2H-Pyran, 2-[4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-3',5'-difluoro[1,1'-biphenyl]-4-yl]-5-ethyltetrahydro-

Endpoint summary

Description of key information

In a subacute toxicity study according to OECD TG 407, the test item was administered daily by oral gavage Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. The NOAEL was considered to be 1000 mg/kg body weight/day (reference 7.5.1 -1).

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:

2007-10-12 to 2008-06-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995-07-27

Deviations:

no

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Weight at study initiation: males: 254 (230 – 291) g and females: 174 (160 – 190) g
- Housing: gang-housed (2 or 3 animals/sex) in type IV Makrolon® cages
- Diet: ad libitum, Provimi Kliba 3433.0
- Water: ad libitum, tap water
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: The diet was checked by an independent laboratory, according to the specifications of the manufacturer. The drinking water was regularly tested according to the German Human Drinking water regulations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.3 – 24.0
- Humidity (%): 34.1 – 54.5
- Photoperiod (hrs dark / hrs light): 12/12

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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

Positive control:

NA

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).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal of the high dose group was killed on day 31 because of moribund condition. All other animals survived the treatment and recovery period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Generally, the body weight and body weight gain were not impaired by the treatment with the test item up to 300 mg/kg. A slight significant decrease of body weight was observed on day 21 in females treated with 1000 mg/kg. Body weight gain of this group was significantly reduced on days 14, 21, and 28 of the treatment. However the values are very small, the effect proofed to be reversible on days 35 and 42 of the recovery period. Therefore, this finding is not considered biologically relevant. The males treated with 1000 mg/kg did not show any effect on body weight or body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was significantly decreased on various time points in all male dose groups and female groups 3 and 4. However, no dose-dependency of the values could be observed, and no difference could be seen on day 28 in male and female animals treated with 1000 mg/kg. Therefore, this finding shows biometrical relevance without a biological match.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was significantly increased in group 4 on all days during administration in the males compared to the control. Females of group 4 showed a significant increase from day 10 – 21 during administration compared to control. After 1 week recovery (day 35) the males and females showed still a significant increase, however thereafter no significant difference from control could be observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

White blood cells:
After 4 weeks of treatment all treated males showed a slight and dose-dependent (not statistically significant) increase of WBC. The number of lymphocytes showed a slight statistically significant decrease in group 2 (100 mg/kg). Females treated with 1000 mg/kg showed an increase of the relative amount of neutrophilic granulocytes together with a decrease of the relative amount of lymphocytes. However, the findings were within the normal laboratory range.
Red blood cells:
After 4 weeks of treatment, male group 2 animals (100 mg/kg) showed a slight statistically significant decrease in hematocrit. At the end of recovery period (week 6) male group 4 rats (1000 mg/kg) showed a slight significant decrease of red blood cells, hemoglobin, and hematocrit together with an increase in the reticulocyte number. However, the findings were within the normal laboratory range.
Of the coagulation parameters the partial thromoplastin time was increased slightly after 4 weeks of treatment (statistically significant), but was normal after 2 weeks of recovery. The values were in the normal laboratory range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In study week 4, mid and high dose treated males showed a slight significant increase of sodium. All treated females showed a slight significant decrease of potassium and calcium. The changes observed were within the normal internal laboratory range of values. All treated males showed a slight increase of cholesterol. All treated females showed slight statistically significant decreases of triglycerides, albumin and A/G ratio. Additionally a slight increase of glucose and urea were observed in group 4 and 3, respectively. The changes observed were within the normal internal laboratory range of values. Changes in bile acid and total protein of the female animals of group 4 are mainly based on the changes seen in 2 individual animals. At the end of the recovery period, values of group 4 were mainly recovered to the levels of control animals. A light decrease of serum enzyme ASAT was observed in male animals of group 3 and is not considered a pathological change. Overall, the changes of serum electrolytes, serum-substrates and –proteins, and serum enzymes are 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.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine weight, specific urinary gravity, and urinalysis did not reveal any pathological alteration in test week 4 and 6.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The total motor activity within 60 minutes was decreased in group 2 and 3 males after 4 weeks of treatment (statistically significant in group 3 versus control). However, the high dose group did not show this decrease because of one individual animal that had increased motor activity whereas all others showed values similar to groups 2 and 3. The females showed a dose-dependent decrease of motor activity, that was statistically significant in groups 3 and 4.

Autonomous nervous system: No effects on lacrimation, salivation, pupil response, number of fecal boluses, feces consistency, number of urine pools, urine stain size, or palpebral closure were seen in any dose group at all time points.
Neuromuscular system: No significant effects on gait, hind limb foot splay, fore and hind limb grip strength, righting reflex, mobility, or muscle tone were observed in all dose groups at all time points. 1 female of group 3 and 2 females of group 4 showed a reduced muscle tone after 28 days of treatment. The general increase of fore and hind limb grip strength reflects then growth of the animals over 4-week investigation period.
Sensomotoric system: No effects on approach response, click response, touch response or tail pinch response could be observed in all dose groups at all time points.
Central nervous system: No effects on ease of removal and handling, arousal, fur appearance, number of raisings or raising behavior could be observed in all dose groups at all time points. After 4 weeks of treatment, catalepsis was observed in 3 males and 2 females of group 2, 2 females of group 3, and 1 male of group 4. These observations were not statistically significant, no dose-dependency could be observed. jThe number of raisings was reduced from day 7 onwards in both genders of all groups. As rats use raising to survey a new environment this reduction was expected since the animals know the observation arena from the first FOB evaluation a week before.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Key result

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

Key result

Critical effects observed:

no

Conclusions:

In this study, the NOEL (no observed effect level) is considered to be 100 mg/kg bw/day because no treatment-related findings were observed at this dose-level. At 300 mg/kg bw/day only motor activity was reduced on day 28 with no behavioral correlate in the FOB. At 1000 mg/kg bw/day 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 bw/day is considered the NOAEL.

Executive summary:

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 bw/day. 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.

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 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 bw/day. Male animals showed a significant decrease at 300 mg/kg bw/day. At 1000 mg/kg bw/day 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 bw/day. 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 bw/day). 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 bw/day 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 bw/day) 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.

100 mg/kg bw/day is considered to be the NOEL as no treatment-related findings were observed at this dose-level. At 300 mg/kg bw/day only motor activity was reduced on day 28 with no behavioural correlate in the FOB. At 1000 mg/kg bw/day 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 bw/day is considered as NOAEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD TG 407

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

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

Oral

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 bw/day. 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.

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 bw/day. Male animals showed a significant decrease at 300 mg/kg bw/day. At 1000 mg/kg bw/day 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 bw/day. 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 bw/day). 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 bw/day 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 bw/day) 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.

100 mg/kg bw/day is considered to be the NOEL as no treatment-related findings were observed at this dose-level. At 300 mg/kg bw/day only motor activity was reduced on day 28 with no behavioural correlate in the FOB. At 1000 mg/kg bw/day 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 bw/day is considered as NOAEL.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The substance is not considered to be classified for repeated dose toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.