Dihydro-3-(tripropenyl)furan-2,5-dione

Endpoint summary

Description of key information

Oral:
28-day study (rats): NOAEL 50 mg/kg bw; target organ: kidney
14-day DRF study (rats): no NOAEL identified
Reproduction/developmental toxicity screening test (rats): NOAEL 50 mg/kg bw, target organ: kidney

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:

weight of evidence

Study period:

2012-03-15 to 2012-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males:145- 163 g, (mean: 155.10 g, ± 20% = 124.08 -186.12 g); females:113 -132 g, (mean: 124.03 g, ± 20% = 99.23- 148.84 g).
- Fasting period before study:
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding; Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected as suggested by the sponsor and on the basis of the test item’s characteristics.
- Concentration in vehicle: 0, 12.5, 37.5, 62.5 mg/ml
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): MKBF 8603V
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical measurements of the samples were performed using GC-MS method. The analytical method was validated during the course of the study.
Furhter, the stability in corn oil was ensured as the substance turned out to be not stable in water and octanol at concentration of below water solubitliy.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once per day

Remarks:

Doses / Concentrations:
0, 50(LD), 150(MD), 250(HD) mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected on the basis of data from a dose range finding studies (BSL study no. 112181A and 114167).
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: at least once a day. Twice daily all animals were observed for morbidity and mortality except for weekends and public holidays when observations were made once daily.
- Cage side observations checked: The health condition of the animals was recorded

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly during the treatment and recovery period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No data

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the first administration and in the last week of the treatment period as well at the end of the recovery period in the recovery animals.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last administration, blood was sampled from all surviving animals of the main study for a haematological evaluation. Blood from the recovery animals was sampled at the end of the recovery period.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All
- Parameters checked: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular, haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last administration, blood was sampled from all surviving animals of the main study for an evaluation of the clinical biochemistry. Blood from the recovery animals was sampled at the end of the recovery period.
- Animals fasted: Yes
- How many animals: All
- Parameters checked: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: specific gravity, nitrite, ph-value (ph), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leucocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure as well as once in the fourth week of exposure
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
Blood Coagulation:
- Time schedule for collection of blood: One day after the last administration, blood was sampled from all surviving animals of the main study for an evaluation of the clinical biochemistry. Blood from the recovery animals was sampled at the end of the recovery period.
- Animals fasted: Yes
- How many animals: All
- Parameters checked: prothrombin time (PT), activated partial thromboplastin time (aPTT)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see tables in the follwing)
HISTOPATHOLOGY: Yes
A histopathological evaluation was carried out on all animals of the control and high dose groups (of the main study) which were sacrificed at the end of the treatment period. Histopathological examinations were extended to animals of the other dose groups and recovery animals, as treatment-related changes were observed in the few organs of high dose group. Therefore, liver, kidney, heart, mesenteric lymph node, axillary lymph node and spleen were also evaluated in all animals of the low and medium dose group sacrificed at the end of the treatment period and in all recovery animals.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Other examinations:

The stability of the test substance in corn-oil and PEG was investigated prior to the animal study. In corn oil the test substance was stable after 4 hours incubation in corn oil at 37°C (Clariant, 2011).

Statistics:

Parameters like body weight Change and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights, Body weight change and food consumption are also presented as figures.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage.
All results are reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).
Analytical results and histopathological findings are presented in separate phase-reports attached to this report.
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. High dose and control values of the recovery groups were compared using a Student’s t-Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

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:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortalities were observed. No clinical signs were observed in any animal during the entire study period.

BODY WEIGHT AND WEIGHT GAIN
No test item related effect was observed; mean values for body weight and body weight change were comparable with respective controls and were within the normal range of variation for this strain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Statistical analysis of food consumption data of male and female animals revealed no test item related effect on food consumption during the whole study period.

FOOD EFFICIENCY
No data

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

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings in any of the animals of this study.

HAEMATOLOGY
In males, at the end of the treatment period and recovery period, No statistically significant difference in any of the haematological parameter was observed and all haematological parameters were within the normal range of variation.
In females, at the end of the treatment period, no statistically significant difference was observed in any of the haematology parameters in treated groups except statistically significant decrease in HB, HCT and increase in WBC in HD group when compared with the controls. There was also statistically significant increase in Lecucocytes observed in HD recovery females.
Besides, all haematological parameters were within the normal range of variation and statistically significant differences are not assumed to be biologically relevant and adverse.

CLINICAL CHEMISTRY
In males, at the end of the treatment period, TBA was decreased in LD, MD and HD group, although statistical significance was achieved only in LD and HD when compared with the controls. As all individual values were within the normal range of variation and due to lack of dose dependency, this statistically significant effect on TBA was not assumed to be biologically or toxicologically relevant and treatment related.
In females, at the end of the treatment period, statistically significant decrease in TP was observed in HD group when compared with the controls.
Besides, all parameters of clinical chemistry were within the normal range of variation for this strain and statistically significant differences between dose and control groups are not assumed to be biologically relevant or adverse.
In recovery males and females, no effect on any of the clinical biochemistry parameter was observed in HD group when compared with the controls.

URINALYSIS
High protein and leukocyte levels were found in the urine of few males including control animals. All urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control group were observed in females and recovery animals.

NEUROBEHAVIOUR
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

ORGAN WEIGHTS
In males, at the end of treatment period, statistically significant decrease in absolute and relative (to brain and body weight) thyroid/parathyroid and pituitary gland weights were observed in HD group when compared with controls. However, statistical analysis of data from animals sacrificed at the end of recovery period revealed decrease in absolute and relative (to body weight) brain weights in HD group when compared to the corresponding control.
In females, statistically significant decrease in group mean absolute kidney, increase in relative (to brain and body weight) spleen and decrease in relative (to brain and body weight) kidney weight was observed in HD group when compared with controls. Absolute and relative (to brain and body weight) organ weight data from females sacrificed at the end of recovery period remained unaffected due to the treatment and all group mean and individual values from treated groups were comparable with the controls.
This observed statistical significant difference for few organs in HD group male and females (especially kidney) sacrificed at the end of treatment period could be considered as treatment related.

GROSS PATHOLOGY
No macroscopic findings were observed in any animal of the study except discoloured dark thymus from one control recovery female (52) and fluid distension of the uterus in one female (60) from high dose recovery group and were assumed to be common background findings in this strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological findings clearly attributable to the test item were seen in the kidney. Treatment induced dose-related multifocal tubular basophilia/vacuolation in the renal cortex and medulla at 150 and 250 mg/kg/day, in some animals accompanied by multifocally dilated tubules. These changes were more prominent in the females and corroborated the renal weight changes noted in this study. They are indicative of degeneration and repair of renal tubular cells and were found to be completely resolved at the end of the recovery period.
In the spleen, axillary lymph node and mesenteric lymph node, histiocytic aggregations were observed in one single male rat treated at 250 mg/kg/day, reaching a marked degree of severity in the mesenteric lymph node. In view of the isolated occurrence of this change a correlation to treatment could not be clearly established and its significance remains unclear.
The dose of 50 mg/kg/day is considered to be the NOEL (No Observed Effect Level) for pathology.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No finding

OTHER FINDINGS
Blood coagulation was not affected in both males and females by the test item.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: test item related histopathological changes in kidneys

Critical effects observed:

not specified

Dose Formulation Analytics

The recoveries of all the test samples were between 74 and 129 % of nominal. The analytical method for the determination of Tripropenyl Succinic Anhydride in corn oil was validated for a concentration range of 10 to 70 mg test item /mL.

Conclusions:

Based on the data in this study, the NOAEL (No Observed Adverse Effect Level) of the test item is considered to be 50 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.

Executive summary:

In this subacute toxicity study, the test item was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 50, 150 and 250 mg/kg body weight/day for a period of 28 days. In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, the animals in the recovery groups were observed for a period of 14 days following the last administration. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The animals were observed each day for signs of toxicity. Body weight was measured twice a week and food consumption was measured weekly. At the end of the treatment period, all main study animals and at the end of recovery period, all recovery animals were sacrificed and subjected to necropsy. Full histopathological evaluation of the tissues was performed on high dose and control animals. In addition, liver kidney, heart, mesenteric lymph node, axillary lymph node and spleen were also evaluated in all animals of the low and medium dose group and in all recovery animals. Organs showing gross alterations were also examined histopathologically.

Haematological and clinical biochemistry examinations were made on blood samples obtained from the overnight fasted main and recovery animals at the terminal sacrifice.

No mortalities, no significant changes, no ophthalmoscopic findings were observed in the study and all animals. No relevant effects were observed in the functional observation battery before and at the end of the treatment period. No test item related effect was observed on body weight, body weight change and food consumption.

In males, no statistically significant difference in any of the haematological parameter was observed. In females, statistically significant decrease in HB, HCT and increase in WBC in HD group were observed. There was also statistically significant increase in Lecucocytes observed in HD recovery females. Blood coagulation was not affected in both males and females by the test item.

In males, TBA was decreased in LD, MD and HD group, although statistical significance was achieved only in LD and HD when compared with the controls. In females, at the end of the treatment period, statistically significant decrease in TP was observed in HD group when compared with the controls.

High protein and leukocyte levels were found in the urine of few males including control animals.

No macroscopic findings were observed in any animal of the study except discoloured dark thymus from one control recovery female and fluid distension of the uterus in one female from high dose recovery group.

In males, statistically significant decrease in absolute and relative thyroid/parathyroid and pituitary gland weights were observed in HD group. In females, statistically significant decrease in group mean absolute kidney, increase in relative spleen and decrease in relative kidney weight was observed in HD group.

Histopathological findings clearly attributable to the test item were seen in the kidney. Treatment induced dose-related multifocal tubular basophilia/vacuolation in the renal cortex and medulla at 150 and 250 mg/kg/day, in some animals accompanied by multifocally dilated tubules. Kidney changes were more prominent in the females and corroborated the renal weight changes noted in this study. They are indicative of degeneration and repair of renal tubular cells and were found to be completely resolved at the end of the recovery period. In the spleen, axillary lymph node and mesenteric lymph node, histiocytic aggregations were observed in one single male rat treated at 250 mg/kg/day.

Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of Tripropenyl Succinic anhydride is considered to be 50 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable and robust

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

Prior to the performance of animal studies the stability of the tripropenyl succinic anhydride was investigated in corn-oil and in PEG, because the test substance undergoes hydrolysis under abiotic condition (see IUCLID 5.2.1). The anhydride was stabile up to 4 hours at 37°C in corn oil and in PEG.

The repeated dose toxicity of tripropenyl succinic anhydride was assessed in a 28 -day study (OECD 407) with a 14 -day dose range finding study in rats, and a reproduction/developmental toxicity sceening test (OECD 421).

In the 28- repeated dose study, the test item was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 0, 50, 150 and 250 mg/kg body weight/day for a period of 28 days according to OECD 407. In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, the animals in the recovery groups were observed for a period of 14 days following the last administration. No mortalities, no significant changes, no ophthalmoscopic findings were observed in the study and all animals. No relevant effects were observed in the functional observation battery. No test item related effect was observed on body weight, body weight change and food consumption.

In males, no statistically significant difference in any of the haematological parameter was observed. In females, statistically significant change of some haematological parameter was observed. Changes of some clinical chemistry parameters were also observed in males and females. No macroscopic findings were observed in any animal of the study except discoloured dark thymus from one control recovery female and fluid distension of the uterus in one female from high dose recovery group.

In males, statistically significant decrease in absolute and relative thyroid/parathyroid and pituitary gland weights were observed in high dose group. In females, statistically significant decrease in group mean absolute kidney, increase in relative spleen and decrease in relative kidney weight was observed in high dose group.

Histopathological findings clearly attributable to the test item were seen in the kidney. Treatment induced dose-related multifocal tubular basophilia/vacuolation in the renal cortex and medulla at 150 and 250 mg/kg/day, in some animals accompanied by multifocally dilated tubules. Kidney changes were more prominent in the females and corroborated the renal weight changes noted in this study. They are indicative of degeneration and repair of renal tubular cells and were found to be completely resolved at the end of the recovery period. Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of Tripropenyl Succinic anhydride is considered to be 50 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.

In the 14 -day dose range finding study, the test item was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 0, 100, 300, 600, 1000 mg/kg bw. The treatment revealed toxicological findings, clinical signs, and mortalities in both male and females. Other predominant toxicologcial findings observed were: Decrease in overall body weight change and food consumption. Dose dependent decrease in male WBC values, a slight dose dependent increase in platelet count. Slightly decrease in ASAT, ALAT and increase in urea, creatanine in females. Slight decrease in AP. Slight decrease in absolute and relative spleen and heart weights in male and females of treated groups. Slightly increase in absolute and relative kidney weights in females. Slightly decrease in absolute and relative epididymides and prostate, seminal vesicles with coagulating gland weights in males.

The repeated dose toxicity potential of Tripropenyl Succinic anhydride was investigated additionally in a reproduction/developmental toxicity screening test in Wistar rat according to OECD 421.In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with50, 150 and 250 mg/kg body weight per day of the test item. One male animal from MD group was found dead on premating day 6 and 8 females from HD group were died on premating day 5, 5, 6, Gestation day 11, 21, 22, 23 and 21, respectively.Biologically significant decrease in body weight and bodyweight change was observed in MD and HD group.No treatment related effect on copulation index and delivery index was observed when compared with controls. These changes were considered to be secondary to the systemic effect.No treatment related gross external findings were observed in pups.At necropsy of male, macroscopic examination of the animals revealed no test item related macroscopic findings in males.With minimal to moderate histopathological findings in the kidneys in females of all groups, effects on kidney weights were considered to be test item related.  

There was no evidence of a direct test item-related effect on male or female reproductive organs in this study.

In conclusion, the repeated dose administration of the test item to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 50, 150 and 250 mg/kg body weight revealed few findings of toxicological relevance and mortalities in MD group male and HD group females.

Based on the data generated from this reproduction/ developmental toxicity screening test with Tripropenyl Succinic anhydride, the no observed adverse effect level (NOAEL) for parental animals was considered to be 50 mg/kg body weight and NOAEL for developmental toxicity of pup was believed to be 250 mg/kg body weight.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study under GLP

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

Based on findings in the 28 -day study, the registered substance tripropenyl succinic anhydride is subject to classification and labeling according to Directive 67/548/EECand Regulation 1272/2008/EC regarding repeated dose toxicity. The registered substance is classified as R48/22 and STOT RE Cat.2 (H373: May cause damage to organ (kidney) through prolonged or repeated exposure (oral).