Amides, C8-18(even-numbered) and C18(unsatd.), N-(2-hydroxypropyl)

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:

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.

Reference 2

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 Day 92. All animals were killed on Day 92 (or Day 91 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 Day 59 and Day 88 and one male treated with read across substance at 100 mg/kg bw/day was killed early on Day 77. One female treated with read across substance at 1000 mg/kg bw/day was found dead on Day 91. 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

Remarks:

Combined repeated dose toxicity study with the reproduction / developmental toxicity screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 04, 2021 to September 08, 2021

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)

Version / remarks:

adopted on 29 July 2016.

Deviations:

yes

Remarks:

Female animals were supplied in the weight range of 197-222 g and not 175-200 g, as indicated in the Study Protocol, while males were in the range of 211-246 g instead of 200-225 g. These deviations had no impact on the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data for this species and strain at ERBC.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animal supply and acclimatization:
A total of 102 Hsd: Sprague Dawley SD rats (45 males and 57 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival, on 28 January 2021, the weight range for each sex was determined (211-246 g for males, 197-222 g for females) and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of approximately 4 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

- Animal husbandry:
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. Drinking water was supplied ad libitum to each cage via water bottles, except in the case of urinalysis investigations. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

-Allocation to groups:
On the day of allocation all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. Furthermore, female animals that exhibited anomalies in the oestrous cycle were not allocated to the main groups. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. At allocation to the study, the body weight variation of animals did not exceed 20% of the mean weight of each sex. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed five per sex per cage. The cages were identified by a label and recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects and or contamination.

Route of administration:

oral: gavage

Details on route of administration:

The test item will be administered orally, by gavage. The oral route has been selected as it is a possible route of exposure of the test item in man.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC

Details on oral exposure:

The required amount of test substance was suspended in the vehicle. The formulations were prepared weekly or daily (concentrations of 10, 30 and 75 mg/mL), according to stability data from ERBC study No. A4126. Concentrations were calculated and expressed in terms of test substance as supplied. The test substance was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4126 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in ERBC validation protocol (r > 0.99; accuracy 80-120%; precision CV < 10%). In ERBC Study no. A4126, 28-hour stability at room temperature and 15-day stability at 2-8°C were verified in the range from 10 to 100 mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (80%-120% for concentration and CV < 10% for homogeneity). The proposed preparation procedure for the test substance was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4126 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (80-120%) and homogeneity (CV < 10%). Samples of the preparations prepared on Weeks 1 and 5 (last week with males and females) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (80-120% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department. The software used for this activity was Empower® 2 Build No. 2154.

Duration of treatment / exposure:

- Males: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing, through the pairing period and thereafter until the day before necropsy, for a total of 33/34 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

- Females: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing and thereafter during pairing, post coitum and post-partum periods until Day 13 post-partum, or the day before sacrifice (for a total of 38 to 65 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Frequency of treatment:

Once daily, 7 days/weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

- Mortality:
Throughout the study, all animals were checked early in the morning and in the afternoon each working day. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day. Severely debilitated animals were observed carefully.

- Clinical signs:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. Observations were performed at the same time interval each day (approximately 5 - 10 minutes and 1.30 - 2 hours post-dose), the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

- Clinical Observations (Functional Observation Battery Tests):
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination (ERBC SOP no. ANI/344). Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. All observations were recorded for individual animals.

- Grip strength and sensory reactivity to stimuli:
Once during the study, towards the end of treatment (during Week 5 for males and Day 12 post partum for females with viable litters, where possible), 5 out of 10 males and 5 out of 10 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength (ERBC SOP
no. ANI/344). Measurements were performed using a computer-generated random order.

- Motor activity assessment (MA):
Once during the study, towards the end of treatment (during Week 4 for males and on Day 12 post partum for females with viable litters where possible), 5 males and 5 females were randomly selected from each group and the motor activity (MA) were measured (for approximately 5 minutes) by an automated activity recording device (ERBC SOP no. ANI/346). Measurements were performed using a computer-generated random order.

- Body weight - Parental animals:
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.

- Food consumption:
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for mated females was measured on gestation Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

- Blood collection for metabolomics: At the end of treatment period, 1 mL was taken from all surviving males and females, under conditions of food deprivation. Blood samples were collected (from the retro-orbital sinus in the males, from the abdominal vena cava in the females) and immediately transferred with a 1 mL pipette tip into an Eppendorf tube containing 10 µL of 10% Titriplex III solution. The tube was immediately closed and gently mixed 5-6 times by inverting it. Blood was kept cool in ice water (approximately 4°C) during the collection period of up to 20 minutes. To separate plasma, the blood was centrifuged at 4°C, 20000 g for 2 minutes. Blood cells and plasma were separated; 0.5 mL of the supernatant plasma layer were pipetted with a 1 mL pipette tip into the second labelled Eppendorf tube and covered with a N2 atmosphere. Tubes were closed with the lid which was wrapped with Parafilm-M, and frozen at -80°Cwithin at maximum 30 minutes after centrifugation, then stored at -80°C until despatch for additional investigations (metabolomics analysis) at an external laboratory. Results of the metabolomic analysis, the corresponding data and their interpretation are the responsibility of the Sponsor and are reported separately.

- Clinical pathology investigations
Blood collection was performed for hormone determination (0.8 mL) from all animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of treatment period, from the 5 males and 5 females (females with viable litters) of each group, under condition of food depriva- tion. No samples were taken for animals sacrificed for humane reasons.
Following haematology and coagulation paramaters were assessed: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count, platelets and prothrobin time. Clinical chemistry parameters assessed corresponds to : alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride, inorganic phosphorus.

-Urinalysis (Only males randomly selected)
During the last week of treatment, individual overnight urine samples were also collected from the same animals selected for clinical pathology investigations (5 males/group, randomly selected). Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. The measurements performed on urine samples are as follows: appearance, volume (manually recorded), specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood.
These parameters were analysed by Menarini Aution Max AX 4280/Aution Eleven AE 4020/Aution Sticks 10EA or ATAGO Refractometer (for Specific gravity), according to in- ternal procedures. The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components.

- Blood collection and thyroid hormone determination (T4 and TSH)
Blood collection for hormone determination was performed from all animals at termination.
Males
Blood samples (approximately 0.8 mL) for hormone determination were collected under isoflurane anaesthesia from the abdominal vena cava . The order of collection was equalised between groups.
Females
As a part of the necropsy procedure, blood samples (approximately 0.8 mL) for hormone determination was withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.

- Immunoanalysis - Thyroid hormone determination (T4 and TSH)
Samples were assayed to determine the serum levels of Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

Sacrifice and pathology:

-Sacrifice/Euthanasia: Parental animals and those that had completed the scheduled test period were killed by exsanguinations under isofluorane anaesthesia. The animal sacrificed for humane reasons was killed with carbon dioxide. Parental males: The males were killed after the mating of all females (up to Day 35 of the study). Parental females: The females with live pups were killed on Day 14 post partum. One female with total litter loss was killed shortly after the occurrence of total litter loss. The females showing no evidence of copulation were killed 25 days after the last day of the mating session. The females which did not gave birth 25 days after positive identification of mating was killed shortly after

- Necropsy: The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

- Organ weights: From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: abnormalities, adrenal glands, bone marrow, aecum, clitoral gland, colon, duodenum, epididymides, eyes, femur, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes, mammary gland, nasal cavity, oessophagus, ovaries, parathyroid gland, pituitary gland, penis, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal column, spinal cord, skeletal muscle, splee, stomatch, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina. The ratios of organ weight to body weight were calculated for each animal.
Tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

- Histopathological examination: After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). A detailed qualitative examination of the testes was performed in all control and high dose group males killed at term. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non- parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was observed in 8 out of 10 males and 9 out of 10 females dosed at 300 and and in all males and females dosed at 750 mg/kg/day with a dose-related frequency (only occasionally in one individual females dosed at 100 mg/kg/day), from the first few days of the pre-mating phase up to termination.
Animals of the control group did not show any sign during the whole treatment period. Piloerection, pallor and swollen neck were also occasionally seen in 1 female dosed at 300 mg/kg/day (animal no. X1680059), while dyspnoea was observed in 1 male dosed at 750 mg/kg/day (animal no. X1680076). Due to the transient occurence and the low incidence, these signs were not considered treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male from Group 3 (dosed at 300 mg/kg/day, no. X1680048) and one male from Group 4 (dosed at 750 mg/kg/day, no. X1680078) were found dead on Days 9 and 13 of the pre- mating phase, respectively. In addition, 1 female from Group 2 (dosed at 100 mg/kg/day, no. X1680033) was sacrificed for humane reasons on Day 22 of the gestation phase. Altogether, these mortalities were not considered treatment-related.
Animal no. X1680048 (300 mg/kg/day) showed salivation and dyspnoea in the days be- fore death. Macroscopically, dark red colour and incomplete collapse of lungs and clear fluid in the thoracic cavity were noted. At microscopic observation, diffuse oedema and haemorrhage of the lungs were observed. Death was considered to be related to misgavage.
Animal no. X1680078 (750 mg/kg/day) showed salivation in the days before death. Macro- scopically, single dark red area of the calvaria and dark red fluid in the cranial and thoracic cavity were observed. At microscopic observation, sub meningeal haemorrhage of the brain, alveolar and interstitial haemorrhage of the lungs, were noted. Death was considered to be related to a traumatic impact of the head following an accidental fall from the cage.
Animal no. X1680033 (100 mg/kg/day), was sacrificed for humane reasons on Day 22 of the gestation phase for difficulty in parturition. Piloerection and pallor were observed prior to sacrifice. No abnormalities were detected at macroscopic observation. At microscopic observation, unilateral cortical hypertrophy was noted. On the basis of the prolonged time without completing the parturition, impaired parturition was considered to be the reason for sacrifice. The gross and microscopic evaluation did not allow to establish the cause of impared parturition. In the absence of a dose-relation and other treatment-related effects, the difficulty in parturition observed in this animal is not considered to be treatment- related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain for male and female animals were generally comparable between the treated and control groups, before and during mating, during gestation and post partum periods.
Before pairing, on Day 15, females receiving 750 mg/kg/day showed a decrease in body weight gain (-91%) compared to the control group, while statistically significant body weight losses were seen in the males dosed at 300 mg/kg/day on Day 15 of mating phase. These isolated changes were followed by a regular growth of the animals. Due to their occasional occurrence and/or in the absence of a dose-relation, these changes were not considered treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both gender during the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
The statistically significant differences recorded between control and treated animals (mean corpuscular haemoglobin concentration in males, reticulocytes and platelets in females) were not dose-related and/or of minimal severity, therefore they were considered to be incidental.
No changes were recorded for coagulation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
A statistically significant decrease of total bilirubin (48%) was recorded in males dosed at 750 mg/kg/day. The decrease of bilirubin has no biological/pathological significance, especially in rats where normal values are low (mean historical control data is 0.04 mg/dL) and the low limit of historical control data is 0 mg/dL.

Endocrine findings:

no effects observed

Description (incidence and severity):

No treatment related changes were observed for thyroid hormone determination in parental males.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were observed in parental males.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in terminal body weight and organ weights when compared to the controls. The increase in relative mean liver weights (+10% for relative liver weight when compared to controls) and brain weight (+7% for relative brain weight when compared to controls) in high dose treated males were not correlated to any histopathological changes and, therefore, they were considered unrelated to treatment.
Any other organ weight variations were in the range of expected spontaneous changes in rats of the same age and considered unrelated to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations at the end of the treatment period. Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment. There were no test item-related microscopic observations in the testis (stage aware evalu- ation on PAS-stained slides).

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

serum/plasma biochemistry

serum/plasma hormone analyses

urinalysis

Key result

Critical effects observed:

no

Please see tables in attachment section.

Conclusions:

Under the study conditions, the NOAEL for general toxicity was considered to be 750 mg/kg/day for males and females.

Executive summary:

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rat according to OECD Guideline 422, in compliance with GLP. Four groups of ten Sprague Dawley rats (males and females) were exposed by oral gavage to increasing concentrations of the test substance (0, 100, 300 and 750 mg/kg/day). All doses were administered by gavage at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, for a total of 38 to 65 days. The following investigations were performed: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five randomly selected animals/sex/group), macroscopic observations, organ weights. Routine histopathological examination was performed in control and high dose groups (five randomly selected animals/sex/group). No mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females at 300 and 750 mg/kg/day. Piloerection, pallor, swollen neck and dyspnoea were also noted in a single male and female animal at 300 and 750 mg/kg bw/day, respectively. These clinical signs were not considered to be treatment-related due to their transient occurrence and low incidence. Neurotoxicity assessment (removal from the home cage and observations in an open arena), motor activity, grip strength and sensory reactivity to stimuli did not reveal changes attributable to the test substance. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. The food consumption was comparable in all groups. No adverse findings were recorded in clinical pathology investigations (haematology including coagulation parameters and clinical chemistry). Hormone analysis did not show any relation to treatment. Urinalysis in male animals did not reveal changes attributable to the test substance. Under the study conditions, the NOAEL for general toxicity was considered to be 750 mg/kg/day for males and females (Longobardi, 2022).

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Dose range finder

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 03, 2020 to December 21, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study is to investigate the toxicity of the test item in rats after daily oral administration for 2 weeks. The data generated will allow selection of dose levels for subsequent toxicological studies.

GLP compliance:

no

Remarks:

This study is a preliminary dose range-finding study and is exempt from compliance with the Principles on Good Laboratory Practice of the OECD. However, it is carried out in a GLP compliant facility.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley (SD) rat is the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Age at study initiation: 7 to 8 weeks old.
- Weight at study initiation: 200 to 225 g for males and 175 to 200 g for females.
- Housing: The animals were housed up to 5 of one sex to a cage.
- Diet: A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019,SettimoMilanese (MI), Italy) was offered ad libitum.
- Water: Free access to tap water.
- Acclimation period: 18 days.

Animal room controls were set to maintain temperature and relative humidity at 22°C±2°Cand 55%±15%, respectively, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered orally, by gavage at a dose volume of 10 mL/kg. Control animals received the vehicle alone at the same dose volume. The dose was administered
to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC

Details on oral exposure:

- Preparation of dosing solutions:
The required amount of test item was suspended in the vehicle and then heated in a water bath at a temperature of 50-60°C for approximately 3 hours. Once dissolved, the suspension was kept under magnetic stirring at room temperature overnight. The preparations were made daily (concentrations of 10, 30 and 100 mg/mL). Concentrations were calculated and expressed in terms of test substance as supplied.

- Dose volume:
10 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

All animals were dosed 7 days a week, for a minimum of 2 consecutive weeks.
All animals were dosed up until the day before necropsy.

Frequency of treatment:

All animals were dosed once a day.

Dose / conc.:

0 mg/kg bw/day (nominal)

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:

4 males and 4 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

The toxicity of Amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl) (abbreviated name: C8-18 and C18-unsatd. MIPA) was investigated, in rats after daily oral administration for 2 weeks. Three groups, each of 4 male and 4 female Sprague Dawley rats, received the test item once daily by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day. A fourth, similarly constituted group received the vehicle alone (0.5% carboxymethylcellulose) and acted as a control.
The animals were observed for mortality check, clinical signs, body weight and food consumption. At termination, all animals were subjected to a detailed macroscopic examination along with organ weights and tissue retention. No histopathological examination was performed. Blood samples were collected at necropsy for metabolomics evaluation.

Observations and examinations performed and frequency:

- Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon.

- Clinical signs
All clinical signs were recorded for individual animals. Once before commencement of treatment and once daily during treatment, each animal was observed and any clinical signs recorded. Observations were performed at the same time intervals each day.

- Body weight
Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

- Food consumption
The weight of food consumed by each cage of rats was recorded at weekly intervals from the start of treatment. The group mean daily intake per rat was calculated.

-Blood collection
Prior to necropsy, 1 mL was taken from the sublingual vein in a randomized sequence from all surviving male and female animals, under conditions of food deprivation.

Sacrifice and pathology:

Animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. All animals, including those found dead,were subjected to necropsy. No histopathological examination was carried out in the first instance.

Other examinations:

Blood samples were collected at necropsy for metabolomics evaluation (ongoing analysis - data will be reported separately under section 13 when available).

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was the only treatment-related clinical sign recorded during the study. This was observed from Day 4 in all animals dosed at 1000 mg/kg bw/day (both sexes) and in a single occasion (Day 12 or 13) in 3 out of 4 males dosed at 300 mg/kg bw/day approximately 10 - 15 minutes post-dose. This sign was no longer present after 1.30 - 2 hours post-dose.
No clinical signs were observed in the remaining males of the mid-dose group (300 mg/kg bw/day) and in both sexes animals treated at 100 mg/kg bw/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male (animal no. E0557004) was found dead on day 13 of the study. At post mortem examination the findings observed in the above mentioned animal were swollen liver, dark colour of mesenteric nodes and multiple dark areas of thymus. The above mentioned findings did not clarify the cause of death. No further mortality occurred during the study

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in body weight, when compared to control animals (both sexes).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No changes in food consumption were recorded in both sexes between treated and control groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

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):

No changes were observed in terminal body weight of treated animals of both sexes sacrificed at term, when compared to controls.
A statistically significant increase in absolute and relative mean liver weight was recorded in high dose treated animals of both sexes (+22% in males and +19% in females for relative weight) and in relative mean liver weight for mid-dose females (+13%). These liver weight changes were considered to be treatment-related.
Any organ weight changes other than those listed above were within the range of occasionally observed and expected spontaneous changes in rats of the same age and considered unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted following gross pathology examination at final sacrifice. Any macroscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

dose level: Highest dose selected for subsequent main toxicological study.

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

The highest dose of 1000 mg/kg/day is not considered to be sufficiently safe for a subsequent longer duration study in Sprague Dawley rats.

Critical effects observed:

no

Conclusions:

Under the study conditions, due to the observed effects, the highest dose of 1000 mg/kg/day was not considered to be sufficiently safe for a subsequent longer duration study in Sprague Dawley rats.

Executive summary:

A preliminary dose range finder was conducted with the test substance to allow selection of dose levels for subsequent main 28 days repeated dose toxicity study. Three groups of Sprague Dawley SD rats (males and females) received the test substance at dose levels of 100, 300 or 1000 mg/kg bw/day once daily for 2 weeks. A fourth similarly constituted group received the vehicle alone (0.5% carboxymethylcellulose) and acted as a control. The animals were observed for mortality, clinical signs, body weight and food consumption. At termination, all animals were subjected to a detailed macroscopic examination along with organ weights determination. No histopathological examination was performed. Blood samples were collected at necropsy for metabolomics evaluation. No treatment-related mortality occurred during the study. Salivation was observed in the animals dosed at 1000 mg/kg bw/day (both sexes) and in a single occasion in the males dosed at 300 mg/kg bw/day. This clinical sign was not considered to be adverse. No effects on body weight and food consumption were seen at any tested dose. A treatment-related, statistically significant, increase in absolute and relative mean liver weight was recorded in animals of both sexes at 1000 mg/kg bw/day and in relative mean liver weight in the females at 300 mg/kg bw/day. No treatment-related changes were noted following gross pathology examination at final sacrifice. In conclusion, under the study conditions, some treatment-related effects (salivation and increased liver weight) were observed in animals dosed with the test substance at 1000 mg/kg bw/day and in the males at 300 mg/kg bw/day. These effects were slight and, as such, were not considered to be adverse in this study. However, the highest dose of 1000 mg/kg bw/day was not considered appropriate for a subsequent longer duration study due to the effects on liver weight (Longobardi, 2021).

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 (14 day)

A preliminary dose range finder was conducted with the test substance to allow selection of dose levels for subsequent main 28 days repeated dose toxicity study. Three groups of Sprague Dawley SD rats (males and females) received the test substance at dose levels of 100, 300 or 1000 mg/kg bw/day once daily for 2 weeks. A fourth similarly constituted group received the vehicle alone (0.5% carboxymethylcellulose) and acted as a control. The animals were observed for mortality, clinical signs, body weight and food consumption. At termination, all animals were subjected to a detailed macroscopic examination along with organ weights determination. No histopathological examination was performed. Blood samples were collected at necropsy for metabolomics evaluation. No treatment-related mortality occurred during the study. Salivation was observed in the animals dosed at 1000 mg/kg bw/day (both sexes) and in a single occasion in the males dosed at 300 mg/kg bw/day. This clinical sign was not considered to be adverse. No effects on body weight and food consumption were seen at any tested dose. A treatment-related, statistically significant, increase in absolute and relative mean liver weight was recorded in animals of both sexes at 1000 mg/kg bw/day and in relative mean liver weight in the females at 300 mg/kg bw/day. No treatment-related changes were noted following gross pathology examination at final sacrifice. In conclusion, under the study conditions, some treatment-related effects (salivation and increased liver weight) were observed in animals dosed with the test substance at 1000 mg/kg bw/day and in the males at 300 mg/kg bw/day. These effects were slight and, as such, were not considered to be adverse in this study. However, the highest dose of 1000 mg/kg bw/day was not considered appropriate for a subsequent longer duration study due to the effects on liver weight (Longobardi, 2021). A highest dose of 750 mg/kg bw/day was therefore selected for the main 28 d repeated dose toxicity study (Longobardi, 2022).

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).

Furthermore, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a combined repeated dose toxicity study with reproduction / developmental toxicity screening was conducted with C8-18 and C18 -unsatd. MIPA in rats according to OECD Guideline 422, in compliance with GLP, to address this REACH endpoint requirement and to further support the read across approach proposed for the FAA category members. In this study, four groups of ten Sprague Dawley rats (males and females) were exposed by oral gavage to increasing concentrations of the test substance (0, 100, 300 and 750 mg/kg/day). All doses were administered by gavage at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, for a total of 38 to 65 days. The following investigations were performed: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five randomly selected animals/sex/group), macroscopic observations, organ weights. Routine histopathological examination was performed in control and high dose groups (five randomly selected animals/sex/group). No mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females at 300 and 750 mg/kg/day. Piloerection, pallor, swollen neck and dyspnoea were also noted in a single male and female animal at 300 and 750 mg/kg bw/day, respectively. These clinical signs were not considered to be treatment-related due to their transient occurrence and low incidence. Neurotoxicity assessment (removal from the home cage and observations in an open arena), motor activity, grip strength and sensory reactivity to stimuli did not reveal changes attributable to the test substance. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. The food consumption was comparable in all groups. No adverse findings were recorded in clinical pathology investigations (haematology including coagulation parameters and clinical chemistry). Hormone analysis did not show any relation to treatment. Urinalysis in male animals did not reveal changes attributable to the test substance. Under the study conditions, the NOAEL for general toxicity was considered to be 750 mg/kg/day for males and females (Longobardi, 2022).

The results are in line with existing data and support similar toxicological profile within and across the different categories. For completeness, it should be noted that small biological variations have been observed at the top dose of 1000 mg/kg bw/day in the dose range findings studies for C8-18 and C18-unsatd. MIPA and C16-18 and C18-unsatd. DEA, but not in the main studies. Taking full advantage of the in vivo studies conducted, additional blood samples were collected in the OECD TG 422 testing programme. These samples are currently undergoing metabolomics analyses to enhance the quality and quantity of data from a biological perspective.

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 Day 92. All animals were killed on Day 92 (or Day 91 for one female). Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically. Epidydimides were collected on Day 92 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 Day 59 and Day 88 and one male treated with read across substance at 100 mg/kg bw/day was killed early on Day 77. One female treated with read across substance at 1000 mg/kg bw/day was found dead on Day 91. 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 present 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 were conducted with a representative short- and a long chain substance of each subcategory (i.e., DEA, MEA, MIPA). The results of the Tier 1 are in line with existing data and support the hypothesis of a similar toxicological profile within and across the different categories. It should however be noted that small biological variations have been observed (e.g., top dose findings for C8-18 and C18-unsatd. MIPA /C16-18 and C18-unsatd. DEA in the dose range-finders).

Taking full advantage of the bridging studies samples, metabolomics analyses arecurrently ongoingto enhance the quality and quantity of data from a biological perspective and to further elucidate the small biological variations observed.The results will be used to refine the Tier 2 testing strategy.

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 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 strategy and current status overview are detailed in two documents entitled‘ECHA-DIAP - FAA testing strategy summary – 24Sep20’ and ‘ECHA-DIAP - FAA testing strategy summary status overview-24Mar22’, both attached in Section 13 of the most recent IUCLID datasets.

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.