Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl

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:

1995-04-20 - 1996-01-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Scientifically valid GLP study on the registered substance itself according to OECD guideline 407, EU method B7 and the Japanese MHW guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare (MHW) Guidelines 1986 for a twenty-eight day repeat dose oral toxicity study as required by the Japanese Chemical Control Law 1973 of the Ministry of International Trade and Industry (MITI) amended 1986.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl

EC Number:

943-016-1

Molecular formula:

Too complex

IUPAC Name:

Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl

Test material form:

liquid: viscous

Details on test material:

- Substance type: pure substance
- Storage condition of test material: room temperature
- Other: description: pale straw-coloured viscous liquid

Test animals

Species:

rat

Strain:

other: Sprague-Dawley strain CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent, UK
- Age at study initiation: 6 weeks
- Weight at study initiation: 116 to 157g (males), 119 to 153g (females)
- Fasting period before study: none
- Housing: in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) ad libitum
- Water (e.g. ad libitum): Mains water ad libitum
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 15, 150, 750 mg active ingredient in 2 mL
- Amount of vehicle (if gavage): 2 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. The formulations were shown to be stable for at least ten days. Formulations were therefore prepared weekly and stored at 4°C in the dark.
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique.
Samples: The test material formulations were diluted with acetone to give a final theoretical test material concentration of approximately 1 mg/ml.
Standards: Standard solutions were prepared in acetone at a nominal concentration of 1 mg/ml.
Procedure: The standard and sample solutions were analysed by GC using the following conditions:
Column :
DB-1 (30 m X 0.25 mm id x 0.25 µm film)
Oven temperature program :
Initial: 120°C for 1 minute
Rate: 10°C for 1 minute
Final: 300°C
Injection temperature : 250°C
Flame ionisation detector temperature : 300° C
Injection volume : 1 µl
Homogeneity Determinations The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking in between sampling. The sampling was performed in triplicate.
Stability Determinations The test material formulations were sampled and analysed initially and then after storage at approximately 4°C in the dark for ten days.
Verification of Test Material Formulation Concentrations: The test material formulations were sampled and analysed within three days of preparation.

Duration of treatment / exposure:

Up to twenty-eight consecutive days.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 15, 150, 750 mg/kg
Basis:
other: dose level of active ingredient in arachis oil

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 15.8, 158, 789 mg/kg
Basis:
other: dose level of test material in arachis oil, incorporating a correction factor for 95% purity

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Results from the 14-day range finding study regarding clinical observations, bodyweight and gross pathology, attached tot he present study report. Rats were treated similarly to the main study, each three rats/sex/dose were treated with either 0, 150, 400 or 1000 mg/kg bw/day of the test item, and Necropsy data, bodyweights and clinical observations were gathered. The following observations were made:

Mortality: One female dosed at 1000 mg/kg/day was killed in extermis approximately two hours and thirty minutes after dosing on Day 3, due to the severe clinical signs of toxicity observed. There were no further deaths during the study

Clinical Observations: Animals dosed at 1000 mg/kg/day showed short-lived increased salivation immediately after dosing from Day 1 onwards. With the exception of an isolated incident of prolonged increased salivation, these animals showed no further signs of toxicity until Day 3, when all six animals underwent a sudden deterioration in condition approximately one hour after dosing. Occasional body tremors were also evident approximately ninety minutes later. In addition, females showed diuresis and fur loss and one of these animals also showed tiptoe gait and red/brown staining around the mouth. This animal was killed in extremis immediately. Occasional body tremors were no longer evident before treatment on Day 4 but the surviving animals showed a heightened response to external stimuli approximately one hour after dosing. This behaviour was especially noticeable amongst the males and was observed daily until the end of Week 1. In addition to these clinical signs, all surviving animals dosed at 1000 mg/kg/day showed fur loss from Day 6 onwards and individual animals showed sporadic incidents of fur wetting together with red/brown staining around the mouth, of the ano-genital region and of the ventral fur.
Animals dosed at 400 mg/kg/day showed short-lived increased salivation immediately after dosing from Day 4 onwards together with isolated incidents of associated fur wetting and red/brown staining around the mouth. In addition, all females showed a heightened response to external stimuli approximately one hour after dosing on Day 6 only. At 150 mg/kg/day, clinical signs of toxicity were confined to short-lived increased salivation, observed immediately after dosing from Day 4 onwards.
Transient increased salivation often accompanies the oral administration of an unpleasant tasting or locally irritant test material formulation and, in isolation, may not be indicative of systemic toxicity.

Bodyweight: Males dosed at 1000 mg/kg/day showed a 58% lower bodyweight gain than controls during the first week of treatment. The adverse effect on bodyweight development regressed slightly amongst these animals during Week 2 although bodyweight gain remained 35% lower than that of controls. Males dosed at 400 mg/kg/day showed a 17% lower bodyweight gain than controls during the study, again with the adverse effect slightly more noticeable during Week 1.
Males dosed at 150 mg/kg/day and females from all three treatment groups showed a similar bodyweight gain to controls during the study.

Necropsy: At terminal kill, the two surviving females dosed at 1000 mg/kg/day showed patchy pallor of the liver whilst two of the males from the same dose group showed a small spleen. One of these males also showed pale adrenals whilst gastric abnormalities were observed for several other surviving animals from this dose group, including liquid gastric contents and sloughing of the glandular mucosa. The female killed in extremis on Day 3 showed no macroscopic abnormalities at necropsy.
Animals dosed at 400 or 150 mg/kg/day showed no toxicologically significant macroscopic abnormalities at terminal kill.
One male dosed at 400 mg/kg/day showed speckled kidneys at necropsy but in the absence of a similar finding amongst animals dosed at 1000 mg/kg/day, this abnormality was considered not to be toxicologically important. The remaining macroscopic abnormalities were confined to the kidneys of one control female and, as such, were clearly of no toxicological significance.

Conclusion:
The dose levels for the main twenty-eight day study were chosen as:
High dose: 750 mg/kg/day
Intermediate dose: 150 mg/kg/day
Low dose: 15 mg/kg/day
plus a control group treated with vehicle only


- Post-exposure recovery period in satellite groups: 14 days

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, early and late during the working period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing and one hour after dosing. An additional observation will be made five hours after dosing on normal working days (not at weekends and on public holidays). Satellite groups will be observed twice daily during the treatment free period (once daily at weekends and on public holidays). All observations will be recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights recorded on Day 0 (the day before the start of dosing) and at weekly intervals thereafter.

FOOD CONSUMPTION:
Dietary intake recorded weekly for each cage group.

FOOD EFFICIENCY:
Weekly food efficiency (bodyweight gain/food intake) will be calculated.

WATER CONSUMPTION:
Monitored daily by visual inspection of water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations will be performed on all animals from Groups 1 to 4 Treatment & Control) at the end of the treatment period (Day 28) and on all animals from Groups 5 to 7 (satellite groups) at the end of the treatment free period (Day 42). Blood samples will be withdrawn from the lateral tail vein. Repeat blood samples may be withdrawn by cardiac puncture prior to necropsy if required.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- The following Parameters were examined: Haemoglobin, Haematocrit, Erythrocyte count, Total leucocyte count, Differential leucocyte count, Erythrocyte indices (mean cell haemoglobin, mean cell volume, mean cell haemoglobin concentration), Clotting time (Hepato Quick), Platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations will be performed on all animals from Groups 1 to 4 Treatment & Control) at the end of the treatment period (Day 28) and on all animals from Groups 5 to 7 (satellite groups) at the end of the treatment free period (Day 42). Blood samples will be withdrawn from the lateral tail vein. Repeat blood samples may be withdrawn by cardiac puncture prior to necropsy if required.
- Animals fasted: No data
- How many animals: all animals
- The following Parameters were examined: Blood urea, Total protein, Albumin, Albumin/Globulin ratio (by calculation), Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Glucose, Triglycerides, Total cholesterol, Gamma glutamyl transpeptidase, Total bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples collected from all animals in Groups 1 to 4 during the final week of dosing, by housing overnight in metabolism cages under normal hydration but without access to food. Urinalysis will be performed on satellite group animals during the final week of the recovery period, where abnormalities are detected in the main groups.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- The following Parameters were examined: Volume, Specific gravity, pH, Protein Glucose, Ketones, Bilirubin, Urobilinogen, Reducing substances, Blood

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

POST MORTEM STUDIES: Carried out on animals dying or killed in extremis during the study and on all animals killed by intravenous sodium pentobarbitone followed by exsanguination at termination.

GROSS PATHOLOGY: Yes
Full external and internal examination of al animals.
Organ weights: Adrenals, Brain, Gonads, Heart, Kidneys, Liver, Pituitary, Spleen
Carried out on all survivors at termination.

HISTOPATHOLOGY: Yes
Samples of the following tissues will be preserved from all animals in buffered 10% formalin:
Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain, Caecum, Colon, Duodenum, Eyes, Gross lesions, 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, Spleen, Stomach, Testes, Thymus, Skin (hind limb), Thyroid/parathyroid, Trachea, Urinary bladder, Uterus
Initially the following tissues from:
a) all animals that die or are killed in extremis during the study
b) all animals in the control and high dose groups (Groups 1 and 4)
will be routinely processed to paraffin wax, sectioned, stained and haematoxylin and eosin and examined microscopically: Adrenals, Spleen, Heart, Testes, Kidneys, Target organs, Liver, Gross lesions
Since there were indications of treatment-related hepatic and renal changes, examination was subsequently extended to include similarly prepared sections of liver and kidneys from all animals in the remaining dose groups.

Statistics:

Evaluation of Data
Data were processed to give group mean values and standard deviations where appropriate.
Absolute and relative organ weights, weekly bodyweight gain, haematological and blood chemical data and quantitative urinalytical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal-Wallis non-parametric analysis of variance and Mann Whitney U-Test.
Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p > 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality: One female dosed at 750 mg/kg/day was killed in extremis on Day 14, due to the severe clinical signs of toxicity observed. Clinical Observations: Animals of either sex dosed at 750 mg/kg/day showed short-lived increased salivation together with associated fur wetting, red/brown staining of the external body surface and, occasionally, noisy respiration, sporadic incidents of pilo-erection and mostly patchy fur loss from Week 2. Predominantly females showed diuresis during the study, together with ventral fur wetting and red/brown staining both of the ventral fur and around the ano-genital region, hunched posture from Day 13. Clinical abnormalities amongst satellite 750 mg/kg/day males regressed immediately following cessation of treatment, Females appeared normal from Day 30 onwards, with the exception of red/brown staining around the ano-genital region which persisted for one animal until Day 35. Animals dosed at 150 mg/kg/day showed short-lived increased salivation immediately after dosing from Day 3 with sporadic incidents of associated fur wetting and red/brown staining around the mouth. Two males showed more prolonged increased salivation during the final two days of treatment. Increased salivation was no longer evident amongst satellite group animals dosed at 150 mg/kg/day immediately following cessation of treatment. Animals dosed at 15 mg/kg/day showed no clinically observable signs of toxicity during the study. One female dosed at 750 mg/kg/day showed a scab in the dorso-cervical region during the study. Rats housed in groups occasionally show minor physical injuries in this region and the finding was considered not to be toxicologically important. One female dosed at 150 mg/kg/day showed a small swelling in the abdominal region during the study but this is a normally-expected, low incidence finding amongst laboratory maintained rats of the strain and age used and was considered not to be of any of any toxicological significance.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality: One female dosed at 750 mg/kg/day was killed in extremis on Day 14, due to the severe clinical signs of toxicity observed. Clinical Observations: Animals of either sex dosed at 750 mg/kg/day showed short-lived increased salivation together with associated fur wetting, red/brown staining of the external body surface and, occasionally, noisy respiration, sporadic incidents of pilo-erection and mostly patchy fur loss from Week 2. Predominantly females showed diuresis during the study, together with ventral fur wetting and red/brown staining both of the ventral fur and around the ano-genital region, hunched posture from Day 13. Clinical abnormalities amongst satellite 750 mg/kg/day males regressed immediately following cessation of treatment, Females appeared normal from Day 30 onwards, with the exception of red/brown staining around the ano-genital region which persisted for one animal until Day 35. Animals dosed at 150 mg/kg/day showed short-lived increased salivation immediately after dosing from Day 3 with sporadic incidents of associated fur wetting and red/brown staining around the mouth. Two males showed more prolonged increased salivation during the final two days of treatment. Increased salivation was no longer evident amongst satellite group animals dosed at 150 mg/kg/day immediately following cessation of treatment. Animals dosed at 15 mg/kg/day showed no clinically observable signs of toxicity during the study. One female dosed at 750 mg/kg/day showed a scab in the dorso-cervical region during the study. Rats housed in groups occasionally show minor physical injuries in this region and the finding was considered not to be toxicologically important. One female dosed at 150 mg/kg/day showed a small swelling in the abdominal region during the study but this is a normally-expected, low incidence finding amongst laboratory maintained rats of the strain and age used and was considered not to be of any of any toxicological significance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males, but not females, dosed at 750 mg/kg/day showed a 15% lower group mean bodyweight gain, considered not to be toxicologically important.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Dietary intake was unaffected by treatment.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex dosed at 750 mg/kg/day showed a slightly reduced food efficiency during the first half of the treatment period, normalizing afterwards. Food efficiency was unaffected by treatment at the remaining dose levels.

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

effects observed, treatment-related

Description (incidence and severity):

Males / females dosed at 750 mg/kg/day showed a 25% / 75% greater group mean water intake during Weeks 3 and 4, the satellite 750 mg/kg/day dose group showed a 47% increase. There was no adverse effect on water consumption at other groups.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the parameters measured.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see freetext below

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

3/5 males and 3/4 females dosed at 750 mg/kg/day showed a diuresis, normalizing amongst the satellite group within 14 days. Animals dosed at 150 or 15 mg/kg/day showed no toxicologically significant changes in the parameters measured.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see freetext below

Gross pathological findings:

no effects observed

Description (incidence and severity):

No changes considered to be of toxicological importance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centrilobular hepatocyte enlargement in relation to treatment was observed for male and for female rats as well as eosinophilic droplets were observed in the epithelium of renal proximal dosed at 750 and at 150 mg/kg/day in males.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality:
One female dosed at 750 mg/kg/day was killed in extremis approximately five hours after dosing on Day 14, due to the severe clinical signs of toxicity observed. There were no further deaths during the study.
Clinical Observations:
Animals of either sex dosed at 750 mg/kg/day showed short-lived increased salivation before or immediately after dosing throughout the treatment period together with associated fur wetting, red/brown staining of the external body surface and, occasionally, noisy respiration. More prolonged increased salivation was also evident amongst females and males from Days 2 and 3 respectively. In addition, animals of either sex showed sporadic incidents of pilo-erection throughout the treatment period and fur loss, which was mostly patchy in nature, was observed from the beginning of Week 2 onwards. Further clinical abnormalities were also observed at this dose level, predominantly amongst the females. Females showed diuresis during the study, mostly towards the end of the first week of treatment, together with associated ventral fur wetting and red/brown staining both of the ventral fur and around the ano-genital region. Hunched posture was also detected from Day 13 onwards, again predominantly amongst the females, and isolated incidents of tiptoe gait, lethargy and chromodacryorrhoea were observed during the study. The female decedent showed similar clinical abnormalities to the other females from this dose group for most of the study but underwent a sudden deterioration in health towards the end of Day 14, showing occasional body tremors approximately five hours after dosing. This animal was killed in extremis without further treatment. Clinical abnormalities amongst satellite 750 mg/kg/day males regressed immediately following cessation of treatment. Females from the same dose group appeared normal in comparison with controls from Day 30 onwards, with the exception of red/brown staining around the ano-genital region which persisted for one animal until Day 35.
Animals dosed at 150 mg/kg/day showed short-lived increased salivation immediately after dosing from Day 3 onwards together with sporadic incidents of associated fur wetting and red/brown staining around the mouth. In addition, two males showed more prolonged increased salivation during the final two days of treatment. Increased salivation was no longer evident amongst satellite group animals dosed at 150 mg/kg/day immediately following cessation of treatment.
Animals dosed at 15 mg/kg/day showed no clinically observable signs of toxicity during the study.
One female dosed at 750 mg/kg/day showed a scab in the dorso-cervical region during the study. Rats housed in groups occasionally show minor physical injuries in this region and the finding was considered not to be toxicologically important. One female dosed at 150 mg/kg/day showed a small swelling in the abdominal region during the study but this is a normally-expected, low incidence finding amongst laboratory maintained rats of the strain and age used and was considered not to be of any of any toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Males dosed at 750 mg/kg/day showed a 15% lower group mean bodyweight gain than controls during the first half of the treatment period. Satellite 750 mg/kg/day males showed a similar reduction in group mean bodyweight gain compared with satellite controls during the same period. There were no further adverse effects on bodyweight development amongst these animals during the remainder of the study and the recovery amongst satellite group males was further evidenced by a statistically significant increase in group mean bodyweight gain in comparison with that of controls during the final week of the treatment-free period.
There was no adverse effect on bodyweight development amongst females dosed at 750 mg/kg/day or amongst animals of either sex from the remaining dose groups that could be attributed to test material toxicity.
Animals treated with the test material showed a statistically significant increase in group mean bodyweight gain in comparison with that of controls on several occasions during the dosing period. Increased bodyweight gain is unlikely to be indicative of systemic toxicity and these findings were therefore considered not to be toxicologically important.

FOOD CONSUMPTION
Dietary intake was unaffected by treatment.

FOOD EFFICIENCY
Animals of either sex dosed at 750 mg/kg/day and those from the corresponding satellite 750 mg/kg/day dose group showed a slightly reduced food efficiency compard with that of controls during the first half of the treatment period. There was no adverse effect on food efficiency amongst these animals during the remainder of the study and food efficiency was unaffected by treatment at the remaining dose levels.

WATER CONSUMPTION
Males and females dosed at 750 mg/kg/day respectively showed a 25% and 75% greater group mean water intake than with controls during Weeks 3 and 4, the period during which water consumption was quantitatively measured. Animals of either sex from the corresponding satellite 750 mg/kg/day dose group showed a 47% increase in group mean water intake compared with satellite controls during the same period. Water intake remained elevated amongst these satellite 750 mg/kg/day animals for at least one week following cessation of treatment but the adverse effect had almost completely regressed by the end of the fourteen day treatment-free period.
There was no adverse effect on water consumption at the remaining dose levels.

HAEMATOLOGY
There were no toxicologically significant changes in the parameters measured.
Females dosed at 750 mg/kg/day showed a marginally increased group mean neutrophil count compared with controls. None of the individual values were outside the normally expected range for rats of the strain and age used (normal range 0.00 - 4.47 x 109/l) and the intergroup difference was considered to be of no toxicological importance. The remaining statistically significant intergroup differences were confined to satellite group animals and involved a reduction in haemoglobin and a reduction in clotting time amongst satellite 750 mg/kg/day males and females respectively, a reduction in mean corpuscular haemoglobin and a reduced neutrophil count amongst satellite 150 mg/kg/day males and a reduction in mean corpuscular haemoglobin concentration amongst satellite 750 and 150 mg/kg/day males. In the absence of any change in these parameters at the end of the twenty-eight day dosing period, the intergroup differences were considered to have arisen fortuitously, mostly due to unusually high control values, and were of no toxicological significance.

CLINICAL CHEMISTRY
Females dosed at 750 mg/kg/day showed an increased group mean alanine aminotransferase (ALAT) concentration compared with controls, with most of the individual values outside the normally expected range for rats of the strain and age used. No such adverse change in ALAT concentration was evident amongst satellite 750 mg/kg/day animals following an additional fourteen days without treatment.
Males dosed at 750 mg/kg/day and animals of either sex dosed at 150 or 15 mg/kg/day showed no toxicologically significant changes in the parameters measured.
Animals of either sex dosed at 750 mg/kg/day and females dosed at 150 mg/kg/day showed a marginally elevated group mean glucose concentration compared with controls. None of the individual values were abnormally high for rats of the strain and age used (normal range: male glucose 126 - 183 mg/dl, female glucose 121 -185 mg/dl) and, as such, the intergroup difference was considered not to be toxicologically important. Males dosed at 750 mg/kg/day also showed a slight reduction in group mean chloride concentration compared with controls together with an increased group mean calcium concentration. Again, none of the individual values were outside the normally expected ranges for rats of the strain and age used (normal range: male chloride 92 - 107 mmol/l, male calcium 2.36 -2.96 mmol/l) and as there was no concomitant effect on either sodium or inorganic phosphorus respectively, these findings were considered unlikely to be associated with test material toxicity. Females dosed at 750 mg/kg/day showed an increased group mean inorganic phosphorus concentration in comparison with controls but in the absence of an adverse effect amongst these animals on any of the other measured plasma electrolytes, this isolated effect was considered not to be indicative of test material toxicity.
Satellite 750 mg/kg/day males and females respectively showed an increased ALAT concentration and an increased cholesterol concentration compared with satellite controls whilst satellite 150 mg/kg/day males showed a slightly reduced sodium concentration. In the absence of an adverse effect on these parameters at the end of the twenty-eight day dosing period amongst animals from the corresponding 750 or 150 mg/kg/day dose groups, these intergroup differences were considered to have arisen fortuitously and were of no toxicological importance.
The remaining statistically significant intergroup differences involved a reduced urea concentration amongst females dosed at 150 mg/kg/day, a reduced potassium concentration and a reduced inorganic phosphorus concentration amongst females dosed at 15 mg/kg/day, a reduced aspartate aminotransferase concentration amongst females dosed at 15 mg/kg/day and amongst animals of either sex dosed at 150 mg/kg/day, and a reduced creatinine concentration amongst females dosed at 750 or 15 mg/kg/day. A dose-response relationship could not be demonstrated for any of these findings and, as such, the intergroup differenes were considered not to be toxicologically significant.

URINALYSIS
Three males and one of the four surviving females dosed at 750 mg/kg/day showed a diuresis, producing a copious volume of urine of reduced specific gravity compared with controls. Diuresis was no longer evident amongst satellite group animals dosed at 750 mg/kg/day following an additional fourteen days without treatment.
Animals dosed at 150 or 15 mg/kg/day showed no toxicologically significant changes in the parameters measured.

ORGAN WEIGHTS
Animals of either sex dosed at 750 mg/kg/day showed an increased group mean liver weight, both absolute and relative to terminal bodyweight, in comparison with controls with many of the individual values outside the normally expected range for rats of the strain and age used. In addition, males dosed at 750 or 150 mg/kg/day showed a slightly increased group mean relative kidney weight compared with controls, with 750 mg/kg/day dose group animals again showing abnormally high individual values. Satellite 750 mg/kg/day females showed a marginally greater group mean relative liver weight than satellite controls following an additional fourteen days without treatment but the intergroup difference was of equivocal toxicological importance because none of the individual values were outside the normally expected range for rats of the strain and age used (normal range: 2.9851 - 4.2370 %) and there was no histopathological evidence of residual centrilobular hepatocyte enlargement amongst these animals.
Females dosed at 150 mg/kg/day and animals of either sex dosed at 15 mg/kg/day showed no toxicologically significant organ weight changes.
Males dosed at 150 mg/kg/day showed a statistically significant increase in group mean absolute kidney weight compared with controls. None of the individual values were abnormally high for rats of the strain and age used in the study (normal range: 1.9470 - 2.9367 g) and, in the absence of a statistically significant increase in this parameter at the 750 mg/kg/day dose level, the intergroup difference was considered not to be toxicologically important.
Females dosed at 750 mg/kg/day showed a slightly reduced group mean brain weight/ both absolute and relative to terminal bodyweight, compared with controls. None of the individual values were outside the normally expected range for rats of the strain and age used (normal range: absolute brain weight 1.7225 -2.0348 g, relative brain weight 0.6784 - 0.9164%) and the intergroup difference was considered to have arisen fortuitously. Group mean absolute adrenal weight was maginally elevated amongst males dosed at 750 or 150 mg/kg/day and group mean relative adrenal weight was also increased amongst males dosed at 750 mg/kg/day. In addition, satellite 750 mg/kg/day animals of either sex showed a statistically significant increase in group mean relative adrenal weight compared with satellite controls following an additional fourteen days without treatment but these findings were considered unlikely to be associated with test material toxicity because most of the individual values were within the normally expected range for these animals (normal range: male absolute adrenal weight 0.0311 - 0.0678 g, male relative adrenal weight 0.0086 - 0.0191%, female relative adrenal weight 0.0186 -0.0354%) and there were no associated morphological adrenal abnormalities detected.
The remaining statistically significant intergroup differences involved an increased group mean absolute spleen weight amongst males dosed at 15 mg/kg/day and an increase group mean absolute heart weight amongst females from the same dose group. In the absence of any dose-response relationship, these findings were considered not to be toxicologically important.

GROSS PATHOLOGY
Three males dosed at 750 mg/kg/day showed speckled kidneys at terminal kill and one of these animals also showed pale kidneys. No toxicologically significant macroscopic abnormalities were detected amongst satellite group males dosed at 750 mg/kg/day following an additional fourteen days without treatment.
Females dosed at 750 mg/kg/day, including the animal killed in extremis on Day 14, and animals of either sex from the remaining dose groups showed no toxicologically significant macroscopic abnormalities at necropsy.
One control female showed dark areas on the right lung at terminal kill on Day 29 and several satellite group animals, including two males from the control group, showed dark areas on either lung at the end of the fourteen day treatment-free period. These findings were considered not to be attributable to test material toxicity and were, instead, probably attributable to a small amount of vehicle entering the respiratory tract during dosing. One satellite 750 mg/kg/day female showed ovaries encased in a fluid-filled sac at necropsy on Day 43 and two males, one from each of the 15 and 150 mg/kg/day dose groups, showed multiple white foci on the liver and hydronephrosis respectively at necropsy on Day 29. These findings represent normally expected, low-incidence lesions amongst laboratory maintained animals of the age and strain used and, in the absence of similar changes amongst 750 mg/kg/day animals at the end of the dosing period, were considered to be of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related hepatic and renal changes were observed:
Liver: Centrilobular hepatocyte enlargement was observed in relation to treatment for male and for female rats dosed at 750 and at 150 mg/kg/day. The condition was generally observed to have regressed amongst satellite 750, and 150 mg/kg/day group animals following an additional fourteen days without treatment.
Kidneys: Eosinophilic droplets were observed in the epithelium of renal proximal tubules for male rats receiving 750, and 150 mg/kg/day. The condition demonstrated partial regression amongst satellite 750, and 150 mg/kg/day animals following an additional fourteen days without treatment. This finding is consistent with the appearance of hydrocarbon nephropathy which is peculiar to male rats.
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.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects; water consumption and compound intake; urinalysis; organ weights; histopathology;

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on overall effects, water consumption, urinalysis, organ weights, and histopathology

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Discussion

The oral administration of the test item to rats by gavage at dose levels of 15, 150 and 750 mg/kg/day, for up to twenty-eight consecutive days, resulted in toxicologically significant changes at dose levels of 750 and 150 mg/kg/day. The changes were largely reversed after a further fourteen days without exposure to the test item.

Most of the observations seen at 750 mg/kg/day could be considered attributable to general stress associated with repeated oral administration of an unpleasant tasting, irritant substance. In particular, increased salivation around the time of dosing together with associated fur wetting and staining were almost certainly a direct response to the dosing procedure. Further observations such as hunched posture, occasional lethargy, chromodacryorrhoea and, possibly, tiptoe gait, might also be associated with dosing stress. Furthermore, an increased water consumption at 750 mg/kg/day was probably a compensatory response to the administration of an unpalatable, irritant substance. There was, however, also a reduction in bodyweight gain for males dosed at 750 mg/kg/day during the first half of the treatment period together with a reduced food efficiency (the ratio of weight gain to food consumption) in either sex. These latter changes could be indicative of effects other than those associated with dosing stress but their transient nature suggests that tolerance to treatment developed after approximately two weeks of dosing. One female, however, showed a sudden deterioration in condition towards the end of Week 2, developing occasional body tremors, and was killedin extremis due to the severity of the clinical observations.

Kidney changes were apparent in males treated with either 750 or 150 mg/kg/day, as evidenced by organ weight and histopathological changes. Kidney weight, relative to terminal bodyweight, was increased although absolute kidney weight was unaffected.

Histopathologically, eosinophilic droplets were apparent in the proximal renal tubules of males receiving 750 or 150 mg/kg/day but not in females. The test substance is hydrocarbon in nature and this finding is consistent with hydrocarbon nephropathy, which is peculiar to male rats. It is therefore unlikely to occur in humans. There was no convincing evidence of renal dysfunction despite diuresis amongst several animals at the highest dose level. In this study, diuresis was most probably a direct response to the increased water intake seen at this dose level. Kidney changes were reversible following cessation of treatment. Relative kidney weight was no longer raised at the end of the fourteen day recovery period and eosinophilic droplet accumulation had regressed both in incidence and severity amongst males dosed at 750 or 150 mg/kg/day.

Doses of 750 mg/kg/day and 150 mg/kg/day resulted in liver changes. Histologically, there was evidence of centrilobular hepatocyte enlargement in either sex at both dose levels. In addition, absolute and relative liver weights for either sex were increased at 750 mg/kg/day but not at 150 mg/kg/day. Increased hepatic activity of this type is considered a normal adaptive biological response to a foreign substance and is usually the result of detoxification mechanisms involving hepatic enzyme induction. The adaptive nature of this response was further demonstrated by its reversibility following an additional two weeks without treatment. The slightly raised plasma alanine aminotransferase concentration amongst females dosed at 750 mg/kg/day may indicate a change in hepatocyte membrane permeability not visible by light microscopy. This enzyme was no longer elevated at the end of the fourteen day recovery period.

Animals dosed at 15 mg/kg/day showed no toxicologically significant changes in the parameters measured and findings at 150 mg/kg/day were considered not to be indicative of serious damage to the health of the animals, as defined by the labelling criteria given in Directive 93/21/EEC.

Justification of No Observed Effect Level

 

The oral administration of the test item to rats, by gavage, for up to twenty-eight consecutive days at dose levels of 15, 150 and 750 mg/kg/day resulted in toxicologically significant changes at 750 and 150 mg/kg/day. Animals dosed at 150 mg/kg/day showed short-lived and prolonged increased salivation, an increased relative kidney weight, accumulation of eosinophilic droplets in the renal proximal tubular epithelium, and centrilobular hepatocyte enlargement. These adverse effects of treatment were also detected amongst animals dosed at 750 mg/kg/day together with one death, severe clinical signs of toxicity, a reduced bodyweight gain, a reduced food efficiency, an increased water consumption, an increased alanine aminotransferase concentration, urinalytical findings characteristic of diuresis, an increased absolute and relative liver weight, and macroscopic changes involving the kidneys. No such changes were detected at a dose level of 15 mg/kg/day and the "No Observed Effect Level" (NOEL) was therefore considered to be 15 mg/kg/day.

Statistical evaluation of the quantitative data showed a significant difference between animals dosed at 15 mg/kg/day and controls in a number of parameters, none of which were considered to be of any toxicological significance:

• The following were confined to the 150 and/or 15 mg/kg/day dose groups and, as such, showed no dose-response relationship.

Increased bodyweight gain during Week 2: females dosed at 150 and 15 mg/kg/day. Reduced plasma potassium concentration: females dosed at 15 mg/kg/day. Reduced plasma inorganic phosphorus concentration: females dosed at 15 mg/kg/day. Reduced plasma aspartate aminotransferase concentration: females dosed at 150 and 15 mg/kg/day

Increased absolute spleen weight: males dosed at 15 mg/kg/day

• Creatinine concentration was reduced amongst females dosed at 15 and 750 mg/kg/day. A dose-response relationship could not be demonstrated because there was no adverse effect on creatinine amongst females dosed at 150 mg/kg/day. Furthermore, there were no other associated changes which might have suggested a relationship with treatment.

Qualitative changes observed at the 15 mg/kg/day dose level but considered not to be indicative of test material toxicity included necropsy and histopathological findings:

• A necropsy finding of multiple white foci on the liver was confined to one male dosed at 15 mg/kg/day. In the absence of a similar finding amongst animals dosed at 150 and/or 750 mg/kg/day, this was considered not to be associated with test material toxicity.

• Morphological changes in animals dosed with 15 mg/kg/day were those commonly observed in laboratory maintained rats of the strain and age used and, since there was no difference in incidence or severity between these animals and controls, all were considered to be without toxicological significance.

Applicant's summary and conclusion

Conclusions:

The study is well-documented and was performed according to OECD guideline 407, EU method B7 and the Japanese MHW guideline, under GLP conditions. Hence, the information provided in the report can be considered reliable. In consequence, the results are suitable for the assessment of toxicity of the test item when applied in repeated doses over 28 days.
Oral administration of the test item to rats, by gavage, at dose levels of 15, 150 and 750 mg/kg/day produced toxicologically significant changes at dose levels of 150 and 750 mg/kg/day. These changes were mostly reversible after an additional fourteen days without treatment. No toxicologically significant changes were observed amongst animals dosed at 15 mg/kg/day and the "No Observed Effect Level" (NOEL) was therefore considered to be 15 mg/kg/day.
The treatment-related changes observed at a dose level of 150 mg/kg/day 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 Directive 93/21/EEC.
Hence, the NOEL of 15 mg/kg/day can be used for the assessment of the test item.

Executive summary:

In a subacute toxicity study (OECD 407, EU method B7, Japanese MHW guideline) the test item was administered to five Sprague-Dawley strain CD rats/sex/dose in by gavage at dose levels of 0, 15, 150, 750 mg/kg bw/day.

 

The results are summarised as follows:

 

Mortality: One female dosed at 750 mg/kg/day was killed in extremis on Day 14, due to the severe clinical signs of toxicity observed. There were no further deaths during the study.

 

Clinical Observations: Animals of either sex dosed at 750 mg/kg/day showed increased salivation before or immediately after dosing from Day 1 onwards together with associated fur wetting, red/brown staining of the external body surface and, occasionally, noisy respiration. More prolonged increased salivation was observed from Day 2 and fur loss was evident from the beginning of Week 2 onwards. Sporadic incidents of pilo-erection were also evident amongst animals of either sex during the study. Further clinical observations at this dose level were observed predominantly amongst the females and included hunched posture, diuresis and, less frequently, lethargy, tiptoe gait and chromodacryorrhea. The female decedent showed similar clinical abnormalities to the other females from the same dose group until Day 14, when occasional body tremors were observed towards the end of the working day. This animal was killed in extremis without further treatment. Clinical abnormalities amongst satellite 750 mg/kg/day males regressed immediately following cessation of treatment and females from the same dose group appeared normal in comparison with controls from Day 30 onwards, except for red/brown staining around the ano-genital region.

Animals dosed at 150 mg/kg/day showed short-lived increased salivation immediately after dosing from Day 3 onwards. Two males also showed more prolonged increased salivation during the final two days of treatment. Increased salivation was no longer evident amongst satellite 150 mg/kg/day animals immediately following cessation of treatment.

Animals dosed at 15 mg/kg/day showed no clinically observable signs of toxicity during the study.

 

Bodyweight: Males dosed at 750 mg/kg/day showed a 15% lower bodyweight gain than controls during the first half of the treatment period. There was no adverse effect on bodyweight development amongst these animals during the remainder of the study.

There was no adverse bodyweight effect amongst females dosed at 750 mg/kg/day or amongst animals of either sex from the remaining dose groups that could be attributed to test material toxicity.

 

Food Consumption: Dietary intake was unaffected by treatment with the test material.

Animals of either sex dosed at 750 mg/kg/day showed a slightly reduced food efficiency compared with that of controls during the first half of the treatment-period. There was no adverse effect on food efficiency amongst these animals during the remainder of the study and food efficiency was unaffected by treatment at the remaining dose levels.

 

Water Consumption: Females and, to a lesser extent, males dosed at 750 mg/kg/day showed an increased water intake in comparison with controls during the treatment period. The adverse effect on water intake regressed gradually amongst satellite 750 mg/kg/day following cessation of treatment.

There was no adverse effect on water consumption at the remaining dose levels.

 

Haematology: There were no toxicologically significant changes in the parameters measured.

 

Blood Chemistry: Females dosed at 750 mg/kg/day showed an increased group mean alanine aminotransferase concentration compared with controls. No such change was evident amongst satellite 750 mg/kg/day females following an additional fourteen days without treatment.

Males dosed at 750 mg/kg/day and animals of either sex dosed at 150 or 15 mg/kg/day showed no toxicologically significant changes in the parameters measured.

 

Urinalysis: Three males and one female dosed at 750 mg/kg/day showed a diuresis, producing a copious volume of urine of reduced specific gravity compared with controls. Diuresis was no longer evident amongst satellite 750 mg/kg/day animals following an additional fourteen days without treatment.

Animals dosed at 150 or 15 mg/kg/day showed no toxicologically significant changes in the parameters measured.

 

Organ Weights: Animals of either sex dosed at 750 mg/kg/day showed an increased liver weight, both absolute and relative to terminal bodyweight, in comparison with controls. Males dosed at 750 or 150 mg/kg/day also showed a slightly increased relative kidney weight compared with controls. Satellite 750 mg/kg/day females showed a marginally elevated relative liver weight in comparison with controls following an additional fourteen days without treatment.

Females dosed at 150 mg/kg/day and animals of either sex dosed at 15 mg/kg/day showed no toxicologically significant organ weight changes.

 

Necropsy: Three males dosed at 750 mg/kg/day showed speckled kidneys at terminal kill and one of these animals also showed pale kidneys. No toxicologically significant macroscopic abnormalities were detected amongst satellite 750 mg/kg/day males following an additional fourteen days without treatment.

Females dosed at 750 mg/kg/day, including the animal killed in extremis on Day 14, and animals of either sex from the remaining dose groups showed no toxicologically significant macroscopic abnormalities at necropsy.

 

Histopathology: Animals of either sex dosed at 750 or 150 mg/kg/day showed centrilobular hepatocyte enlargement. Males dosed at 750 or 150 mg/kg/day also showed accumulations of eosinophilic droplets in the renal proximal tubular epithelium. Centrilobular hepatocyte enlargement had mostly regressed amongst satellite 750 or 150 mg/kg/day animals following an additional fourteen days without treatment. Accumulations of eosinophilic droplets in the kidneys showed only partial regression amongst males from either satellite dose group.

Animals dosed at 15 mg/kg/day showed no toxicologically significant morphological lesions.

 

Based on overall effects, water consumption, urinalysis, organ weights, and histopathology, NOAEL is 150 mg/kg bw. The NOEL is 15 mg/kg bw/day.

 

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study (OECD 407, EU method B7, Japanese MHW guideline) in the rat.