2-(2-oxo-5-(1,1,3,3-tetramethylbutyl)-2,3-dihydro-1-benzofuran-3-yl)-4-(1,1,3,3-tetramethylbutyl)phenyl acetate

Endpoint summary

Description of key information

28d subacute study: NOAEL = 1000 mg/kg; Notox 1999, OECD Guideline Study. No treatment-related findings noted at any of the concentrations tested.

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:

24.06. - 21.10.1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

NOTOX B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands

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 Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 6 weeks
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding.
- Diet: standard pelleted laboratory animal diet, ad libitum (from Carfil Quality BVBA, Oud-Turnhout, Belgium).
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing. Adjustment was made for specific gravity of vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of representative formulations, prepared after termination of the in-life phase of the study, were analysed to check stability over 4 hours and homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The representative formulations were prepared on the day of analysis and according to the same method as used for preparation of the formulations for dosing purposes.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Once daily for 28 days, 7 days per week. The times of dosing per day varied between 7.32 am and 12.47 pm.

Remarks:

Doses / Concentrations:
0, 50, 150, 1000 mg/kg bw/day (m/f)
Basis:
other: nominal

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 264408).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28.

FOOD CONSUMPTION: weekly

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes, overnight (with a maximum of 20 hours), but water was provided
- How many animals: all rats/sex/group
- Parameters checked: Erythrocyte count/RBC; Haemoglobin/HB; Haematocrit/HCT; Mean corpuscular volume/MCV; Mean corpuscular haemoglobin/MCH; Mean corpuscular haemoglobin concentration/MCHC; Platelet count; Red cell distribution width/RDW; Total leucocyte count/WBC; Differential leucocyte count/SEG; Prothrombin time/PT; Partial thromboplastin time/PTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Animals fasted: Yes, overnight (with a maximum of 20 hours), but water was provided
- How many animals: all rats/sex/group
- Parameters checked: Alanine aminotransferase; Aspartate aminotransferase; Bilirubin, total; Cholesterol, total; Creatinine; Glucose; Urea; Protein, total; Protein, albumin; Alkaline phosphatase; Sodium; Potassium; Chloride; Calcium; Phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: During week 4 of treatment, the following tests were
performed on all animals:
- hearing ability
- pupillary reflex
- static righting reflex
- grip strength

Sacrifice and pathology:

GROSS PATHOLOGY:
All animals surviving to the end of the observation period were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

ORGAN WEIGHTS:
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus.

HISTOPATHOLOGY:
Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, and all gross lesions of all animals were examined by a pathologist. All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement:
Adrenal glands; Aorta; Brain; Caecum; (Cervix); (Clitoral gland); Colon; Duodenum; Epididymides; (Eyes with optic nerve and Harderian gland); (Female mammary gland area); (Femur including joint); Heart; Ileum; Jejunum; Kidneys; (Larynx); (Lacrimal gland, exorbital); Liver; Lung, infused with formalin; Lymph nodes - mandibular, mesenteric; (Nasopharynx); Oesophagus; Ovaries; Pancreas; Peyer's patches (jejunum, ileum) if detectable; Pituitary gland; (Preputial gland); Prostate gland; Rectum; (Salivary glands - mandibular, sublingual); Sciatic nerve; (Seminal vesicles); (Skeletal muscle); (Skin); Spinal cord -cervical, midthoracic, lumbar; Spleen; Sternum with bone marrow; Stomach; Testes; Thymus; Thyroid including parathyroid; (Tongue); Trachea; Urinary bladder; Uterus; (Vagina); All gross lesions

Statistics:

The following statistical methods were used to analyse the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Test statistics were calculated on the basis of exact values for means and pooled variances.

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. One female (no.27, receiving 150 mg/kg bw/d) died after blood sampling and prior to necropsy, which was considered to be an accidental death.

There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment.
Incidental findings that were noted included scabs (neck), alopecia (neck and legs) and red staining (neck). These findings were considered to be within the range of biological variation for rats of this age and strain and housing under the conditions in this study. Salivation was incidentally observed during the study in treated animals. Salivation is often noted in these types of studies following oral gavage treatment and is considered to be related to multiple intra-oesophageal intubation, and possibly also related to the taste of the test substance. Based on the incidence in this study, no toxicological significance was attached to the observation of salivation.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain of treated females was slightly lower over the 4-week treatment period than that seen for control females. Minor levels of statistical significance, when compared to controls were achieved on day 22 for females, receiving 50 and 1000 mg/kg bw/d and persisted on day 28 in females at 50 mg/kg bw/d only. However, the changes in body weight gain did not result in statistical significant differences in body weights of treated females, when compared to controls. Moreover, in comparison with the historical background data, the body weight gain of control females between day 8 and 22 was slightly higher than expected. Therefore, -also taking into account the absence of a clear dose response relationship, the toxicological significance of the (statistical significant) changes in body weight gain in females were doubted. The body weights and body weight gain of treated males remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION
There were no differences in food consumption before or after allowance for body weight between treated and control animals.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment. Levels of statistical significance were found for the (slightly decreased) values for mean corpuscular haemoglobin (low dose males) and for the mean corpuscular haemoglobin concentration (mid dose males and females). Based on the absence of a dose response relationship and the fact that all values were within the range of historical background data these changes were considered to have arisen by chance and, to be of no toxicological significance.

CLINICAL CHEMISTRY
There were no differences between control and treated rats that were considered to be related to treatment with the test substance. Levels of statistical significance were found for the (slightly decreased) values for chloride (mid dose males) and for the (slightly increased) values for glucose (low dose females) and albumin (mid dose females) concentration. Based on the absence of a dose response relationship and the fact that all values were within the range of historical background data these changes were considered to have arisen by chance and to be of no toxicological significance. In 3/5 females receiving 150 mg/kg bw/d, increased levels of urea and to a minor extent increased levels of creatinine were noted, indicative of possible effects in the kidneys of these animals. As a consequence, the mean value for urea (and not for creatinine) achieved a level of statistical significance in these females, although a large standard deviation was seen. However, no changes were observed in the kidneys in organ weights and at macroscopic examination in these animals. Based on the absence of effects in urea and creatinine at the higher dose of 1000 mg/kg bw/d, the changes were considered not to be induced by treatment. Moreover, the increased levels of urea were only of a minor degree and of no pathological importance. From the data obtained in this study, the cause of these changes could not be established.

ORGAN WEIGHTS (organ-to-body-weight ratios)
Organ weights and organ weights:body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidental findings, comprised pelvic dilation of the kidneys (control male), cyst in the uterus (low dose female) and a hernia (diaphragm, mid dose female) and were considered to be within the range of biological variation for rats of this age and strain and not to represent a change of toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

FUNCTIONAL OBSERVATIONS
Any findings noted and the variation in motor activity did not indicate a relation with treatment.
A markedly increased motor activity, recorded by both the upper and lower sensors, was noted in one female (receiving 50 mg/kg bw/d). In the absence of a dose-dependent relationship this was considered to have occurred by chance without any toxicological relevance.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related findings up to and including the high dose level of 1000 mg/kg bw/d.

Critical effects observed:

not specified

Analysis of dose preparations.

The accuracy, homogeneity and stability (over 4 hours) of the formulations of the test substance in propylene glycol were considered of an acceptable level for preparations used in these types of studies. The accuracy of one of the duplicate mid dose samples showed a value of 79%, which is below the range of acceptability (NOTOX criteria: 90-110% accuracy). Taking into account the accuracy determined in the other sample and in the low and high dose samples, this value was considered an incidental outlier and excluded from interpretation.

Conclusions:

There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment. From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day was concluded.

Executive summary:

In a GLP-compliant subacute in vivo study (Notox 1999) performed according to OECD guideline 407, the test material was administered by gavage to three groups, each of five male and five female SPF-bred Wistar rats, for twenty-eight consecutive days, at dose levels of 50, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Propylene glycol). No substance-induced mortality occurred during the study period. One female receiving 150 mg/kg/day died after blood sampling and prior to necropsy, which was considered to be an accidental death. There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. No treatment related differences were observed for body weights, body weight gain and food consumption. Select parameters of haematology and clinical biochemistry were changed and reached levels of statistical significance. However, based on the absence of dose response relationships and the fact that all values were within the range of historical background data these changes were considered to have arisen by change and of no toxicological significance. Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidental findings, comprised pelvic dilation of the kidneys (control male), cyst in the uterus (low dose female) and a hernia (diaphragm, mid dose female) were considered to be within the range of biological variation for rats of this age and strain and not to represent a change of toxicological significance. Organ weights and organ weights : body weight ratios of treated animals were considered to be similar to those of control animals. There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats. From the results presented in this report, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day was concluded.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

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

In a GLP-compliant subacute in vivo study (Notox 1999) performed according to OECD guideline 407, the test material was administered by gavage to three groups, each of five male and five female SPF-bred Wistar rats, for twenty-eight consecutive days, at dose levels of 50, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Propylene glycol). No substance induced mortality occurred during the study period. One female receiving 150 mg/kg/day died after blood sampling and prior to necropsy, which was considered to be an accidental death. There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. Salivation was incidentally observed during the study in treated animals. Salivation is often noted in these types of studies following oral gavage treatment and is considered to be related to multiple intra-oesophageal intubation, and possibly also related to the taste of the test substance. Based on the incidence in this study, no toxicological significance was attached to the observation of salivation. Body weight gain of treated females was slightly lower over the 4-week treatment period than that seen for control females. However, the statistical significant changes in body weight gain did not result in statistical significant differences in body weights of treated females, when compared to controls. Moreover, in comparison with the historical background data, the body weight gain of control females between day 8 and 22 was slightly higher than expected. Therefore,-also taking into account the absence of a clear dose response relationship, the toxicological significance of the changes in body weight gain in females were doubted. There were no differences in food consumption before or after allowance for body weight between treated and control animals. Select parameters of haematology and clinical biochemistry were changed and reached levels of statistical significance. However, based on the absence of dose response relationships and the fact that all values were within the range of historical background data these changes were considered to have arisen by change and of no toxicological significance. In 3/5 females receiving 150 mg/kg/day, increased levels of urea and to a minor extent increased levels of creatinine were reported. However, no changes were observed in the kidneys in organ weights and at macroscopic examination in these animals. Based on the absence of effects in urea and creatinine at the higher dose of 1000 mg/kg/day, the changes were considered not to be induced by treatment. Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidental findings, comprised pelvic dilation of the kidneys (control male), cyst in the uterus (low dose female) and a hernia (diaphragm, mid dose female) were considered to be within the range of biological variation for rats of this age and strain and not to represent a change of toxicological significance. Organ weights and organ weights : body weight ratios of treated animals were considered to be similar to those of control animals. There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

From the results presented in this report, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day was concluded.

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

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for repeated dose oral toxicity is not warranted under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose oral toxicity is not warranted under Regulation (EC) No.1272/2008.