Reaction products of Dihydro-3-(tetrapropenyl)furan-2,5 dione with propane-1,2,diol

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2016 to 15 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was not designed to meet any particular regulatory requirements and is a range-finder for further study.
The purpose of the study was to assess the systemic toxic potential of the test material by oral administration to Han Wistar rats for 14 days at initial test concentrations of 100, 300 and 1 000 mg/kg/day. During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken.

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

Limit test:

no

Test material

Test material information

1

Reference substance name:

2-(tetrapropenyl)succinic acid, monoester with propane-1,2-diol

EC Number:

257-836-6

EC Name:

2-(tetrapropenyl)succinic acid, monoester with propane-1,2-diol

Cas Number:

52305-09-6

Molecular formula:

C17H30O5 - C21H38O5

IUPAC Name:

2-[2-(2-hydroxy-1-methylethoxy)-2-oxoethyl]tetradecanoic acid

Test material form:

solid

Details on test material:

- Apperance: Yellow solid block
- Storage: Room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST rat.

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Animal ages ranged from 74 to 96 days at start of treatment, specifically:
Group 1 and 2: 74 to 84 days
Group 3: 81 to 91 days
Group 4: 86 to 96 days
- Weight at study initiation: Weight ranged from 296 to 369 g for male animals and 197 to 241 g for female animals; specifically:
Group 1 and 2: Males: 296 to 356g; females: 197 to 232 g.
Group 3: Males: 335 to 369 g; females: 213 to 229 g.
Group 4: Males: 351 to 365g; females: 209 to 241 g.
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Males and females were blocked by sex and the cages constituting each group were dispersed in a single battery so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The position of the cage batteries in the room were changed weekly, following a rotation plan, to further minimise possible effects of spatial variations. Animals were caged in groups of three (groups 1 to 3) or two (group 4) of the same sex.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 14 to 26 days before commencement of treatment; specifically:
Group 1 and 2: 14 days before commencement of treatment.
Group 3: 21 days before commencement of treatment.
Group 4: 26 days before commencement of treatment.
On day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ± 20 % of the mean for the appropriate sex. No replacements were required in Groups 1 to 3. No check was performed for Group 4, since no spare animals were remaining.
During the acclimatization period observations of animals and their cages were recorded at least once per day.

DETAILS OF FOOD AND WATER QUALITY
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 40-70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated. At least 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was chosen to simulate the conditions of potential human exposure. The test item was administered by gavage.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- Method of preparation: Starting with the lowest concentration, the required amount of test item was weighed into a suitable container. Approximately 50 % of vehicle was added; it was placed in a water bath and heated up to 40 °C if necessary. It was then stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous. The formulation was refrigerated (nominally 2 - 8 °C).
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- Frequency of preparation: Weekly

VEHICLE
- Concentration in vehicle: Formulated concentrations of 20, 60, 200, and 100 mg/mL.
- Amount of vehicle: Dose volume of 5 mL/kg/day. Individual dose volumes were calculated from the most recently recorded scheduled body weight.

Analytical verification of doses or concentrations:

no

Remarks:

No formulation analysis was performed on this study.

Details on analytical verification of doses or concentrations:

No formulation analysis was performed on this study.
Before the commencement of treatment the suitability of the proposed mixing procedures was determined as part of another study. The homogeneity of the test item in the vehicle (at concentrations of 2 and 200 mg/mL) was achieved following refrigerated (2 – 8 °C) storage for up to 15 days and at ambient temperature (nominally 21 °C) storage for up to seven days.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

3 animals per sex per dose for the animals dosed at 100, 300 and 1 000 mg/kg/day and 2 animals per sex per dose for the animals dosed at 500 mg/kg/day.

Control animals:

yes, historical

Details on study design:

DOSE SELECTION RATIONALE
Doses were selected based on the results from an acute oral toxicity study in the rat. In this acute toxicity study, a single oral dose of the test item at 2 000 mg/kg (given at a dose volume of 10 mL/kg using arachis oil as the vehicle) was associated with marked clinical signs comprising hunched posture, tip-toe gait, pilo-erection, lethargy, diarrhoea, dehydration and hypothermia and body weight loss necessitating the euthanasia of the animal. Macroscopic findings at necropsy comprised gaseous stomach, haemorrhagic non-glandular and gastric mucosa of the stomach and dark liver and kidneys. At 300 mg/kg, hunched posture was observed for up to two hours after dosing. There were no further signs and no macroscopic findings were apparent at necropsy on completion of the observation period. Consequently, it was considered that that the LD50 was > 300 < 2 000 mg/kg.
Considering the limited information available, it was considered appropriate to use a cautious approach to dosing and treatment commenced initially at 100 and 300 mg/kg/day, with treatment for Group 3 commencing one week later. Following 6-days of dosing at 100 and 300 mg/kg/day, there was no overt evidence of toxicity (food consumption was lower than that recorded pre-treatment, approximately 80 % of pre-dose, but without dose-relationship and there was no clear effect on body weight) and, therefore, a high dose level of 1 000 mg/kg/day for Group 3 was considered appropriate.
Following two days of dosing at 1 000 mg/kg/day, this treatment group was terminated because of excessive toxicity. Since no overt evidence of toxicity was apparent at 300 mg/kg/day, the spare animals ordered for the study (two males and two females) were used to investigate a dose level of 500 mg/kg/day.
The range of dose levels was selected to provide sufficient information for dose level selection for the associated 4-week toxicity study.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes.
Signs are considered in two parts: Detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’ and extended changes in condition, classified as ‘clinical signs’.
Signs associated with dosing are presented for each animal that showed signs, providing detail of type of sign, day of occurrence and information on the duration of the sign applicable.
Clinical signs are presented for each animal, providing detail of type of sign, day of occurrence and information on the duration of the sign applicable.
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

Signs associated with dosing:
Detailed observations were recorded daily at the following approximate times in relation to dose administration:
- Pre-dose.
- After completion of dosing each group.
- One to two hours after dosing.
- As late as possible in the working day.
Additional observations were performed on Day 2 on Group 3 to monitor the condition of the animals.

Clinical signs:
A detailed weekly physical examination was performed on each animal to monitor general health.

MORTALITY
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded before treatment commenced on Day -3 (Groups 1 to 3 only) and Day -1, on the day that treatment commenced (Day 1), twice weekly thereafter throughout the study and before necropsy.
Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period.

FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for three days before treatment started (Groups 1 to 3 only) and twice weekly for throughout the study.
As there was only one cage per group and sex, only individual cage consumption values are presented.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

Sacrifice and pathology:

NECROSCOPY
All animals were subject to a necropsy. Only the thoracic and abdominal cavities were opened. The cranial cavity was not opened as there were no observations during the study to indicate a possible neurotoxic action. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in fixative. The retained tissues were checked before disposal of the carcass.
The organs weighed and tissue samples fixed. The spleen, liver and kidneys were weighed and fixed (left and right kidney weighed individually). Any tissues with abnormalities were also fixed. Tissues were preserved in 10 % Neutral Buffered Formalin.

METHOD OF SACRIFICE
Carbon dioxide asphyxiation with subsequent exsanguination.

ORGAN WEIGHTS
Organ weights were presented both as absolute and relative for terminal body weight, using the weight recorded on the day of necropsy.

Statistics:

No statistical analysis of the data was performed on this study.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 100 or 300 mg/kg/day was well-tolerated. Salivation was observed from Day 5 for males and females receiving 300 mg/kg/day and there was an isolated occurrence on Day 5 for two males receiving 100 mg/kg/day. Chin rubbing was also occasionally observed for animals receiving 300 mg/kg/day. These signs became apparent on completion of dosing and were usually absent by 1 to 2 hours after dosing.
Salivation is often a common occurrence in studies where the test material is administered by oral gavage and is not usually considered of toxicological importance.
Vocalization was also recorded on Days 6, 13 and 15 for the majority of females receiving 100 or 300 mg/kg/day. This was considered not to preclude the use of these doses in future studies.
Treatment at 500 or 1 000 mg/kg/day was not tolerated. Following the second dose administration at 1 000 mg/kg/day, animals showed signs comprising elevated, swaying and/or unsteady gait, decreased activity, irregular and slow breathing, piloerection, partially closed eyes and one female became prostrate. This group was terminated on Day 2 because of the severity of the signs observed.
Treatment at 500 mg/kg/day was tolerated for the first 4-days of treatment, but animals showed deterioration in condition following the fifth dose. Signs observed comprised piloerection, abnormally cold to the touch, decreased activity and hunched posture. It was considered that this dose level would not be tolerated for further doses and, therefore, this group was terminated on Day 5 for welfare reasons.

Mortality:

no mortality observed

Description (incidence):

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gains (Days 1 to 14) were variable with no clear effect of treatment for animals receiving 100 or 300 mg/kg/day.
Bodyweight stasis or small bodyweight losses were observed for animals which received 500 mg/kg/day (Days 1 to 4) and for animals which received 1 000 mg/kg/day (Day 1 compared to terminal bodyweight on Day 2).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for animals receiving 100 or 300 mg/kg/day was lower than that recorded pre-treatment (approximately 74-83 % pre-dose) but there was no clear dose-relationship and this may have been influenced by the calorific value of the corn oil vehicle.
Food consumption measured over the first 4 days of treatment for animals receiving 500 mg/kg/day was lower than expected compared to pretreatment values of the other groups. Pretreatment food consumption was not recorded for this group as they were not initially assigned to the study.
Food consumption could not be assessed for animals receiving 1 000 mg/kg/day which were killed prematurely on Day 2.

Food efficiency:

not examined

Description (incidence and severity):

not examined

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

no effects observed

Description (incidence and severity):

A visual assessment of water consumption did not reveal any treatment-related effect

Ophthalmological findings:

not examined

Description (incidence and severity):

not examined

Haematological findings:

not examined

Description (incidence and severity):

not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not examined

Urinalysis findings:

not examined

Description (incidence and severity):

not examined

Behaviour (functional findings):

not examined

Description (incidence and severity):

not examined

Immunological findings:

not examined

Description (incidence and severity):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no clear effects of treatment on organ weight for animals receiving 100, 300, 500 or 1 000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination at necropsy for animals given 1 000 mg/kg/day, killed prematurely on Day 2 of treatment, revealed findings in the stomach (distended appearance, abnormal contents and depressions, thickening and irregular surface of the non-glandular mucosa) and intestinal tract (distended ileum, jejunum and/or caecum and abnormal contents (gas or thin yellow fluid) in the ileum and jejunum). In addition, a dark pancreas was recorded for one male and all three females and a dark liver for one female.
Macroscopic examination at necropsy for animals given 500 mg/kg/day, killed prematurely on Day 5 of treatment, also revealed findings in the stomach (abnormal contents, distended appearance, irregular surface and thickening of the non-glandular mucosa) and intestinal tract (abnormal contents in the duodenum, ileum and jejunum, distended jejunum and abnormal color of the colon, duodenum, ileum, and jejunum. A dark pancreas was recorded in one male and two females.
For animals killed on completion of the 14-day treatment period, depressions on the non-glandular mucosa was recorded for one male which received 300 mg/kg/day and thickened non-glandular mucosa was recorded for one male and two females which received 300 mg/kg/day.
There were no treatment-related macroscopic findings for animals which received 100 mg/kg/day.

Neuropathological findings:

not examined

Description (incidence and severity):

not examined

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not examined

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not examined

Other effects:

not examined

Description (incidence and severity):

not examined

Details on results:

It is concluded that the oral (gavage) administration the test material to Han Wistar (RccHanTM;WIST) rats was well tolerated at doses up to 300 mg/kg/day.
Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively. The main target organs were the stomach and small intestine.
Whilst changes in the stomach were apparent at 300 mg/kg/day, they were not associated with any adverse clinical signs or any effect on bodyweight gain or food consumption. Based on the findings in this study, 300 mg/kg/day was considered to be a suitable high dose level in the forthcoming 28-day study.

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

not specified

Treatment related:

not specified

Any other information on results incl. tables

Discussion

The test material was administered orally by gavage to Han Wistar (RccHanTM;WIST) rats for up to 14 days at dose levels of 100, 300, 500 and 1 000 mg/kg/day.

The doses of 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated in the premature termination of the animals on Day 5 and 2, respectively. Necropsy findings largely comprised abnormalities of the stomach (distended appearance, abnormal colour, irregular surface, depressions or thickening of the non-glandular mucosa) and intestinal tract (abnormal content, abnormal colour and/or distended appearance predominantly in the ileum, jejunum and duodenum). In addition, dark appearance of the pancreas was reported for animals receiving 500 or 1 000 mg/kg/day and a dark liver for one female receiving 1 000 mg/kg/day.

The dose of 300 mg/kg/day was well tolerated with no deaths, minor transient signs and no effects on bodyweight gain or food consumption. Necropsy examination revealed changes in the stomach comprising thickening and depressions on the non-glandular mucosa for one male and thickening of the non-glandular mucosa for two females.

There were no findings of significance at 100 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, administration of the test material to Han Wistar (RccHanTM;WIST) rats through the oral route (gavage) was well tolerated at doses up to 300 mg/kg/day. Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of the test material by oral gavage administration to Han Wistar rats for 14 days.

The initial doses selected for this study were 100, 300 and 1 000 mg/kg/day. Males and females at 1 000 mg/kg/day were killed for reasons of animal welfare on Day 2 of dosing due to the severity of signs observed. An additional two males and two females were assigned to the study and dosed at 500 mg/kg/day to gain further information on the dose response.

During the study, clinical condition, body weight, food consumption, visual water consumption assessment, organ weight and macropathology investigations were undertaken.

Following the second dose administration at 1 000 mg/kg/day, signs observed included elevated, swaying and/or unsteady gait, decreased activity, irregular and slow breathing, piloerection, partially closed eyes and one female became prostrate. All animals were killed prematurely on Day 2 because of the severity of the signs observed.

Necropsy examination revealed findings in the stomach (distended appearance, abnormal contents and depressions, thickening and irregular surface of the non-glandular mucosa) and intestinal tract (distended ileum, jejunum and/or caecum and abnormal contents in the ileum and jejunum). A dark pancreas was reported for one male and all three females and a dark liver for one female.

Treatment at 500 mg/kg/day was tolerated for the first 4-days of treatment, but animals deteriorated following the fifth dose. Signs comprised piloerection, abnormally cold to the touch, decreased activity and hunched posture. Bodyweight gain and food consumption were low. It was considered that this dose level would not be tolerated for further doses and, therefore, this group was terminated on Day 5 for welfare reasons.

Necropsy examination revealed findings in the stomach (abnormal contents, distended appearance and irregular surface and thickening of the non-glandular mucosa) and intestinal tract (abnormal contents in the duodenum, ileum and jejunum, distended jejunum and abnormal color of the colon, duodenum, ileum, and jejunum). A dark pancreas was recorded in one male and two females.

Treatment of animals with 300 mg/kg/day was well-tolerated and there were no deaths. The only signs comprised transient salivation and chin rubbing. There were no effects on bodyweight gain or food consumption. There was no clear effect of treatment on organ weights. Necropsy examination revealed changes in the stomach comprising thickening and depressions on the non-glandular mucosa for one male and thickening of the non-glandular mucosa for two females.

With the exception of an occasional incidence of transient salivation, the appearance and behavior of the animals were unaffected by treatment dose of 100 mg/kg/day treatment and there were no deaths. There were no effects on bodyweight gain, food consumption or organ weights and no findings at necropsy.

It is concluded that the oral (gavage) administration of the test material to Han Wistar (RccHanTM;WIST) rats was well tolerated at doses up to 300 mg/kg/day.

Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively. The main target organs were the stomach and small intestine.

Whilst changes in the stomach were apparent at 300 mg/kg/day, they were not associated with any adverse clinical signs or any effect on bodyweight gain or food consumption. Based on the findings in this study, 300 mg/kg/day was considered to be a suitable high dose level in the 28-day study.