1,4-dioxacyclohexadecane-5,16-dione

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1-10-1997 to 14-11-1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

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

Test material

Test material information

1

Reference substance name:

1,4-dioxacyclohexadecane-5,16-dione

EC Number:

259-423-6

EC Name:

1,4-dioxacyclohexadecane-5,16-dione

Cas Number:

54982-83-1

Molecular formula:

C14H24O4

IUPAC Name:

1,4-dioxacyclohexadecane-5,16-dione

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 47 male and 47 female healthy CD rats of Sprague-Dawley origin (CrI: CD® BR VAF PLUSTM) was received from Charles River (UK) Ltd, Margate, Kent, England on 24 September 1997.
The rats were 28 ± 1 days old, in a weight range of 69 -79 g on arrival. A seven day acclimatisation period was allowed between delivery of animals and start of treatment.
All rats were initially caged in groups of five according to sex in metal cages with wire mesh floors.
Animal room temperature was maintained at 19 -22°C and relative humidity was maintained at 40 to 62% RH. These parameters were continuously monitored using a Kent Clearspan MI05 7-day chart recorder. Air exchange was set at approximately 15 air changes per hour and lighting was controlled to provide 12 hours artificial light (0700 -1900 hours) in each 24-hour period.
The health status of all animals was monitored throughout the acclimatisation period, to ensure that the rats selected for final assignment to the study were satisfactory.
On arrival each animal was weighed and sixty rats were randomly allocated to five groups, two groups consisting of ten males and ten females and three groups consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.
Each rat was identified within each cage by earmark and the cage number was tattooed on the leg of each animal in the cage.
Following the commencement of treatment spare animals were removed from the study. No further investigations were performed on these animals.
The cages (each containing five rats) were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test substance was administered by oral gavage to rats of Groups 2 to 5 inclusive using a syringe and rubber catheter at a dose volume of 5 ml/kg/day. Control animals received the vehicle (corn oil) alone at the same dose volume (5 ml/kg/day) by oral gavage. Each animal received a constant dosage based on its most recently recorded bodyweight. Animals were treated once daily. Prior to dosing, the test substance formulations for Groups 2 to 5 were mixed by inversion and stirred magnetically for at least 10 minutes prior to dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations of Zenolide in test formulations analysed during the study were within 8% of nominal concentrations. confirming the accuracy of formulation. The homogeneity and stability of 97-211-01 in the corn oil formulation was confumed at nominal concentrations of 1 mg/ml and 200 mg/ml. The results confirm that Zenolide produces a homogeneous solution in the corn oil formulation, which can be maintained for up to 4 hours while magnetically stirred and successfully resuspended following ambient temperature storage for 2 days and refrigerated storage for 2 days. The mean concentration at each time-point remained close to nominal (within ±2%) confirming that the formulations were homogeneously distributed between the two bottles and stable (with respect to the level of concentration) during ambient temperature storage for 2 days and refrigerated storage for 2 days. The linearity of detector response was confmned for Zenolide over the concentration range 20 -100 μg/ml. The precision of injection was confumed for six replicate injections of standard solutions containing Zenolide at concentrations of 20 μg/ml and 100 μg/ml, for which the coefficients of variation were less than 0.5%. The procedural recovery data obtained during analytical procedure validation confirm the intra-and inter-day accuracy and precision: a mean procedural recovery value of 97.1% (CV=O.76%, n=10) was obtained for 1 mg/ml and 101.6% (CV=1.00%, 0=10) for 200 mg/ml. Results for the analysis of test samples were corrected for the cumulative mean procedural recovery value at analysis. The limit of detection was determined as 0.06 mg/ml (1 ml of test formulation diluted to 20 ml), using the operating parameters defined in this procedure. The specificity of the GLC assay was demonstrated by the absence of a charasteristic retention time for Zenolide in the control sample chromatogram.

Duration of treatment / exposure:

28 days, daily, followed by a 2-week recovery period

Frequency of treatment:

Animals were treated once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

Group 1: control animals; 10 male and 10 female
Group 2: 15 mg/kg/day; 5 male and 5 female
Group 3: 150 mg/kg/day; 5 male and 5 female
Group 4: 400 mg/kg/day; 5 male and 5 female
Group 5: 1000 mg/day/kg; 10 male and 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

On arrival each animal was weighed and sixty rats were randomly allocated to five groups, two groups consisting of ten males and ten females and three groups consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.
The high dosage (1000 mg/kg/day) was selected on the basis of a preliminary oral toxicity investigation performed at this laboratory (Huntingdon Life Sciences report number IFF 264/973760). The low intermediate and low dosage levels (15 and 150 mg/kg/day) were selected to comply with the most up-todate EEC recommendations for labelling 28-day studies with the R48 risk phase. The high intermediate dose (400 mg/kg/day) was included to provide additional information because of the large gap between the high dose level and the low intermediate dosages.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

Clinical signs: All animals were observed daily (at least three times per day during the dosing phase) for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals.

Bodyweight: All rats were weighed one week prior to the start of dosing (Week -1) and subsequently at twice weekly intervals throughout the study.

Food consumption: The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

Water consumption: Daily monitoring by visual appraisal was maintained throughout the dosing period. There was no evidence of a treatment-related effect on water intake, thus, gravimetric measurement was not performed.

Clinical pathology: Each rat was placed in a urine collecting cage overnight prior to collection of samples, without access to food or water. Overnight urine samples were collected and blood was withdrawn under isofluorane anaesthesia from the orbital sinus of the five male and five female rats selected from Groups 1 and 5 and rats of Groups 2, 3 and 4. These samples were collected after 4 weeks of treatment (Day 30). Investigations were also carried out for remaining rats after the two-week recovery period (Recovery Day16). Blood and urine sampling, when undertaken on Recovery Day 16, followed a similar procedure to that carried out on Day 30.
The collected blood samples were divided as follows: EDTA anticoagulant tubes for haematological investigations; Citrate anticoagulant tubes for coagulation tests; Heparin anticoagulantmicrotainer tubes for biochemical tests (Microtainer, brand plasma separator tube, Becton Dickinson, Rutherford, New Jersey, USA). All tubes were then mechanically agitated for at least five minutes and the microtainer tubes were subsequently centrifuged for a minimum period of two minutes (3000g).
- Haematology: The following parameters were analysed: Packed cell volume, Haemoglobin, Red blood cell count, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Mean corpuscular haemoglobin, Total white cell count, Differential WBC count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelet count, Prothrombin time, Activated Partial Thromboplastin Time, Cell Morphology: the most common morphological changes (anisocytosis, micro/macrocytosis, variation in colour, hypo/hyperchromasia, left shift, atypical/blast cells (In case of atypical/blast cells, or other abnormalities, confirmation or a written description from a blood film were made.
- Biochemistry: The following parameters were analysed: Glucose, Total Protein, Albumin, Globulin, Albumin/Globulin ratio, Urea, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-Glutamyltransferase, Total bilirubin, Sodium, Potassium,
Calcium, Chloride, Inorganic phosphorus, Cholesterol
- Urinalysis: The following parameters were analysed: Volume, Appearance, pH, Specific Gravity, Protein, Total reducing substances, Glucose, Ketones, Bile pigments, Haem pigments. Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscopic slide. The deposit is examined for the presence of the following: Epithelial cells, Polymorphonuclear leucocytes, Mononuclear leucocytes, Erythrocytes, Organisms, Renal tubule casts, Sperm, Other abnormal constituents, Crystals

Sacrifice and pathology:

After 4 weeks of treatment and following clinical pathology investigations, surviving animals selected for post treatment sacrifice were killed on Day 31 by carbon dioxide asphyxiation and a complete autopsy undertaken. These rats received their last dose on Day 30, the day prior to sacrifice. The order of sacrifice was determined using a pre-set cage sequence which allowed for satisfactory intergroup comparison. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination. Remaining rats of Groups 1 and 5 were killed and examined in a similar manner on Day 45 (Recovery Day 17) following a two-week post-treatment observation period; these rats received their last dose on Day 28.

GROSS PATHOLOGY
The macroscopic appearance of the tissues of all rats were recorded and samples of the following tissues were preserved. Eyes were preserved in Davidson's fixative. Testes/epididymides were fixed in Bouin's solution and then transferred to 70% alcohol. All other tissues were preserved in 10% buffered formalin
- adrenals*, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), femur (for bone marrow and sections) with joint, Harderian gland head (to preserve nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal's gland), heart* ileum, jejunum, kidneys* larynx, liver* lungs (with bronchi), lymph nodes (mandibular and mesenteric), mammary glands, oesophagus, optic nerve, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary glands, sciatic nerves, seminal vesicles, skeletal muscle, skin, spinal cord spleen*, stomach, sternum, testes including epididymis* (Bouin's as fixative), thymus (where present), thyroids (with parathyroid), tongue, trachea, urinary bladder, uterus (with cervix), vagina, any macroscopically abnormal tissue*
*Tissues required for histopathological examination for all rats

ORGAN WEIGHT
The following organs from each animal were dissected free of fat and weighed: adrenals, liver, seminal vesicles, brain, ovaries, spleen, kidneys, prostate, testes and epididymides. Paired organs were weighed together

HISTOPATHOLOGY
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (m.p. 56°C); sections were cut at 4 µm and stained with haematoxylin and eosin. A transverse section of each testis and a full longitudinal section of each epididymis were cut as near as possible to 2 µm and stained with PAS (Periodic Acid Schiff}-haematoxylin. Microscopic examination ofthe testes was made with reference to the stages of the cycle of the seminiferous epithelium. Microscopic examination of prepared slides (from tissues indicated under Gross pathology) was carried out for all animals.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. The following sequence of statistical tests were used for bodyweight gains, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode >75%), proportion of values different from the mode was analysed, (Fisher 1950) and (Mantel 1963). Otherwise:
- A test was applied to test for heterogeneity of variance between treatments, (Bartlett 1937). Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, an analysis of rank was used, (Kruskal and Wallis 1952/3).
- Except for pre-dose data, analyses of variance were followed by Student's t test and Williams' test (Williams' 1971/2) for a dose-related response, only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the nonparametric equivalents of these tests, (Shirley 1977).
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For most parameters, the appropriate covariate was the same parameter at pre-dose. For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate when the within-group relationship between organ weight and bodyweight was significant at the 10% level in an attempt to allow for differences in bodyweight which may have influenced the organ weights (Angervall, and Carlstrom, 1963). Significant differences between control animals and those treated with the test substance were expressed at 5%.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Post-dosing salivation was observed in all treated groups. At 1000 and 400 mg/kg/day, the post-dosing salivation was accompanied by the associated finding of post-dosing wet coat. These signs were generally evident immediately after dosing and had ceased in all but a few instances by the one hour post-dosing observation reading. At 15 mg/kg/day, clear post-dosing salivation was observed in three males on Day 6 at the zero hour post-dosing observation time only. At 150 mg/kg/day, brown post-dosing salivation was observed in one male on Day 6 and in one female on Day 7 at the zero hour post-dosing observation time only. At 400 mg/kg/day, post-dosing salivation, occasionally brown in colouration, was observed from Day 4 in the majority of animals. This sign was occasionally accompanied by the associated finding of post-dosing wet coat. At 1000 mg/kg/day, post-dosing salivation, occasionally brown in colouration, was observed from Day 3 in the majority of animals. This sign was frequently accompanied by the associated finding of post-dosing wet coat. Throughout the recovery period there were no clinical signs apparent. The clinical findings of post-dosing salivation and associated post-dosing wet coat are not uncommon in orally dosed rat studies and are considered to be associated with unpalatability of the test substance rather than toxicity. Therefore, whereas in this study these clinical signs are considered to be related to treatment with the test material, they are not considered to be of toxicological importance.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences from control for bodyweight gain in any of the treatment groups during the treatment or recovery periods. Bodyweight gain for all treated groups was generally comparable with controls and there was no effect of treatment during the treatment or recovery periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intake for all treated groups was generally comparable with controls and there was no effect of treatment during the treatment or recovery periods.

Food efficiency:

no effects observed

Description (incidence and severity):

Efficiency of food utilisation was comparable with controls for all treated groups and there was no effect of treatment during the treatment or recovery periods.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects. Differences from control, including those attaining statistical significance (increased MCHC, MCH and platet count at the end of the treatment period; increased RBC count and decreased MCHC at the recovery period), were minor in degree and were not considered to be related to treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

On Day 30, group mean GOT levels were lower than concurrent controls for males and females receiving 1000 mg/kg/day and females only receiving 400 mg/kg/day. On Recovery Day 16, there was no similar finding apparent. These lower GOT levels are not considered to be of toxicological importance as lower levels than controls were seen which are not considered to be associated with cell damage. Additionally, the mean and all individual GOT values for these treatment groups were within the background range for this parameter (GOT, mU/ml, female rat: 58 (46 -72), GOT, mU/ml, male rat: 61 (47 -94), figures are median (5-95 percentile)). Slight increases in sodium and chloride were noted among females receiving 1000 or 400 mg/kg/day. A similar finding was not apparent in males. Increased kidney weights were observed in these animals at post mortem however there were no macroscopic or histologic changes associated with treatment. Similar differences were not apparent following recovery. These slight increases in sodium and chloride are not considered to be related to treatment. Other differences from control, including those attaining statistical significance (increased albumin glucose and sodium and decreased GPT, urea and potassium at the end of the treatment period; decreased creatinine at the recovery period), were minor in degree and were not considered to be related to treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no treatment-related effects. The statistically significant decrease in urinary pH for 1000 mg/kg/day females at the end of the treatment period was minor in degree and was not considered to be related to treatment. The mean and majority of individual urinary pH values for this treatment group were within background range for this parameter (urinary pH, female rat: 6.5 (5.8 -8.1) figures are median (5-95 percentile)).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Group mean bodyweight-adjusted kidney weights were higher for females receiving 1000 or 400 mg/kg/day in comparison with controls, the differences attaining statistical significance. A similar finding was apparent for males at these dosages but to a lesser (not statistically significant) degree. These increases were not apparent following the recovery period. No macroscopic or histopathologic changes were observed in the kidneys of females receiving 1000 or 400 mg/kg/day which were associated with treatment. Therefore the increased kidney weights for 1000 and 400 mg/kg/day females were not considered to be of toxicological importance. All individual kidney weight values were within background range for this parameter (kidney weight, grams, female rat: 1.97 (1.49 -2.62), figures are median (5 -95 percentile)). Group mean bodyweight-adjusted spleen weights were lower for females receiving 1000 mg/kg/day in comparison with controls. However, no dosage relationship was evident and a similar finding was not apparent for males. This difference was not apparent for the recovery period. The lower spleen weights for 1000 mg/kg/day females were not considered to be related to treatment. The mean and majority of individual spleen weight values for this treatment group were within background range for this parameter (spleen weight, grams, female rat: 0.52 (0.39 -0.67), figures are median (5 -95 percentiles))

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination performed at both the terminal and recovery post mortems revealed no changes associated with treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were detected. Minor microscopic changes reported (including isolated unilateral changes in testis and epididymis of one 150 mg/kg/day male at the end of the treatment period and one 1000 mg/kg/day male at the recovery period and occurrences of mineralisation at the corticomedullary junction in the kidney of control and treated females at the end of treatment and recovery periods) were considered to be spontaneous in origin and of no toxicological importance. No microscopic findings were seen in the kidneys of females receiving 1000 or 400 mg/kg/day which were considered to be associated with the increased bodyweight-adjusted kidney weights for these treatment groups.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 407, GLP), the NOAEL was determined to be >=1000 mg/kg bw/day.

Executive summary:

In a 28-days repeated dose toxicity study with a 2-week recovery period, the test substance was administered orally to rats at dosages of 15, 150, 400 or 1000 mg/kg bw/day. Control animals received the vehicle, corn oil, alone. No adverse effects considered to be associated with treatment were observed for mortality, clinical signs, bodyweight, food consumption, efficiency of food utilisation, haematology, and urinalysis. Lower glutamic oxalacetic transaminase (GOT) levels were noted in females receiving 1000 or 400 mg/kg bw/day and males receiving 1000 mg/kg bw/day. However, these findings are not considered to be of toxicological importance as reduced plasma GOT levels are not considered to be associated with cell damage. These findings were also not apparent at day 16 of recovery. Slight increases in plasma sodium and chloride levels were seen among females receiving 1000 or 400 mg/kg bw/day. Also these findings were not apparent at recovery and are not considered to be of toxicological importance. Relative kidney weights were statistically significant for females receiving 1000 or 400 mg/kg bw/day in comparison with controls. A similar finding was apparent for males at these dosages but not statistically significant different. These increases were not apparent following the recovery period. In addition, there were no macroscopic or histopathological changes associated with treatment, hence these findings are not considered to be of toxicological importance. As there were no adverse treatment-related findings in this study, it is concluded that 1000 mg/kg bw/day, the highest dose tested, represents the NOAEL when administered orally for at least 28 days.