4-oxo-4-(p-tolyl)butyric acid adduct with 4-ethylmorpholine

Endpoint summary

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:

13.02.1995 - 08.08.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Sprague-Dawley strain CD rats were obtained from Charles River (UK) Limited, Manston, Kent. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for eight days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) 134 to 179 g, and the females weighed 129 to 157 g, and were approximately five to six weeks old.
The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water, except during urine collection when food was withdrawn overnight. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was used. Mains water was supplied from polycarbonate bottles attached to the cage. the diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of the hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity were maintained within target ranges of 21 +/- 2 °C and 55 +/- 15% respectively. Occasional deviations from these nominal values were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to dose groups using random letter tables and the group mean bodyweights were then determined to ensure similarity between the dose groups. The cage distribution within the holding rack was also determined.
The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories. Colour coded cage labels were used to assist recognition of dose groups.

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg/day of arachis oil B. P.. One satellite high dose male developed severe respiratory problems on Day 28 and, as a consequence, remained undosed for the final day of treatment. Animals from satellite groups 5 and 6 were maintained for a further fourteen days treatment-free period following termination of treatment.

Vehicle:

arachis oil

Details on oral exposure:

For the purpose of the study the test material was prepared at the appropriate concentrations as a suspension in arachis oil B. P.. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. The results show the formulations to be stable for at least ten days. Formulations were therefore prepared weekly and stored at 4 °C in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test material formulation and were analysed for concentration at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within +/- 11% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg/day of arachis oil B. P.. One satellite high dose male developed severe respiratory problems on Day 28 and, as a consequence, remained undosed for the final day of treatment. Animals from satellite groups 5 and 6 were maintained for a further fourteen days treatment-free period following termination of treatment.

Observations and examinations performed and frequency:

Observations
Clinical observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends and on public holidays. During the treatment-free period, satellite group animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.
Bodyweight
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28 and, in the case of satellite group animals, on Days 35 and 42. Bodyweights were also recorded at necropsy.
Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.
Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from Groups 1 to 4 at the end of the treatment period (Day 28) and on all satellite group animals (Groups 5 and 6) at the end of the treatment-free period (Day 42). Blood samples were obtained prior to necropsy on Day 29 by cardiac puncture. Animals were not fasted prior to sampling.
Urinalysis was performed on all animals from Groups 1 to 4 during Week 4. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Enthrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices -mean corpuscular haemoglobin (MCH)
-mean corpuscular volume (MCV)
-mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count -neutrophils (Neut)
-lymphocytes (Lymph)
-monocytes (Mono)
-eosinophils (Eos)
-basophils (Bas)
Platelet count (PLT)
Clotting (prothrombin) time (CT) was assessed by 'Hepato Quick' using samples collected into sodium citrate solution (0.11 mol/l).
Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot. Prot)
Albumin
Albumin/Globulin (A/G)
ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic Phosphorus (P)
Gamma glutamyl transpeptidase (γ-GT)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Triglycerides (Tri)
Total cholesterol (Chol)
Total bilirubin (Bili)
Urinalysis
The following parameters were measured on freshly collected urine.
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Reducing substances
Blood
Organ Weights
The following organs, removed from animals that were killed at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Gonads
Heart
Kidneys
Liver
Pituitary
Spleen
Histopathology
Samples of the following tissues were removes from all animals and preserved in buffered 10% formalin:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain
Caecum
Colon
Duodenum
Eyes
Gross lesion
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs
Lymph nodes (cervical and mesenteric)
Muscle (skeletal)
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spleen
Stomach
Testes
Thymus
Thyroid/parathyroid
Trachea
Urinary bladder
Uterus
All tissues were despatched to Experimental Pathology Services, Willow Court, Netherwood Road, Rotherwas, Hereford, UK for processing. The following preserved tissues from all control and high dose group animals (Groups 1 and 4) were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination:
Adrenals
Heart
Kidneys
Liver
Spleen
Macroscopically observed lesions were also processed
Microscopic examination was conducted by the Study Pathologist. All findings were entered into ROELEE84 pathology computerisation system for tabulation and report production.
Since there were indications of treatment-related hepatic changes, examination was subsequently extended to include similarly prepared sections of liver from all remaining test and control group males.

Sacrifice and pathology:

Pathology
On completion of the dosing period or, in the case of satellite group animals, at the end of the treatment-free period; all animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal, 60 mg/ml; May and Baker Limited, Dagenham, Essex, UK) followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High dose animals of either sex showed transient increased salivation approximately two minutes after dosing from Day 4 onwards. There were a few isolated incidents of prolonged increased salivation one hour after dosing together with red/brown staining around the mouth, snout, ano-genital region or of the fur. Incidents of noisy respiration were observed in the majority of high dose animals from Day 3 onwards together with occasional signs of decreased respiratory rate. Such observations concerning excessive visible salivation and respiratory pattern changes are commonly reported when the test material formulation has an unpleasant taste or is slightly irritant and are considered not to be indicative for toxicity. One satellite high dose male, however, developed severe respiratory problems from Day 27, including decreased respiratory rate, noisy, laboured and gasping respiration. As a consequence this animal remained undosed for the final day of treatment.
Satellite high dose animals showed a swift recovery upon cessation of treatment with a regression in clinical signs seen during the treatment-free period. Noisy respiration was noted in the adversely affected male on Day 36 only whilst three females were reported as having noisy respiration from Day 35 to Day 37 continuing in one of these animals throughout the remaining study period.
No clinically observable signs were detected in the other dose groups throughout the treatment period. One low dose male showed dorso-cervical fur loss from Days 1 to 5 followed by scab formation in the same region from Days 17 to 25. Dorso-cervical scab formation was also noted in one satellite high dose male from Day 11 to Day 15. Physical injuries of this nature are occasionally seen when animals are housed for prolonged periods and are of no toxicological importance.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose males showed a reduction in bodyweight gain throughout the treatment period which achieved statistical significance for Weeks 1 through to Week 4. This reduction was generally reflected in satellite high dose males although statistical significance was only achieved for Week 3. A statistically significant reduction in bodyweight development was noted for both main and satellite high dose females during the first week of treatment only when compared with controls.
No adverse effect on bodyweight development was detected in the other dose groups. The remaining statistically significant difference detected between test and control groups was confined to an increase in bodyweight gain for satellite high dose females during Week 6. Such an increase cannot be regarded as an adverse effect of treatment and, as a result, was considered to be of no toxicological relevance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

High dose males showed reduced dietary intake throughout the treatment period when compared with that of controls and food efficiency was also slightly reduced. Satellite high dose males showed reduced food consumption during Week 1 only with a reduction in food efficiency noted during Week 3. A treatment-related effect on dietary intake was also seen for high dose females but was confined to Weeks 1 and 2 for main group animals only. Food efficiency was reduced during Week 1 for both main and satellite high dose females with the satellite animals continuing to show reduced food efficiency during Week 3. A complete recovery was seen in these animals throughout the treatment-free period with dietary intake and food efficiency comparable with satellite controls during Weeks 5 and 6.
Animals in the remaining dose groups showed no adverse effects on food consumption during the treatment period.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

High dose males showed reduced dietary intake throughout the treatment period when compared with that of controls and food efficiency was also slightly reduced. Satellite high dose males showed reduced food consumption during Week 1 only with a reduction in food efficiency noted during Week 3. A treatment-related effect on dietary intake was also seen for high dose females but was confined to Weeks 1 and 2 for main group animals only. Food efficiency was reduced during Week 1 for both main and satellite high dose females with the satellite animals continuing to show reduced food efficiency during Week 3. A complete recovery was seen in these animals throughout the treatment-free period with dietary intake and food efficiency comparable with satellite controls during Weeks 5 and 6.
Animals in the remaining dose groups showed no adverse effects on food consumption during the treatment period.

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no overt intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in haematological parameters measured.
Statistically significant increases in erythrocyte count (RBC) and clotting time (CT) were detected for high dose males in comparison with controls whilst platelet count (PLT) was reduced for high dose females. All values for these parameters were within the respective normal ranges (male RBC; 6.8 - 8.4 x 1012/l; male CT; 23 - 32 secs, female PLT; 816 - 1394 x 109/l) and, as isolated, negligible changes all were considered to be without toxicological significance. A slight but statistically significant reduction in mean corpuscular haemoglobin was detected for high dose females in comparison with controls. In the absence of any other changes in erythrocyte indices and in view of the minimal level of statistical significance achieved (p < 0.05), the reduction was considered to be a consequence of slightly elevated control values rather than an effect of treatment. Statistically significant increases in mean corpsular volume and clotting time and a reduction in mean corpsular haemoglobin concentration were detected for satellite high dose females at the end of recovery period. No such changes were apparent in main high dose females after twenty-eight days of treatment and these were , therefore, considered to be of no toxicological importance.
The remaining statistically significant difference detected between test and control groups was confined to an increase in neutrophil count for low dose females. No dose relationship was detected and the increase was, therefore, regarded as fortuitous.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood Chemistry
A statistically significant increase in alanine aminotransferase was detected for high dose animals of either sex with three individual male and female values outside the respective normal ranges for rats of this strain and age. Aspartate aminotransferase and plama bilirubin levels were also significantly elevated for high dose males or females respectively when compared with controls. Plasma triglyceride levels were also elevated for animals of either sex and, although the group mean for males did not achieve statistical significance, one value was outside the normally expected range for this sex. A similar effect was noted for high dose female alkaline phosphatase levels. An increase in the group mean was apparent when compared with controls, although statistical significance was not achieved, but one value was outside the normal range. High dose females also showed a slight but statistically significant reduction in plasma cholesterol. All values were within the normal range for rats of this strain and age (41 - 98 mg/dl) and the reduction was considered to be a result of slightly elevated control values rather than a direct effect of treatment. Satellite high dose females showed a reduction in total plasma protein but a similar change was not evident for main high dose animals at the end of the dosing period and the reduction was, therefore, considered to be fortuitous. The remaining statistically significant differences between test and control groups were confined to low or intermediate dose females and, as such, showed no dose-related response.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

High dose males showed an increase in urine volume and reduction in specific gravity which achieved statistical significance for both parameters when compared with controls. High dose satellite males recovered upon cessation of treatment to reveal no treatment-related intergroup differences. A statistically significant increase in specific gravity was noted, but this did not correlate with the changes seen for main high dose group males and was, therefore, considered to be fortuitous.
There were no treatment-related changes in the urinalytical parameters measured for high dose females or animals of either sex in the remaining treatment groups.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

High dose females showed a statistically significant increase in relative liver weight in comparison with controls with two of the individual values outside the normal ranges for rats of this strain and age. No such effect was demonstrated in satellite high dose females after fourteen days with no treatment.
There were no other changes in the organ weights measured which could be considered toxicologically significant. The statistically significant reductions seen in absolute liver, pituitary and spleen weight for high dose males were probably a consequence of the reduced bodyweight gain seen in these animals at the end of the treatment period and were not, therefore, a direct response of treatment. A statistically significant increase in brain weight, relative to bodyweight, was detected for high dose females but all values were within the normal range (0.6784 - 0.9164%) and, in view of the minimal level of statistical significance achieved (p<0.05), the increase was considered to be fortuitous. Satellite high dose males showed a significant reduction in relative liver weight after fourteen days without treatment whilst satellite high dose females showed an increase in relative kidney weight. Neither change was seen for main high dose animals at the end of the dosing period and, as such, both were considered not to be toxicologically important.
The remaining statistically significant differences detected between test and control groups were confined to low dose animals and, as such, showed no dose-related response.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy
No treatment-related macroscopic abnormalities were detected.
The incidental findings recorded at necropsy were consistent with those normally expected in laboratory maintained rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Generalised hepatocyte enlargement was observed in relation to treatment for rats of either sex dosed at 1000, 150 and 15 mg/kg/day. Such a change is commonly observed in the rodent liver following the administration of xenobiotics. The entirely adaptive nature of the condition amongst satellite 1000 mg/kg/day animals following an additional fourteen days without treatment.
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

< 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Key result

Critical effects observed:

no

Conclusions:

Oral administration of the test material to rats for a period of twenty-eight consecutive days, at dose levels of up to 1000 mg/kg/day, resulted in treatment-related effects at dose levels of 15, 150 and 1000 mg/kg/day. The "No Observed Effect Level" (NOEL) is, therefore, considered to be less than 15 mg/kg/day.
The toxicologically significant findings at 150 or 15 mg/kg/day were confined to minimal adaptive liver changes. These were considered not to be indicative of serious damage to the health of the animals as defined by the criteria given in the EC labelling guide of Commission Directive 93/21/EEC.

Executive summary:

The oral administration of the test material by gavage for a period of twenty-eight consecutive days resulted in treatment related changes at dose levels of 1000, 150 and 15 mg/kg/day.

During the dosing period animals treated with 1000 mg/kg/day showed increased salivation approximately two minutes and, occasionally up to one hour after dosing, together with associated fur staining and incidents of noisy respiration interspersed with sporadic signs of decreased respiratory rate. Such observations are commonly reported when the test material formulation has an unpleasant taste or is slightly irritant and are considered not to be indicative of toxicity. One satellite high dose male, however, developed severe respiratory problems from Day 27 and, as a consequence remained undosed for the final day of treatment. Bodyweight gain and food consumption were adversely affected for high dose males throughout the treatment period and, to a lesser extent, for high dose females during Weeks 1 and 2. Urinalytical investigations revealed an increased urine volume of reduced specific gravity for high dose males. Such effects would normally be expected with an unpleasant tasting test material formulation as a result of the animals drinking more. In the absence of an effect on water consumption, or any evidence of renal impairment, however, the aetiology of this change is unclear.

Blood chemical changes were detected for high dose animals of either sex. These included increases in alanine aminotransferase and triglycerides for males and females, increased aspartate aminotransferase for males and increased bilirubin and alkaline phosphatase for females, all of which suggest an adverse effect on the liver. Relative liver weights were elevated for high dose females and microscopic examination of liver sections revealed changes identified as generalised hepatocyte enlargement. This morphological change is often seen in rodent liver following treatment with xenobiotics and, in the absence of any associated degenerative or inflammatory changes, is considered to be an adaptive response. The increase in levels of plasma enzymes of hepatic origin, however, suggest a change in hepatocellular integrity at the high dose level, which must be regarded as toxicologically important.

A dose-related hepatic response extended into the intermediate and low dose group. Three males and four females treated with 150 mg/kg/day showed slight or minimal hepatocyte enlargement whilst only one male and one female treated with 15 mg/kg/day showed minimal hepatocyte enlargement. This was not associated with an effect in any of the other parameters measured and, was considered to be entirely adaptive in nature, not representing any adverse effect on the health of the animals treated at these levels.

Effects of treatment with the test material were apparently reversible since satellite high dose animals showed a partial regression in the condition after two weeks without treatment.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subacute

Species:

rat

System:

hepatobiliary

Organ:

liver

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