Propanoic acid, 2-[4-[4,6-bis([1,1'-biphenyl]-4-yl)-1,3,5-triazin-2-yl]-3-hydroxyphenoxy]-, isooctyl ester

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:

11-Nov-2002 to 05-June-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD test guideline 407) Uterus weights were not recorded at necrospy. Not all recommended organs/tissues were examined histopathologically.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 2

Reference Type:

other: repot summary

Title:

Unnamed

Year:

2003

Report date:

2003

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:

revised 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Roxicity Study, 2002)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-090-3

EC Name:

-

Cas Number:

204848-45-3

Molecular formula:

C44 H43 N3 O4

IUPAC Name:

Propanoic acid, 2-[4-[4,6-bis([1,1'-biphenyl]-4-yl)-1,3,5-triazin-2-yl]-3-hydroxyphenoxy]-, isooctyl ester

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent, England
- Age at study initiation: between 33 and 37 days, selected rats were approximately 6 weeks old when treatment commenced
- Weight at study initiation: 97.1 - 244.8 g (males) and 156.6 - 195.6 g (females)
- Housing: cages were made of stainless steel with stainless steel grid floors. Cages and undercage trays holding absorbent paper were changed at appropriate intervals.
- Diet: free access to pelleted SDS Rat and Mouse No. 1 Maintenance Diet
- Water: free access to tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 23 °C
- Humidity: 40 - 70%
- Air changes: minimum of 15 air changes per hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: From: 15-Jan-2003 To: 06-Mar-2003

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% (w/v) methylcellulose in water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered in 1% (w/v) methylcellulose in water for formulation. The required amount of test substance was initially ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added to the test substance and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. A series of suspensions at the required concentrations was prepared by dilution of individual weighings of the test substance. All suspensions were prepared freshly each week and stored at 4 °C in the dark. Formulations were allowed to warm up to room temperature prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The analytical procedure was validated for the test substance in aqueous methylcellulose (1% w/v) with respect to the specificity of the chromatographic analysis, linearity of detector response, precision of injection, limit of detection, method accuracy and precision.
- Concentration in vehicle: The mean concentrations in the test formulation analysed during the study were within +1.3% and -13.3% of nominal values (nominal concentration: 0, 1.5, 15, 100 mg/L), confirming the accuracy of formulation.
- Amount of vehicle (if gavage): 10 mL/kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity and concentration of the test item in the dose preparations was determined in samples taken from the first preparation. The analytical method involved dissolution in tetrahydrofuran and dilution using ethyl acetate followed by reverse phase high performance liquid chromatographic analysis with ultra-violet detection (320 nm). Sample concentrations were determined with reference to extemal standards prepared in the concentration range 1 - 5 µg/mL.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Basis: actual ingested

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

Basis: actual ingested

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

Basis: actual ingested

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

5 rats in the main study (all dose level) and 5 satellite rats for recovery assessment (control and high dise group only)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose level at 1000 mg(kg bw/day was selected on the basis of a 7-day preliminary toxicity study (CBG 860/030005). The intermediate and low dose levels were selected on the basis of the key dosages relative to the EU labelling requirements..
- Rationale for selecting satellite groups: according to the guideline
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (twice daily for mortality)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were observed at least twice each day from arrival to termination. During the treatment period all animals were observed at intervals after dosing each day to monitor any signs of ill health and behavioural changes or reaction to treatment. Records of these post dose checks were made daily during the treatment period. A detailed physical examination was performed weekly during pre-treatment, Weeks 1, 2, 3, and 4 and Recovery Weeks 1 and 2.

BODY WEIGHT: Yes
- Time schedule for examinations:
All rats were weighed Week - 1 , prior to dosing on Day 1 and on Days 8, 15, 22 and 28 (prior to overnight starvation for clinical pathology) of the main study. Recovery animals were weighed on Days 1, 8 and 14 of the recovery period. In addition the bodyweights of all animals were recorded prior to necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Daily monitoring by visual appraisal was maintained throughout the treatment period. No formal measurements were made.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from all main group animals on Day 29 and from all recovery group animals on Day 15 of the recovery period, prior to termination.
- Anaesthetic used for blood collection: Yes; using isoflurane
- Animals fasted: overnight, prior to sampling
- Parameters: red blood cell count, total white cell count, platelet count, mean cell haemoglobin concentration, differential white cell count, blood cell morphology including differential white cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from all main group animals on Day 29 and from all recovery group animals on Day 15 of the recovery period, prior to termination.
- Animals fasted: overnight, prior to sampling
- Parameters: glucose, urea, creatinine, albumin, total protein, albumin/globulin ratio, total bilirubin, alkaline phosphatase activity, alanine aminotransferase activity, aspartate aminotransferase activity, gamma-glutamyl transferase activity, sodium, potassium, chloride, phosphorus (as phosphate), calcium, cholesterol, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: urine samples were collected from all main group animals on Day 29 and from all recovery group animals on Day 15 of recovery.
- Metabolism cages used for collection of urine: No data
- Animals fasted: yes, during urine collection
- Parameters: colour, volume, appearance, specific gravity, pH, glucose, ketones, bilirubin, protein, blood; microscopy

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Grip strength assessments were performed during Week 4 (treatment) and 6 (recovery).
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / loco motor activity

Sacrifice and pathology:

NECROPSY
On completion of the 4-week treatment period (Day 29), all surviving main group rats were humanely killed. Following a further 2 week recovery period all surviving Recovery rats were killed on Day 43. All animals were killed by carbon dioxide asphyxiation.

ORGAN WEIGHTS:
The following organ weights were recorded on the scheduled dates of necropsy:
Brain, Thymus, Heart, Kidneys, Liver, Adrenals, Spleen, Testes, Epididymides, Ovaries, seminal vesicles

GROSS PATHOLOGY: Yes
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY: Yes
Microscopic examination of prepared slides (from the following tissues: adrenals, aorta, brain (cerebellum, cerebrum and midbrain sections), caecum, colon, duodenum, epididymides eyes, femur (with joint), Harderian glands, head, heart (including auricular and ventricular regions), ileum (including peyer's patches), jejunum, kidneys, lachrymal glands, liver (section from all main lobes), lungs (including bronchi), lymph nodes (mandibular and mesenteric), mammary area, oesophagus, optic nerves, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerves (only one examined), seminal vesicles, skeletal muscle - thigh, skin, spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), spleen, stemum, stomach, testes, thymus, thyroid (with parathyroids), tongue, trachea, urinary bladder, utems (with cervix), vagina, other macroscopically abnonnal tissue) was carried out for all main group animals of Groups 1 and 4. In addition, adrenals, epididymides, heart, kidneys, liver, spleen, testes and other macroscopically abnormal tissue were examined for all main group animals from Groups 2 and 3 and all recovery animals.

Other examinations:

none

Statistics:

- For bodyweight, neurobehavioural screening, haematology, blood chemistry, organ weight and pathology data:
If 75% of the data (across all groups) were the same value c, then a frequency analysis was applied. Treatment groups were compared using a Mantel test for a trend in proportions (Mantel 1963) and also pairwise Fisher's Exact tests (Fisher 1973) for each dose group against the control both for i) values < c versus values >= c, and for ii) values <= c versus values > c, as applicable.
If Bartlett's test for variance homogeneity (Bartlett, 1937) was not significant at the 1% level, then parametric analysis was applied. If the F1 test for monotonicity of dose-response (Healey, 1999) was not significant at the 1% level, Williams' test for a monotonic trend (Williams, 1971, 1972) was applied. If the F1 test was significant, suggesting that the dose-response was not monotone, Dunnett's test (Dunnett, 1955, 1964) was performed instead.
If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, then non-parametric tests were applied. If the H1 test for monotonicity of dose-response (Healey, 1999) was not significant at the 1% level, Shirley's test for a monotonic trend (Shirley, 1977) was applied. If the H1 test was significant, suggesting that the dose-response was not monotone, Dunn's test (Dunn, 1964) was performed instead. Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, analysis of variance was performed using terminal bodyweight as covariate when the within group relationship between organ weight and bodyweight was significant at the 10% level (Angervall and Carlstrom, 1963), in an attempt to allow for differences in bodyweight which might influence the organ weights.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical findings observed during the study that were considered to be related to treatment with the test substance.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the period Day 1 to 28 males treated at 1000 mg/kg bw/day showed a slightly lower mean bodyweight gain compared with controls. No statistical significance was achieved, individual values for this group were within the concurrent control range and there was no similar difference noted for females treated at 1000 mg/kg bw/day, therefore this difference from control was considered not to be attributable to treatment. During treatment males treated at 15 and 150 mg/kg/day and all treated females showed mean bodyweight gains which were, generally comparable with control values. During the recovery period previously treated males showed a mean bodyweight gain which was comparable with control. Whilst, the females previously treated at 1000 mg/kg bw/day showed a higher mean bodyweight gain compared with control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In the period Week 1 to 4, the mean food intake for males treated at 1000 mg/kg bw/day was slightly lower compared with controls. During this period males treated at 15 and 150 mg/kg bw/day and all treated females showed mean food intakes that were generally comparable with control. During the recovery phase, males previously treated at 1000 mg/kg bw/day still showed lower mean food intake compared with control of a similar magnitude to that seen during treatment. The mean food intake for previously treated females was comparable with controls.

Food efficiency:

no effects observed

Description (incidence and severity):

All treated and previously treated groups showed comparable efficiencies of food utilisation in comparison with controls which indicates that the lower bodyweight gain noted for males at 1000 mg/kg bw/day was due to the lower food consumption and not toxicity.

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):

On Day 29 males at 150 and 1000 mg/kg bw/day showed statistically significantly lower mean platelet values compared with controls with a dosage-relationship evident. In addition, males treated at 1000 mg/kg bw/day showed slight but statistically significantly higher mean Hct values compared with controls. Similar differences from controls were not noted for females for either parameter. As there was no concomitant effect on MCHC or MCV for males at 1000 mg/kg bw/day and the majority of individual Hct values were within or very similar to the concurrent control range the slightly higher Hct mean values were not considered attributable to treatment.
At the end of the recovery period, the Hct and platelet values for males previously treated at 1000 mg/kg bw/day were similar to controls. In addition, mean cell haemoglobin concentration (MCHC) was statistically lower than control for males previously treated at 1000 mg/kg bw/day and activated partial thromboplastin time (APTT) was statistically significantly lower than control for females previously treated at 1000 mg/kg bw/day at this time. However, the lower MCHC was not associated with any effects on the Hct or Hb and was not seen during the treatment period and therefore was not considered to be due to treatment. APTT values for previously treated females did show some overlap with control values and in the absence of any effect during treatment this difference was not considered attributable to previous treatment. There were no other differences from controls which were considered to be attributable to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

On Day 29 all treated males showed higher mean calcium values compared with controls with statistical significance attained. However, there was no dosage-relationship and females showed lower mean calcium values compared with controls with statistical significance attained at 1000 mg/kg bw/day and therefore this change was not considered to be treatment-related. In addition, males at 1000 mg/kg bw/day showed higher mean phosphorus values compared with controls with statistical significance attained. A similar difference from control was not noted for females. Females at 1000 mg/kg bw/day showed lower mean alanine amino-transferase activity values compared with controls with statistical significance attained and all treated females showed statistically significant but not dosage related lower mean total protein values compared with controls. However, similar differences from controls for these parameters were not noted for males, the magnitude of the differences from controls was minor and the majority of individual values for the females were within the concurrent control range and therefore these changes were not considered to be treatment-related. At the end ofthe recovery period, the mean calcium, phosphorus, alanine amino-transferase and total protein values were similar to controls. The mean urea and glucose levels were statistically significantly lower than control for males previously treated at 1000 mg/kg bw/day and mean sodium and chloride levels were significantly higher than control for males previously treated at 1000 mg/kg bw/day at the end of the recovery period. However, the differences from control were small and were not noted in females or during treatment and therefore they are not considered to be associated with previous treatment and thus not of toxicological importance. There were no other differences from controls which were considered to be attributable to treatment.

Endocrine findings:

not specified

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 29 males treated at 1000 mg/kg bw/day showed a statistically significant lower mean pH value compared with controls. In addition, females treated at 150 and 1000 mg/kg bw/day showed statistically significant but not dosage-related lower mean specific gravity values compared with controls. However, similar differences from controls for these parameters were not noted in the opposite sex and were not considered to be treatment-related. At the end of the recovery period, the pH and specific gravity values for animals previously treated at 1000 mg/kg bw/day were comparable with controls. There were no other differences from controls which were considered to be attributed to treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no clinical signs or changes in behaviour that were considered indicative of neurotoxicity.
Assessment of the sensory reactivity, grip strength and activity parameters in Week 4 and grip strength in recovery Week 2 did not reveal any treatment related differences between the control and treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 29 males receiving 150 and 1000 mg/kg bw/day showed statistically significantly lower bodyweight-adjusted mean testes weights, compared with controls. However, no dosage-relationship was evident, absolute values were comparable with controls and no pathology findings were observed for the testes, therefore this finding is not considered to be related to treatment. At the end of the recovery period, the results from animals previously treated at 1000 mg/kg bw/day were comparable with controls. There were no other differences from controls which were considered to be attributed to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

After four weeks of treatment:
The macroscopic examination revealed no lesions attributable to treatment with the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was an increased incidence of oestrus recorded in the vagina of females receiving 1000 mg/kg/day when compared with controls. However the relationship to treatment is uncertain given the small number of rats per group together with the expected variation in the stage of the oestrus cycle.

Incidental findings: There were no findings which related to the lower bodyweight-adjusted mean testes weight recorded in males receiving 150 mg/kg bw/day or 1000 mg/kg bw/day of the test substance compared with their control group.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Increased cellularity of the marginal zone in the spleen was noted in occasional females where it was graded as minimal or slight and occurred at a marginally increased incidence in treated females. There was no dose relationship and there were no differences in the group mean spleen weight of rats receiving the test substance when compared with controls, and no notable difference in haematological parameters in females.

Other effects:

no effects observed

Effect levels

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 test item-related adverse effects up to and including the high dose level of 1000 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

1000 mg/kg/day was classed as the No Observed Adverse Effect Level (NOAEL).

Executive summary:

Based on a previous range finder study, a subacute repeated dose toxicity study with daily oral administration to male and female rats was performed (according to OECD guideline 407 and GLP) to assess the systemic toxicity of the test material. A subsequent 2-week recovery period was allowed for selected animals to assess the potential for reversibility of any treatment-related changes. The substance was administered by oral gavage, once daily, to three groups of five male and five female rats for twenty-eight consecutive days at dosage levels of 15, 150 or 1000 mg/kg/day. Bodyweights, food consumption and clinical observations were recorded during the study for all animals. Neurobehavioural screening was performed at regular intervals during the study. Blood and urine samples for clinical investigations were taken prior to termination and all animals were killed and examined macroscopically on Day 29 (main study animals) and on Day 43 (recovery animals). At the scheduled necropsy selected organ weights were recorded and a wide range of tissues were preserved. Histopathological examination of specified tissues was then undertaken.

There were no deaths and no treatment-related clinical signs including neurobehavioural signs noted during the study. The Day 29 haematology investigations revealed males at 150 and 1000 mg/kg showed lower mean platelet values compared with controls. At the end of the recovery period, the platelet values for males previously treated at 1000 mg/kg/day were similar to controls. The Day 29 blood chemistry investigations revealed males at 1000 mg/kg/day showed higher mean phosphorus values compared with controls. At the end of the recovery period, the phosphorus values for males previously treated at 1000 mg/kg/day were similar to controls. The Day 29 urinalysis investigations revealed no treatment-related effects. There were no differences in organ weights compared with control that were considered to be attributable to treatment. The macroscopic and microscopic examination of animals killed at the end of the treatment or recovery periods revealed no treatment-related effects.