isostearic acid monoisopropanolamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 09, 1999 to May 28, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HanIbm:WIST (SPF)

Details on species / strain selection:

Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: RCC Ltd Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf / Switzerland
Acclimatisation: 7 days
Age when treated: 6 weeks
Body weight: males: 135-153 g (mean 144 g) and females: 115-133 g (mean 123 g)
Temperature: 21 +/- 3°C and relative humidity: 40-70%
Light period: 12 hour light/dark cycle
Diet: pelleted standard diet Provimi Kliba 3433 , ad libitum and water: community tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

The test substance formulations were prepared weekly. The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken during acclimatization. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the sponsor.

Duration of treatment / exposure:

28 days and the reversibility of treatment-related changes was assessed after a treatment-free 14 days recovery period.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 1)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 2)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 3)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 4)

No. of animals per sex per dose:

Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

In this subacute toxicity study, the test substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 days. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated for 28 days and then allowed a 14-days treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, livers were examined to establish a no-effect level.

Positive control:

-

Observations and examinations performed and frequency:

- Observations for mortality/viability were recorded twice daily. The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28; and once daily during days 29-42 (recovery). The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter. During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The test was performed approximately 1 hour after application. NB. The results of the Functional Observational Battery are presented under week 4.
- The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer. Body weights were recorded weekly during pretest, treatment and recovery and before each necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

- Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AGF 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded. Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

- Blood samples for hematology (hematology, methemoglobin and coagulation) and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

- The following organ weights were recorded on the scheduled dates of necropsy:Brain, Thymus, Spleen, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides

The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

Sacrifice and pathology:

- after 4 weeks
- after 6 weeks (post recovery)
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution:
- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain (telencephalon, cerebellum, pons)
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin’s solution)
- Esophagus
- Eyes with optic nerve (fixed in Davidson’s solution)
- Harderian gland (fixed in Davidson’s solution)
- Heart
- Ileum, with Peyer’s patches
- Jejunum with Peyer’s patches
- Kidneys
- Larynx
- Lacrimal gland (exorbital)
- Liver
- Lungs (infused with formalin at necropsy)
- Lymph nodes (mesenteric, mandibular)
- Mammary gland area
- Nasal cavity
- Ovaries
- Pancreas
- Pituitary gland
- Prostate gland
- Rectum
- Salivary glands (mandibular, sublingual)
- Sciatic nerve
- Seminal vesicles
- Skeletal muscle
- Skin
- Spinal cord (cervical, midthoracic, lumbar)
- Spleen
- Stomach
- Testes (fixed in Bouin’s solution)
- Thymus
- Thyroid (incl. parathyroid gland)
- Tongue
- Trachea
- Urinary bladder (infused with formalin at necropsy)
- Uterus
- Vagina
- Gross lesions

Statistics:

- Group means were calculated according to the definition of any mean value using the individual values per animal and the number of animals.
- The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clinical laboratory data:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher’s exact-test was applied to macroscopic findings.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical signs were evident in any animal of any group during daily observation or during weekly observation (pretest and weeks 1-3). No test substance-related clinical signs were evident in any animal of any group during the Functional Observational Battery performed at week 4. Salivation, slight to moderate in degree, was observed in one male treated with 200 mg/kg bw/day (treatment days 18-23) and in up to three males treated with 1000 mg/kg bw/day (two males on treatment day 17, three males on treatment days 18-23). Slight salivation was noted in up to two females treated with 1000 mg/kg bw/day (two females on treatment day 17, one female on treatment days 18-19). In view of the infrequent occurrence of this finding, it was considered to be incidental. During pretest, slight hyperactivity was noted in one male foreseen for treatment with 200 mg/kg bw/day. Moderate hyperactivity was noted in one male and one female of the control group during week 3 of treatment. All other animals were without abnormalities.

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled study periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related differences in body weight development were noted at any dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A transient reduction of food consumption was noted at 200 mg/kg bw/day and 1000 mg/kg/day. When compared with the control and pretest values, test substance-related inappetence was noted in males and females treated with 200 mg/kg bw/day and 1000 mg/kg bw/day during treatment week 1. This finding was also noted in the latter group during treatment week 2. During subsequent weeks, food consumption compared favorably. No effects upon food consumption were noted in rats treated with 50 mg/kg bw/day. During the 14-day recovery period, the mean daily food consumption of males treated with 1000 mg/kg bw/day exceeded that of the controls. This was considered to be a compensatory reaction. The females treated with 1000 mg/kg bw/day consumed slightly less feed during the recovery period when compared with the control females.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences of a few hematology parameters to control values were noted in males and females (higher leukocyte count in males treated with 1000 mg/kg bw/day, higher ratio of middle fluorescent reticulocytes in males treated with 200 mg/kg bw/day, higher absolute lymphocyte count in males treated with 1000 mg/kg bw/day, lower mean corpuscular hemoglobin and lower mean corpuscular hemoglobin concentration in females treated at 1000 mg/kg bw/day, shorter activated partial prothrombin times in females treated at 1000 mg/kg bw/day). None of the observed differences were supported by dose-response relationships, by similar findings in the opposite sex or by concomitant differences in related parameters, and therefore were considered to be incidental. All remaining differences to the control values at the end of the recovery period were also considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A small number of test substance-related differences to the control values were noted. These differences were restricted to males and females treated with the test substance at 1000 mg/kg bw/day and included higher activities of alanine aminotransferase and alkaline phosphatase, higher phosphorus levels (1000 mg/kg bw/day), higher albumin levels higher albumin/globulin ratio. A number of statistically significant differences to the control values were noted. In the absence of dose-response relationships, similar findings in the other sex or concomitant changes in related parameters, the following differences were considered to be incidental:
Higher plasma glucose levels in males (1000 mg/kg bw/day), higher creatinine levels in males (1000 mg/kg bw/day); lower bilirubin levels in females (50 mg/kg bw/day and 1000 mg/kg bw/day), lower plasma cholesterol levels in males (1000 mg/kg bw/day), higher triglyceride levels in males (50 mg/kg bw/day) and females (200 mg/kg bw/day and 1000 mg/kg bw/day), higher phospholipid levels in males (50 mg/kg bw/day), lower activity of aspartate aminotransferase in females (200 mg/kg bw/day), lower lactate dehydrogenase in males (50 mg/kg bw/day) and higher lactate dehydrogenase levels in females (1000 mg/kg bw/day), higher calcium levels in males (1000 mg/kg bw/day), lower chloride levels in males (1000 mg/kg bw/day), increased total protein in males (50 mg/kg bw/day), higher globulin levels in males (50 mg/kg bw/day), lower albumin/globulin ratio in males (50 mg/kg bw/day). All remaining differences to the control values at the end of the recovery period were also considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 4 weeks: The absolute and relative liver weights of male and female rats treated with 1000 mg/kg bw/day were markedly higher than those of the controls. The differences were generally statistically significant and considered to be related to the treatment with the test substance. The slightly lower thymus weights in males treated with 200 mg/kg bw/day or 1000 mg/kg bw/day (thymus/body weight ratio) were not present in the females and these differences were considered to be incidental. All other organ weights were considered to be unaffected by treatment when compared with the test substance.

After 6 weeks: No differences of statistical significance were evident in the males or females when compared with the control animals. The higher liver weights noted in males and females treated previously with 1000 mg/kg bw/day were largely absent after recovery.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings attributed to treatment with the test substance were not evident during necropsy. All macroscopic findings recorded were unremarkable and within the range of spontaneous alterations which may be seen in rats of this age and strain. They consisted of renal pelvis dilation, reddish discoloration or discolored foci in several organs, incompletely collapsed lungs, thickened thyroid glands and dilated uterine horns filled with fluid.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Grip strength and locomotor activity: No test substance-related differences between groups were ascertained

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings which were test substance-related consisted of hepatocellular hypertrophy at minor degrees and hepatocellular cytoplasmic eosinophilia in both sexes treated with 1000 mg/kg bw/day. These findings were not accompanied by any inflammatory or degenerative lesion. There were no such findings evident in animals sacrificed after a 14-day recovery period.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

For detailed results tables and figures, kindly refer to the attached background material section of the IUCLID.

Conclusions:

Under the study conditions, the systemic NOAEL in rats was established at 200 mg/kg bw/day (equivalent to 188.2 mg a.i./kg bw/day).

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, isoC18 MIPA (94.1% active), according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. The substance was administered daily by oral gavage to Wistar rats at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 d. The groups comprised 5 animals per sex which were sacrificed after 28 d of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated for 28 d and then allowed a 14 d treatment-free recovery period, after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during Week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post-mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. No test substance-related clinical signs were noted at any dose level. Body weights and food consumption were unaffected by treatment. Treatment-related findings were generally restricted to animals at 1000 mg/kg bw/day. These included higher activities of alanine aminotransferase and alkaline phosphatase, higher phosphorus levels, higher albumin levels and higher albumin/globulin ratio. Absolute and relative liver weights of animals at 1000 mg/kg bw/day showed test substance-related increases which were reversible after recovery. Liver changes consisting of hepatocellular hypertrophy at minor degrees of severity and eosinophilia of the hepatocellular cytoplasm were noted at 1000 mg/kg bw/day. These findings were considered to be caused by enzymatic changes and due to an adaptive metabolic response to a xenobiotic. The findings were resolved completely after the 14 d recovery period. Under the study conditions, the systemic NOAEL in rats was established at 200 mg/kg bw/day (Braun, 1999).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting 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.

Reference 3

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/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). Based on the results of the read across study, a similar NOAEL value is expected for the test substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic 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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

chronic 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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

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 test substance, isoC18 MIPA (94.1% active), according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. The substance was administered daily by oral gavage to Wistar rats at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 d. The groups comprised 5 animals per sex which were sacrificed after 28 d of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated for 28 d and then allowed a 14 d treatment-free recovery period, after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during Week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post-mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. No test substance-related clinical signs were noted at any dose level. Body weights and food consumption were unaffected by treatment. Treatment-related findings were generally restricted to animals at 1000 mg/kg bw/day. These included higher activities of alanine aminotransferase and alkaline phosphatase, higher phosphorus levels, higher albumin levels and higher albumin/globulin ratio. Absolute and relative liver weights of animals at 1000 mg/kg bw/day showed test substance-related increases which were reversible after recovery. Liver changes consisting of hepatocellular hypertrophy at minor degrees of severity and eosinophilia of the hepatocellular cytoplasm were noted at 1000 mg/kg bw/day. These findings were considered to be caused by enzymatic changes and due to an adaptive metabolic response to a xenobiotic. The findings were resolved completely after the 14 d recovery period. Under the study conditions, the systemic NOAEL in rats was established at 200 mg/kg bw/day (Braun, 1999).

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).

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 (LOAEL = 100 mg/kg bw/day), whereas in females, the NOAEL was considered as 300 mg/kg bw/day (Mortier, 2018).

Justification for classification or non-classification

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