Reaction mass of N-(2-hydroxypropyl)decan-1-amide and N-(2-hydroxypropyl)octanamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 09, 1999 to May 28, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-540-9

EC Name:

-

Molecular formula:

C21H43NO2

IUPAC Name:

N-(2-hydroxypropyl)-16-methylheptadecanamide

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanIbm:WIST (SPF)

Details on species / strain selection:

Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

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

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 1)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 2)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 4)

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

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

Positive control:

-

Examinations

Observations and examinations performed and frequency:

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

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

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

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

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

Sacrifice and pathology:

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

Statistics:

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled study periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Neuropathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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