Trans-2-[4'-[Difluor(3,4,5-trifluorphenoxy)methyl]-3',5'-difluor[1,1'-biphenyl]-4-yl]-5-propyl-tetrahydro-2H-pyran

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:

2007-10-12 to 2008-06-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to Good Laboratory Practice (GLP) and followed the OECD Guidelines for the Testing of Chemicals, No. 407.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

1995-07-27

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

96/54/EC

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

EC Number:

616-651-4

Cas Number:

787582-75-6

Molecular formula:

C26H2 lF702

Constituent 2

Reference substance name:

APUQU-2-F

IUPAC Name:

APUQU-2-F

Details on test material:

Batch: E06000594
Purity (GC): 99.9 %

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: Rat, HsdCpb:WU
Breeder: Harlan Winkelmann GmbH, Borchen, Germany

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.25 % aqueous hydroxypropyl methylcellulose (Methocel K4M Premium)

Details on oral exposure:

oral administration by gavage, once daily, 7 d /week
Volume of administration: 5 mL/kg body weight
Volume of administration per animal was calculated using DATATOX

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test material in 0.25% aqueous hydroxypropyl methylcellulose (Methocel K4M Premium):
Before start of study: stable and homogenous for at least 7 days
After start of the study: stable for 14 days.
Test material suspensions were prepared weekly.
Analyses performed in week 1 and 4 of administration from samples (3 concentrations): within acceptance limits (90 - 110%) of nominal concentration. In control samples concentrations were less than LOQ (0.014 g/L at dilution factor 100) of analytical method

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 times a week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

10 f + 10 m in control and high dose group; 5 f + 5 m in low and mid dose group

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered orally by gavage, once daily, 7 times a week
for 4 weeks to 3 groups of male and female HsdCpb:WU Wistar rats at doses of 100, 300,
or 1000 mg/kg. A similarly constituted control group received the vehicle, 0.25% aqueous
hydroxypropyl methylcellulose (Methocel® K4M Premium) and served to generate
contemporary control data.
The control and high dose groups consisted of 10 male and 10 female rats each. The low
and mid dose groups consisted of 5 male and 5 female rats each. At the end of the
treatment period 10 (5 males and 5 females) rats were scheduled for necropsy. The
remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period. The rats
were gang-housed under conventional conditions at the Merck KGaA Institute of Toxicology.

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

Observations/Measurements (Frequency)
- Appearance and behaviour (daily)
- Mortality (daily)
- Motor activity (day 28)
- Functional observational battery Predose ((day 0), day 7, day 28)
- Body weight (once a week)
- Food consumption (once a week)
- Water consumption (twice a week)
- Hematology (week 4, week 6)
- Clinical chemistry (week 4, week 6)
- Urinalysis (week 4, week 6)

Sacrifice and pathology:

All surviving rats and all rats that died in the course of the study were necropsied and examined for gross pathological alterations. The surviving rats were killed by anesthesia with a carbon dioxide air mixture and exsanguination after opening of the abdominal vessels. All findings were recorded, reported and archived.

Other examinations:

In all animals but those that were killed or die in the course of the study, the body and organs weights are recorded. Based on the absolute organ weights the relative organs weights (related to 100 g body weight) were calculated.

Statistics:

All parameters were analysed separately for each sex and time. Absolute body weight, body weight gain (differences to baseline values on day 0), food and water consumption, and organ weights - relative and absolute - of the dose groups were compared with those of the control, using the multiple two-sided Dunnett-Test (1955, 1964). For the evaluation of the clinico-chemical parameters and the hematological parameters, the Wilcoxon rank sum test (Hollander, 1973) was used to make pairwise comparisons of the dose groups with the control group. The correction for multiple testing was done according to Bonferroni-Holm (Holm, 1979).

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 specified

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

effects observed, treatment-related

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Water consumption was significantly increased over the entire treatment period in male group 4 animals (1000 mg/kg), and from day 10-21 in female group 4 animals. However, this change was reversible within the 2 week recovery period.
Organ weights showed increased absolute and relative liver weights in high dose females (1000 mg/kg) after 28 days of treatment. The males showed only slightly increased relative liver weights after 28 days. A tendency to increased relative liver weights was already detectable in the intermediate dose group. No histopathological correlate was found.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: some reduced motor activity at the 30 mg/Kg bw/d

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

There was no substance related mortality in this study. On test day 31 (day 3 of the recovery period), one female high dose animal (1000 mg/kg) was killed because of its moribund condition. Histopathologic evaluation showed that this animal had suffered a traumatic injury of the spinal cord that is not treatment-related. All other animals were kept until their schedule main or recovery kill.

During the daily clinical observations, several symptoms of discomfort were seen in some high dose animals (males and females), e.g. laboured respiration, piloerection, sunken flanks. 2 high dose males showed convulsions or trembling once (day 13) during the treatment. Since these findings were only seen on isolated occasions, a relation to treatment remains equivocal.

The functional observational battery (FOB) was performed in designated animals on day 0, day 7 and day 28 of treatment. No significant changes were noted in the autonomous, neuromuscular, sensomotoric or central nervous domain at any time point. Body temperature was reduced significantly on day 28 in the high dose females but was still in the physiological range. Motor activity was measured in designated animals on day 28. A dose-dependent significant decrease of activity was seen in female rats at 300 and 1000 mg/kg. Male animals showed a significant decrease at 300 mg/kg. At 1000 mg/kg this decrease could not be observed because of a high interindividual variation. However, no behavioural correlate was seen in the FOB. Body weight and body weight gain were not impaired by treatment with the test item at doses up to 1000 mg/kg. Food consumption showed some variation, but was not significantly different between treatment groups and control. Water consumption was significantly increased over the entire treatment period in male group 4 animals (1000 mg/kg), and from day 10-21 in female group 4 animals. This change was reversible within the 2 week recovery period.

Hematological evaluation after 4 weeks of treatment with the test item revealed some slight significant changes of white and red blood count and coagulations parameters. However, all hematological values were in the range of the normal internal laboratory range of values and the findings are not considered treatment-related. Some slight changes at the end of the recovery period were also in the range of the normal internal laboratory range of values and not considered treatment-related.

Clinical chemistry after 4 weeks of treatment with the test item showed some statistically significant deviations from the control. However, overall the changes of serum electrolytes, serum-substrates and –proteins, and serum enzymes were very slight and in the range of the normal internal laboratory range of values. At the end of the recovery these slight changes could no longer be observed. Urine weight, specific urinary gravity, and urinalysis did not reveal any toxicologically relevant changes.

At gross pathology only sporadic and spontaneous alterations were diagnosed. In summary, up to doses of 1000 mg/kg no treatment-related findings were detected in the organs examined. Organ weights showed increased absolute and relative liver weights in high dose females (1000 mg/kg) after 28 days of treatment. The males showed only slightly increased relative liver weights after 28 days. A tendency to increased relative liver weights was already detectable in the intermediate dose group. No histopathological correlate was found. Histological examination of group 1 and 4 main kill animals showed no treatment-related findings in the organs and tissues examined. Only findings considered to be spontaneous and sporadic in nature were detected.

Applicant's summary and conclusion

Conclusions:

100 mg/kg were considered to be the NOEL (no observed effect level) because no treatment-related findings were observed at this dose-level.
At 300 mg/kg only motor activity was reduced on day 28 with no behavioral correlate in the FOB.
At 1000 mg/kg some minimal clinical findings were observed, a slight reduction of motor activity (day 28) without a correlate in the FOB, slight changes of body weight, body weight gain (females), and water consumption were noted. These findings proved to be reversible. No treatment-related histopathological findings were diagnosed in the organs examined in the dose groups. Therefore, 1000 mg/kg is considered the NOAEL.

Executive summary:

Study Design

The test item was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of male and female HsdCpb:WU Wistar rats at doses of 100, 300 or 1000 mg/kg. A control group received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium). The control and high dose groups consisted of 10 male and 10 female rats each. The low and mid dose groups consisted of 5 male and 5 female rats each. At the end of treatment period 10 rats (5 males and 5 females) were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period.

Results

No substance related mortality in this study. On test day 31 (day 3 of the recovery period), one female high dose animal (1000 mg/kg) was killed because of its moribund condition. Histopathologic evaluation showed no treatment-relation. All other animals were kept until their schedule main or recovery kill. During daily clinical observations, several symptoms of discomfort were seen in some high dose animals (males and females), e.g. laboured respiration, piloerection, sunken flanks. The functional observational battery (FOB) was performed in designated animals on days 0, 7 and 28 of treatment. No significant changes were noted in the autonomous, neuromuscular, sensomotoric or central nervous domain at any time point. Body temperature was reduced significantly on day 28 in the high dose females but was still in the physiological range. Motor activity was measured in designated animals on day 28. A dose-dependent significant decrease of activity was seen in female rats at 300 and 1000 mg/kg. Male animals showed a significant decrease at 300 mg/kg. At 1000 mg/kg this decrease could not be observed because of a high interindividual variation. However, no behavioural correlate was seen in the FOB. Body weight and body weight gain were not impaired by treatment at doses up to 1000 mg/kg. Food consumption showed some variation, but no significantly difference between treatment groups and control. Water consumption was significantly increased over the treatment period in males (1000 mg/kg) and from day 10-21 also in females (1000 mg/kg). This change was reversible within the 2 week recovery period. Haematological evaluation revealed slight significant changes of white and red blood count and coagulations parameters, but all values were in the range of normal internal laboratory range of values, the findings are not considered treatment-related. Even if clinical chemistry showed some deviations from control, the changes of serum electrolytes, serum-substrates and –proteins, and serum enzymes were very slight and in range of normal internal laboratory range of values. At the end of the recovery these slight changes could no longer be observed. Urine weight, specific urinary gravity, and urinalysis did not reveal any toxicologically relevant changes. At gross pathology only sporadic and spontaneous alterations were diagnosed. Up to doses of 1000 mg/kg no treatment-related findings were detected in the organs. Organ weights showed increased absolute and relative liver weights in high dose females (1000 mg/kg) after 28 days of treatment. Males showed slightly increased relative liver weights, a tendency to increased relative liver weights already detectable in the intermediate dose group. No histopathological correlate was found. Histological examination of group 1 and 4 main kill animals showed no treatment-related findings in the organs and tissues examined.

Conclusions

100 mg/kg considered to be the NOEL (no observed effect level) as no treatment-related findings were observed at this dose-level. At 300 mg/kg only motor activity was reduced on day 28 with no behavioural correlate in the FOB. At 1000 mg/kg minimal clinical findings observed, slight reduction of motor activity, without correlate in FOB, slight changes of body weight, body weight gain (females), and water consumption were noted. These findings proved to be reversible. No treatment-related histopathological findings diagnosed in the organs the dose groups. Therefore, 1000 mg/kg is considered as NOAEL.