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Description of key information

Oral: NOAEL (rat, 28d, OECD 407) ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted September 1996
Deviations:
no
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
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
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
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.
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.
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.
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.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirement set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An oral repeated dose toxicity study was performed with 2-Hexyl-2 ‘-hydroxy-5 ‘methyldecananilide (AF-654) according to OECD Guideline 407 (van Otterdijk, 2004b). Wistar rats (5/sex/dose) were administered 50, 150 and 1000 mg/kg bw/day of the test substance in propylene glycol by gavage for 28 consecutive days. Animals in the control group received the vehicle propylene glycol. Cage side observations, mortality, body weight and food consumption were recorded during the study period. In the last week of exposure a neurobehavioral examination was performed. Haematology and clinical chemistry were assessed at terminal sacrifice before gross pathology and histopathology was done.

There were no mortalities during the study period. Restless behaviour noted among 5/5 high dose females in week 3 had resolved in week 4. Quick breathing was observed in 1/5 males at 50 mg/kg bw/day in week 4 and in 1/5 females at 50 mg/kg bw/day in week 1 as well as in 1/5 males and 1/5 females at 1000 mg/kg bw/day in week 2. Rales were noted in 1/5 males at 50 mg/kg bw/day occasionally during the whole study period and in 1/5 males and 1/5 females at 1000 mg/kg bw/day once in week 4. In 1/5 males and 1/5 females at 50 mg/kg bw/day and in 1/5 males at 1000 mg/kg bw/day the respiratory problems were associated with other signs including lethargy, hunched posture, piloerection, pallor, lean appearance and / or ptosis. One male animal at 150 mg/kg bw/day showed laboured respiration, rales and gasping at the end of week 2 Although these clinical findings were considered treatment-related, they occurred only incidental in a few animals and without a clear dose-relationship. Mean body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period. There were no changes of toxicological significance at performance of functional observations, food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination and organ weight determination.

Histopathological lesions noted among the treatment groups only included inflammation of the trachea in 1/5 males each in the 50 and 1000 mg/kg bw/day dose group, inflammation of the esophagus 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 and minimal fibrosis of the capsule of the thymus in 1/5 male animals in the 1000 mg/kg bw/day dose group. Since these histopathological lesions were of a minimal to slight nature and occurred in a few animals only without a dear 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/oesophagus, 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.

No treatment-related effects were observed up to and including the highest dose level tested in the 28-day toxicity study. Therefore, the no observable adverse effect level (NOAEL) for AF-654 is considered to be ≥ 1000 mg/kg bw/day for male and female rats.

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
There is only one study available.

Justification for classification or non-classification

The available data on the repeated dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.