Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

Endpoint summary

Description of key information

A 28-day repeat dose toxicity study using the oral route by gavage in the rat, conducted according to the OECD 407 guideline under GLP.
There were no adverse effects or target organs of toxicity noted following 28 days of oral administration of the test article. Based on these results, the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day.
A 7-day repeat dose toxicity study using the oral rote by gavage in the rat, conducted according to a range-finder standard procedure under GLP.

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:

01 July 2008 to 13 November 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD 407 test guideline.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: 7 weeks
- Weight at study initiation: males 186 to 212g, females 147 to 173g
- Housing: Suspended stainless steel cages
- Diet (e.g. ad libitum): PMI Certified Rodent Chow ad libitum
- Water (e.g. ad libitum): municipal water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 50+/- 20
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12 hours light:12 hours dark

IN-LIFE DATES: From: 29 July 2008 To: 26 August 2008

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For Group 1 , control article (distilled water) was dispensed as received. Test article dosing formulations were prepared at appropriate concentrations to meet dosage level requirements. For each dose level, an appropriate amount of the test article was weighed into a beaker. An appropriate amount of the control article was added and the mixture was sonicated for approximately 45 minutes and then gently hand shaken for approximately 1 minute. A stir bar was added and the mixture was stirred at low speed for approximately 30 minutes, gently hand shaken for approximately 1 minute, sonicated for approximately 30 minutes, gently hand shaken for approximately 1 minute, and stirred at low speed for approximately 30 minutes. The dosing
formulations were prepared weekly, stored refrigerated at 5 rt 3"C, and dispensed into amber glass containers. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes prior to dosing. The physical description was recorded for all groups. Group 1 (control article) was a clear colorless solution, Group 2 was a cloudy colorless mixture, and Groups 3 and 4 were cloudy, pale yellow mixtures following preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water
- Concentration in vehicle: 0, 6, 25, 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:

Dose formulation analysis was performed by HPLC.

Dose formulation analysis were taken as follows:
Week 1 Week 2
Concentration all groups all groups
Homogeneity Groups 2 and 4 None
Stability None None

Concentration: Four 5-mL samples were collected from each dosing formulation for concentration analysis on Weeks 1 and 4. For Week 1, samples were collected from Groups 1 and 3, and the homogeneity results from Groups 2 and 4 were averaged and utilized as the concentration results.

Storage and shipment: All samples were stored frozen (-20 i 10°C) and protected from light. Duplicate sets of samples were shipped overnight on dry ice to the dose formulation analytical laboratory for analysis. On Day 28, the back-up samples from Week 4 for Group 4 were shipped to the analytical laboratory
for analysis. The remaining sample sets were maintained at the Testing Facility and discarded after acceptance of the analytical results.

Analytical method
Standard Solutions: Standard solutions of reference standard were prepared in diluent (acetonitri1e:dimethylsulfoxide(50:50 v/v)) covering the nominal concentration range of 1.50 to 2.50 mg/mL.

Spiked Samples: Spiked samples were prepared in vehicle at nominal concentrations of 5.00 and 100 mg/mL. Each was diluted with diluent to give nominal concentrations of 1.75 and 2.00 mg/mL, respectively

Study Samples: Samples received on dry ice from Study No. VKA00146 were stored at approximately -20°C until analysis. Samples were analyzed within 1 day of receipt. Prior to analysis, the study samples were thawed at room temperature. The samples at nominal concentrations of 6,25 and 100 mg/mL were diluted with diluent to give nominal concentrations within the range of the calibration curve. The control samples were diluted equivalent to the 6 mg/mL sample.
All samples were analyzed in duplicate.
For concentration verification, mean measured concentrations within *l5% of their nominal concentrations were considered acceptable. Homogeneity was considered acceptable if the relative standard deviation for the samples of each group analyzed was 55%.

Column: HPLC system Agilent Technologies 1100 series
Data capture system: Waters Corporation Empower 2
Column: Luna phenyl hexyl, 3 µm (30 x 4.6 mm id)
Column temperature: 25°C
Mobile phase gradient elution: Eluant A - 0.1% formic acid in water; Eluant B - tetrahydrofuran:acetone (90:10 v/v)
Time (min) %B
0 50
2 50
2.1 100
3.5 100
3.6 50
5 50
Flow rate: 1.5 L/min
Pressure: 30 PSI
Tube temperature: 70°C
Nebuliser temperature: 90°C
Gain: 5
Injection volume: 40 µL
Tray temperature: 4°C
Reference standard retention time: ~3.2 min

System Suitability: The reproducibility of the chromatographic system was determined by injecting a calibration standard solution, at a nominal concentration of 2.00 mg/mL in triplicate, at the beginning and at the end of the chromatographic run.
A coefficient of variation (CV) of =<3% in peak area and a difference of ±10% between the average response for the standards injected at the end compared with those injected at the beginning were considered acceptable.

All study samples analyzed were within the acceptance criteria of ± 5% of their nominal concentrations, except Week 4, Group 4 (121 and 126%); therefore as part of the investigation, the retention samples were analyzed (two samples analyzed in duplicate), and the results (102% for all four replicates) were within the acceptance criteria. The investigation demonstrated that Week 4, Group 4 dose formulations were most likely within specification.
The relative standard deviation for the samples analyzed for homogeneity determination were =< 5%.

Duration of treatment / exposure:

28 Days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 60, 250, 1000 mg/kg bw//day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selcted by the Sponsor based on the results of a 7-day range-finding study.
- Rationale for animal assignment (if not random): random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to treatment, weekly and on the day of scheduled termination

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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 / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to dosing and during last week of dosing
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes ( isoflurane)
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Full functional observation battery

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (standard procedures)
HISTOPATHOLOGY: Yes (standard organs)

Statistics:

Inferential statistical analyses were performed for the toxicity phase animals using the Compaq Alpha DS 10 Computer. The following parameters and end points were analyzed: body weights; body weight changes; food consumption; hematology, coagulation, clinical chemistry, and urinalysis (specific gravity, pH, and total volume); organ weights; and parametric, ranked, and count FOB data.
Each data set was subjected to a statistical decision tree. Data sets for each interval were initially analyzed for homogeneity of variance using Levene's test followed by the Shapiro-Wilk test for normality. A p < 0.001 level of significance was required for each test to reject the null hypothesis.
If both Levene's test and the Shapiro-Wilk test were not significant, a single-factor parametric ANOVA' was applied, with animal grouping as the factor, using a p < 0.05 level of significance.
If the parametric ANOVA was significant at p < 0.05, Dunnett's test was used to identify statistically significant differences between the control group and each test article-treated group using a minimum significance level of p < 0.05.
If either Levene's test and/or the Shapiro-Wilk test were significant, then the Kruskal-Wallis non-parametric ANOVA6 was applied, with animal grouping as the factor, using a p < 0.05 level of significance. If the non-parametric Kruskal-Wallis ANOVA was significant at p < 0.05, Dum's test was used to identify statistically significant differences between the control group and each test article-treated group using a minimum significance level of p < 0.05.
Descriptive (categorical) and quanta FOB data were analyzed by Fisher's Exact test'. If significance was detected, a group by group comparison proceeded using Chi-square test'.

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:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

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:

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:

Dose Formulation Analysis
Stability was assessed at concentrations encompassing those used in this study ( i e . , approximately 5 and 100 mg/mL). Samples were determined to be stable for 4 days at room temperature, up to 14 days at approximately 4°C, and up to 14 days at approximately -20°C. Samples obtained for concentration verification analysis were within the acceptable limits of ± 15% error. Homogeneity was calculated by determining the percent relative standard deviation (RSD) of samples taken from the top, middle, and bottom of the test article dosing preparations.
All results were within the acceptable range of =<5% RSD. The values obtained were 4.7% and 0.7% RSD for the 6 and 100 mg/mL dosing formulations, respectively.

Survival
There were no mortalities during this study.

Clinical Observations
There were no treatment-related clinical observations noted during the study. Incidental clinical observations of hair loss were noted throughout the test groups, including the vehicle control animals.

Full Functional Observational Battery
There were no test article-related or statistically significant differences noted in any of the hnctional observational battery parameters.

Body Weights
Male and female body weight growth curves are presented in the attached document.
There were no toxicologically meaningful effects on body weights or body weight changes during the study. There were no statistically significant differences in mean body weights for males and females or in body weight changes for females. A single statistically significant increase in mean body weight change was noted for the Group 3 males for Days 1 to 8. No other changes in mean body weight gain were noted.

Food Consumption
There were no test article-related or statistically significant differences in mean food consumption noted for the males and females during the study.

Ophthalmology
There were no differences among the dose groups that indicated a test article-related effect.

Clinical Pathology
Hematology and Coagulation
Statistically significant differences in mean hematology and coagulation parameters are presented in the attached document.
There were no test-article related effects on hematology parameters. There were several statistically significant differences noted in the hematology data for the female animals on Day 29. The majority of the differences occurred in the 250 and 1000 mg/kg/day groups; however, all of the mean values remained within the Testing Facility’s historical control range.
A slight statistically significant decrease in platelet count was noted in the female animals administered 1000 mg/kg/day on Day 29. Although the difference in this parameter compared to the controls appeared to be test article related, the results did not reach a level of toxicological significance and there was no corresponding change in males for this parameter. The remaining differences in hematology values noted were not considered toxicologically meaningful as they were not observed in a dose-related manner and the absolute values remained well within the Testing Facility’s normalized historical control range.
Clinical Chemistry
Statistically significant differences in mean clinical chemistry parameters are presented in the attached document.
There were no toxicologically meaningful differences noted in mean clinical chemistry parameters on Day 29. The majority of the differences noted occurred in the low- and mid-dose groups, did not follow a dose-dependent pattern, and the absolute values remained well within the Testing Facility’s normalized historical control range.
Urinalysis
There were no test article-related or statistically significant differences in mean urinalysis parameters noted for the males and females.

Gross Necropsy
Gross (internal) necropsy findings are summarised in the attached document
There were no test article-related gross necropsy findings noted at the end of the dosing period. There were a number of incidental gross necropsy findings on Day 29; however, the findings observed were noted sporadically throughout the dose groups, including the control group and were limited in number. Therefore, these findings were not considered toxicologically meaningful.

Organ Weights
There were no statistically significant differences in mean organ weight data noted for females.
Statistically significant differences in mean organ weight data for male groups are presented in the attached document.
Organ weight differences in the males included increases in the absolute and/or relative weights of the pituitary, kidney, thyroid, and spleen. Statistically significant increases in pituitary, kidney, and spleen were noted for the 250 mg/kg/day dose group. In addition, differences in mean organ weight relative to brain weight included an increase in the thyroid weight for the 1000 mg/kg/day group, increases in kidney weight for the 250 and 1000 mg/kg/day groups, and increases in pituitary weight for the 60 and 250 mg/kg/day dose groups. None of these differences were considered toxicologically meaningful as there was no correlation between these organ weight differences and any histopathological findings.

Histopathology
Once daily administration of the test substance by oral gavage at doses of 60,250, and 1000 mg/kg/day for 28 days was well tolerated. All animals survived to the scheduled terminal euthanasia (Day 29). There were few gross pathology and histopathology findings and none of the findings were considered to be test article related.

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: No test substance related mortalities, clinical signs, haematological or clinical chemistry findings, or histopathological observations.

Critical effects observed:

not specified

Conclusions:

Administration of the test substance by once daily oral gavage was well tolerated in rats at levels of 60,250, and 1000 mg/kg/day. There were no adverse effects or target organs of toxicity noted following 28 days of oral administration of the test article. Based on these results, the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day .

Executive summary:

Introduction

In an OECD Guideline No. 407 study the potential toxicity of the test substance was evaluated when administered to rats by once daily oral gavage for a minimum of 28 days.

Methods

The study design include four dose groups, dosed with 0, 60, 250 or 1000 mg/kg bw/day. The following variables and end points were evaluated in this study: clinical signs, full functional observational battery (FOB), body weights, body weight changes, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations.

Results

There were no mortalities or clinical observations of toxicity noted during this study. Likewise, there were no differences noted in any of the fimctional observational battery parameters examined at the conclusion of the dosing period. There were no statistically significant or toxicologically meaningful differences in mean body weight, body weight gain, or food consumption observed during this study. In addition, there were no toxicologically meaningful differences in the hematology, coagulation, clinical chemistry, or urinalysis parameters noted at study termination. A few gross necropsy findings were noted at study termination; however, these findings were considered incidental in nature and in no way related to treatment. There were no test article-related changes in organ weights or test article-related histopathological findings.

Conclusion

Administration of the test substance by once daily oral gavage was well tolerated in rats at levels of 60,250, and 1000 mg/kg/day. There were no adverse effects or target organs of toxicity noted following 28 days of oral administration of the test article. Based on these results, the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch grade 1. GLP study conducted according to OECD 407 guideline
Klimisch grade 1 GLP study conducted according to standrad range-finder protocol.

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

KEY: 28 -day toxicity study

Introduction

The purpose of this study was to evaluate the potential toxicity of the test substance when administered to rats by once daily oral gavage for a minimum of 28 days.

Methods

The study design included four dose groups, dosed with 0, 60, 250 or 1000 mg/kg bw/day. The following variables and end points were evaluated in this study: clinical signs, full functional observational battery (FOB), body weights, body weight changes, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations.

Results

There were no mortalities or clinical observations of toxicity noted during this study. Likewise, there were no differences noted in any of the functional observational battery parameters examined at the conclusion of the dosing period. There were no statistically significant or toxicologically meaningful differences in mean body weight, body weight gain, or food consumption observed during this study. In addition, there were no toxicologically meaningful differences in the hematology, coagulation, clinical chemistry, or urinalysis parameters noted at study termination. A few gross necropsy findings were noted at study termination; however, these findings were considered incidental in nature and in no way related to treatment. There were no test article-related changes in organ weights or test article-related histopathological findings.

Conclusion

Administration of the test substance once by daily oral gavage was well tolerated in rats at levels of 60,250, and 1000 mg/kg/day. There were no adverse effects or target organs of toxicity noted following 28 days of oral administration of the test article. Based on these results, the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day.

Supporting: 7-day toxicity study

Introduction

The purpose of this study was to evaluate the potential toxicity of the test substance when administered to rats by once daily oral gavage for a minimum of 7 days.

Methods

The study design include four dose groups, dosed with 0, 125, 250, 500 or 1000 mg/kg bw/day. The following variables and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, and clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis).

Results

There were no mortalities or clinical observations of toxicity noted during this study. There were no statistically significant or toxicologically meaningful differences in mean body weight, body weight gain, or food consumption observed during this study. In addition, there were no toxicologically meaningful differences in the hematology, coagulation, clinical chemistry or urinalysis data noted at study termination.

Conclusion

Administration of the test substance by once daily oral gavage was well tolerated in rats at levels up to 1000 mg/kg/day.

Justification for classification or non-classification

No classification for repeat dose is required as there were no adverse effects at up to and including the maximum recommended dose level of 1000 mg/kg bw/day.