINATION: No


Other: Groups of 5 male and 5 female rats kept for an additional 4 month recovery period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
the following tissues/organs were examined:
adrenal gland (2)
aorta thoracica
brain (cornu ammonis)
coagulating gland with seminal vesicle
epididymis (2)
eye with optic nerve (2)
heart
intestine, large
intestine, small
kidney (2)
liver
lungs
lymph node (cervical) (1)
lymph node (mesenteric) (1)
mucles
oesophagus
ovary (2)
pancreas
prostate
salivary glands (mandibular,
parotid and sublingual gland)
skin
spleen
stomach
testicle (2)
thymus
thyroid (2) (incl. parathyroids)
tongue
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)



HISTOPATHOLOGY: Yes
see gross pathology
Statistics:
't' test used for statistical analysis of all parameters except organ weight (U-test)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
The doses up to 1500 mg/kg bw/day were tolerated by all animals without lethality. The body weight gain and total increase of body weights did not differ from the control group. Absolute and relative organ weights showed no significant changes in the substance groups compared to the control group, accept the organ weight of the adrenal gland which is increased for the females of group 3 (250 mg/kg) and 4 (750/1500 mg/kg bw). This result is considered of no relevance. The pathological, histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose independent reversible local findings were restricted to the fore stomach mucose of the highest group (hyperplastic and cellular changes in the forestomach but not dose-related and also found in controls. Attributed to the use of olive oil as carrier. Effects disappeared during 4 month recovery period). The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterine, GGT, GOT, GPT and LDH did not show any irregular signs. Slight alterations of phosphate in the highest group were noted and regarded as dose/compound related but not critical effect.
Key result
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: biologically relevant effects observed on any of the parameters recorded at any dose , also test animals treated with 1500 mg/kg bw /d (Days 15-28) showed no adverse effect
Key result
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the 28 d NOAEL for systemic toxicity was considered to be > 750 mg/kg bw.
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the read across substance, amides C12-18 and C18-unsatd. N-(hydroxyethyl), to rat according to OECD Guideline 407. Groups of 10 male and 10 female rats were orally gavaged with the substance diluted in olive oil 5 days per week for 28 d at doses of 0, 70, 250, 750 (Days 1 – 14) and 1,500 (Days 15-28) mg/kg bw. Clinical signs, bodyweight, hematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. The doses up to 1500 mg/kg body weight/day were tolerated by all animals without lethality. The body weight gain and total increase of body weights did not differ from the control group. The pathological, histological evaluation did not show significant compound related gross or microscopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose independent reversible local findings were restricted to the fore stomach mucosa of the highest group. The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterol, GGT, GOT, GPT and LDH did not show any irregular signs. Slight alterations of phosphate in the highest group were noted and regarded as dose/compound related but not critical effect. Under the study conditions, the 28 d NOAEL for systemic toxicity was therefore considered to be >750 mg/kg bw (Gloxhuber and Potokar, 1983).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral (28 day)

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to design based on OECD Guideline 407. Groups of 10 male and 10 female Wistar rats were orally gavaged with the substance diluted in olive oil, 5 d/week for 28 d at doses of 0, 70, 250, 750 (Days 1-14) and 1500 (Days 15-28) mg/kg bw/d. Clinical signs, bodyweight, haematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. No treatment-related adverse effects were observed at any of the doses. Changes in the forestomach at some doses including controls were attributed to the use of olive oil and found to be reversible after end of exposure. Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day (Potokar, 1983).

Oral (Combined repeated dose and reproductive/developmental screen)

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. 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 p.c. and lactation on Days 1 and 5 p.p. 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 p.p. 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 p.p. 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. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. 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 pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg/day bw and in most of the animals at 100 mg/kg/day bw (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/day bw 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 hematology parameters in any group and sex. An effect of the read across substance treatment on mean motor activity data at 300 and/or 1000 mg/kg/day bw was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg/day bw 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 bw and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg/day bw (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 bw or in males. At histopathology at 1000 mg/kg/day bw, 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 bw, 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 bw. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013).

Also, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a combined repeated dose toxicity testing/reproductive and developmental screening according to OECD Guideline 422 is planned with C8-18 and C18 -unsatd. MIPA in order to further support the read across approach proposed for the FAA category members. 

Oral (90 day)

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 408, in compliance with GLP. The substance diluted in corn oil was administered by gavage to groups of male and female Sprague-Dawley rats (10/sex/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/day for 13 weeks (at constant administration volume of 5 mL/kg bw). Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. A full clinical examination was performed once a week. Functional and neurobehavioral tests were performed before the first dosing and in the last week. Body weight was recorded at pre-dose and once a week. Food consumption was measured weekly. Ophthalmological examination was performed before the first dosing and during the last week. Blood samples for haematology parameters and clinical chemistry analysis and urine were collected on d92. All animals were killed on d92 (or d91 for one female). Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically. Epidydimides were collected on d92 for sperm analysis. 5 males treated with read across substance (2 males at 300 mg/kg bw/day and 2 males at 1000 mg/kg bw/day) were found dead between d59 and d88 and one male treated with read across substance at 100 mg/kg bw/day was killed early on d77. One female treated with read across substance at 1000 mg/kg bw/day was found dead on d91. On the days before death, there were no particular clinical signs but on the day of the death, there was mainly bradypnoea or polypnoea or absence or decrease in spontaneous locomotor activity. There was dose related statistically significant higher ALT, AST and ALP activities in males treated with read across substance at 300 and 1000 mg/kg bw and statistically significant higher ALT activity in females treated at 1000 mg/kg bw/day. There were statistically significant changes at 1000 mg/kg bw/day, including higher liver weight in males and females, higher adrenals weight in males, lower thymus weight in males, and higher testes and epididymides weight in males. There were also bone marrow microscopic changes. No lesions at histopathology examination suggested the cause of death; however similar effects as those seen at the highest dose was noted such as higher hepatic enzymes activities or mild periacinar hepatocytic hypertrophy. In other animals treated at 100 mg/kg bw/day, there were no changes in hepatic plasma enzyme and no microscopic changes. Whatever the tested doses, the substance induced mortality, a lower food consumption and lower body weight gain in males. The male gender seems to more sensitive than female. Under the study conditions, a NOAEL could not be determined in males, whereas in females, the NOAEL was considered as 300 mg/kg bw/day (Mortier, 2018).

Additional considerations 

Based on an in-depth analysis of the available information (see read-across justification in Section 13 of the IUCLID dataset), it is the FAA consortium’s hypothesis that a read-across within and across MEA, DEA and MIPA derived alkanolamides is scientifically plausible and justified. While there is at this point no evidence putting the read-across hypothesis in question, the FAA consortium recognizes some limitations, predominantly related to the quality of individual endpoint studies (including quality of the test substance characterisations) and existing higher Tier endpoint data gaps. After discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), the FAA Consortium agrees to the need to strengthen the toxicology data-based link between and within the DEA, MEA and MIPA subcategories.

In view of this, the FAA Consortium decided to proceed with a 3-Tier testing strategy. In Tier 1, a series of bridging studies according to OECD TG 422 will be conducted with a representative short- and a long chain substance of each subcategory (i.e., DEA, MEA, MIPA).

The objective of Tier 2 will be to generate a complete set of higher toxicology data for a selected >1000 tpa substance (i.e., OECD TG 408/443/414 (rats)/414 (2nd species). The testing in Tier 2 will be conducted with C8-18 and C18-unsatd. DEA, the substance that is generally perceived to be the most critical from a reproductive toxicity point of view based on the classification of DEA.

Tier 1 and 2 proposed studies have been included in the present dossier update. Should these suggest significant differences in type and strength of effects between the DEA, MEA and MIPA subcategories, therefore not supporting the current read across justification, further testing may be initiated. The Consortium’s strategy is detailed in a document entitled ‘ECHA-DIAP -FAA testing strategy summary – 24Sep20’ attached in Section 13 of the IUCLID dataset.

Justification for classification or non-classification

Based on the results of repeated dose oral toxicity studies in rodents, the test substance does not require classification according to CLP (EC 1272/2008) criteria.