Reaction mass of N, N'-hexane-1,6-diylbis [12-hydroxyoctadecanamide] and 12-hydroxy-N-[6-[1-oxoalkyl)amino] hexyl ] octadecanamide

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:

09 March - 06 April 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Directive 96/54/EEC, B.7 OECD 407 (1995) OPPTS 870.3050, Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): AD-1000
- Substance type: white powder
- Physical state: powder
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing: Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilised sawdust as bedding material and paper as cage-enrichment. No cage-enrichment was provided during overnight activity monitoring.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 – 23.4
- Humidity (%): 43 - 92
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.


DIET PREPARATION
- Rate of preparation of diet (frequency): daily within 4 hours prior to dosing
- Storage temperature of food: At ambient temperature


VEHICLE: Propylene glycol
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information from the sponsor.
- Concentration in vehicle: 0, 50, 100, 1000 mg/kg/day
- Amount of vehicle (if gavage): 5 ml/kg body weight

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 100, 1000 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a 5-day dose range finding study
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality / Viability: At least twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily.


BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28.


FOOD CONSUMPTION:
- Weekly


WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes; iso-flurane anaesthesia
- Animals fasted: Yes
- How many animals: all
- Parameters examined: White blood cells, Differential leucocyte count, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin
Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets
Clotting Potential: Prothrombin time, Activated Partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: all
- Parameters c were examined.
Clinical Biochemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea
Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment, the following tests were performed on all animals
- Dose groups that were examined: all
- Battery of functions tested:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes Tissues mentioned within brackets were not examined microscopically as there were no signs of toxicity or target organ involvement.
Adrenal glands
Aorta
Brain [cerebellum, mid-brain, cortex]
Caecum
Cervix
(Clitoral gland)
Colon
Duodenum
Epididymides
(Eyes with optic nerve [if detectable] and
Harderian gland)
(Female mammary gland area)
(Femur including joint)
Heart
Ileum
Jejunum
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
(Preputial gland)
Prostate gland
Rectum
(Salivary glands - mandibular, sublingual)
Sciatic nerve
(Seminal vesicles)
(Skeletal muscle)
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid [if detectable]
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions

Organ Weights
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Spleen
Testes
Thymus

HISTOPATHOLOGY: Yes
All organs and tissue samples, as mentioned in the histopathology report were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all animals of the control and the highest
dose group
- all gross lesions

Other examinations:

none

Statistics:

The following statistical methods were used to analyse the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test2 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 was applied to frequency data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.

There were no clinical signs of toxicity noted over the 28-day observation period.
Incidental findings that were noted included alopecia and scabs in group 3 and 4 females. These findings are commonly noted in rats of this age and strain that are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted in all male groups, and in control females and females at 50 mg/kg/day.


BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain were considered to have been unaffected by treatment.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

OPHTHALMOSCOPIC EXAMINATION
Hearing ability was normal

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Minor statistically significant differences were considered to have occurred by chance as no dose-response was apparent and/or means remained well within the historical control range. These changes comprised higher relative neutrophil counts in males at 50 and 1000 mg/kg/day, lower relative lymphocyte counts in males at 50 and 1000 mg/kg/day and lower platelet counts in females at 50 mg/kg/day.


CLINICAL CHEMISTRY
There were no differences noted between control and treated rats that were considered to be related to treatment with AD-1000.
The statistically significant lower glucose and total bilirubin values in males at 150 and 50 mg/kg/day respectively were considered to have arisen by chance as a treatment-related distribution was absent.


NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment.


ORGAN WEIGHTS
No toxicologically significant changes were noted regarding absolute and/or relative organ weights.
The statistically significant lower spleen and liver weight at 50 mg/kg/day in males and females respectively were considered to have occurred by chance as means remained well within the historical control range and no dose-response relationship was present.


GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant macroscopic alterations.
Incidental findings among control and/or treated animals included red foci on the thymus and red discolouration of the thymus, enlargement of the mandibular lymph nodes and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance. No macroscopic abnormalities were noted in males at 50 mg/kg/day and higher.


HISTOPATHOLOGY:
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Wistar rats were treated with AD-1000 for 28 consecutive days by oral gavage at dose levels up to 1000 mg/kg/day.
There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination that were considered to be an effect of treatment.
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for AD-1000 of 1000 mg/kg/day was established.