Reaction mass of N,N’-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide), Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]- and Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2017 - 19 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 21 September 1998

Deviations:

yes

Remarks:

All females on Day 50 received a higher dose amount than required due to technician error. Dose amounts were correct for remainder of study. There were no adverse effects in any female on this day and no treatment related findings overall on the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

EC Number:

204-613-6

EC Name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Cas Number:

123-26-2

Molecular formula:

C38H76N2O4

IUPAC Name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecanamide)

Constituent 2

Reference substance name:

Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Molecular formula:

C38H76N2O3

IUPAC Name:

Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Constituent 3

Reference substance name:

Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]-

Molecular formula:

C38H76N2O3

IUPAC Name:

Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]-

impurity 1

Reference substance name:

N-(2-aminoethyl)-12-hydroxyoctadecanamide

IUPAC Name:

N-(2-aminoethyl)-12-hydroxyoctadecanamide

impurity 2

Reference substance name:

12-hydroxystearic acid

EC Number:

203-366-1

EC Name:

12-hydroxystearic acid

Cas Number:

106-14-9

Molecular formula:

C18H36O3

IUPAC Name:

12-hydroxyoctadecanoic acid

impurity 3

Reference substance name:

9,12-dihydroxy-N-(2-(12-hydroxyoctadecanamido)ethyl)octadecanamide

IUPAC Name:

9,12-dihydroxy-N-(2-(12-hydroxyoctadecanamido)ethyl)octadecanamide

impurity 4

Reference substance name:

Octadecanamide, 12-hydroxy-N-[2-[(1-oxohexadecyl)amino]ethyl]-

IUPAC Name:

Octadecanamide, 12-hydroxy-N-[2-[(1-oxohexadecyl)amino]ethyl]-

impurity 5

Reference substance name:

N-(2-(12-hydroxyoctadecanamido)ethyl)octadec-12-enamide

IUPAC Name:

N-(2-(12-hydroxyoctadecanamido)ethyl)octadec-12-enamide

impurity 6

Reference substance name:

(ethane-1,2-diylbis(azanediyl))bis(18-oxooctadecane-18,7-diyl) bis(12-hydroxyoctadecanoate)

IUPAC Name:

(ethane-1,2-diylbis(azanediyl))bis(18-oxooctadecane-18,7-diyl) bis(12-hydroxyoctadecanoate)

Test material form:

solid: particulate/powder

Details on test material:

Test material is 100% registered material EC# 701-269-3. Constituents listed above.

Specific details on test material used for the study:

Physical State/Appearance: Off white powder
Purity: 100%
Batch Number: S/99/17
Storage Conditions: Stored in ambient temperature in darkness, used/formulated in light
Expiry Date: 30 March 2021

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were acclimatized for eight days during which time their health status was assessed. At the start of treatment the males weighed 197 to 232g, the females weighed 146 to 175g, and were approximately six weeks old. The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. The animals were allowed free access to food and water. A pelleted diet was used. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the substance formulations were taken on four occasions and analyzed for concentration. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Low

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Intermediate

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Clinical Observations: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Body Weight: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption: Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

Water Consumption: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Functional Observations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

Functional Performance Tests: Motor Acivity, Forelimb/Hindlimb Grip Strength, Sensory Reactivity, Ophthalmoscopic Examination.

In-Life Sampling and Analysis included:
Haematology:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)

Sacrifice and pathology:

Necropsy: On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights:
Adrenals
Ovaries
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus
Liver

Histopathology:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint) (Retained only and not processed)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Caecum
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides (preserved in Modified Davidson’s fluid)
Skin
Esophagus
Spinal cord (cervical, mid-thoracic
Eyes
and lumbar)
Gross lesions (where applicable)
Spleen
Heart
Stomach
Ileum (including Peyer’s patches)
Testes (preserved in Modified Davidson’s fluid)
Jejunum
Thymus
Kidneys
Thyroid/Parathyroid
Liver
Tongue (Retained only and not processed)
Lungs (with bronchi)
Trachea
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary glands
Uterus (with cervix)
Muscle (skeletal) (Retained only and not processed)
Vagina

Pathology: Microscopic examination was conducted by the Study Pathologist.

Statistics:

Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidences of increased salivation were evident in five males treated with 1000 mg/kg bw/day throughout the majority of the treatment period and in one male treated with 100 mg/kg bw/day on two occasions. An observation of this nature is commonly observed following the oral administration of an unpalatable substance formulation and is considered not to represent an adverse effect of treatment.

Noisy respiration was also evident in three males treated with 1000 mg/kg bw/day between Days 52 and 90, in one female treated with 1000 mg/kg bw/day on two occasions, in one male and two females treated with 100 mg/kg bw/day on one occasion and in one male and one female treated with 10 mg/kg bw/day on one occasion only. At these low frequencies and distributions, this is considered to indicate possible difficulties in dosing particular animals on isolated occasions and not indicative of systemic toxicity.

Incidental observations, showing no dose related response and considered to be unrelated to test item administration included one control male had noisy respiration on Day 43, one male treated at 100 mg/kg bw/day with an open wound on the left shoulder between Days 9 and 11, which then became a scab between Days 12 and 39 and a male treated with 10 mg/kg bw/day was observed to have misaligned posture, holding its head tilted to the left from Day 13 to termination.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control female was found dead on Day 75. At necropsy this female had black colored contents in the cecum, colon, duodenum, ileum, jejunum and stomach and a pale liver with dark patches. There were no changes at histopathology to account for the death or the macroscopic changes, however, lymphoid necrosis/atrophy/depletion was present along with cortical hypertrophy in the adrenal glands which indicate poor clinical condition/stress.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 1000 and 100 mg/kg bw/day showed statistically significantly higher (p<0.05-0.01) body weight gains during Week 2, with males treated at 1000 mg/kg bw/day also showing statistically significantly higher body weight gains during Weeks 4 (p<0.01), 5 (p<0.05) and 7 (p<0.01). An increase in body weight gain is considered not to be an adverse effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Total leukocyte count was statistically significantly lower (p<0.05) in all treated males. With the exception of one individual male treated at 100 mg/kg bw/day, all individual values for treated males were within the historical control range. Mean corpuscular hemoglobin concentration and lymphocytes for males treated with 1000 mg/kg bw/day were statistically significantly lower (p<0.05) compared to controls. For mean corpuscular hemoglobin concentration, four of the individual control values exceeded the historical control range, whereas all of the individual values for males treated with 1000 mg/kg bw/day were within the historical control range. For lymphocytes, although all individual control values were within the historical control range, only one individual value for males at 1000 mg/kg bw/day was below this range. As the majority of values were within the historical control normal ranges, and in the absence of any histopathological correlates, these intergroup differences were considered to be of no toxicological significance.

Eosinophils were statistically significantly lower (p<0.05) than controls for males treated with 10 mg/kg bw/day. All individual values were within the historical control range and in the absence of any similar findings in males treated with 100 or 1000 mg/kg bw/day, the intergroup difference was considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated at 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in calcium concentration. All of the individual values were within the historical control range and in the absence of any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance. Bilirubin levels were also statistically significantly higher (p<0.05) than controls in males treated with 100 and 1000 mg/kg bw/day. However, two individual control values were below the historical control range and only one individual value for males at 1000 mg/kg bw/day was above the historical control range. All values for males treated with 100 mg/kg bw/day were within the historical control range. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Occasional incidences of noisy respiration were evident in one male treated with 1000 mg/kg bw/day during the final two weeks of treatment, but this is likely to be due to slight difficulties in dosing this particular animal on these occasions and is not indicative of true systemic toxicity.

The incidental clinical finding of mis-aligned posture was evident in one male treated with 10 mg/kg bw/day from Week 2 assessments onwards.

Motor activity assessment did not indicate any effect of treatment for either sex at 10, 100 or 1000 mg/kg bw/day. There was no effect of treatment at 10, 100 or 1000 mg/kg bw/day on forelimb or hindlimb grip strength.

There were no treatment-related changes in sensory reactivity. Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 10, 100 or 1000 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 100 and 10 mg/kg bw/day showed statistically significant increases (p<0.05) in absolute and body weight relative kidney weights in comparison with controls. The majority of individual values were within historical control ranges and in the absence of any similar findings in males treated with 1000 mg/kg bw/day or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Both absolute and body weight relative liver weights were statistically significantly higher (p<0.05) for males treated with 100 and 1000 mg/kg bw/day compared to controls. However, the majority of these individual values were within historical control normal ranges, a true dose related response for relative weights was not evident and there were no histopathological correlates. The intergroup differences were therefore considered not to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Red or dark coloration of the lungs was observed for one female at 10 mg/kg bw/day, three males and one female treated with 100 mg/kg bw/day and two males treated with 1000 mg/kg bw/day. Such findings are common in this type of study and were considered to be unrelated to treatment with the substance.

Increased pelvic space in both kidneys was observed in one control female and mottled kidneys was observed in one control male. In the absence of treatment, these were considered to be incidental findings. One female treated with 100 mg/kg bw/day had a dark liver and one female treated with 10 mg/kg bw/day had a dark patch on the liver. In the absence of a similar effect at 1000 mg/kg bw/day or any associated histopathological correlates, these findings were considered incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Centrilobular hypertrophy was noted in the liver of one male treated with 1000 mg/kg bw/day, however, due to the low incidence and minimal severity after ninety days of treatment this is considered not to be related to the administration of the substance. No changes were noted which could account for the weight increase noted in the liver of males and all are considered to be incidental.

Histopathological findings: neoplastic:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of the substance, for ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 1000 mg/kg bw/day did not result in any adverse treatment-related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be >1000 mg/kg bw/day.

Executive summary:

Male and female Wistar rats (10/sex/dose) were administered the substance (dissolved in Arachis oil BP) by oral gavage at doses of 0 (vehicle only), 10, 100 or 1000 mg/kg bw/day once daily for 90 consecutive days. The study was conducted according to OECD Guideline 408 (Subchronic Oral Toxicity - Rodent: 90 Day Study) and in accordance with GLP. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. No mortality occurred and no treatment-related clinical signs were observed during the study. Incidences of increased salivation were evident in males treated with 1000 mg/kg bw/day throughout the majority of the treatment period and in males treated with 100 mg/kg bw/day albeit to a lesser extent. Noisy respiration was also evident in three males treated with 1000 mg/kg bw/day between Days 52 and 90 and in one female treated with 1000 mg/kg bw/day, one male and two females treated with 100 mg/kg bw/day and one male and one female treated with 10 mg/kg bw/day on one occasion only. There were no toxicologically significant changes in the behavioral parameters measured. There were no treatment-related changes in functional performance. There were no treatment-related changes in sensory reactivity. No adverse effect on body weight development was evident in treated animals when compared to controls. No effect on food consumption or food efficiency was evident in treated animals when compared to controls. Visual inspection of water bottles did not reveal any intergroup differences. Ophthalmoscopic examination of animals of both sexes from the control and surviving 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences. There were no toxicologically significant effects detected in the hematological or blood chemistry parameters examined. No treatment-related findings were reported at post mortem macroscopic observations and histopathological examination. No toxicologically significant effects were detected in the organ weights measured. In conclusion, no treatment-related changes, which could be considered adverse, were observed in male and female rats following dosing with the substance, when administered by oral gavage for 90 consecutive days at the dosages of 10, 100 and 1000 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for this study was >1000 mg/kg bw/day.