Oxazolidine, 3-butyl-2-(1-ethylpentyl)-

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:

2005-03-23 to 2005-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 27th, 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 30th 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-660-0

EC Name:

-

Cas Number:

165101-57-5

Molecular formula:

C14H29NO

IUPAC Name:

3-butyl-2-(heptan-3-yl)-1,3-oxazolidine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5-8 weeks old
- Weight at study initiation: male: 144-173 g; female: 139-163 g
- Fasting period before study: no
- Housing: in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet: Rodent 5LF2 (pellets), ad libitum
- Water: mains water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

dried

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in dried Arachis oil BP. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. The formulations were stable for at least 14 day. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Incozol 2 in the test material formulations was determined by gas chromatography (GC) using an external standard technique. The test material formulations were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/mL. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/mL.

The standard and sample solutions were analysed by GC using the following conditions:

GC system: Agilent Technologies 5890, incorporating
autosampler and workstation
Column: DB-1 (15 m x 0.53 mm id x 1.5 µm film)
Oven temperature program: initial 70 °C for 1 mins
rate 20 °C/min
final 250 °C for 1 mins
Injection temperature: oven track on column injection
Flame ionisation detector temperature: 200°C
Injection volume: 1 µL
Retention time: 5.6 min

Duration of treatment / exposure:

28

Frequency of treatment:

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.:

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

No. of animals per sex per dose:

five animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selection was based on a preliminary range finding study in the rat (14 days repeated dose toxicity study)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on Days 3, 10, 16 and 23, all animals were observed for signs of behavioural toxicity. Observations were carried out from approximately two hours after dosing on each occasion.
- observation sites: Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on day 1 and at weekly intervals thereafter. Bodyweights were also performed prior to terminal kill.

FOOD CONSUMPTION: Yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters checked: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Animals fasted: No
- How many animals: all
- Parameters checked: Urea Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: every day
- Dose groups that were examined: animals of one sex
- Battery of functions tested: sensory activity, Forelimb/Hindlimb grip strength, motor activity

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:

Adrenals, Liver, Brain (including cerebrum, cerebellum and pons), Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus

For Histopathology samples of the following tissues were removed from all animals, plus any gross lesions, and preserved in buffered 10% formalin:

Adrenals, Aorta (thoracic), Bone and bone marrow (femur including stifle joint), Bone and bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical), Lymph nodes (mesenteric), Muscle (skeletal), Oesophagus, Ovaries, Parathyroid, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary) Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical), Spleen, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder, Uterus

All tissues were despatched to the Test Site (histology processing) for processing (Principal Investigator: N Candy). The tissues shown below, plus any gross lesions, from all control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination:

Adrenals, Bone and bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical), Lymph nodes (mesenteric), Ovaries, Parathyroid, Urinary bladder, Uterus, Prostate, Rectum, Spleen, Stomach, Testes, Thymus, Thyroid, Trachea, Sciatic nerve, Seminal vesicles, Spinal cord (cervical)

Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg/day dose group animals.

Other examinations:

No

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. All data was summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Reduced alkaline phosphate levels were detected for animals of either sex treated with 1000 and 150 mg/kg bw/day, extending into the female 15 mg/kg bw/day dose group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Elevated kidney and liver weights both absolute and relative to terminal bodyweights were detected for animals of either sex treated with 1000 mg/kg bw/day.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centrilobular hepatocyte enlargement was observed in relation to treatment for males treated with 1000 mg/kg bw/day. One female treated with 1000 mg/kg bw/day was similarly affected.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study. Increased salivation was detected immediately after dosing and on occasion, up to one hour after dosing, for animals of either sex treated with 1000 mg/kg bw/day from Day 4 onwards. Increased salivation was also observed, on occasion, prior to dosing at the high dose and was also observed immediately post-dose for one female treated with 150 mg/kg bw/day on Day 18. In addition, isolated instances of staining around the mouth, snout and fur were also observed. Females treated with 1000 mg/kg bw/day showed isolated incidents of hunched posture from Days 18 to 20. These observations were most probably attributed to the administration of a slightly irritant or unpalatable test material formulation and not a consequence of systemic toxicity. The incidental observation of staining around the ano-genital region for one 1000 mg/kg bw/day female was isolated and as such, was considered to be of no toxicological importance. One female treated with 1000 mg/kg bw/day and one female treated with 15 mg/kg bw/day displayed damage to the tail tip. These physical injuries were unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN

Males treated with 1000 and 150 mg/kg bw/day showed statistically significant reductions in bodyweight gains (p<0.01) in comparison to controls, during the final week of treatment. Incidentally, females treated with 1000 mg/kg bw/day showed a statistically significant increase in bodyweight gain in comparison to controls (p<0.01). The toxicological significance of these findings is unclear due to the contradictory data, but considered not to represent an adverse health effect.

FOOD CONSUMPTION AND COMPOUND INTAKE

No adverse effect on dietary intake was detected throughout the study.

FOOD EFFICIENCY

Food efficiency was observed to be lower for treated males during the final week of treatment, in comparison to controls. Incidentally, food efficiency for 1000 mg/kg bw/day females was increased during the final week of treatment, in comparison to controls. Because these effects were contradictory and only present in the final week of treatment, the toxicological significance of this finding was unclear but was considered not to represent an adverse effect of treatment.

WATER CONSUMPTION AND COMPOUND INTAKE

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

HAEMATOLOGY

There were no treatment-related changes detected in the haematological parameters measured. The statistically significant reductions in haemoglobin, mean cell haemoglobin and mean cell haemoglobin concentration detected throughout the male treatment groups, were considered attributed to higher than expected control values, and in the absence of a convincing dose-related response were considered to be of no toxicological significance.

CLINICAL CHEMISTRY

No toxicologically significant blood chemical changes were detected. Statistically significant reductions in alkaline phosphatase level were detected for animals of either sex treated with 1000 and 150 mg/kg bw/day, extending into the female 15 mg/kg bw/day dose group. The reductions may be a result of altered metabolism attributed to the administration of a xenobiotic and was considered to be adaptive and of no toxicological importance. The statistically significant reductions in chloride concentration detected for males treated with 1000 and 150 mg/kg bw/day and for females treated with 1000 mg/kg bw/day, in the absence of a convincing electrolyte imbalance, were considered to be without toxicological significance. The statistically significant reduction in inorganic phosphate detected for 1000 mg/kg bw/day males and the increase in this parameter detected for 1000 mg/kg bw/day females was of minimal significance (p<0.05) and considered to have arisen incidentally. The statistically significant reduction in blood urea detected for animals of either sex (p<0.01), in the absence of any associated histopathological correlates, was considered to be of no toxicological significance. Additionally, the reduction in alanine aminotransferase levels detected for females treated with 15 mg/kg bw/day, was of minimal significance (p<0.05) and in the absence of a dose-related response, was considered to have arisen fortuitously.

NEUROBEHAVIOUR

There were no treatment-related changes in the functional performance parameters measured. Statistical analysis of the data revealed no significant intergroup differences. There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

ORGAN WEIGHTS

Animals of either sex treated with 1000 mg/kg bw/day showed elevated liver weights, both absolute and relative to terminal bodyweights, however, statistical significance was only achieved for males(p<0.01). Elevated kidney weights, both absolute and relative to terminal bodyweights were also detected for animals of either sex treated with 1000 mg/kg bw/day (males: p<0.05, females: p<0.01). Females treated with 1000 mg/kg bw/day showed statistically significant reductions in brain weight, both absolute and relative to terminal bodyweight. The significance was minimal (p<0.05) and in the absence of any histopathological correlates, this finding was considered to have arisen incidentally. No such effects were detected for animals of either sex treated with 150 or 15 mg/kg bw/day. The statistically significant increase in thymus weight, both absolute and relative to terminal bodyweight, detected for 15 mg/kg bw/day males, in the absence of a dose-related response, was considered to be of no toxicological significance.

GROSS PATHOLOGY

No treatment-related macroscopic abnormalities were detected at terminal kill. One female treated with 1000 mg/kg bw/day showed reddened lungs and one male treated at this dose level showed pallor of both kidneys. These isolated findings were considered to be incidental and of no toxicological importance. No treatment related macroscopic abnormalities were detected for animals of either sex treated with 150 or 15 mg/kg bw/day. One control female incurred damage to the left eye during the removal procedure. This was attributed to technical error and unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC

The following treatment-related changes were observed:
LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for males treated with 1000 mg/kg bw/day. One female was similarly affected, but hepatocyte enlargement does occasionally occur spontaneously and there is insufficient evidence of an effect in females in this instance. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

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

Basis for effect level:

other: highest dose tested

Remarks on result:

other: 1000 mg/kg bw/day resulted in microscopic hepatic changes at 1000 mg/kg bw/day and reduced alkaline phosphatase levels detected at all dose levels. These changes were entirely adaptive in nature and considered not to represent an adverse health effect.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material Incozol 2 to rats for a period of twenty-eight consecutive days at dose levels of 15, 150 and 1000 mg/kg bw/day resulted in microscopic hepatic changes at 1000 mg/kg bw/day and reduced alkaline phosphatase levels detected at all dose levels. These changes were entirely adaptive in nature and considered not to represent an adverse health effect. The NOAEL was therefore 1000 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity of the test material according to EU Method B.7 and OECD TG 407. The test material was administered by oral gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (dried Arachis oil BP).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths during the study. No clinically observable signs of toxicity were detected. No treatment related effects were detected with regard to behavioural, functional performance and sensory reactivity assessment. Body weight gain and food consumption showed no adverse effects. No intergroup differences were detected while examining water consumption. In haematology no effects were detected relating to the treatment. For clinical chemistry reduced alkaline phosphate levels were detected for animals of either sex treated with 1000 and 150 mg/kg bw/day, extending into the female 15 mg/kg bw/day dose group. Elevated kidney and liver weights, both absolute and relative to terminal bodyweights, were detected for animals of either sex treated with 1000 mg/kg bw/day. No such effects were detected at 150 or 15 mg/kg bw/day. No abnormal macroscopic alterations were detected during necropsy. Histopathology showed centrilobular hepatocyte enlargement in relation to treatment for males treated with 1000 mg/kg bw/day. One female treated with 1000 mg/kg bw/day was similarly affected. The NOAEL was therefore considered to be 1000 mg/kg bw/day (Safepharm, 2005).