Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-08-2001 to 12-02-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Reference: Commission Directive 96/54/EC
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: US EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, (2000).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: February 2000 ; signature: April 2000
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: At room temperature under nitrogen in the dark.
- Other: Clear colourless

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD) IGS BR
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 5 to 7 weeks
- Weight at study initiation: males 121 – 157 g and females 114 – 144 g; individuals were randomly allocated to treatment groups using a randomization procedure.
- Fasting period before study: None
- Housing: polypropylene grid-floor cages with wooden chew blocks for environment enrichment ; group housed (5 per group) by sex. During locomotor investigations were housed individually.
- Diet (e.g. ad libitum): ground rodent diet number 1, certified (recognised supplier), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: ground rodent diet, certified (recognised supplier) – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. A known amount of test item was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a QE200 mixer. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a H800 mixer. Dietary admixtures were prepared prior to treatment. The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use. The test item was incorporated into the diet at concentrations of 225, 2250 and 15000 ppm

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

IN-LIFE DATES: From: 17-10-2001 to 14-11-2001

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. A known amount of test item was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a QE200 mixer. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a H800 mixer. Dietary admixtures were prepared prior to treatment. The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use. The test item was incorporated into the diet at concentrations of 225, 2250 and 15000 ppm
Vehicle:
unchanged (no vehicle)
Remarks:
PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Monthly (dietary admixtures were determined to be stable for a period of at least seven weeks).
- Mixing appropriate amounts with (Type of food): Basal diet (number 1 Certified) with test item.
- Storage temperature of food: Room temperature, diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use.
- Stability under test conditions: Stable. Dietary admixtures were determined to be stable for a period of at least seven weeks. The stability and homogeneity of the test material formulations were determined. Results indicated that the mean prepared admixture concentrations were within ± 4% of the nominal concentration.
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The formulated diet analysis consisted of GC FID analysis with external calibration. The method was calibrated by using calibration standards of the test item response between nominal concentrations of 225 ppm and 15000 ppm within a dedicated dietary formulation analysis report attached to the full study report. The dietary admixtures were extracted with acetonitrile to give a final, theoretical test item concentration of approximately 10 µg/mL. Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 10 µg/mL. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With linearity = 0.999 between 4 μg/mL and 15 μg/mL. Accuracy and precision was confirmed and mean procedural recovery was 102% (n=5 ; CV = 0.99%) at 229 ppm and 99% at 14990 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in diet. Dietary admixtures were determined to be stable for a period of at least seven weeks. The stability and homogeneity of the test material formulations were determined. Results indicated that the mean prepared admixture concentrations were within ± 4% of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
225 ppm
Remarks:
Low – Group
Dose / conc.:
2 250 ppm
Remarks:
Intermediate – Group
Dose / conc.:
15 000 ppm
Remarks:
High – Group
Dose / conc.:
24 mg/kg bw/day (actual dose received)
Remarks:
Low – Group ; mean achieved dose
Dose / conc.:
216 mg/kg bw/day (actual dose received)
Remarks:
Intermediate – Group ; mean achieved dose
Dose / conc.:
1 486 mg/kg bw/day (actual dose received)
Remarks:
High – Group ; mean achieved dose
No. of animals per sex per dose:
5 per sex per dose (5 male / 5 female)
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 225, 2250 and 15000 ppm) were chosen based upon the results obtained in a 14 day preliminary study (available in the full study report) which utilised dose levels as follows: 0 (control) mg/kg (0 ppm) and 1000 mg/kg (15000 ppm).
- Rationale for animal assignment (if not random): Randomly assigned.
- Rationale for selecting satellite groups: Not applicable.
- Post-exposure recovery period in satellite groups: Not applicable.
- Section schedule rationale (if not random): Random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily. During treatment, twice daily on days 1 to 2; three times daily on day 3, twice daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily. During treatment, twice daily on days 1 to 2; three times daily on day 3, twice daily thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes.
- Body weight gain % was determined.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals and/or end of recovery period (week 6) for satellite recovery groups.
- Anaesthetic used for blood collection: Yes. isoflurane (recognised supplier)
- Animals fasted: Yes. Overnight (18 hours).
- How many animals: All animals
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium, high fluorescence), Leukocyte count, total, Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count, Methemoglobin Heinz bodies, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals and/or end of recovery period (week 6) for satellite recovery groups.
- Animals fasted: Yes. Overnight (18 hours).
- How many animals: All animals
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin, total Cholesterol, total Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein, total Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes.
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals and/or end of recovery period (week 6) for satellite recovery groups.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. Overnight (18 hours).
- Parameters checked: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Kidneys, Liver, Ovaries

HISTOPATHOLOGY: Yes
- Organs and tissues examined and/or preserved in neutral buffered 10% formalin (where appropriate): Adrenals, Aorta (thoracic), Oesophagus, Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Colon, Salivary glands (submaxillary), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, Eyes, Skin (hind limb), Gross lesions, Spinal cord (cervical), Heart, Spleen, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, Liver, Thyroid/parathyroid, Lungs (with bronchi), Trachea, Lymph nodes (cervical and mesenteric), Urinary bladder, Muscle (skeletal), Uterus.
- Other: Additional investigations of liver, kidney and urinary bladder were made in all individuals in all treatment groups.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
The following statistical methods were used to analyse: Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight):
• dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance
• where homogenous variances observed: homogenous pairwise comparisons were conducted using Dunnett's test
• Where the Levene’s test showed unequal variances: non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No adverse clinical signs of toxicity were noted during the observation period.

At 225 ppm: one female had a missing tail tip. This was considered an incidential finding.

There were no treatment-related changes in behavioral assessments, functional performance or sensory reactivity.
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm: there was a statistically significant reduction in bodyweight relative to control for males and females during the first week of treatment. Recovering thereafter.

No effects were observed at 225 ppm and 2250 ppm treatment levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm: there was a slight reduction in dietary intake for females throughout the study period compared to controls.

No effects were observed at 225 ppm and 2250 ppm treatment levels.

At 15000 ppm: Food efficiency (the ratio of bodyweight gain to dietary intake) was also impaired in both males and females. Confined to Week 1 only.

No effects were observed at 225 ppm and 2250 ppm treatment levels.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
See body weight and weight changes and food consumption sections.

At 15000 ppm: Food efficiency (the ratio of bodyweight gain to dietary intake) was also impaired in both males and females. Confined to Week 1 only.

No effects were observed at 225 ppm and 2250 ppm treatment levels.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Description (incidence and severity):
There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm: A statistically significant increase in dotting (prothrombin) time was detected in males.

No other effects were observed.

No effects were observed at 225 ppm and 2250 ppm treatment levels.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse clinical chemistry changes were noted during the observation period.

At 15000 ppm: A statistically significant reduction in plasma bilirubin and increase in plasma cholesterol (p<0.05) were detected for males, together with an increase in plasma total protein (p<0.05) observed for females. In isolation all were considered during the study, to have arisen fortuitously and were of no toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in behavioral assessments, functional performance or sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm: males and females showed an increased liver weight, both absolute and relative to terminal bodyweight. Statistical significance was achieved for males only but all relative liver weights were outside the respective normal ranges for both males and females. Relative kidney weight was also elevated in males.

Females showed an increased relative adrenal weight (p < 0.01) and reductions in relative ovary and spleen weights (p <0.01 and 0.001 respectively). In the absence of any histopathological correlations these changes were not considered toxicologically relevant.

No treatment related effects were observed at 225 ppm and 2250 ppm treatment levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were observed.

Incidental findings were considered to be of no toxicological significance, due to no dose-responses and being consistent with findings commonly seen post-mortem.

A single female in control group: had a grossly enlarged thymus due to thymic tumour. Unrelated to treatment.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in behavioral assessments, functional performance or sensory reactivity.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm:
Liver: Centrilobular hepatocyte enlargement was observed in relation to treatment in males and females. No effects were observed at 225 ppm and 2250 ppm treatment levels. The centrilobular hepatocyte enlargement was considered an adaptive response.

Kidneys: Globular accumulations of eosinophilic material were observed in the tubular epithelium of three males and two males at 2250 ppm. This finding is consistent with the appearance of hydrocarbon nephropathy, which results from the excessive accumulation of a2-microglobulin in renal proximal tubular epithelial cells. a2-Microglobulin is found only in the proximal tubular epithelium of adult male rats.

Urinary Bladder: Treatment-related hyperplasia of the transitional epithelium, occasionally with associated subepithelial inflammatory cell infiltrates, was observed for males and females. Epithelial hyperplasia was also observed in the urinary bladder of one control female, one 225 ppm male, and one 2250 ppm female rat as a spontaneous finding.

No other effects were observed and considered toxicologically relevant at 225 ppm and 2250 ppm treatment levels.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected.

Incidental findings were considered to be of no toxicological significance, due to no dose-responses and being consistent with findings commonly seen post-mortem.

A single female in control group: had a grossly enlarged thymus due to thymic tumour. Unrelated to treatment.
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 2 250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
>= 216 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: mean achieved dose

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 2250 ppm (equivalent to a mean achieved dose of 216 mg/kg body weight per day).
Executive summary:

The study was performed according the requirements of OECD TG 407 and EU method B.7 guidelines under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day dietary study in Sprague-Dawley Crl:CD (SD) IGS BR strain rats. Three groups, each comprising five male and five female rats, received test item at dietary concentrations of 225, 2250 and 15000 ppm (equivalent to mean achieved dosage of 24, 216 and 1486 mg/kg/day). A control group of five males and five females was dosed with basal diet. Chemical analyses of the prepared diet was conducted during the study to assess accuracy, homogeneity and stability. Analyses confirmed that dietary admixtures were determined to be stable for a period of at least seven weeks at room temperature. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated. Gross necropsy and histopathological evaluation was also performed. There was no mortality, relevant clinical signs, behavioural abnormalities, functional performance and sensory reactivity abnormalities found during the study. Males and females at 15000 ppm showed a reduction in bodyweight gain during. the first week of treatment compared with controls. A slight reduction in dietary intake was detected for females treated with 15000 ppm throughout the study period compared with controls. Food efficiency was also impaired for males/females during week 1 at 15000 ppm. These observations was absent from lower dose levels. There was no effect on water consumption. Within haematology an increase in clotting (prothrombin) time was detected for males treated with 15000 ppm compared with controls, absent from lower dose levels. There was no effect on blood chemical parameters. At 15000 ppm males/females an increased liver weight, both absolute and relative to terminal bodyweight. Relative kidney weight was also elevated for males only. No effects were seen at lower dose levels. There was no treatment related macroscopic abnormalities. Within histopathology at 15000 ppm, males and females indicated liver: centrilobular hepatocyte enlargement and urinary bladder: hyperplasia of the transitional epithelium, occasionally with associated subepithelial inflammatory cell infiltrates. These effects were absent from lower dose levels. The centrilobular hepatocyte enlargement was considered an adaptive response. Within controls epithelial hyperplasia was also observed in the urinary bladder of one control female, one 225 ppm male, and one 2250 ppm female which was considered as a spontaneous finding. In kidneys: globular accumulations of eosinophilic material were observed in the tubular epithelium of three male rats treated with 15000 ppm and for two male rats treated with 2250 ppm. It was considered the findings is consistent with the appearance of hydrocarbon nephropathy, which results from the excessive accumulation of alpha-2-microglobulin in renal proximal tubular epithelial cells. alpha-2-Microglobulin is found only in the proximal tubular epithelium of adult male rats. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 2250 ppm (equivalent to a mean achieved dose of 216 mg/kg body weight per day) for males/females.