2-Hexyl-2 ‘-hydroxy-5 ‘methyldecananilide

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:

03 Mar - 31 Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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)

Version / remarks:

adopted 27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted September 1996

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (adopted July 2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): AF-654
- Physical state: white powder
- Lot/batch No.: #CE-201
- Expiration date of the lot/batch: 01 Jan 2005
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Crl:(WI) BR

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: approximately 6 weeks
- Weight at study initiation: females (mean): approx. 159 g; males (mean): approx. 203 g
- Housing: group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors, during activity monitoring animals were individually housed overnight in Makrolon plastic cages type III with sterilized sawdust provided as bedding
- Diet: standard pelleted laboratory animal diet (Altromin (code VFR-1), Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7 - 25.3
- Humidity (%): 29 - 93
- 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 approximately 4.25 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. Formulations were placed on a magnetic stirrer during dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at the testing laboratory
- Amount of vehicle (if gavage): 10 mL/kg bw (actual dose volumes were calculated weekly according to the latest body weight)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared on day 7 were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 4 hours was also determined (highest and lowest concentration).
Test substance formulations in propylene glycol were noted as stable for at least 4 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 90-100% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150, 1000 mg/kg bw/day
Basis:
other: nominal by gavage

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. In this study, 3 females/group were treated with the test substance in propylene glycol at dose concentrations of 150 or 1000 mg/kg bw/day. No mortality occurred and no adverse clinical signs were noted. Slight reduction in body weight and in food consumption was observed in animals receiving 1000 mg/kg bw/day. No effects on liver/kidney weights and no abnormalities in macroscopic examinations were recorded for the 150 and 1000 mg/kg bw/day groups.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter (except inadvertently on day 28), this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present; Maximum grade 3 or 4: grade 1 slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

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

FOOD CONSUMPTION:
- Food consumption: Yes, daily

WATER CONSUMPTION: Yes
- Time schedule for examinations: subjective appraisal maintained during the study, but no quantitative investigation

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: erythrocytes count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin (MCHC) concentration, platelet count (PLT), red celI distribution width (RDW), total leucocytes count (WBC), differential leucocyte count (WBC), neutrophils (NEUT), Iymphocytes (LYMPHO), monocytes (MONO), eosinophils (EOS), basophils (BASO), prothrombin time (PT), partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: alanine aminotransferase (ALAT), alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), bilirubin total, chloride, cholesterol total, creatinine, glucose, phosphorus (INORG.PHOS), protein total, protein albumin, urea, calcium, potassium, sodium

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all dose groups
- Battery of functions tested:
- hearing ability (HEARING), pupillary reflex (PUPIL LIR), 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, organ weights: adrenal glands, liver, brain, spleen, epididymides, testes, heart, thymus, kidneys
HISTOPATHOLOGY: Yes, samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:
adrenal glands, pancreas, peyers patches (jejunum, ileum) if detectable, aorta, pituitary gland, brain (cerebellum, mid-brain, cortex), (preputial gland), caecum, prostate gland, (cervix), rectum, (clitoral gland), (salivary glands - mandibular, sublingual), colon, sciatic nerve, duodenum, (seminal vesicles), epididymides, (skeletal muscle), (eyes with optic nerve and harderian gland), (skin), (female mammary gland area), spinal cord -cervical, midthoracic, lumbar, (femur including joint), spleen, heart, sternum with bone marrow, ileum, stomach, jejunum, testes, kidneys, thymus, (larynx), thyroid including parathyroid, (lacrimal gland, exorbital), (tongue), liver, trachea, lung, infused with formalin, urinary bladder, lymph nodes - mandibular, mesenteric, uterus, (nasopharynx), (vagina), oesophagus, ovaries, all gross lesions
(tissues mentioned in brackets were not examined as there were no signs of target organ involvement)

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (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 Student’s t-test was applied for motor activity data.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

50 mg/kg bw/day: incidential cases of quick breathing or rales (m, f), in one case with lethargy, hunched posture, piloerection, lean appearance and ptosis; 100 mg/kg bw/day: laboured respiration, rales and gasping (1 m); 1000 mg/kg bw/day: restless behaviour (all females week 3), incidential cases of quick breathing or rales (m, f) in one case with hunched posture (non adverse)

Mortality:

mortality observed, treatment-related

Description (incidence):

50 mg/kg bw/day: incidential cases of quick breathing or rales (m, f), in one case with lethargy, hunched posture, piloerection, lean appearance and ptosis; 100 mg/kg bw/day: laboured respiration, rales and gasping (1 m); 1000 mg/kg bw/day: restless behaviour (all females week 3), incidential cases of quick breathing or rales (m, f) in one case with hunched posture (non adverse)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

50 mg/kg bw/day: incidental cases of slight weight loss in week 1 or 4 (m,f); 150 mg/kg bw/day: incidental cases of slight weight loss in week 4 (f); 1000 mg/kg bw/day: incidental cases of slight weight loss in week 4 (m); (non adverse)

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:

not examined

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:

effects observed, treatment-related

Description (incidence and severity):

acute inflammation and epithelial ulceration of trachea (50 mg/kg), chronic inflammation of oesophagus (50, 150, 1000 mg/kg), chronic inflammation of the trachea (1000 mg/kg), minimal fibrosis of the capsule of the thymus (1000 mg/kg) (non adverse)

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. Restless behaviour was observed among all high dose females in week 3. Signs of respiratory problems were apparent in some of the treated animals. Quick breathing was noted for one male rat at 50 mg/kg bw/day in week 4 and for one female rat at 50 mg/kg bw/day in week 1 as well as for one male and female animal at 1000 mg/kg bw/day at the end of week 2. Rales were noted in one male rat at 50 mg/kg bw/day intermittently during the study and in one male and female rat at 1000 mg/kg/day on a single day of week 4. In 1/5 males and in 1/5 females in the 50 mg/kg bw/day and in 1/5 males in the 1000 mg/kg bw/day group the respiratory problems were associated with other signs including lethargy, hunched posture, piloerection, pallor, lean appearance and/or ptosis. Laboured respiration, rales and gasping were noted for one male rat at 150 mg/kg bw/day at the end of week 2.
Alopecia, scales, scabs, wounds, chromodacryorrhoea, broken upper incisors, watery discharge from the eyes, lethargy and diarrhoea (one control male noted on single days only) and a broken tail are not uncommon for rats of this age and strain which are housed and treated under the conditions in this study. At the observed incidence these signs were not considered to be related to treatment with the test substance. No clinical signs were noted among control females.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period. Slight weight loss was noted for some animals among the dose groups (i.e. one male rat each at 50 mg/kg bw/day and at 1000 mg/kg bw/day in week 4, one female rat at 50 mg/kg bw/day in week 1, and two female rats at 150 mg/kg bw/day in week 4). This was considered to be of an incidental nature and in the absence of a dose-related incidence regarded to be of no toxicological significance.

FOOD CONSUMPTION
There were no changes in food intake that were considered to be significant in toxicological terms. A slightly lower food intake was measured for females at 50 mg/kg bw/day in week 1, also after correction for body weight. Relative food consumption of treated females was generally lower than their controls in weeks 3 and 4. As these findings did not show a dose related response, no toxicological relevance was ascribed to these changes.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment. Individual increases of neutrophil counts with concurrently reduced lymphocyte counts were noted among the dose groups without a treatment related distribution. This shift in type of white blood cells was considered to be a secondary non-specific response to stress. The statistically significant lower prothrombin time in females at 150 mg/kg bw/day occurred in the absence of a dose-related response. These changes were therefore considered to be of no toxicological significance.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters. Higher aspartate aminotransferase activity values were obtained for one female at 150 mg/kg bw/day and one female at 1000 mg/kg bw/day, while a higher alkaline phosphatase activity was noted for another high dose female. Means did not attain a level of statistical significance, and these deviations occurred in the absence of a clear dose response relationship. Statistically significant changes included higher potassium levels in males at 50 and 1000 mg/kg bw/day, higher sodium levels in females at 1000 mg/kg bw/day, and lower potassium and inorganic phosphate levels in females at 150 mg/kg bw/day. These changes were not related to the dose and/or were within the normal range for this type of study. These alterations were therefore considered to be of no toxicological significance.

ORGAN WEIGHTS
No toxicologically significant changes were noted regarding organ weights and organ to body weight ratios. Statistically significant changes included lower absolute brain weights of animals at 150 mg/kg bw/day, and lower absolute testes weights in males at 50 mg/kg bw/day. In the absence of a dose-related response, these changes were considered to have occurred by chance and therefore considered not to be a sign of toxicity.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations. Incidental findings among control and/or treated animals included foci on the lungs, discolouration of the liver, mandibular lymph node or thymus, reduced size of the testes, enlarged mandibular lymph nodes, fractures of the bone, fluid in the uterus, and alopecia. 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.

HISTOPATHOLOGY: NON-NEOPLASTIC
Potential treatment-related microscopic observations consisted of findings in trachea, esophagus and thymus. In the trachea slight chronic inflammation (adventitial) was noted in 1/5 males at 1000 mg/kg bw/day and slight tracheal acute inflammation and epithelial ulceration was observed in 1/5 males at 50 mg/kg bw/day. In the esophagus minimal to slight chronic inflammation (adventitial or muscle layers) was seen in 2/5 males and 2/5 females at 1000 mg/kg bw/day, 1/5 males and 1/5 females at 150 mg/kg bw/day and 1/5 males at 50 mg/kg bw/day. In the thymus minimal fibrosis of the capsule was noted in 1/5 males at 1000 mg/kg bw/day.
The histopathological lesions noted among the dose groups (except controls) in the trachea, esophagus and thymus were, in some animals, associated with weight loss and/or respiratory problems and other signs of ill health, i.e. lethargy, hunched posture and piloerection. Since these histopathological lesions were of a minimal to slight nature and were evident in a few animals only without a clear dose-related incidence, these signs were considered to have occurred secondary to minor trauma during gavage administration. Since no signs of respiratory problems or inflammatory changes in the trachea/esophagus, and no weight loss were noted in control animals, the possibility that the test substance formulations were irritating secondary to minor trauma induced by the gavage administration could not be excluded. However, based on the above, it was concluded that the test substance administration had not resulted in primary local toxicity.
In all groups a number of histopathological observations were recorded but this was considered to be within the normal range and severity of background alterations that may be seen in untreated animals of this age and strain.

NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with AF-654, when compared to control animals. No explanation could be given for the high standard deviation from the mean sensor counts in females. This variation did not correlate to the clinical signs observed, and was not related to the dose. The statistically significant lower motor activity of males at 50 mg/kg bw/day as recorded by the low sensors occurred in the absence of a dose-response relationship. Therefore, these variations were not considered to be of toxicological relevance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to and including the highest dose tested.

Target system / organ toxicity

Critical effects observed:

not specified