Dodecanedioic acid, 1,12-bis[2-[4-(4,6-diphenyl-1,3,5-triazin-2-yl)-3-hydroxyphenoxy]ethyl] 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:

4th April - 28th June (main study) or 12th July (recovery study) 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Official notice of J MHLW, METI and ME (21 November 2003), YAKUSHOKUHATSU No. 1121002, SEIKYOKU No. 2, KANPOKIHATSU No. 03112002.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,12-bis({2-[4-(4,6-diphenyl-1,3,5-triazin-2-yl)-3-hydroxyphenoxy]ethyl}) dodecanedioate

EC Number:

939-042-8

Cas Number:

1482217-03-7

Molecular formula:

C58H56N6O8

IUPAC Name:

1,12-bis({2-[4-(4,6-diphenyl-1,3,5-triazin-2-yl)-3-hydroxyphenoxy]ethyl}) dodecanedioate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): T-1620L
- Physical state: pale yellow powder
- Analytical purity: 99.9%
- Lot/batch No.: OF1211
- Expiration date of the lot/batch: 30 April 2014
- Storage condition of test material: RT in the dark
- Other:

Specific details on test material used for the study:

- Appearance: Pale yellow powder
- Storage conditions: Room temperature (Ca. 20°C) in the dark.
- Supplier: Sponsor
- Batch number: OF1211
- Expiry date: 30th April 2014
- Purity: 99.9%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Reputable commercial supplier
- Age at study initiation: 38 to 44 days
- Weight at study initiation: males 184g to 225g, Females 152g to 203g
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: Rat and Mouse No. 1 Maintenance Diet available ad libitum
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40 - 70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12h light/12h dark

IN-LIFE DATES: From: To: 4th April to 28th June (Main study) or 12th July (Recovery study) 2013

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose 10 mL/kg.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.

Vehicle:

other: 1% w/v aqueous methylcellulose

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: use in acute toxicity testing and 7-day preliminary study to 28-day study
- Concentration in vehicle: 3, 30 or 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A 7-day preliminary study (CVJ0180) was performed and a report attached to the report for 28-day toxicity study report as an annex which describes the analytical and sampling procedures used and details the results obtained for:
The validation of the analytical procedure (FIA/M040/13) established at the testing lab for the determination of T-1620L in 1% w/v aqueous methylcellulose formulations.
The homogeneity and stability, determined with respect to the level of concentration, of T-1620L in 1% w/v aqueous methylcellulose at nominal concentrations of 1 mg/mL and 100 mg/mL.
The concentrations of T-1620L in test formulations analysed during the study.

The conclusion to the analytical report states that the analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, linearity of detector response, precision of injection, accuracy and precision.
The homogeneity and stability was confirmed for T-1620L in 1% w/v aqueous methylcellulose formulations at nominal concentrations of 1 mg/mL and 100 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. The storage times represented the maximum time from preparation to completion of administration.
The mean concentrations of T-1620L in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

28 daily doses

Frequency of treatment:

One dose administration per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

5 male and 5 female rats per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses used in this study (0, 30, 300 and 1000 mg/kg/day) were set following a review of the results of a 7 day oral preliminary study (Huntingdon Life Sciences study CVJ0180) and in conjunction with the sponsor. In that study, doses up to 1000 mg/kg/day were well tolerated with no death or adverse signs of toxicity. Accordingly, doses of 0, 30 (threshold for Category 1 classification (EEC 1272/2008 and UN GHS Systems)), 300 (threshold for Category 2 classification (EEC 1272/2008 and UN GHS Systems)) and 1000 mg/kg/day were selected for this study

- Rationale for animal assignment (if not random): Random

- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: checked in table [No.1] were included. evidence of ill-health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment and during each week of treatment

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment commenced (Day -7), on the day that treatment commenced (Day 1), on Days 8, 15, 22 and 28 of the treatment period and on Days 1, 8 and 14 of the recovery period and before necropsy


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (after 28th dose) for all main study animals and Day 15 of recovery (all recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters: Checked in table [No.6] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (after 28th dose) for all main study animals and Day 15 of recovery (all recovery animals)
- Animals fasted: Yes
- How many animals: All animals
- Parameters: Checked in table [No.7] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 (all main study animals) and week 2 of recovery (all recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: Checked in table [No.8] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Effects on sensory reactivity, grip strength and motor activity were all measured

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. Details of the exact methods used are presented in the report.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

small effects incidental to treatment noted

Mortality:

no mortality observed

Description (incidence):

small effects incidental to treatment noted

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slightly high haematocrit, haemoglobin concentration and red blood cell count were apparent at the end of the treatment period in both sexes receiving 1000 mg/kg/day. Similar differences were not apparent after 15 days off dose.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slight effects seen on plasma calcium and plasma glucose which were not seen at the end of the recovery period, demonstrating a full recovery had occurred.

Urinalysis findings:

no effects observed

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths and the general appearance and behaviour of the animals was unaffected by treatment.
Irritable behaviour, vocalisation upon handling, coat hair loss and/or encrustations that were seen in a small number of treated animals are commonly observed findings in this strain of rat and were, therefore, considered incidental.

BODY WEIGHT AND WEIGHT GAIN
There was no effect of treatment on body weight gain.
The overall body weight gain (Week 0 to 4) was slightly high, when compared with the Controls, for animals which received 300 or 1000 mg/kg/day but there was no dose-response in the magnitude of change and in the case of animals at 1000 mg/kg/day reflected a pre-treatment trend of slightly higher weight gain than the Control animals. Consequently, this finding was not attributed to treatment.

FOOD CONSUMPTION
There was no effect of treatment on food consumption.
Higher total food intake was apparent for all treated male groups, but this difference from controls was also apparent in the pre-treatment week and is consequently not considered to be related to treatment.

WATER CONSUMPTION
A visual assessment of water intake did not reveal any treatment-related effect.

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY
Slightly high haematocrit, haemoglobin concentration and red blood cell count were apparent at the end of the treatment period in both sexes receiving 1000 mg/kg/day (1.03 – 1.04X Control) with statistical significance attained in females for haematocrit and haemoglobin concentration. Similar differences were not apparent after 15 days off dose.
Other inter-group differences from Controls, including those which attained statistical significance, were generally minor, confined to one sex, lacked any clear dose-response or, in the case of those seen in the recovery period, were not similarly affected at the end of the treatment period and were therefore considered to be due to normal biological variation. Such differences included the slightly high platelet count or extended prothrombin time seen at the end of the recovery period in females or males, respectively, which had previously received 1000 mg/kg/day, where no similar statistically significant increase was apparent at
the end of the treatment period.

CLINICAL CHEMISTRY
The biochemical investigation on Day 28 revealed a marginal increase (up to 1.04X Control) in plasma calcium for males and females which received 1000 mg/kg/day. All individual values at this dose level were within the background control range for this parameter (90-percentile range; 2.50 to 2.77 mmol/L for males (n=282) and 2.50 to 2.82 mmol/L for females (n=258)), however, so this finding was considered of no toxicological significance. At the end of the recovery period, the calcium concentrations for males and females which had previously received 1000 mg/kg/day were similar to the Control values, demonstrating
that full recovery had occurred.
Plasma glucose concentration was slightly low (0.88 X Control) for males which received 1000 mg/kg/day but this finding was of uncertain relationship to treatment since no similar finding was evident in females at this dose level. At the end of the recovery period, the glucose concentrations for males which had previously received 1000 mg/kg/day were similar to the Control values.
All other inter-group differences from Controls, including those which attained statistical significance, were generally minor, confined to one sex only or lacked any clear doseresponse and were, therefore, considered to be due to normal biological variation. Such differences included the low albumin to globulin ratio (which was due to a slight decrease in albumin concentration) seen in females which received 300 mg/kg/day where no similar effect was seen at 1000 mg/kg/day in females or at any dose level in males and the high urea concentrations apparent for all treated groups of females but with no dose relationship, no statistical significance and no similar difference in males.

URINALYSIS
Examination of the urine in Week 4 did not reveal any clear treatment-related findings.
Urinary pH was statistically significantly high (1.11 X Control) for females which received 1000 mg/kg/day with a similar finding (1.09 X Control) also being present at the end of the recovery period. No similar trends were seen in males at 1000 mg/kg/day, however, and so this finding had an uncertain relationship to treatment.
The high (1.9 X Control) urinary volume seen at the end of the recovery period in females which had previously received 1000 mg/kg/day was considered a chance finding related to the slightly low urinary output of the Control females; no statistically significant increase in urinary volume was seen at the end of the 4 week treatment period.

NEUROBEHAVIOUR
Sensory reactivity was not affected by treatment.
Grip strength was not affected by treatment.
Motor activity scores were not affected by treatment.
The total low and high beam break scores were slightly high for all treated groups of males but these inter-group differences were considered to be fortuitous and not related to treatment.

ORGAN WEIGHTS
Analysis of organ weights after 4 weeks of treatment revealed slightly low (maximum reduction of 0.73 X Control for absolute weight but without any dose-relationship) absolute and adjusted thymus weights for males which received 300 or 1000 mg/kg/day but no similar trend was seen in females. At the end of the recovery period, absolute and adjusted thymus weights were marginally low for males which had previously received 1000 mg/kg/day but the difference (0.91 X Control) was less than that seen at the end of the treatment period and did not attain statistical significance.

GROSS PATHOLOGY

The macroscopic examination performed after 4 weeks of treatment or 2 weeks of recovery revealed no test substance related lesions.
The incidence and distribution of all findings were consistent with the common background of macroscopic changes seen at these laboratories.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histopathological examination performed after 4 weeks of treatment or 2 weeks or recovery revealed no findings attributable to treatment.
The incidence and distribution of all findings were consistent with the common background of microscopic changes seen at these laboratories.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no target organs identified at histopathological examination. Some minor changes were seen in a few clinical pathology parameters or thymus weight but none were considered adverse in nature.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It is concluded that the oral administration of T-1620L to Sprague-Dawley (Crl:CD(SD)) rats for 4 weeks at dosages up to 1000 mg/kg/day was well tolerated. There were no target organs identified at histopathological examination. Some minor changes were seen in a few clinical pathology parameters or thymus weight but none were considered adverse in nature. Consequently, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1000 mg/kg/day.