2-hexyldecanoic acid [4-(6-tert-butyl-7-chloro-1H-pyrazolo[1,5-b][1,2,4]triazol-2-yl)phenylcarbamoyl]methylester

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:

24 Feb - 23 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 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, Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, adopted July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): UM-235
- Physical state: white powder
- Lot/batch No.: #SI-1
- Expiration date of the lot/batch: 01 Jan 2005
- Stability under test conditions: stable in vehicle for a least 4 hours
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Crl:(WI) BR strain

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: all animals were within ± 20 % of the sex mean
- Housing: group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors; during activity measurements animals were housed individually overnight in Macrolon plastic cages (type III) with sterilized sawdust provided as bedding
- Diet: standard pelleted laboratory animal diet (Altromin code VRF-1, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 23.6
- Humidity (%): 30 - 70
- 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.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at the contract laboratory, the test substance is not soluble in water
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 1 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of the dose preparations (all concentrations). Stability in the vehicle over 4 hours was also determined. Quantitative analysis was based on the largest peak in the HPLC-chromatogram of UM-235. For the three dose concentrations measurements were 94 - 97% of target concentration. The dose formulations were found to be homogenous and stable for at least 4 hours.

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: based on a preliminary range-finding test. In this study, 3 females/group were treated with the test substance dissolved in propylene glycol at dose concentrations of 150 or 1000 mg/kg bw/day for 5 consecutive days. No mortality occured. No adverse clinical signs and effects on body weight, food consumption and liver/kidney weights were observed. Macroscopic examination revealed watery cysts on the overies in one animal of the 150 mg/kg bw/day dose group. In the 1000 mg/kg bw/day dose group, fluid in the uterus of two animals and discolouration of both kidneys in one female were observed. These findings are common in studies of this type and length and are not considered to be treatment-related.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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, performed outside of the home cage in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 1, 8, 15, 22 and 28

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 28, immediately prior to scheduled post-mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all animals
- Parameters checked: erytrocytes count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin concentration, platelet count, partial thromboplastin time, prothrombin time, red cell distribution width, total leucocytes count, differential leucocytes count (neutrophiles (NEUT), lymphocytes (LYMPHO), monocytes (MONO), eosinophiles (EOS), basophils (BASO))

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post-mortem examination
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all animals
- Parameters checked: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin (total), calcium, chloride, cholesterol (total), creatinine, glucose, potassium, phosphorus, protein (total), protein (albumin), sodium, urea

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all dose groups
- Battery of functions tested (functional observations - FOB): grip strengths / motor activity (12 hours during overnight for individual animals, using computerized monitoring system) / other: static righting reflex, hearing ability, pupillary reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (the following organ weights (and terminal body weight) were recorded from the animals: adrenal glands, brain, epididymides, heart, kidneys, liver spleen, testes, thymus)
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, aorta, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, duodenum, epididymides, eyes with optic nerve 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), sciativ nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthorarcic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions

Other examinations:

organ weights and histotechnology

Statistics:

The following statistical methods were used to analyse the data:
1) If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) (C.W. Dunnett, A Multiple Comparison Procedure for Comparing Several Treatments with a control, Journal of the American Statistics Association 50, 1096 - 1121, 1955) 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 (R.R. Sokal and F.J. Rohlf, Bio,etry, W.H. Freeman, San Francisco, 1981) was applied for motor activity data.
2) The Steel-test (many-to-one rank test) (R.G. Miller, Simultaneous Statistical Interference, Springer Verlag, New York, 1981) was applied when the data could not be assumed to follow a normal distribution.
3) The exact Fisher-test (R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh, 1950) 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 continous 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:

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):

not examined

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:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occured during the study period. No treatment-related clinical signs were observed.
(General clinical signs noted included: scabs, focal erythema, alopecia and salivation, all findings which are commonly observed in rats of this age and strain which are housed under the conditions in this study.)

BODY WEIGHT AND WEIGHT GAIN
The body weights and body weight gain were comparable between treated animals and control animals.
Significant reduced body weight gain was noted on day 22 in females dosed at 50 mg/kg bw/day, however, in the absence of a dose effect relationship and the recovery on day 28 these findings were considered toxicologically irrelevant (see table 1).

FOOD CONSUMPTION AND FOOD EFFICIENCY
Food consumption was comparable between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes were observed in the haematologic parameters of treated rats when compared to controls.

CLINICAL CHEMISTRY
There were no differences noted between control and treated rats that were considered to be related to treatment with the test substance. Values of sodium (50 mg/kg bw/day dose group), potassium (50 and 150 mg/kg bw/day dose group) and total protein (150 mg/kg bw/day dose group) in treated males and alkaline phosphatase and glucose in females treated at 150 mg/kg bw/day, achieved a level of statistical significance when compared to controls, were considered to have occured as a result of slightly high or low control values. Lower albumin levels in all treated males were observed. In the absence of a treatment-related distribution or corroborative findings in the opposite sex, considered to be of no toxicological significance (see table 2).

NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with the test substance, when compared to control animals. The variation in motor activity did not indicate a relation with treatment (no dose effect relationship, see table 3).

ORGAN WEIGHTS
Organ weights and organ/body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.
Incidental findings among control and treated animals included diaphragmatic hernia of the liver, size reduction in seminal vesicles, red discoloration and foci in the thymus, pelvic dilation of the kidney, enlarged mandibular lymph nodes and cysts on 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. Watery fluid in the uterus is related to a stage in the oestrus cycle and is a normal finding.

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 occured at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Body Weight Gain (%) - Summary

		Control	50 mg/kg	150 mg/kg	1000 mg/kg
Treatment (males)
DAY1 (Week1)	mean	0	0	0	0
	standard deviation	0.0	0.0	0.0	0.0
	N	5	5	5	5
DAY8 (Week2)	mean	36	35	37	32
	standard deviation	2.9	3.5	1 .9	2.9
	N	5	5	5	5
DAY15 (Week3)	mean	67	66	69	66
	standard deviation	7.5	5.2	3.6	3.6
	N	5	5	5	5
DAY22 (Week4)	mean	88	92	91	86
	standard deviation	11.4	5.0	7.7	6.4
	N	5	5	5	5
DAY28 (Week4)	mean	104	109	109	92
	standard deviation	12.7	7.6	11.0	18.3
	N	5	5	5	5
Treatment (females)
DAY1 (Week1)	mean	0	0	0	0
	standard deviation	0.0	0.0	0.0	0.0
	N	5	5	5	5
DAY8 (Week2)	mean	15	13	16	17
	standard deviation	1.3	4.2	3.1	3.2
	N	5	5	5	5
DAY15 (Week3)	mean	33	24	30	32
	standard deviation	4.7	4.8	2.4	9.1
	N	5	5	5	5
DAY22 (Week4)	mean	45	35*	42	42
	standard deviation	5.4	5.8	3.4	5.8
	N	5	5	5	5
DAY28 (Week4)	mean	55	51	55	52
	standard deviation	8.6	9.2	5.8	3.5
	N	5	5	5	5

*/** Dunnett-test based on pooled variance significant at 5 % (*) or 1 % (**) level

Table 2: Results of Clinical Biochemistry

		control	50 mg/kg	150 mg/kg	1000 mg/kg
End of Treatment (males)
ALAT U/L	mean	31.9	31.7	27.1	29.1
	standard deviation	2.4	3.4	2.3	4.3
	N	5	5	5	5
ASAT U/L	mean	69.6	77.4	67.2	72.0
	standard deviation	6.0	8.9	6.5	2.9
	N	5	5	5	5
ALP U/L	mean	132	109	110	129
	standard deviation	24	18	12	22
	N	5	5	5	5
Bilirubin µmol/L	mean	2.4	2.2	2.2	2 .3
	standard deviation	0.3	0.4	0.2	0.3
	N	5	5	5	5
Urea mmol/L	mean	7.0	6.7	7.0	6.8
	standard deviation	0.8	1.1	0.7	0.8
	N	5	5	5	5
Creatinine µmol/L	mean	38.5	38.6	38.6	39.4
	standard deviation	2.3	1.0	3.2	1.9
	N	5	5	5	5
Glucose mmol/L	mean	6.51	7.30	6.77	6.57
	standard deviation	0.81	0.73	0.84	0.99
	N	5	5	5	5
Cholesterol mmol/L	mean	1.78	1.66	1 .99	1.68
	standard deviation	0.23	0.23	0.33	0.53
	N	5	5	5	5
Sodium mmol/L	mean	139.3	137.5*	138.5	138.3
	standard deviation	1.1	1.2	0.5	1.0
	N	5	5	5	5
Potassium mmol/L	mean	3.54	3.81*	3.80*	3.76
	standard deviation	0.07	0.21	0.11	0.13
	N	5	5	5	5
Calcium mmol/L	mean	2.93	2.86	2.89	2.87
	standard deviation	0.05	0.01	0.08	0.09
	N	5	5	5	5
Chloride mmol/L	mean	100	100	100	100
	standard deviation	1	1	1	1
	N	5	5	5	5
Inorganic Phosphorus mmol/L	mean	2.78	2.63	2.64	2.66
	standard deviation	0.26	0.09	0.28	0.14
	N	5	5	5	5
Total Protein g/L	mean	61.5	59.9	58.2*	62.3
	standard deviation	1.8	1.0	2.2	0.9
	N	5	5	5	5
Albumin g/L	mean	32.5	30.9 **	30.7**	31.1*
	standard deviation	0.7	0.2	1.1	0.3
	N	5	5	5	5

End of Treatment (females)
ALAT U/L	mean	30.0	33.7	29.6	28.0
	standard deviation	8.2	3.2	4.3	3.7
	N	5	5	5	5
ASAT U/L	mean	78.6	75.4	75.1	66.6
	standard deviation	7.7	9.0	6.8	7.4
	N	5	5	5	5
ALP U/L	mean	105	80	72*	82
	standard deviation	21	18	10	17
	N	5	5	5	5
Bilirubin µmol/L	mean	2.7	2.8	2.2	2.5
	standard deviation	0.6	0.3	0.5	0.4
	N	5	5	5	5
Urea mmol/L	mean	7.8	8.5	8.2	7.3
	standard deviation	0.9	1.1	1.3	1.0
	N	5	5	5	5
Creatinine µmol/L	mean	40.6	41.3	44.6	38.9
	standard deviation	0.9	2.4	4.4	2.4
	N	5	5	5	5
Glucose mmol/L	mean	6.05	6.47	7.00*	6.02
	standard deviation	0.26	0.50	0.61	0.56
	N	5	5	5	5
Cholesterol mmol/L	mean	1.31	1.32	1.38	1.75
	standard deviation	0.42	0.11	0.38	0.23
	N	5	5	5	5
Sodium mmol/L	mean	136.7	137.1	136.1	136.6
	standard deviation	1.4	1.5	1.4	0.4
	N	5	5	5	5
Potassium mmol/L	mean	3.58	3.71	3.66	4.25
	standard deviation	0.13	0.17	0.21	0.92
	N	5	5	5	5
Calcium mmol/L	mean	2.78	2.76	2.77	2.65
	standard deviation	0.11	0.09	0.04	0.37
	N	5	5	5	5
Chloride mmol/L	mean	101	103	102	101
	standard deviation	1	2	1	1
	N	5	5	5	5
Inorganic Phosphorus mmol/L	mean	2.29	2.16	2.23	2.26
	standard deviation	0.20	0.13	0.08	0.16
	N	5	5	5	5
Total Protein g/L	mean	58.4	58.2	57.4	61.2
	standard deviation	2.7	2.7	3.3	1.2
	N	5	5	5	5
Albumin g/L	mean	32.2	33.5	31.9	32.9
	standard deviation	1.1	1.5	1.3	0.4
	N	5	5	5	5

*/** Dunnett-test based on pooled variance significant at 5 % (*) or 1 % (**) level

Table 3: Motor activityx measurement summary

		control	50 mg/kg	150 mg/kg	1000 mg/kg
males week4
total high sensor count	mean	3779	7017	3317	2803
	standard deviation	2198	5128	1272	710
	N	5	4	5	5
total low sensor count	mean	7206	6044	7385	5970
	standard deviation	1278	1707	2502	1314
	N	5	5	5	5
females week4
total high sensor count	mean	4209	5580	4406	4600
	standard deviaton	961	5720	2277	4494
	N	4	4	5	5
total low sensor count	mean	8702	7955	9228	7138
	standard deviation	2221	1444	2567	2069
	N	5	5	5	5