2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1H-pyrrolo[1,2-b][1,2,4] triazole-7-carboxylic acid 2,6-di-tert-butyl-4-methylcyclohexylester

Endpoint summary

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:

02 Feb - 30 Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study. The weight of the uterus and ovaries were not recorded.

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. EPA 712-C-00-366, 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: 179-211 g (range, males), 143-172 g (range, females)
- Housing: animals were housed in groups of 5 per sex in stainless steel suspended cages with wire mesh floors (55 cm x 34 cm x 21.5 cm). During overnight activity monitoring animals were housed individually in Macrolon plastic cages (type III, height 15 cm) with sterilised sawdust (Woody Clean bedding type 3/4, Tecnilab-BMI B.V., Someren, the Netherlands) as bedding.
- Diet: standard pelleted laboratory animal diet (code VRF-1, from Altromin, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7-25.3 (actual range)
- Humidity (%): 32-93 (actual range; temporary fluctuations above 70% were due to cleaning of the facilities)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 Feb 2004 To: 30 March 2004

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 before dosing and were homogenised to visually acceptable levels. Adjustment was made for the specific gravity of the vehicle. The 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): 5 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 the formulations were analysed once in week 1 to check the accuracy of preparations (all doses), and the stability and homogeneity (highest and lowest dose) of the test substance in the formulations. The formulations were stirred during the sampling. Duplicate samples for the determination of stability were taken at 50% height of the formulation and stored for 4 hours. For the determination of the accuracy and homogeneity, duplicate samples were taken at 90% height, at 50% height and at 10% height of the formulation. The analytical concentration was determined for all the samples by HPLC analysis. The actual concentrations were found to be 97-101% of nominal, which represents an acceptable level of accuracy and homogeneity. In addition the substance formulation propylene glycol was noted as stable (<10% difference between time t=0 and t=4) at room temperature and exposed to light for at least 4 hours.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
50, 150 and 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: A range-finding study was performed, in which 3 male rats/dose were administered 150 and 1000 mg/kg bw/day for 5 days (project No. 400185). No treatment-related effects were observed on clinical signs, body weight, food consumption and organ weight, and no treatment-related findings were reported from the necropsy. The NOAEL was set at 1000 mg/kg bw/day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, once daily for detailed clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes. Observations were made outside the home cage in a standard arena.
- Time schedule: once before the first exposure on Day 1, and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once prior to exposure on Day 1, and thereafter on Day 8, 15, 22 and 28

FOOD CONSUMPTION:
- Food consumption for each treatment group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was measured weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No, only a subjective appraisal was maintained during the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy on Day 28, drawn from the retro-orbital sinus
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight for a maximum of 20 hours
- How many animals: 5/sex/dose
- Parameters examined: red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, red blood cell distribution width, white blood cells, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy on Day 28, drawn from the retro-orbital sinus
- Animals fasted: Yes, overnight for a maximum of 20 hours
- How many animals: 5/sex/dose
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, protein (total), albumin, bilirubin (total), urea, creatinine, glucose, cholesterol, potassium, sodium, calcium, chloride, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 of the study for males and females
- Dose groups that were examined: all dose groups
- Battery of functions tested:
Standard functional observational battery (FOB) including grip strength measurements, auditory response, static righting reflex, pupillary reflex; and motor activity, measured as number of movements per hour over a 12-hour period, with a computerised motor activity monitoring system (Pearson Technical Services, Debenham, Stowmarket, England). Each animal was tested individually.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were necropsied and the tissues/organs subjected to macroscopic examination.
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus.

HISTOPATHOLOGY: Yes. Samples of the tissues/organs listed below were collected from all animals and fixed in neutral phosphate buffered 4% formaldehyde. Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, and all gross lesions of all animals, were prepared and examined. Slides were stained with hematoxylin and eosin. Tissues mentioned between brackets were not examined as there were no signs of target organ involvement.

Samples collected from: adrenal glands, aorta, brain - cerebellum, mid-brain and 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 and mesenteric, (nasopharynx), oesophagus, ovaries, pancreas, Peyer’s patches- jejunum and ileum - if detectable, pituitary gland, (preputial gland), prostate gland, rectum, (salivary glands - mandibular and sublingual), sciatic nerve, (seminal vesicles), (skeletal muscle), (skin), spinal cord - cervical, midthoracic and lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, (tongue), trachea, urinary bladder, uterus, (vagina), all gross lesions

Statistics:

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:

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:

effects observed, treatment-related

Description (incidence and severity):

all dose groups, females: increased basophil level (non-adverse)

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 occurred. One female in the low-dose group had clonic spasms on Day 18-19, but this is not considered to be treatment-related. Incidental cases of alopecia, scabs and chromodacryorrhoea were noted at all dose levels and in control animals. These effects are frequently observed during subacute studies performed in the rat and are therefore not treatment-related. No other clinical signs were observed in the animals.

BODY WEIGHT AND WEIGHT GAIN
The body weight and bodt weight gain was comparable between the control and treatment groups during the whole study period.

FOOD CONSUMPTION
The food consumption was comparable between the control and treatment groups during the whole study period.

HAEMATOLOGY
The level of basophils was significantly decreased in females at all dose levels (see Table 1). The values fall within the historical control range (Technical Bulletin, Charles River Laboratories, Spring 1998). Furthermore, no effects were seen in the males, leading to the conclusion that this is not a treatment-related effect.
Increases of neutrophil counts with concurrently reduced lymphocyte counts were seen in females among the dose groups without a treatment related distribution (see Table 1). This shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance.
Minor statistically significant differences in mean corpuscular haemoglobin concentration in males of the mid-dose group when compared to control animals (see Table 1) were considered to be an incidental occurence and not to represent a change of biological significance.

CLINICAL CHEMISTRY
A significant increase in the inorganic phosphorus level in the females of the low- and mid-dose groups was noted (see Table 2). As the increase was around 10% compared with the control value and no significant increase was noted in the high-dose females, the effect is not considered to be treatment-related. Changes in the levels of potassium and glucose in the low- or mid-dose group of one sex are considered to be incidental occurrences in the absence of a treatment-related distribution or corrobative findings in the opposite sex (see Table 2).

NEUROBEHAVIOUR
There were no significant differences in grip strength measurements, auditory response, static righting reflex and pupillary reflex between the control group and treatment groups. In the males of the mid-dose group, the number of motor activity measurements (low sensor) were significantly decreased. As no similar effect was observed in the measurements by the high sensor or in the high-dose group and in the absence of supportive clinical signs, this is considered to be an incidental occurrence.

ORGAN WEIGHTS
The absolute and relative spleen weight was significantly decreased in females of the mid-dose group. As no similar decrease was seen in the high-dose group, the results is not considered to be treatment-related. In females of the low- and mid-dose group, a significant increase in the absolute weight of the adrenal gland was noted. No increase was observed in the female high-dose group or in the males, leading to the conclusion that this is an incidental occurrence.

GROSS PATHOLOGY
2, 1, 0 and 2 females in the control, low-, mid- and high-dose group, respectively, had fluid in the uterus. This is a normal observation during a certain part of the estrus cycle of the female rat.
Incidental findings among control and treated rats included pelvic dilation of the kidneys, nodules in the epididymides, adrenal glands grown together with kidney, dark red discolouration and foci of the thymus, dark red discoloured and enlarged mandibular lymph nodes. 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
No treatment-related effects were observed in the control or treatment groups.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No substance-related effects were observed up to and including the highest dose level

Critical effects observed:

not specified

Table 1: Haematology results

	Group (mg/kg bw/day)
	Males				Females
	Control	50	150	1000	Control	50	150	1000
Mean corpuscular hemoglobin concentration, % (mean±SD)	19.73±0.30	19.45±0.12	19.10±0.21**	19.66±0.26	19.55±0.34	19.60±0.33	19.74±0.36	19.69±0.21
Neutrophils, % of white blood cells (mean±SD)	19.3±10.6	17.6±4.8	25.2±3.8	19.2±5.7	12.6±4.8	17.8±7.2	24.5±6.7*	18.8±10.2
Basophils, % of white blood cells (mean±SD)	0.3±0.1	0.2±0.1	0.3±0.1	0.4±0.1	0.4±0.0	0.2±0.1*	0.2±0.1*	0.2±0.1*
Lymphocytes, % of white blood cells (mean±SD)	76.4±11.7	77.8±5.2	68.6±5.6	76.6±6.5	82.6±4.0	75.7±8.8	69.9±6.4*	76.0±11.2

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 2: Clinical chemistry results

	Group (mg/kg bw/day)
	Males				Females
	Control	50	150	1000	Control	50	150	1000
Glucose (mmol/L)	6.07±0.23	6.72±0.52*	6.52±0.32	5.75±0.40	6.70±0.32	6.55±0.54	6.58±0.39	6.58±0.49
Inorganic phosphate (mmol/L)	2.69±0.30	2.71±0.37	2.70±0.23	2.59±0.20	2.27±0.11	2.53±0.14*	2.52±0.11*	2.43±0.16
Potassium (mmol/L)	3.66±0.19	3.80±0.02	4.24±0.48**	3.83±0.15	3.99±0.18	3.90±0.22	3.94±0.25	3.89±0.13

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

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

A 28-day oral repeated dose toxicity study was performed according to OECD guideline 407 and in compliance with GLP, using 2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1 H-pyrrolo[1 ,2-b][1 ,2,4]triazole-7-carboxylic acid-2,6-di-tert-butyl-4-methyl-cyclohexyl ester (UC-141) (Hooiveld, 2004). Wistar Crl:(WI) BR rats (5/sex/dose) were administered 50, 150 and 1000 mg/kg bw/day by gavage, 7 days/week, over a period of 28 days. Animals in the control group received the vehicle propylene glycol.

There was no mortality and no treatment-related clinical signs were observed during the study period. The body weight, body weight gain and food consumption was comparable between the control group and treatment groups during the whole study period. In females at all dose levels, the percentage of basophils (percentage of total lymphocyte count) was significantly decreased. However, the values fall within the historical control range (Technical Bulletin, Charles River Laboratories, Spring 1998) and no corrobative findings were seen in the opposite sex, leading to the conclusion that this is not a treatment-related effect. No treatment-related changes in clinical chemistry values were noted. There were no treatment-related differences in the neurobehavioural parameters between the control group and treatment groups. The treatment did not affect the absolute and relative organ weights. No unusual findings were reported during the gross pathology and at histopathological examinations no microscopic findings were recorded, which could be attributed to treatment with the test substance. Based on the lack of toxicologically relevant effects up to and including the highest dose level, the NOAEL is considered to be ≥ 1000 mg/kg bw/day.

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