Registration Dossier
Registration Dossier
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EC number: 240-539-0 | CAS number: 16484-77-8
Endpoint summary
Administrative data
Description of key information
Short-Term Repeated Dose Toxicity, Oral: Mellert (2003a)
Under the conditions of the study there are clear indications of peroxisome proliferation in both liver and kidney at the 400 and 800 ppm inclusion rates. At 100 ppm there was a clear effect in the kidney, but not the liver, for both sexes, this is important as it reinforces the kidney as the primary organ affected, and shows that there was already significant proliferation at the high dose in the 2 year study.
Short-Term Repeated Dose Toxicity, Oral: Beimborn & Leibold (2003)
Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1 500 and 2 200 ppm. No test-material related clinical findings were observed.
Short-Term Repeated Dose Toxicity, Oral: Beimborn & Leibold (2004)
Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 570, 760 and 950 ppm. No test-material related clinical findings were observed.
Short-Term Repeated Dose Toxicity, Oral: Mellert (2003b)
Under the conditions of the study the results are clearly indicative of peroxisome proliferation in both liver and kidney.
Short-Term Repeated Dose Toxicity, Oral: Kirsch (1986)
Under the conditions of the study, the no effect level is between 50 and 400 ppm for both the racemate and the isomer.
Sub-Chronic Repeated Dose Toxicity, Oral: Mellert et al. (1995)
Under the conditions of this study the no effect level for neurotoxicity was therefore above 3 000 ppm in females and above 2 500 ppm in males. The NOAEL for systemic toxicity was 75 ppm (about 6 mg/kg bw/day).
Sub-Chronic Repeated Dose Toxicity, Oral: Rondot (1998)
Under the conditions of the study no behavioural change which could be attributed to the ingestion of the test materials was observed. A dose-related reduction of the growth rate was however noted. No ocular lesion was observed. The histological examination of the eyes of all animals at the end of the study revealed no treatment-related lesion.
Sub-Chronic Repeated Dose Toxicity, Oral: Reinert (1998)
Under the conditions of the study the no-effect level was 200 ppm for both the racemate and the isomer.
Sub-Chronic Repeated Dose Toxicity, Oral: Mellert (1993)
Under the conditions of thestudy, the no observed adverse effect level in this study is for male animals 100 ppm and for female animals slightly below 100 ppm
Chronic Repeated Dose Toxicity, Oral: Bachmann et al. (1997)
Under the conditions of the study it can be stated that the test material administered with the diet to Beagle dogs in a dose of 600 ppm led to retarded body weight change and to slight decreases in haemoglobin concentrations and haematocrit values in the peripheral blood in the males and to slight decreases in inorganic phosphate and calcium in the females.
No toxicologically relevant findings occurred in the male and female dogs receiving 180 ppm and 60 ppm of the test material.
The no observed adverse affect level (NOAEL) for male and female Beagle dogsunder the chosen test conditions is 180 ppm (approx. 5 mg/kg bw/day).
Chronic Repeated Dose Toxicity, Oral: Kuehborth et al. (1988)
Under the conditions of the study a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups was observed, while the kidney weights of the female animals remained uninfluenced. Thereore the NOAEL for male rats was 20 ppm (equivalent to 1.1 mg/kg bodyweight/ day) and for female rats was 400 ppm (equivalent to 27.9 mg/kg bodyweight/ day).
Short-Term Repeated Dose Toxicity, Dermal: Allan et al. (1993)
Under the conditions of the study it was considered that 1 000 mg/kg bw/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The test material was administered to groups of 4 male and 4 female Wistar rats at dietary concentrations of 0, 100, 400 and 800 ppm for 4 weeks. Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. At the end of the administration period, cyanide-insensitive palmitoyl-CoA-oxidation activity (PALCOA) was examined in liver and kidney as an indicator for peroxisome proliferation.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- CrlGlxBrlHan:WI
- Details on species / strain selection:
- Wistar rats were selected since this strain was used in the previously conducted long-term study.
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 11 weeks
- Housing: Housed singly in type DK Ill stainless steel wire mesh cages
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: On the day of arrival the animals were subjected to a 7 day acclimatisation period during which they received ground diet and drinking water ad libitum
DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical and microbiological contaminants by the supplier.
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.)
- Route of administration:
- oral: feed
- Details on route of administration:
- The oral route was selected as this was used previously.
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: For each concentration (with exception of the control feed), the test material was weighed out and thoroughly mixed with a small amount of diet in a beaker. Subsequently, a premix was prepared in a Hobart mixer by adding an appropriate amount of dry diet and mixing for about 3 min. Then appropriate amounts of dry diet, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a Ruberg EM 100 laboratory mixer. The test material preparations were prepared once at the start of the study. The diet in the food hoppers was changed weekly.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test material in the diet over 33 days at room temperature was demonstrated prior to the study. This exceeded the interval from diet preparation to the end of the feeding period.
Homogeneity analyses of the test material preparations were demonstrated in samples of the high and low concentration at the start of the administration period. These samples also served for concentration control analyses. Additional concentration control analyses were performed with samples drawn from the mid concentration at the start of the administration period.
The stability of the test material in the diet for 33 days at room temperature was verified
Sample Preparation for Analysis
Ten gram (10 g) samples were weight directly into flasks. 20 mL doubly distilled water was added to each sample. After 20 min standing at ambient temperature, the samples were extracted twice with 35 mL of acetonitril by shaking 30 min at ambient temperature.
The combined extracts were filtered and diluted with acetonitrile ad 100 mL. Aliquot of these extracts were used directly for HPLC analysis.
Percent recoveries of the test material were 85.8 ± 1.3 % (n=4) and 85.2 ± 1.7 % (n=4) in fortified feed samples containing 101.2 ppm and 1 006 ppm of the test material.
Analytical Method
HPLC with external calibration
Column: Nucleosil 120 5C 18, 250 x 3 mm
Eluant:
50 % Acetonitrile + 0.5 M Sulfuric Acid (1 000 + 5 mL)
50 % Doubly distilled water+ 0.5 M Sulfuric Acid (1 000 + 5 mL)
Flow rate: 0.8 mL/min
Detection: UV, 230 nm
Concentration control analysis and analysis of homogeneity
The results obtained for the homogeneity and concentration control analysis of the test material in feed are summarised:
Nominal concentration: 100 mg/kg: Mean % of nominal concentration = 101.4 (SD 1.6)
Nominal concentration: 800 mg/kg: Mean % of nominal concentration = 100.8 (SD 1.4)
Considering the low standard deviation in the homogeneity analysis, it can be concluded that the test material was distributed homogeneously in feed. The mean values of the test material in feed were found to be in the range of 100.8 - 101.4 % of the nominal concentration. These results demonstrated the correctness of the concentrations of the test material in feed. - Duration of treatment / exposure:
- About 4 weeks.
- Frequency of treatment:
- The test material was administered daily in the diet.
- Dose / conc.:
- 100 ppm
- Remarks:
- Mean daily test material intake: 6.15 mg/kg bw/day (males); 7.2 mg/kg bw/day (females)
- Dose / conc.:
- 400 ppm
- Remarks:
- Mean daily test material intake: 24.5 mg/kg bw/day (males); 29.1 mg/kg bw/day (females)
- Dose / conc.:
- 800 ppm
- Remarks:
- Mean daily test material intake: 50.7 mg/kg bw/day (males); 58.7 mg/kg bw/day (females)
- No. of animals per sex per dose:
- Four per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Rationale for animal assignment:
Prior to the start of test material administration the animals were distributed according to weight among the individual test groups, separated by sex. The list of randomisation instructions was compiled with a computer. The weight variation of the animals used did not exceed 10 percent of the mean weight of each sex.
- Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for overt signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, all animals were checked daily for any abnormal clinical signs.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomise the animals. During the administration period the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals.
FOOD CONSUMPTION AND COMPOUND INTAKE:
Individual food consumption was determined weekly and calculated as mean individual food consumption (g/animal/day).
The mean daily intake of test material (group means) was calculated based upon individual values for body weight and food consumption.
Substance intake for day x = (FCx x C) /BWx
Where:
BWx = body weight on day x [g]
FCx = mean daily food consumption on day x [g]
C = concentration in the diet on day x [mg/kg]
WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: Yes
- Animals fasted: Not specified
- How many animals: All animals
- Parameters checked:
The rate of cyanide-insensitive palmitoyl-CoA-oxidation in liver and kidney homogenate was measured in all animals per test group and sex with an automatic analyser (Cobas Fara II; Roche, Mannheim, Germany). An automatic analyser (Hitachi 917; Roche, Mannheim, Germany) was used to examine the protein contents.
The following parameters were determined:
• Cyanide-insensitive palmitoyl-CoA-oxidation activity in liver
• Cyanide-insensitive palmitoyl-CoA-oxidation activity in kidney
• Total protein in liver
• Total protein in kidney
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- All animals were sacrificed by decapitation under CO2-anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
GROSS PATHOLOGY: Yes
Liver and kidney samples were taken at necropsy. The subsequent analyses of the liver and kidney homogenates were carried out in a randomised sequence. The lists of randomisation instructions were compiled with a computer using a random number generator. The following weights were determined:
1. Anaesthetised animals
2. Liver
3. Kidneys
Organ / Tissue preservation list
The following organs/ tissues were preserved in neutral buffered 4 % formaldehyde:
1. Liver (routine sections only)
2. Left kidney
No further processing was carried out on the preserved material.
HISTOPATHOLOGY: No - Statistics:
- Means and standard deviations of each test group were calculated for several parameters.
Body weight, food consumption: A comparison of each group with the control group was performed using Dunnett's test (two-sided) for the hypothesis of equal means.
Clinical pathology parameters: Non-parametric one-way analysis using Kruskall-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
Means and standard deviations of each test group were calculated for weight parameters. The number of animals per group was too low for the pathology software to perform further statistical analyses. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- All animals survived the study period in apparent good health.
One animal in the control group (No. 19) showed a palpable mass in the skin (left flank). This finding was clearly incidental and not related to treatment. - Mortality:
- no mortality observed
- Description (incidence):
- All animals survived the study period in apparent good health.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No test material-related findings were seen.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No test material-related findings were obtained.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No overt changes in volume were observed.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- After 4 weeks of test material administration significantly increased cyanide-insensitive palmitoyl-CoA-oxidation activity (PALCOA) values were found in the liver homogenates of the high dose males and mid and high dose females. In the kidney homogenates significant increases in PALCOA activity were observed in the mid and high dose males. In the treatment groups females a tendency towards increased PALCOA values was seen.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- In all treated males, the absolute kidneys weights were increased (up to 16 % above control). Although there was no dose-response relationship, this might be related to treatment.
In all treated males, the relative kidneys weights were increased (up to 19 % above control). Although there was no dose-response relationship, this might be related to treatment. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Gross lesions
Kidneys: A "pelvic dilation" was noted in one dose 2 female.
Mammary gland: A "mass" was recorded in one female control. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Dose descriptor:
- NOAEL
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not measured/tested
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 400 ppm
- System:
- hepatobiliary
- Organ:
- kidney
- liver
- Treatment related:
- yes
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- Conclusions:
- Under the conditions of the study there are clear indications of peroxisome proliferation in both liver and kidney at the 400 and 800 ppm inclusion rates. At 100 ppm there was a clear effect in the kidney, but not the liver, for both sexes, this is important as it reinforces the kidney as the primary organ affected, and shows that there was already significant proliferation at the high dose in the 2 year study.
- Executive summary:
The repeated dose toxicity of the test material was assessed via the oral route and in compliance with GLP.
The test material was administered to groups of 4 male and 4 female Wistar rats at dietary concentrations of 0, 100, 400 and 800 ppm for 4 weeks. Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. At the end of the administration period, cyanide-insensitive palmitoyl-CoA-oxidation activity (PALCOA) was examined in liver and kidney as an indicator for peroxisome proliferation.
Increased PALCOA values were seen in the liver of the high dose animals of both sexes as well as in the kidney of the high and mid dose males and of all treated females.
The PALCOA-value of one control animal was measured as 0.00 mU/mg protein. The integrity of this result cannot be assured. Therefore, this value was eliminated from the statistical evaluation. Due to the low number of control animals, the increases in kidney PALCOA activity in the treated females, although biologically meaningful, were not statistically significant.
The absolute and relative weights of the kidneys were increased in all treated males; this, however, is not considered to be an adverse effect but rather an adaptation to increased functional demand.
Under the conditions of the study there are clear indications of peroxisome proliferation in both liver and kidney at the 400 and 800 ppm inclusion rates. At 100 ppm there was a clear effect in the kidney, but not the liver, for both sexes, this is important as it reinforces the kidney as the primary organ affected, and shows that there was already significant proliferation at the high dose in the 2 year study.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 May 1994 to 23 August 1994
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-1 (90-Day Oral Toxicity)
- Version / remarks:
- 1984
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: U.S. EPA: Pesticide Assessment Guideline, Subdivision F; Neurotoxicity Screening Battery; NTIS, pp. 13 - 27, 1991.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EC Commission Directive 87/302/EEC of November 18, 1987; Part B: Methods for the determination of Toxicity Subchronic Oral Toxicity Test.
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Chbb: THOM (SPF)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: All animals were 42 days old at the start of the test material administration (i.e. day 0 for each subset).
- Weight at study initiation: At the start of the administration period (day 0) the groups had a mean body weight of: Male animals: 176 (156 - 201) g; female animals: 145 (114 - 158) g.
- Housing: The rats were housed singly in type DK III stainless steel wire cages except during motor activity measurements when they were housed in polycarbonate cages with wire covers.
- Diet: Ad libitum (except during motor activity measurements and for about 16 - 20 hours before sacrifice).
- Water: Ad libitum.
- Acclimation period: On the days of arrival, an acclimatisation period started, in which the animals were accustomed to housing and diet.
DETAILS OF FOOD AND WATER QUALITY:
The food used in the, study was assayed for chemical and microbiological contaminants.
The drinking water is regularly assayed for chemical contaminants as well as for the presence of microorganisms by a contract laboratory.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00 - 18.00 h, 12 hours dark from 18.00 - 6.00 h).
- Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: The test material was weighed out and thorougly mixed with a small amount of food in a beaker. Subsequently a premix was prepared in a BOSCH household mixer by adding an appropriate amount of food and mixing for about 3 min. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a GEBR. LODIGE laboratory mixer. The mixtures were prepared in intervals for which the stability of the test material in the diet was guaranteed. The stability of the test material in the diet was proven with a comparable batch before the start of the study. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Concentration control analyses were performed with samples taken at the start of the administration period and after 8 weeks of administration.
The stability of the test material in the diet was proven for a period of 33 days at room temperature.
The homogeneous distribution of the test material in the diet was proven in a previous study with the test material using the same mixing procedure and comparable concentrations in the diet.
Analytical checks of the concentrations confirmed that the concentrations were within an acceptable range (between 93.8 % and 110.3 % of the target concentrations). - Duration of treatment / exposure:
- 3 months
- Frequency of treatment:
- Daily
- Dose / conc.:
- 3 000 ppm
- Remarks:
- Females only, ca. 240 mg/kg body weight per day.
- Dose / conc.:
- 2 500 ppm
- Remarks:
- Males only, ca. 189 mg/kg body weight per day.
- Dose / conc.:
- 500 ppm
- Remarks:
- Males and females, ca. 38 mg/kg body weight/day.
- Dose / conc.:
- 75 ppm
- Remarks:
- Males and females, ca. 6 mg/kg body weight/day.
- No. of animals per sex per dose:
- 15 per sex per dose, separated into subgroups of 5 per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
The doses were chosen on the basis of previous studies.
- Rationale for animal assignment: The animals were randomly assigned to the groups based upon body weight, separated by sex prior to the first functional observational battery. The randomisation list was drawn up by a computer.
- Rationale for selecting satellite groups: As this study was conducted as a combined study (subchronic toxicity and neurotoxicity), the following subgroups consisting of 5 males and 5 females per dose were used:
Section A: Used for functional observational battery, motor activity measurement, perfusion fixation and neuropathology.
Section B: Used for functional observational battery, motor activity measurement, clinical chemistry, haematology, urinalyses, immersion fixation and histopathology according to guidelines for subchronic toxicity.
Section C: Used for clinical chemistry, haematology, urinalyses, immersion fixation and histopathology according to guidelines for subchronic toxicity.
In order to balance the groups for functional observational batteries and motor activity measurements, the study was conducted with 4 subsets (Section A males, Section B males, Section A females, Section B females). Within each subset, animals of control, low, mid and high dose group were tested again in randomised order, whereas randomisation was not based upon body weight. The randomisation list was drawn up by a computer.
a) All animals were examined on the same day after start of dosing. Time of testing was therefore identical for all animals;
b) For each examination animals from all test groups (including control) could be used;
c) The examinations for all subsets could be performed on the same time of the day, thus diurnal effects could be neglected. - Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The general state of health concerning mortality or moribund state of the animals was checked twice a day (Monday to Friday) and once a day (Saturdays, Sundays and public holidays). The animals were additionally examined in detail and palpated once a week.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was determined before the first neurofunctional test in order to randomise the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter in weekly intervals. Body weight was also determined on the days, when functional observational batteries were carried out. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as the body weight change.
FOOD CONSUMPTION: Yes
- The food consumption of the animals was determined weekly. The values were calculated as grams per animal per day.
- Intake of the test substance: The mean daily intake of the test material (group means) was calculated based upon individual values for body weight and food consumption using the equation below:
Substance intake for day x = (FCx x C) / BWx
Where:
BWx = Body weight on day x (g)
FCx = mean daily food consuption on day x (g)
C = ppm in the diet (mg/kg)
FOOD EFFICIENCY: Yes
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption using the equation below:
Food efficiency for day x = ((BWx - BWy) / FCy to x ) x 100
Where:
BWx = Body weight on day x (g)
BWy = Body weight on day y (last weighing date before day x) (g)
FCy to x = Mean food consumption from dau y to day x; calculated as mean daily food consumption on day x multiplied with the number of days from day y to day x (g)
WATER CONSUMPTION: Yes
- Time schedule for examinations: In the early phase of the study, water consumption was checked daily by visual inspection of the water bottles for any overt changes in volume. As water consumption was obviously higher in high dose animals, the water consumption was determined quantitatively by weighing the water bottles once a week from day 21 onwards (representative values over 4 days in males and 3 days in females). The values were calculated as grams per animal per day.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start and at the end of the administration period.
- Dose groups that were examined: The eyes of each 10 males and 10 females of the control and high dose groups were examined for pathological changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, FRG).
HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: The following examinations were carried out in 10 animals per test group and sex of sections B and C.
- Parameters checked: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (H1E model, by Bayer, Munich, FRG):
- leukocytes
- erythrocytes
- haemoglobin
- haematocrit
- mean corpuscular volume
- mean corpuscular haemoglobin
- mean corpuscular haemoglobin concentration
- platelets
The differential blood count were evaluated visually.
Clotting analyses: The clotting analyses were carried out using a ball coagulometer (KC 10 A model, by Amelung, Lemgo, FRG) and the results transferred to the computer. The following parameter was determined:
- prothrombin time (Hepato Quick's test)
CLINICAL CHEMISTRY: Yes
- Animals fasted: No
- How many animals: The following examinations were carried out in 10 animals per test group and sex of sections B and C.
- Parameters checked: An automatic analyser (Hitachi 737; by Boehringer, Mannheim, FRG) was used to examine the clinicochemical parameters.
Chloride was measured with a chloride analyser (model 925; by Ciba-Corning, Fernwald, FRG).
The following parameters were determined:
1. Enzymes
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase
- serum-ϒ-glutamyltransferase
2. Blood chemistry
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol
- magnesium
URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes. For urinalysis the individual animals were transferred to metabolism cages and urine was collected overnight.
- Animals fasted: Yes, food and water was withdrawn.
- Parameters checked: With the exception of volume, colour, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test RL, by Boehringer, Mannheim, FRG) and a reflection photometer (Urotron RL9 model by Boehringer, Mannheim, FRG) .
The specific gravity was determined using an urine refractometer. The sediment was evaluated microscopically.
The following examinations were carried out:
- volume
- colour
- turbidity
- nitrite
- pH
- protein
- glucose
- ketones
- urobilinogen
- bilirubin
- blood
- specific gravity
- sediment
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At days -7, 22, 50 and 85 functional observational batteries and measurements of the motor activity were carried out.
- Battery of functions tested:
A functional observational battery (FOB) was carried out in all animals of Section A and B on days -7, 22, 50 and 85, starting each time at about 10:00 a.m.
The FOB consisted of 4 parts, starting with passive observations, without disturbing the animals, followed by removal from the home cage and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests as well as quantitative measurements were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. The findings were ranked according to the degreee of severity, if applicable. In order to ensure that the observer was unaware of the treatment groups the tattoo numbers of the animals were covered with adhesive labels outside the cage, and examinations were carried out in randomised order.
- Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behaviour of the animals.
Attention was paid to: Posture, tremors, convulsions, behaviour, defecation, urination, general observations (all other abnormal findings).
- Open field observations:
The animals were transferred to a standard arena (62 x 62 cm with sides of 25 cm high) and observed for at least 2 minutes.
The following parameters were examined: Fur, skin colour, posture, salivation, respiration, activity, arousal level, vocalisation, tremors, convulsions, bizarre behavior, impairment of gait, lacrimation, exophthalmus, number of rearings within 2 minutes.
Thereafter, the animals were removed from the standard arena and subjected to following sensorimotor or reflex tests: hyperesthesia, abdominal tension, palpebral closure, winking reflex, pupil size, pupillary reflex, pinna reflex, audition ("startle response"), olfaction, pain perception ("tail pinch"), coordination of movements ("righting response"), vision ("visual placing response").
Then quantitative parameters were determined:
Grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, motor activity measurement.
Motor activity was measured on the same day as FOB was performed. The measurement was performed using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The cages were cleaned prior to each use. The animals were put into the cages in a randomised order. The measurements started at about 2.00 p.m. and the number of beam interrupts were counted over 18 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack. Measurements ended exactly 1.5 hours therafter. During the measurements the animals received no food and no water.
IMMUNOLOGY: No - Sacrifice and pathology:
- At the end of the administration period 5 animals per dose and sex (Section A) were sacrificed by perfusion fixation and subjected to neuropathological examinations. The remaining 10 animals per dose and sex (Section B and C) were sacrificed by decapitation under CO2 anesthesia and subjected to gross pathological assessment, followed by histopathological examinations.
All animals were sacrificed after a fasting period (withdrawal of food) for about 16 - 20 hours. - Statistics:
- See below.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- One mid dose male animal showed skin lesion from day 21 to day 56 and one low dose female animal showed tooth anomaly from day 49 onwards till the end of the study. Both findings were assessed as being incidental and not related to test material administration.
No other abnormal clinical symptoms were observed. - Mortality:
- no mortality observed
- Description (incidence):
- No animal died during the conduct of the study.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In high dose males and females, body weights were impaired statistically significant on most of the days. The values on day 91 were about 18 % below controls in males and about 14 % below controls in females.
Corresponding body weight change values were about 28 % and 29 % below controls, respectively.
No substance-related effects were seen in mid and low dose males and females. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- In high dose males and females food consumption was reduced with statistical significance on several days. The impairment was about 4 % - 19 % in males and about 6 % - 19 % in females.
In mid and low dose groups no statistically or biologically relevant deviations were seen. - Food efficiency:
- effects observed, treatment-related
- Description (incidence and severity):
- Food efficiency was impaired statistically significant in high dose males on days 14, 21, 35, 49 and 56 and in high dose females on day 35. This was assessed as being substance-related.
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- In high dose males and females water consumption was increased with statistical significance on all measurement days (except day 56, males). The increase was about 13 % - 55 % in males and about 47 % - 87 % in females when compared to controls.
Water consumption was also increased in mid dose females, the values being about 4 % - 20 % above controls. Although not statisitcally significant, a substance-related effect cannot be excluded.
In mid dose males and in low dose males and females no statistically or biologically relevant deviations were seen. - Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No substance related effects were obtained. All findings were spontaneous in nature and within the biological variation in this strain of rats.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- After a treatment period of 3 months high dose males and females had statistically significantly lower red blood cell counts, haemoglobin concentrations and haematocrit values than controls.
Slightly but statistically significantly decreased erythrocytes, haemoglobin and haematocrit values were also found in mid dose males.
No significant changes in the morphology of the red blood cells were seen in the differential blood count of treated animals of either sex.
Haematology examinations revealed no changes in white blood cell counts and in the differential white blood cell count of the treated animals.
Clotting analyses
There are no treatment-related changes in the clotting parameters measured. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Enzymes
In the sera of high dose animals statistically significantly increased alkaline phosphatase activities were measured. Furthermore, alanine aminotransferase activities were statistically significantly increased in high dose females.
Blood chemistry
Statistically significantly decreased calcium, total protein, globulin, triglyceride and cholesterol concentrations were detected in the sera of high dose males and females.
In mid dose males triglyceride and cholesterol levels were also decreased. Moreover, statistically significantly increased urea concentrations were found in high dose animals of either sex and increased creatinine levels were measured in high dose males. In high dose females serum chloride was slightly lowered. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Males of the high dose group excreted statistically significantly increased amounts of transitional epithelial cells. No other test material-induced changes were noted in the urinalyses of the treated males and females.
There is one additional statistically significant difference in the results of urinalysis (males; low dose group; urinary crystals) which is considered to be of no toxicological concern, because this deviation is incidental, inconsistent when compared with the other sex, and lacks dose-response relationship. - Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Home cage observations:
One mid dose male animal showed skin lesion on day 50. This was assessed as being incidental. No other abnormalities were obtained in any of the test groups when observing the animals in their home cages.
Open field observations:
One mid dose male animal showed skin lesion on day 50 and one low dose female animal showed tooth anomaly on days 50 and 85. Both findings were incidental.
Sensorimotor tests/Reflexes:
No substance-related effects were obtained.
Quantitative observations:
In high dose females rearing was decreased statistically significant on day 85. However, as there was no dose-response relationship (the values of mid dose females were even increased) and as only females were affected, this was assessed as being incidental and not substance-related.
In low dose males, grip strength of hindlimbs was slightly, but statistically significantly decreased on day 85. However, as there was no dose-response relationship and as only males were affected, this minor deviation was assessed as being incidental and not substance-related.
Motor activity measurement
Regarding the overall motor activity (summation of all intervals) no statistically significant deviation was seen in any of the treatment groups.
Comparing the single intervals with the control group, some few statistically significant deviations of single intervals (either increased or decreased values) occurred in various groups on different test days. However as these deviations from controls occurred only sporadically and without dose-response relationship, they were assessed as being incidental and not treatment-related. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In high dose males and females, substance-related findings were seen in the liver (increase in weight in both sexes, alterations of hepatocytes with cytoplasmic eosinophilia and granular cytoplasm in both sexes, centrolobular hypertrophy of hepatocytes in males).
Additionally, substance-related findings were seen also in the adrenal cortex of 8 males and 10 females (presence of fine lipid vacuoles; lipid storage). - Neuropathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Macroscopy and histopathology of selected organs/tisues and locations of the central and peripheral nervous system did not reveal any substance-related neuropathological alterations.
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 6 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- clinical signs
- food consumption and compound intake
- gross pathology
- haematology
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 38 mg/kg diet
- System:
- hepatobiliary
- Organ:
- adrenal glands
- kidney
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Under the conditions of this study the no effect level for neurotoxicity was above 3 000 ppm in females and above 2 500 ppm in males. The NOAEL for systemic toxicity was 75 ppm (about 6 mg/kg bw/day).
- Executive summary:
The sub-chronic toxicity of the test material following repeated dose administration of the test material via the oral route was assessed according to OECD Test Guideline 408 and in compliance with GLP.
The test material was administered to groups of 15 male and 15 female Wistar rats for 3 months at dietary concentrations of 0 ppm, 75 ppm, 500 ppm, 2 500 ppm (males only) and 3 000 ppm (females only). Based upon the results of a range finding study, different high concentrations were selected for males and females.
Food consumption and body weight were determined at least once a week. Water consumption was determined once a week from day 21 onwards. A check of the general state of health of the rats was made at least daily. Furthermore, the animals were thoroughly examined and palpated once a week. In addition to these general clinical examinations, functional observational batteries and measurements of motor activity were carried out in 10 animals per group and sex before the start of the administration period, and in the 4th, 8th and 13th week of the administration period. Before the commencement and at the end of the administration ophthalmological examinations were carried out in 10 males and 10 females of control and high dose groups. Haematological and clinic-chemical examinations as well as urinalyses were carried out at the end of the administration period in 10 animals per sex and dose. Five animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations. All other animals were subjected to gross-pathological assessment, followed by histopathological examinations.
The following findings were obtained and assessed as being related to the test substance administration:
3 000 ppm (females; about 240 mg/kg bw/day) and 2 500 ppm (males; about 189 mg/kg bw/day):
- Reduced food consumption in both sexes.
- Increased water consumption in both sexes.
- Decreased body weights, resulting in reduced values of about 18 % (males) and 14 % (females) on day 91.
- Decreased body weight change, resulting in reduced values of about 28 % (males) and 29 % (females) on day 91.
- Reduced food efficiency in males and females.
- Decrease in red blood cells, haemoglobin, haematocrit, calcium, total protein, globulins, triglycerides and cholesterol in both sexes.
- Decrease in chloride in females increase in alkaline phosphatase and urea in both sexes.
- Increase in serum creatinine and transitional epithelial cells in the urine of males.
- Increase in alanine aminotransferase in females.
- Increase of absolute and relative liver weights in both sexes.
- Alterations of hepatocytes with cytoplasmic eosinophilia and granular cytoplasm in both sexes.
- Centrolobular hypertrophy of hepatocytes in males (2/10).
- Presence of fine lipid vacuoles (lipid storage) in the adrenal cortex of 8/10 males and 10/10 females.
500 ppm (about 38 mg/kg bw/day):
- Increased water consumption in females.
- Decrease in red blood cells, haemoglobin, haematocrit, triglycerides and cholesterol in males.
75 ppm (about 6 mg/kg bwday):
- No substance-related findings.
In conclusion, the oral administration of the test material led to signs of toxicity at dietary concentrations of 3 000 ppm (females), 2 500 ppm (males) and 500 ppm (both sexes). Target organs were liver and adrenals. Toxicity was further characterised by an anaemic process and slight functional impairment of kidneys. Lipids were also lowered in either sex. No signs of neurotoxicity were detected.
Under the conditions of this study the no effect level for neurotoxicity was therefore above 3 000 ppm in females and above 2 500 ppm in males. The NOAEL for systemic toxicity was 75 ppm (about 6 mg/kg bw/day).
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 14 January 2003 to 10 February 2003
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for Testing of Chemicals; Method No. 417 Toxicokinetics
- Version / remarks:
- 1984
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability of the radiolabelled test material in the vehicle (0.5 % aqueous carboxymethylcellulose supplemented with 1 % Cremophor) was confirmed in all experiments. The stability of non-radiolabelled test material in feed at ambient temperature for at least 14 days was demonstrated previously. The homogeneity and achieved concentrations were verified analytically in all experiments. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- CrlGlxBrlHan:WI
- Details on species / strain selection:
- Recognised by international guidelines as the recommended test system. Study results will be used in relation to already available data from the same test system.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: About 7 weeks at the day of first administration of unlabelled material.
- Housing: During acclimatisation and prior to the experiment in Macrolon Cages Type Ill (2 animals per cage); during pretreatment and plasma kinetics experiments in steel wire mesh cages (1 animal per cage).
- Diet: Ad libitum prior to and during experiments.
- Water: Tap water ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.). - Route of administration:
- other: Oral gavage and diet
- Vehicle:
- other: diet (diet administration), 0.5 % Carboxymethylcellulose/ 1 % Cremophor in double-distilled water (gavage administration)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Material for oral administration via gavage: In order to achieve the required concentration and specific activity, the respective amount of non-radiolabelled test material was added to the respective amount of the stock solution of radiolabelled test material in acetone and the organic solvent was evaporated to dryness, 0.5 % Carboxymethylcellulose/ 1 % Cremophor in double-distilled water was added and filled up to the final volume. Prior to administration the preparation was sonicated and stirred to produce a homogeneous suspension. Samples were taken to check the amount of radioactivity in the preparation and to demonstrate the stability, homogeneity and the achieved concentration of the test material in the preparation.
- About 1 mL of the test material preparation per 100 g bw was administered to rats by gavage.
DIET PREPARATION
- Rate of preparation of diet: Material for oral administration via feed:
For each concentration, the test material was weighed out and thoroughly mixed with a small amount of food in a beaker. Subsequently a premix was prepared in a household mixer by adding an appropriate amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a laboratory mixer.
On day 15, mixed feed was substituted by control feed at 6:00 a.m. for 24 hours in order to avoid interference of unlabelled test material in feed with the radiolabelled test material given via gavage. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- HPLC ANALYSIS OF TEST SUBSTANCE PREPARATIONS
Feed mixtures:
Feed samples were preincubated with double-distilled water and than extracted twice with acetonitrile. Aliquots of these extracts were used for HPLC analysis. The homogeneity and achieved concentration of the test material in feed was checked via HPLC under the following conditions:
Column: Nucleosil 100, 5 C18, 250 x 4 mm
Eluent:
60 % Acetonitrile + 0.5 M Sulfuric acid (1 000 mL + 5 mL)
40 % Double-distilled water+ 0.5 M Sulfuric acid (1000 mL+ 5 mL)
Flow: 1 mL/min
Detection: UV-Extinction at 230 nm
Radioactive Preparations
The homogeneity and achieved concentration of the test material in 0.5 % Carboxymethylcellulose in double-distilled water as well as the radiochemical purity of 14C-test material were checked via HPLC (HP 1050 series) under the following conditions:
Column: Nucleosil 120, 7C18, 250 x 4 mm
Eluent: Acetonitrile / double-distilled water / 0.5 M Sulfuric acid (500/500/5 (v/v/v))
Flow: 1.2 mL/min
Detection: UV-Extinction at 230 nm HPLC Radioactivity Monitor LB 509 (Cell: VG 150 U4D)
Preparation of samples and measurement of radioactivity
Aliquots of plasma samples were mixed with scintillation cocktail (Hionic-Fluor, Packard) and counted for 10 minutes in a liquid scintillation counter (LSC; Wallac type 1409) and the disintegration rate corrected by the respective background. - Duration of treatment / exposure:
- 14 consecutive days plus gavage on Day 15.
- Frequency of treatment:
- Daily
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- Experiment 1
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Remarks:
- Experiment 2
- Dose / conc.:
- 40 mg/kg bw/day (nominal)
- Remarks:
- Experiment 3
- Dose / conc.:
- 80 mg/kg bw/day (nominal)
- Remarks:
- Experiment 4
- Dose / conc.:
- 120 mg/kg bw/day (nominal)
- Dose / conc.:
- 180 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 4 males per experiment
- Control animals:
- no
- Details on study design:
- - Dose selection rationale: In order to clarify whether changes in toxicokinetic parameters occurred in the intended dose range which was selected using the results from previously performed studies of the subacute, sub-chronic and chronic toxicity of the test material, plasma kinetics were investigated after oral administration at the following six dose levels:
Dose 1: 10 mg/kg bw radiolabeled gavage dose, corresponding to 125 ppm in feed for feeding period.
Dose 2: 20 mg/kg bw radiolabeled gavage dose, corresponding to 250 ppm in feed for feeding period.
Dose 3: 40 mg/kg bw radiolabeled gavage dose, corresponding to 500 ppm in feed for feeding period.
Dose 4: 80 mg/kg bw radiolabeled gavage dose, corresponding to 1 000 ppm in feed for feeding period.
Dose 5: 120 mg/kg bw radiolabeled gavage dose, corresponding to 1 500 ppm in feed for feeding period.
Dose 6: 180 mg/kg bw radiolabeled gavage dose, corresponding to 2 200 ppm in feed for feeding period.
The dietary concentrations in the present study were designed to reflect those which achieve a similar mean daily intake in mg/kg bw (i.e. 10, 20... 180 mg/kg bw/day) in a sub-chronic toxicity study. Thus, the achieved dose was expected to exceed target for the early phase of this 14 day study but approach it more closely towards the end. The single dose of radiolabelled material was at the specific level. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs were recorded daily at workdays during acclimatisation and application period.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: In all groups body weight was determined weekly.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes.
- Time schedule: In all groups body feed consumption was determined weekly. Feed consumption was calculated using the mean body weight of the respective feeding period.
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
OTHER: Blood samples (100 - 200 μL) were taken from the retroorbital sinus at the following times after administration: 1; 2; 4; 6; 8; 12; 24; 48 hours. Plasma samples were checked for total radioactivity. - Sacrifice and pathology:
- GROSS PATHOLOGY: No
HISTOPATHOLOGY: No - Statistics:
- Analysis of kinetic data was performed based on the group mean values using the PC program system TOPFIT Version 2.0
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No test material related clinical findings were observed.
- Mortality:
- not specified
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1 500 and 2 200 ppm.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- In the first week of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding week, i.e. the week before radioactive dosing, feed doses were closer to target at all dose levels.
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Dose descriptor:
- NOAEL
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- not measured/tested
- Critical effects observed:
- no
- Conclusions:
- Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1500 and 2 200 ppm. No test-material related clinical findings were observed.
- Executive summary:
The repeated dose toxicity of the test material was assessed as part of an in vivo toxicokinextics study according to OECD Test Guideline 417 and in compliance with GLP.
Four male rats were fed non-radiolabelled test material for 14 days at dietary concentrations of 125, 250, 500, 1 000, 1 500, and 2 200 ppm and the daily equivalent by a single gavage dose of radiolabelled test material at day 15.
As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1 500 and 2 200 ppm.
In the first week of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding week, i.e. the week before radioactive dosing, feed doses were closer to target at all dose levels.
No test-material related clinical findings were observed.
Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1 500 and 2 200 ppm. No test-material related clinical findings were observed.
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 February 1995 to 21 February 1996
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 452 (Chronic Toxicity Studies)
- Version / remarks:
- 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 83-1 (Chronic Toxicity)
- Version / remarks:
- 1984
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japan MAFF Testing Guidelines
- Version / remarks:
- 1985
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EC Commission Directive 87/302/EEC
- Version / remarks:
- 1988
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Details on species / strain selection:
- Purebred Beagle dogs were selected since this non-rodent species is recommended in the respective test guidelines and since there is extensive experience available in the laboratory with this strain of dogs.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: The animals were aged approximately 8 to 9 months at the beginning of the administration period.
- Weight at study initiation: At the beginning of the study the mean body weight (and range) was as follows:
Male animals: 10.5 (8.8 - 11.9) kg
Female animals: 9.6 (6.9 - 11.4) kg
- Fasting period before study: On the days before blood and urine collection and necropsy, the feeding bowls were usually kept in the kennel until about 14.00 h on the same day (fasting period of at least 16 hours), whereas, on the other days, they remained there until about 7.30 h the following day (adaptation and administration period).
- Housing: The dogs of each test group were housed singly in kennels.
- Diet: Each dog was offered a food ration of 700 g each day during the adaptation and administration period (in the late morning). This consisted of 350 g of powdered food pellets which was made into a paste with 350 mL drinking water in a food bowl immediately before the administration to each dog.
- Water: Blended water, which is completely demineralised water adjusted with drinking water to about 2° German hardness, was available ad libitum (automatic watering device) throughout the study (adaptation and administration period) except the time intervals during which the dogs were housed in metabolism cages for urine sampling; during this period each dog received about 500 mL drinking water. Possible further exceptions were the time intervals during which the animals' water supply was temporarily switched from blended water to drinking water because of chlorination of the container holding the blended water.
- Acclimation period: Eight days before administration started the animals were transferred from the breeding and the adaptation period began. From the 7th day before the beginning of administration onward the body weight of each individual animal was determined weekly and the food consumption daily.
DETAILS OF FOOD AND WATER QUALITY:
- Food analyses: The food used in the study was assayed for chemical and for microbiological contaminants.
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food was found to be suitable. Fed. Reg. Vol. 44, No. 91 of May 9, 1979, p. 27 354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The amount of microorganisms did not exceed 10^5/g food.
- Drinking water analyses: The drinking water has been regularly assayed for chemical contaminants as well as for the presence of microorganisms. The range of hardness and the microbial contamination of the blended water were checked by the above mentioned institutions. The completely demineralised water used for the preparation was obtained from Frankenthal drinking water.
On the basis of the analytical findings the drinking water and blended water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, Dec. 05, 1990) served as a guideline for maximum tolerable contaminants.
ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): The illumination corresponded to the natural day/night rhythm, with additional artificial light as required during working hours. - Route of administration:
- oral: feed
- Details on route of administration:
- The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Vehicle:
- water
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: New mixtures of test material and food were usually prepared at two-weekly intervals.
- Mixing appropriate amounts with: For the preparation of the mixtures of test material and food, the weighed test material was intensely mixed with a small portion of the food in a beaker using a spatula. Subsequently, a premix was prepared in a BOSCH mixer. This premix was then adjusted to the concentration desired with the appropriate amount of food and mixed in a laboratory mixer of GEBR LÖDIGE for about 10 minutes.
- Storage temperature of food: Not specified
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analyses of the test material preparations
The stability of the test material in the diet of rats for a period of 33 days was demonstrated in a comparable batch.
The stability of the test material in an aqueous medium (0.5 % carboxymethyl-cellulose-suspension) over a period of 24 hours was verified analytically.
The homogeneity of the test material preparations was proven towards the start of the administration period at the highest and lowest concentration. These analyses served also as concentration control.
Further concentration control analyses were carried out with samples of all concentration drawn at the start of the administration period and thereafter at about 3-month intervals as well as about 4 weeks before the end of the administration period.
Stability Analyses
The stability of the test substance in food was demonstrated for a period of 33 days at room temperature. The results of the stability analyses of the test
substance in aqueous CMC-Suspension (0.5 %) confirmed the stability at room temperature for at least 24 hours.
Homogeneity analyses
The homogeneity of the mixtures was verified at concentrations of 60 ppm and 600 ppm.
Concentration control analyses
The concentration control analyses revealed that the values were in the expected range of all target concentrations:
60 ppm: 95.1 – 105.8 % recovery rates
180 ppm: 93.5 – 99.8 % recovery rates
600 ppm: 96.2 – 102.5 % recovery rates
Results
The homogeneity analyses of the samples from August 6, 1990 yielded mean concentrations and relative standard deviations of 84.2 % (± 7.2 %) and 98.8 % (± 4.88 %) for the 500 mg/100 mL and 1 500 mg/100 mL-samples.
The results for the 1500 mg/100 mL-samples confirm the theoretical values and the homogeneity of the CMC-mixture. The results of the 500 mg/100 mL-samples deviate more than 10 % from the theoretical value and the coefficient of variance was more than ± 5 %.
The concentration control analyses yielded values of 95.2 % (500 mg/100 mL-samples), 95.7 % (samples from August 6, 1990) and 92.4 % for the 1 000 mg/100 mL-samples and 97.7 % (1 500 mg/100 mL samples) of the nominal concentrations. These results confirm the theoretical concentrations.
At the beginning of the stability test and after 3 and 24 hours three samples each were taken out of the mixing container and analysed. The mean concentrations found·were 99.4 % and 99.2 % of the initial concentration after 3 and 24 hours.
The results confirm the stability of the test material in CMC-Suspension at room temperature during at least 24 hours. - Duration of treatment / exposure:
- About 12 months
- Frequency of treatment:
- Daily
- Dose / conc.:
- 60 ppm
- Remarks:
- corresponding to a test material intake of ca. 1.8 mg/kg bw/day (males) and ca. 2.0 mg/kg bw/day (females)
- Dose / conc.:
- 180 ppm
- Remarks:
- corresponding to a test material intake of ca. 5.2 mg/kg bw/day (males) and ca. 5.7 mg/kg bw/day (females)
- Dose / conc.:
- 600 ppm
- Remarks:
- corresponding to a test material intake of ca. 18.3 mg/kg bw/day (males) and ca. 19.0 mg/kg bw/day (females)
- No. of animals per sex per dose:
- Five per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
The test material has already been studied in dogs. The results of this study were taken into account when choosing the doses for the present study. In a 3-month toxicity study each 4 animals per sex and dose received the test material daily for about 13 weeks as addition to the diet in doses of 0, 4, 16 or 64 mg/kg body weight. The following findings were obtained, and assessed or discussed in relation to the test material:
64 mg/kg body weight/day
- One animal showed ulcera in the buccal mucosa, a purulent conjunctivitis, pale mucous membranes, poor condition and decreased food consumption
- markedly decreased body weight gain in all animals
- decrease in haemoglobin content, packed cell volume and red blood cell counts
- moderate increase in serum urea values
- increase in Phenol-red and BSP-retention
- pH of the urine slightly increased
- increased relative heart, liver and kidney weights
- three dogs showed brown discolored fat tissue in pericardium and/or mesenterium
16 mg/kg body weight/day
- slightly decreased body weight gain
- low values for haemoglobin content, packed cell
- volume and red blood cell counts
- pH of the urine slightly increased
4 mg/kg body weight/day
- no substance-related findings
Thus, the following concentrations were selected for the present study:
60 ppm as the expected "no observed adverse effect level"
180 ppm as the intermediate dose level
600 ppm as the dose level with expected toxic effects
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Rationale for animal assignment: Before the first administration the dogs to be used in the study were allocated to the 4 different test groups. Allocation of the animals was randomised (random number generator). A subsidiary condition for the randomisation was an approximately equal mean body weight in the individual groups. - Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined at least once each working day for any evident signs of toxicity and, if signs occurred, several times daily.
In order to prepare tables of the clinical symptoms observed, the data were entered manually into the computer systems. According to the particular health status of the individual animals, sometimes several different clinical signs were documented during one week. As the tables of individual values represent summaries of all clinical findings observed during one week, different symptoms (including "nothing abnormal detected") can occur during one observation period.
A check was made for any moribund or dead animals twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and on public holidays).
BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined starting on day -7 (beginning of the adaptation period). During the further course of the study, body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals.
The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The food consumption of the animals was determined daily, starting on day -7 (beginning of the adaptation period).
The animals were offered food each day in the late morning. Unconsumed food was then reweighed the following morning and subtracted from the original amount of food offered.
Exceptions from this procedure occurred on the days before blood and urine collection and necropsy. On these days, the feeding bowls were usually kept in the kennel until about 14.00 h on the same day (fasting period of at least 16 hours).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: The amount of test material (in mg) per kg body weight consumed daily was calculated for each individual animal at the same time at which body weight was determined using the following formula:
(FCx x D) / BWx
Where:
FCx = daily consumption of food (in g) on day x of the study divided by 2 [since one half of the food ratio consisted of powdery diet and the other half of water]
D = dose in ppm
BWx =bodyweight on day x of the study (in g)
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes. Food efficiency was calculated for each animal at weekly intervals on the basis of body weight changes and the total amount of food consumed during this period, using the formula below:
(BWx – BWx-7 / FC) x 100
Where:
BWx = Body weight on day x (in g)
BWx-7 = Body weight on day x - 7 (in g)
FC = total daily food consumption (in g) over periods of 7 days (i.e. days 0 - 6; 7 – 13 and so on) divided by 2 [since one half of the food ratio consisted of powdery diet and the other half of water.
Due to technical reasons food efficiency values are given only up to study day 357.
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the beginning of the study and at the end of the administration periog all dogs to be used in the study were examined for changes of their eyes using a KOWA-RC 2 fundus camera.
- Dose groups that were examined: All dogs to be used in the study
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken from the vena cephalica antebrachii in the morning from fasted, unanesthetised animals. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomised sequence. The list of randomisation instructions was compiled with a computer using a random number generator.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: Five animals per test group and sex.
- Parameters checked: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (H 1 E model, by Bayer, Munich, FRG): Leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular heamoglobin, mean corpuscular haemoglobin concentration, platelets.
Differential blood smears and reticulocytes were evaluated visually.
The data obtained were transferred to a computer (VAX; supplied by DEC, Munich, FRG).
The clotting analyses were carried out using a ball coagulometer (KC 10 A model, by Amelung, Lemgo, FRG) and the results transferred to the computer.
The following parameters were determined:
- activated partial thromboplastin time
- prothrombin time (Quick's test)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken from the vena cephalica antebrachii in the morning from fasted, unanesthetised animals. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomised sequence. The list of randomisation instructions was compiled with a computer using a random number generator.
- Animals fasted: Yes
- How many animals: Five animals per test group and sex.
- Parameters checked: An automatic analyser (Hitachi 737; by Boehringer, Mannheim, FRG) was used to examine the clinicochemical parameters.
Chloride was measured with a chloride analyser (model 925; by Ciba-Corning, Fernwald, FRG).
The following parameters were determined: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-ϒ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium.
URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes. For urinalysis the individual animals were transferred to metabolism cages and urine was collected overnight. The urine samples were evaluated in a randomised sequence.
- Animals fasted: Yes, withdrawal of food; about 500 mL water.
- Parameters checked: With the exception of volume, colour, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test RL, by Boehringer, Mannheim, FRG) and a reflection photometer (Urotron RL9 model by Boehringer, Mannheim, FRG).
The specific gravity was determined using an urine refractometer.
The sediment was evaluated microscopically.
The following examinations were carried out: Volume, colour, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment.
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes.
The animals were anesthetised and killed by exsanguination from the cervical and brachial vessels. The exsanguinated animals were necropsied and assessed by gross pathology.
The weights of brain, liver, kidneys, adrenal glands, testes, epididymides, ovaries, and thyroid glands (with parathyroid glands) from all animals sacrificed at scheduled dates were determined. For calculation of relative organ weights, the final body weight measured in the animal house was entered off-line into the computer system.
HISTOPATHOLOGY: Yes.
The following organs or tissues were fixed in 4 % formaldehyde solution:
- all gross lesions, brain, pituitary gland, thyroid glands, parathyroid glands, thymus, trachea, lungs, heart, aorta, salivary glands (mandibular and parotid glands), liver, gallbladder, spleen, kidneys, adrenal glands, pancreas, testes, epididymides, ovaries, oviducts, uterus/vagina, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, mesenteric and axillary lymph nodes, skeletal muscle, sciatic nerve, spinal cord (cervical, thoracic and lumbar cord), sternum with sternal bone marrow, femur with articular surface of knee joint and bone marrow, eyes, prostate gland, female mammary gland, skin.
After the organs were fixed, histotechnical processing and examination by light microscopy were performed. - Statistics:
- Means and standard deviations were calculated for the variables (food consumption, body weight, body weight change, intake of the test substance and food efficiency) for the statistical evaluation.
Body weight data: Non-parametric one-way analysis using Kruskall-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Mann Whitney U-test (two-sided) for the equal medians.
Statistics of clinical pathology:
Means and standard deviations of each test group were calculated for several parameters.
Clinical pathology parameters, except differential blood count: Non-parametric one-way analysis using Kruskall-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Mann Whitney U-test (two-sided) for the equal medians.
Urinalysis, except volume, colour and turbidity: Pairwise comparison of each dose group with the control group using Fisher' s exact test for the hypothesis of equal proportions
Statistics of pathology:
Means and standard deviations of each test group were calculated for the variables of terminal body weight and of absolute and relative organ weights (related to terminal body weight) of the animals in each test group. Further statistical analyses were performed.
Weight parameters: Non-parametric one-way analysis using Kruskall-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the Wilcoxon test for the hypothesis of equal medians. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Throughout the entire administration period the animals showed no clinical symptoms which could be causally related to the administration of the test material.
The diarrhoea observed in one female dog of test group 2 (180 ppm) and in one male and two females of test group 3 (600 ppm) is judged as being incidental and not related to the test material administered, since diarrhoea occurred only sporadically (once or twice) and at scattered times throughout the study.
Vomitus was observed in one female of test group 1 (60 ppm) and in one female of test group 2 (180 ppm) without any relation to the doses, since none of the high dose group animals (600 ppm) showed this clinical finding. Moreover, one male control animal was affected by vomitus, too.
The above mentioned findings are assessed as being incidental, spontaneous in nature and not test material-related. - Mortality:
- no mortality observed
- Description (incidence):
- There were no deaths during the 12-month study period.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The single occurrence of a statistically significantly increased mean body weight change on study day 49 in the male animals of test group 1 (60 ppm) is regarded to be incidental and not related to the test material administration due to its isolated nature and the missing dose response relationship. Comparing to the control, there was no statistically significant deviation of the body weight or body weight change in any of the other dose groups (males and females). However, regarding the mean body weight change of the male animals in test group 3 (600 ppm), it was stagnant at the beginning of the study (study days 14 - 49) and in the following course of the study body weight change remained slightly reduced when compared to the concurrent control group. For the female animals of test group 3 (600 ppm) a temporary and marginal effect on body weight change in the form of slightly retarded body weight gain was noted at the very beginning of the study, i.e. from study days 0 until study day 42.
However, in the course of the study body weight gain values of the females of the highest dose group even exceeded those of the concurrent control group. Therefore, the effect on body weight gain in the females at the beginning of the study was not regarded as being of toxicological relevance.
Whereas a slight influence of the test material on body weight change of the male animals of test group 3 (600 ppm) cannot be ruled out when taking into account that there is no effect of the test material on the food consumption of the animals of either dose group. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- During the entire study period all males and most of the females of test groups 0, 1, 2 and 3 consumed all the food offered.
The occasional lack of appetite observed in one female of test group 3 (600 ppm) is unrelated to the administration of the test material since one female
control animal was affected, too. Hence, the lack of appetite can be regarded as an incidental finding.
A few animals - irrespective of the test groups - had not consumed all the food on days when they were fasted (fasting period of at least 16 hours) i.e. before blood and urine collection and before necropsy.
There is no evident effect of the test material on the food consumption of the animals.
The mean food consumption (in %) during the administration period was 100 % for males and females in all groups.
The amount of test material (in mg) per kilogram body weight consumed daily by the animals was calculated at the same time at which the body weight
was determined.
The average test material intake was for the (male and female) animals of test group 1 (60 ppm) about 2 mg/kg body weight, for test group 2 (180 ppm) about
5 mg/kg body weight and for test group 3 (600 ppm) about 19 mg/kg body weight. - Food efficiency:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Although a slight influence of the test material on body weight change was observed in the animals of test group 3 (600 ppm), there is no evident effect of the test material on the food efficiency of the animals of either dose group.
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- The ophthalmological examinations of all animals carried out before the beginning and towards the end of the administration period with a KOWA-RC 2 fundus camera revealed no pathological changes which can be attributed to the test material administration.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- On day 87 decreased haemoglobin concentrations and haematocrit values were found in the peripheral-blood of the high dose males. At the 185 day interval only reduced haemoglobin concentrations were detected in the high dose males. The other haematological examinations revealed no treatment-related changes in the males. In the females no treatment-related findings were observed in the haematology parameters.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Enzyme examinations revealed no treatment-related changes in the sera of either sex.
No test material-related alterations were seen in the blood chemistry examinations of the males. In the females decreased concentrations of inorganic phosphate and calcium were found in the sera of the high dose animals on day 90. The other blood chemistry examinations in the females revealed no treatment-related changes. - Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- No treatment-related changes were seen in the urinanalytical parameters measured.
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Absolute weights
The mean weight of the liver was significantly decreased in males of group 1, revealing no dose response relationship. This was-regarded to be unrelated to treatment.
The mean weight of the brain was significantly decreased in females of group 2, revealing no dose response relationship. This was regarded to be unrelated to treatment.
The other mean absolute weight parameters did not show significant differences when compared with the control group.
Relative weights (related to terminal body weight)
The mean weight of the liver was significantly decreased in males of group 1. There was no indication of a dose response relationship. This was regarded to be unrelated to treatment.
The other mean relative weight parameters did not show significant differences when compared with the control group. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A few gross lesions were noted in male and/or female animals. They were located in the lungs (focus, discolouration), testes (reduced organ size), ovaries (enlarged), adrenal cortez (focus), thyroid glands (unilateral cyst, unilateral gray or white discoloration), and adipose tissue (focal necrosis).
All these gross lesions are regarded to be spontaneous in origin and hence unrelated to treatment as they almost all occurred either singly, or only in the control group, or at comparable incidences in treated and control animals. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The vast majority of the gross lesions could be correlated with a meaningful microscopic finding.
Only for the unilateral reduced size of a testis of a control male (animal No. 5) and the focal unilateral grey or white discolouration noted in the thyroid glands of a male of group 2 (animal No. 13) and of another male of dose group 3 (animal No. 16) no microscopic correlate could be found in the corresponding histopathological slides. These gross lesions were regarded to have occurred spontaneously, anyway. Hence a relationship to treatment can be excluded as no treatment related microscopic findings were obtained in the testes or thyroid glands, respectively.
Histopathology did not reveal treatment related findings or a target organ. The vast majority of the microscopic findings recorded were either single observation in the control group alone, and/or in dose groups 1 and 2, alone, or in combinations with the control group, or they were seen in all or almost all groups at a comparable incidence and graded severity, or at an incidence that lacks a dose response relationship.
In the tubular epithelia of the kidneys of all male dogs and of almost all female dogs, the presence of an intracytoplasmatic granular pigment was noted. According to the tinctorial properties of this pigment (light yellow brown colour in haematoxylin and eosin stained slides) it is assumed that it represents lipofuscin. The graded severity of the pigment deposition was minimal (grade 1) to slight (grade 2), with a minimal grade in the control animals and a predominantly slight degree in males and females of dose group 3.
A few microscopic findings occurred in treated males and/or females of dose group 3, alone, or in dose group 3 and additionally in dose group 2 and, sometimes additionally in dose group 1, but not in the control group of either sex:
Slight (grade 2) focal unilateral tubular degeneration in the kidneys of a top dose male dog (animal No. 16); slight (grade 2) focal atrophy in the prostate gland of a male of groups 1 (animal No. 10), 2 (animal No. 11), and 3 (animal No. 19); cystic corpora lutea in the ovaries of three top dose females (animal Nos. 36, 37, 40); oedema in the interstitium of the female mammary gland of a high dose female dog (animal No. 36). - Histopathological findings: neoplastic:
- not specified
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There are additional statistically significant intergroup differences in the results of clinical pathology testing. These deviations are marginal, incidental or inconsistent, when compared with the other sex, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 180 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- haematology
- Remarks on result:
- other: 180 ppm corresponds to a test material intake of ca. 5.2 mg/kg bw (males), ca. 5.7 mg/kg bw/day (females) and ca. 5.4 mg/kg bw/day (combined).
- Critical effects observed:
- no
- Conclusions:
- Under the conditions of the study it can be stated that the test material administered with the diet to Beagle dogs in a dose of 600 ppm led to retarded body weight change and to slight decreases in haemoglobin concentrations and haematocrit values in the peripheral blood in the males and to slight decreases in inorganic phosphate and calcium in the females.
No toxicologically relevant findings occurred in the male and female dogs receiving 180 ppm and 60 ppm of the test material.
The no observed adverse affect level (NOAEL) for male and female Beagle dogs under the chosen test conditions is1 80 ppm (approx. 5 mg/kg bw/day). - Executive summary:
The chronic toxicity from oral administration of the test material was assessed according to OECD Test Guideline 542 and in compliance with GLP.
The test material was administered to groups of 5 male and 5 female purebred Beagle dogs in the diet at concentrations of 0, 60, 180 and 600 ppm over a period of about 12 months. Food consumption of the animals was determined daily and their body weight once a week; the dogs' state of health was checked each day. Clinical chemistry and haematological examinations as well as urinalyses were carried out once before and three times during the administration period. Ophthalmological examinations were carried out 7 days before the beginning of the administration period and on study day 363. All animals were subjected to complete gross and histopathological examination.
The following findings were obtained and assessed as being test material-related:
600 ppm (approx. 19 mg/kg bw/day):
- Slight but not statistically significant influence on body weight gain in the males (especially from study days 14 - 49 mean body weight change was stagnant and/or retarded; thereafter the animals gained weight but still in a reduced manner);
- Decrease in haemoglobin and haematocrit in the males;
- Decrease in calcium and inorganic phosphate in the females.
180 ppm (approx. 5 mg/kg bw/day):
- No substance-induced changes.
60 ppm (approx. 2 mg/kg bw/day):
- No substance-induced changes.
Under the conditions of the study it can be stated that the test material administered with the diet to Beagle dogs in a dose of 600 ppm led to retarded body weight change and to slight decreases in haemoglobin concentrations and haematocrit values in the peripheral blood in the males and to slight decreases in inorganic phosphate and calcium in the females.
No toxicologically relevant findings occurred in the male and female dogs receiving 180 ppm and 60 ppm of the test material.
The no observed adverse affect level (NOAEL) for male and female Beagle dogs under the chosen test conditions is 180 ppm (approx. 5 mg/kg bw/day).
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 09 December 2003 to 18 February 2004
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for Testing of Chemicals; Method No. 417 Toxicokinetics
- Version / remarks:
- 1984
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test material in the solvent/dispersant/vehicle/test medium: The stability of the radiolabelled test material in the vehicle (0.5 % aqueous carboxymethylcellulose) was confirmed in all experiments. The stability of non-radiolabelled test material in feed at ambient temperature for at least 33 days was demonstrated previously.
- The homogeneity and achieved concentrations were verified analytically in all experiments. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- CrlGlxBrlHan:WI
- Details on species / strain selection:
- Recognised by international guidelines as a recommended test system. Study results will be used in relation to already available data from the same test system.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: About 8 weeks at the day of first administration of unlabelled material
- Housing: During acclimatisation and prior to the experiment in Macrolon Cages Type Ill (2 animals per cage); during pre-treatment and plasma kinetics experiments in steel wire mesh cages (1 animal per cage).
- Diet: Ad libitum prior to and during experiments.
- Water: Tap water ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.). - Route of administration:
- other: Gavage and diet
- Details on route of administration:
- Dose 1: 190 ppm non-radiolabelled test material in feed (corresponding to about 15 mg/kg bw) averaged over 60 consecutive days; Single oral dose of 14C-test material at 15 mg/kg bw at day 61.
Dose 2: 380 ppm non-radiolabelled test material in feed (corresponding to about 30 mg/kg bw) averaged over 60 consecutive days; Single oral dose of 14C-test material at 30 mg/kg bw at day 61.
Dose 3: 570 ppm non-radiolabelled test material in feed (corresponding to about 45 mg/kg bw) averaged over 60 consecutive days; Single oral dose of 14C-test material at 45 mg/kg bw at day 61.
Dose 4: 760 ppm non-radiofabelled test material in feed (corresponding to about 60 mg/kg bw) averaged over 60 consecutive days; Single oral dose of 14C-test material at 60 mg/kg bw at day 61.
Dose 5: 950 ppm non-radiolabelled test material in feed (corresponding to about 75 mg/kg bw) averaged over 60 consecutive days; Single oral dose of 14C-test material at 75 mg/kg bw at day 61. - Vehicle:
- other: diet (diet administration), 0.5 % carboxymethylcellulose in double-distilled water (gavage administration)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Material for oral administration via gavage:
In order to achieve the required concentration and specific activity, the respective amount of non-radiolabelled material was added to the respective amount of the stock solution of radiolabelled material in acetone and the organic solvent was evaporated to dryness; 0.5 % carboxymethylcellulose in double-distilled water was added to the residue and filled up to the final volume. Prior to administration the preparation was sonicated and stirred to produce a homogeneous suspension. Samples were taken to check the amount of radioactivity in the preparation and to demonstrate the stability, homogeneity and the achieved concentration of the test substance in the preparation.
DIET PREPARATION
- Mixing appropriate amounts with: For each concentration, the test material was weighed out and thoroughly mixed with a small amount of food in a beaker. Subsequently a premix was prepared in a household mixer by adding an appropriate amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a laboratory mixer. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Method type(s) for identification: HPLC
HPLC Analysis of Feed Mixtures
Feed samples were preincubated with double-distilled water and than extracted twice with acetonitrile. Aliquots of these extracts were used for HPLC analysis. The homogeneity and achieved concentration of the test material in feed was checked via HPLC under the following conditions:
Column: Nucleosil 100, 5 C18, 250 mm x 4 mm
Eluent:
50 % Acetonitrile + 0.5 M Sulfuric acid (1 000 mL + 5 mL)
50 % Double-distilled water+ 0.5 M Sulfuric acid (1 000 mL+ 5 mL)
Flow: 1 mL/min
Detection: UV-Extinction at 230 nm
HPLC Analysis of Radioactive Preparations
The homogeneity and achieved concentration of the test matrial in 0.5 % carboxymethylcellulose in double-distilled water as well as the radiochemical purity of 14C-test material were checked via HPLC (HP 1050 series) under the following conditions:
Column: Nucleosil 120, 7C18, 250 mm x 4 mm
Eluent: Acetonitrile / double-distilled water / 0.5 M Sulfuric acid (500/500/5 (v/v/v))
Flow: 1.2 mL/min
Detection: UV-Extinction at 230 nm HPLC Radioactivity Monitor LB 509 (Cell: VG 150 U4D)
- Preparation of samples and measurement of radioactivity: Aliquots of plasma samples were mixed with scintillation cocktail (Hionic-Fluor, Packard) and counted for 10 minutes in a liquid scintillation counter (LSC; Wallac type 1409) and the disintegration rate corrected by the respective background. - Duration of treatment / exposure:
- 60 consecutive days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 15 mg/kg bw/day (nominal)
- Remarks:
- Experiment 1
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- Experiment 2
- Dose / conc.:
- 45 mg/kg bw/day (nominal)
- Remarks:
- Experiment 3
- Dose / conc.:
- 60 mg/kg bw/day (nominal)
- Remarks:
- Experiment 4
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 4 males per dose
- Control animals:
- no
- Details on study design:
- - Dose selection rationale:
Dose selection for this study was based on information gathered in a previous kinetic study. Plasma kinetics were investigated after oral administration at the following five dose levels:
Dose 1: 15 mg/kg bw radiolabelled dose (gavage) corresponding to 190 ppm in feed.
Dose 2: 30 mg/kg bw radiolabelled dose (gavage) corresponding to 380 ppm in feed.
Dose 3: 45 mg/kg bw radiolabelled dose (gavage) corresponding to 570 ppm in feed.
Dose 4: 60 mg/kg bw radiolabelled dose (gavage) corresponding to 760 ppm in feed.
Dose 5: 75 mg/kg bw radiolabelled dose (gavage) corresponding to 950 ppm in feed.
The dietary concentrations in the present study were designed to reflect those which achieve a similar mean daily intake in mg/kg bw in a subchronic toxicity study (i.e. 15, 30, 45, 60, and 75 mg/kg bw/day). Thus, the achieved dose was expected to exceed target for the early phase of this 60 day study but approach the target more closely towards the end of the study. The single dose of radiolabelled material was at the specified level.
- Rationale for animal assignment: The animals were selected based on health status and to provide a narrow range of body weights; animals were assigned to the groups randomly, i.e. without conscious bias. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs were recorded daily on workdays (Monday- Friday) during acclimatisation and application period.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: In all groups, body weight was determined weekly.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: In all groupsw, feed consumption was determined weekly. Feed consumption was calculated using the mean body weight of the respective feeding period.
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
OTHER: Blood samples (100 - 200 μL) were taken from the retroorbital sinus at the following times after administration: 1; 2; 4; 6; 8; 12; 24; 48 hours. Plasma samples were checked for total radioactivity. - Sacrifice and pathology:
- GROSS PATHOLOGY: No
HISTOPATHOLOGY: No - Statistics:
- Analysis of kinetic data was performed based on the group mean values using the PC program system TOPFIT Version 2.0.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No test material related clinical findings were observed.
- Mortality:
- not specified
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 570, 760, and 950 ppm.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- During the first month of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding month, i.e. the month before radioactive dosing, feed doses were closer to target at all dose levels.
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Dose descriptor:
- NOAEL
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- not measured/tested
- Critical effects observed:
- no
- Conclusions:
- Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 570, 760 and 950 ppm. No test-material related clinical findings were observed.
- Executive summary:
The repeated dose toxicity of the test material was assessed as part of an in vivo toxicokinextics study according to OECD Test Guideline 417 and in compliance with GLP.
Four male rats were fed non-radiolabelled test material for 60 days at dietary concentrations of 190, 380, 570, 760, and 950 ppm and the daily equivalent by a single gavage dose of radiolabelled test material at day 61.
As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 570, 760, and 950 ppm.
During the first month of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding month, i.e. the month before radioactive dosing, feed doses were closer to target at all dose levels.
No test material related clinical findings were observed.
Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 570, 760 and 950 ppm. No test-material related clinical findings were observed.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 03 December 2002 to 09 January 2003
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The test material was administered to groups of 4 male and 4 female B6C3F1 mice at dietary concentrations of 0, 250, 750 and 2 500 ppm for 4 weeks. Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. At the end of the administration period, cyanide-insensitive palmitoyl-CoA-oxidation (PALCOA) was examined in liver and kidney as an indicator for peroxisome proliferation.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Remarks:
- 86C3F1/Crl
- Details on species / strain selection:
- B6C3F1 mice were selected since this strain was used in the previously conducted long-term study.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 11 weeks of age
- Housing: The mice were housed singly in Makrolon® cages, type M I with wire cover (floor area about 200 cm^2).
- Diet: Ad libitum except during the fasting period.
- Water: Ad libitum
- Acclimation period: On the day of arrival the animals were subjected to an 8 day acclimatisation period during which they received ground diet and drinking water ad libitum for eight days.
DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical and microbiological contaminants by the supplier. The drinking water is regularly assayed for chemical contaminants as well as for the presence of microorganisms by a contract laboratory.
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of May 9, 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 5 x 10^5 /g food.
On the basis of the analytical findings the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, December 5, 1990) served as a guideline for maximum tolerable contaminants.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C.
- Humidity (%): 30 - 70 % relative humidity.
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.).
- Route of administration:
- oral: feed
- Details on route of administration:
- The oral route was selected since this was used previously.
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): For each concentration (with exception of the control feed), the test material was weighed out and thoroughly mixed with a small amount of diet in a beaker. Subsequently, a premix was prepared in a Hobart mixer by adding an appropriate amount of dry diet and mixing for about 3 min. Then appropriate amounts of dry diet, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a Ruberg EM 100 laboratory mixer. The mixtures were prepared once before start of the study. The diet in the food hoppers was changed weekly. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test material in the diet over 33 days at room temperature was demonstrated prior to the study. This exceeded the interval from diet preparation to the end of the feeding period.
Homogeneity analyses of the test material preparations were demonstrated in samples of the high and low concentration at the start of the administration period. These samples also served for concentration control analyses. Additional concentration control analyses were performed with samples drawn from the mid concentration at the start of the administration period.
The stability of the test material in the diet for 33 days at room temperature was verified.
The homogeneity and achieved concentration of the test material preparations were satisfactory. The recovery rates were in an acceptable range (95.5 % - 96.9 % of the target concentrations
CONCENTRATION CONTROL ANALYSIS AND ANALYSIS OF THE HOMOGENEITY
Vehicle: Feed "Kliba lab diet for rat-hamster-mouse".
Storage conditions of the samples until analysis: Freezer
SAMPLE PREPARATION FOR ANALYSIS
Ten gram (10 g) samples were weight directly into Erlenmeyer flasks. 20 mL doubly distilled water was added to each sample. After 20 min standing at ambient temperature, the samples were extracted twice with 35 mL of acetonitrile by shaking 30 min at ambient temperature. The combined extracts were filtered and diluted with acetonitrile ad 100 mL.
Aliquot of these extracts were used directly for HPLC analysis.
ANALYTICAL METHOD
HPLC with external calibration
Column: Nucleosil 120 5C 18, 250 x 3 mm
Eluant:
50 % Acetonitrile + 0.5 M Sulfuric Acid (1 000 + 5 mL)
50 % Doubly distilled water+ 0.5 M Sulfuric Acid (1 000 + 5 mL)
Flow rate: 0.8 mL/min
Detection: UV, 230 nm
Considering the low standard deviation in the homogeneity analysis, it can be concluded that the test material was distributed homogeneously in feed.
The mean values of the test material in feed were found to be in the range of 96.1 - 96.9 % of the nominal concentration.
These results demonstrated the correctness of the concentrations of the test material in feed. - Duration of treatment / exposure:
- The test material was administered for about 4 weeks.
- Frequency of treatment:
- The test material was administered daily in the diet
- Dose / conc.:
- 250 ppm
- Remarks:
- Mean daily test material intake: 57.9 mg/kg bw/day (males); 87.0 mg/kg bw/day (females)
- Dose / conc.:
- 750 ppm
- Remarks:
- Mean daily test material intake: 165.0 mg/kg bw/day (males); 251.4 mg/kg bw/day (females)
- Dose / conc.:
- 2 500 ppm
- Remarks:
- Mean daily test material intake: 681.0 mg/kg bw/day (males); 955.9 mg/kg bw/day (females)
- No. of animals per sex per dose:
- 4 per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Rationale for animal assignment:
Prior to the start of test material administration the animals were distributed according to weight among the individual test groups, separated by sex. The list of randomisation instructions was compiled with a computer. The weight variation of the animals used did not exceed 10 % of the mean weight of each sex.
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for overt signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, all animals were checked daily for any clinically abnormal signs.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomise the animals. During the administration period the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Individual food consumption was determined weekly and calculated as mean individual food consumption (g/animal/day).
- The mean daily intake of test material (group means) was calculated based upon individual values for body weight and food consumption.
(FCx x C) / BWx
Where:
BWx = Body wieght on day x [g]
FCx = Mean daily food consumption on day x [g]
C = Concentration in the diet on day x [mg/kg]
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked: The rate of cyanide-insensitive palmitoyl-CoA-oxidation in liver and kidney homogenate was measured in all animals per test group and sex with an automatic analyser (Cobas Fara II; Roche, Mannheim, Germany). An automatic analyser (Hitachi 917; Roche, Mannheim, Germany) was used to examine the protein contents.
The following parameters were determined:
• Cyanide-insensitive palmitoyl-CoA-oxidation activity in liver (Kinetic UV-test, 334 nm, 37 °C)
• Cyanide-insensitive palmitoyl-CoA-oxidation activity in kidney (Kinetic UV-test, 334 nm, 37 °C)
• Total protein in liver (Biuret method)
• Total protein in kidney
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- All surviving animals were sacrificed after a fasting period (withdrawal of food) for at least 16 - 20 hours.
GROSS PATHOLOGY: Yes.
Liver and kidney samples were taken at necropsy. The subsequent analyses of the liver and kidney homogenates were carried out in a randomised sequence. The lists of randomisation instructions were compiled with a computer using a random number generator.
All animals were sacrificed by decapitation under CO2-anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The following weights were determined:
1. Anaesthetised animals
2. Liver
3. Kidneys
The following organs/ tissues were preserved in neutral buffered 4 % formaldehyde:
1. Liver (routine sections only)
2. Left kidney
No further processing was carried out on the preserved material.
HISTOPATHOLOGY: Yes.
The livers of all animals were blocked, sliced and stained using both H&E as well as antibody PMP 70 for the detection of peroxisomes.
Histotechnical processing of the liver was carried out using paraplast embedding and staining with Hematoxylin and Eosin (HE) and PMP70 immunohistochemistry on four animals per group for the control 250 ppm, 750 ppm and 2500 ppm groups.
The slides were evaluated by light microscopy. An initial semiquantitative assessment of the PMP70 immunohistochemistry of control and high dose animals according to the following grading scheme was carried out:
+: Very low number of positively labelled, intracytoplasmic globules in hepatocytes.
++: Moderate number of positively labelled, intracytoplasmic globules in hepatocytes.
++(+): Moderate to high number of positively labelled, intracytoplasmic globules in hepatocytes.
After this approach, a quantitative analysis of PMP70 positive cytoplasmic labeling area of the hepatocytes of all animals was carried out according to the following procedure:
At a magnification of 1 000-fold (oil immersion), 10 areas from two liver lobes (5 per lobe) were randomly selected but had to be located close to the central veins approximately 2 - 3 cells apart from the central vein lumina). Images were taken and these images were used for further measurements. This procedure was selected to ensure that especially centrilobular areas (Rappaport zone 3) are considered for measurement as peroxisome proliferation is initiated in centrilobular located hepatocytes.
In the selected measurement fields, an areal measurement of positively labeled, cytoplasmic areas of hepatocytes was carried out and was related to the whole liver measurement field in per cent.
In addition, a counting of the number of labelled cytoplasmic areas of hepatocytes in the measurement field was performed as well. Roughly, this will represent the number of peroxisomes. However, it can not be excluded that due to the limited resolution at the light microscopic level one area may represent one or more peroxisomes.
For the quantitative analysis, the Leica Q500 image analysis system was used. - Statistics:
- Means and standard deviations of each test group were calculated for several parameters.
Body weight, food consumption: A comparison of each group with the control group was performed using Dunnett’s test (two-sided) for the hypothesis of equal means.
Clinical pathology parameters: Non-parametric one-way analysis using Kruskal-Wallis test (two sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
Means and standard deviations of each test group were calculated for weight parameters. The number of animals per group was too low for the pathology software to perform further statistical analyses. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No test material-related findings were seen.
- Mortality:
- no mortality observed
- Description (incidence):
- All animals survived the study period in apparent good health.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Male mice from the mid and high dose groups showed a small (not statistically significant) reduction in weight gain during weeks 2 and 3, but the differences were substantially recovered during week 4.
Female mice in the high dose group showed a progressive reduction in weight gain from week 2 so that by termination these mice were some 10% lighter than controls . The difference did not attain statistical significance at any point. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Male mice from all test groups showed a substantial reduction in food consumption during the first two weeks of the study, although this attained statistical significance only for the first week in the group fed 750 ppm in the diet. After this the consumption returned to near control levels for the high dose group but remained depressed at the mid and low doses.
By contrast female mice showed no consistent test material-related change. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No overt changes in volume were seen.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- After 4 weeks of test material administration significantly increased cyanide-insensitive palmitoyl-CoA-oxidation activity (PALCOA) values were found in the liver homogenates of the high dose males and mid and high dose females. In the kidney homogenates dose-related, significant increases in PALCOA activity were observed in all treatment groups of male and female animals.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The absolute and relative liver weight was increased in the high dose males and females.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Liver: An "enlarged" organ was recorded for all male and female high dose animals.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Light microscopy of HE stained liver slides showed a moderate (grade 3) to extreme (grade 5) hypertrophy of hepatocytes in all high dose groups (2 500 ppm) with tissue areas showing a centrilobular or more panlobular accentuation. The hypertrophic hepatocytes do also exhibit a moderate (grade 3) cytoplasmic eosinophilia in these animals. Treatment with 250 or 750 ppm led to no compound-induced morphological changes in the HE stains.
The semiquantitative assessment of the immunohistochemical stain for peroxisomes (PMP70) revealed an increase of positively staining, intracytoplasmic globules in the hepatocytes of the 2 500 ppm test groups giving evidence of a peroxisomal proliferation. All zones of the liver lobules were involved, with a varying accentuation of centrilobular or peripherolobular areas. Peroxisomal proliferation seems to be slightly more pronounced in the females, when compared with the male high dose test animals.
The quantitative assessment of positively stained, intracytoplasmic area size and number of areas related to the measurement field revealed an increase in area size as well as in the number of positively stained areas. This increase shows a clear dose-dependency and is already significantly present in the females of the low dose group. In males, a clear trend in the low dose group is already present as well and regarded to be a biologically relevant increase. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Dose descriptor:
- NOAEL
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not measured/tested
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the study the results are clearly indicative of peroxisome proliferation in both liver and kidney.
- Executive summary:
The aim of the study was to obtain additional information on the toxicological profile of the test material, especially with regard to peroxisome proliferation.
The test material was administered to groups of 4 male and 4 female B6C3F1 mice at dietary concentrations of 0, 250, 750 and 2 500 ppm for 4 weeks. Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. At the end of the administration period, cyanide-insensitive palmitoyl-CoA-oxidation (PALCOA) was examined in liver and kidney as an indicator for peroxisome proliferation.
Increased PALCOA values were seen in the liver of the high dose males and high and mid dose females as well as in the kidney of all treated male and female animals.
The absolute and relative liver weights were increased in high dose males and females; this, however, is not considered to be an adverse effect but rather an adaptation to increased functional demand.
Under the conditions of the study the results are clearly indicative of peroxisome proliferation in both liver and kidney.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- (1983)
- Deviations:
- yes
- Remarks:
- Extension of the study duration
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Chbb = THOM (SPF)
- Age at study initiation: 42 days.
- Weight at study initiation: The mean weight of the rats used was as follows 7 days after they had been supplied: Male animals: 135.4 g (125 - 146 g); female animals: 123.6 g (111 - 138 g). The mean weight of the rats used was as follows at the beginning of administration: Male animals: 188.7 g (167 - 208 g); female animals: 145.6 g (131 - 167 g)
- Housing: During the study period the rats were housed singly in DK III-type stainless steel wire mesh cages, floor area about 900 cm² (Becker & CO, Castrop-Rauxel, Germany).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 7 days
DETAILS OF FOOD AND WATER QUALITY:
- Analysis of feed: The feed used in the study was assayed for contaminants. In view of the duration of use and the results on the content of contaminants the food used was verified to be suitable. The Proposed EPA Guidelines of May 9, 1979, Fed. Reg. Vol. 44, No. 91, page 27354 were used as the basis for maximum acceptable contaminants.
- Analysis of drinking water: The drinking water is regularly assayed for contaminants by the municipal authorities of Frankenthal and by the Department of Water Chemistry and Technical Services of BASF Aktiengesellschaft. The drinking water was verified to be suitable on the basis of the results. The Drinking Water Regulation of Jan. 31, 1975 was used as the guideline.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity
- Air changes (per hr): No data (fully air conditioned rooms).
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours). - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Mixing appropriate amounts with: Various amounts, depending on the test group, of the racemate and isomer were added to the ground feed. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- At the beginning of the study 3 samples of each test material formulation was analysed to verify the correctness of the concentration and homogeneity of the mixture and the stability of the test material in the food for 10 days. At the originally scheduled end of the study 1 further sample of each concentration was analysed to confirm the concentration.
Method: After extraction the concentration of the test material was determined by thin layer chromatography.
The examinations carried out at the beginning and end of the study confirmed the correctness of the concentration. The stability of the test material for 12 days in the feed was verified; the values of the stability analysis after 12 days differed by < 10 % from the values on the day that the feed was mixed. The homogeneous distribution of the test material in the feed was also confirmed; the analytical values did not differ by > 10 %. - Duration of treatment / exposure:
- 49 days
The duration of the 28 days study was extended by 21 days of dosing with the purpose to follow an observed effect in clinical chemistry found in the 400 ppm group at day 23. - Frequency of treatment:
- Fed continuously in diet.
- Dose / conc.:
- 50 ppm
- Remarks:
- Racemate
- Dose / conc.:
- 400 ppm
- Remarks:
- Racemate
- Dose / conc.:
- 50 ppm
- Remarks:
- Isomer
- Dose / conc.:
- 400 ppm
- Remarks:
- Isomer
- No. of animals per sex per dose:
- 10 per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: 400 ppm was chosen as the dose with a toxic effect since in an earlier study a reduction in body weight and an increase in the relative kidney weight were observed after the administration of 400 ppm test material in the feed. 50 ppm proved to be the no effect level in this study. In a further study the test material was administered for 7 months via the feed also at a dose level of 400 ppm. Increased relative kidney and liver weights as well as signs of anaemia were found. The administration of 100 ppm also resulted in increased kidney weights. Therefore 50 ppm was chosen as the no effect level for the present study.
- Rationale for animal assignment: On the day they were supplied at an age of 28 days the rats were split up into test groups strictly at random. - Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: The state of health was checked daily. Each time that the animals were weighed they were also inspected and palpated.
A check for mortality was made twice daily (Mondays to Fridays) and once daily (Saturdays, Sundays and public holidays).
BODY WEIGHT: Yes
- Time schedule for examinations: Once a week. All the animals were weighed once weekly (Wednesdays). The body weight of each rat was determined on the same day of the week and at the same time of the day.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Once a week. In order to determine the amount of food consumed, the contents of the food bowl were weighed and subtracted from the amount offered. The food consumption was determined over the course of a week (weighing of food offered on Wednesdays and weighing of food bowl contents on Wednesdays) during the acclimatisation and administration periods.
The mean amount of test material (in mg) ingested per day was calculated per kg body weight for each week of the study according to the following formula:
(FC . D) / [(BWx + BWx+7) / 2]
Where:
FC = Mean daily food consumption (g) during a week of the study (from day x to day x+7)
D = Dose in ppm
BWx = Mean body weight on day x of the study
BWx+7 = Mean body weight on day x + 7 of the study
HAEMATOLOGY: Yes
- Time schedule for collection of blood: With the exception of the differential blood counts and reticulocyte smears, the blood samplings and the subsequent analysis of the blood and plasma samples were carried out in random sequence 23 and 43 days after the beginning of administration. The blood required was taken from the retroorbital venous plexus. In each case blood sampling took place in the morning between 7:00 and 12:00 hours.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 animals per test group and sex.
- Parameters examined: The following parameters were determined using a particle counter: Haemoglobin, erythrocytes, haematocrit, mean haemoglobin content per erythrocyte, mean cell volume, mean corpuscular haemoglobin concentration, platelets and leukocytes.
The differential blood count and the reticulocytes were evaluated with an automatic differentiator.
The clotting analyses was carried out using a coagulator. The following parameters was determined: Thromboplastin time (Hepato Quick test).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood samplings and the subsequent analysis of the blood and plasma samples were carried out in random sequence 23 and 43 days after the beginning of administration. The blood required was taken from the retroorbital venous plexus. In each case blood sampling took place in the morning between 7:00 and 12:00 hours.
- Animals fasted: Not specified
- How many animals: 10 animals per test group and sex.
- Parameters examined: An automatic analyser (GSA II, Greiner Electronics) was used for investigating the clinico-chemical blood parameters. The following parameters were determined: Total bilirubin, creatinine, urea, sodium, potassium, total protein, glucose, inorganic phosphate, calcium, chloride, cholesterol and albumin
The enzyme activities in the plasma were determined with an automatic enzyme analyser. The following enzyme activities were determined: Glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and alkaline phosphatase. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, all animals
The animals were sacrificed by decapitation under CO2 anesthesia and subjected to gross-pathological assessment.
Liver, kidneys, testes and adrenals were weighed.
The following organs or tissues were fixed in 4 % formaldehyde solution: Liver, kidneys, spleen, adrenals, heart, testes, all gross lesions and sternum with marrow.
After fixation, processing, examination was conducted by light microscopy.
HISTOPATHOLOGY: Yes, all organs of control and high dose groups. For liver and kidneys all dose groups were evaluated. - Statistics:
- Means and standard deviation were calculated for the variables food consumption, body weight and body weight change of the animals of each test group in the statistical evaluation of the study; means and the standard deviation were calculated for the statistical evaluation of the variables absolute organ weights of the animals of each test group.
The following were compared:
control with groups 1 and 2 (racemate)
control with groups 3 and 4 (isomer)
For the parameters body weight change and absolute organ weights examination for statistical significance was carried out using at test generalized by WILLIAMS for the simultaneous comparison of several dose groups with a control group.
Blood and plasma examinations: After statistical adjustment (NALIMOV criterion) means and standard errors were calculated. For the purpose of testing the significance, the individual dose groups were compared with the control group using the t-test. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No signs occurred at any time during the administration period that could be attributed to the administration of the test material.
- Mortality:
- no mortality observed
- Description (incidence):
- No animal died during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The body weight gain of the rats of teat groups 1 - 4 exhibited no test material-induced impairment in comparison with the control animals
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- As regards the amount of food consumed there were no differences between the untreated control animals and the rats of test groups 1 - 4
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- On the basis of the plausibility criteria the significant changes can be denied any test material-induced relevance.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Cholesterol: The reduction in the plasma cholesterol level detected in the female animals after both blood samplings (23 and 43 days after the beginning of administration) in test groups 2 and 4 (400 ppm racemate and 400 ppm isomer) is probably of a test material-induced origin. In the case of the male animals, however, only after blood sampling 1 (23 days after the beginning of administration) in test group 4 (400 ppm isomer) were there significantly reduced cholesterol values that can no doubt be also ascribed to the test material administered. On account of the absence of any concomitant signs, however, it is difficult to assign these findings to a specific clinical picture.
Urea, creatinine and calcium: 23 days after the beginning of administration (blood sampling 1) the female animals of test group 4 (400 ppm isomer) were found to have signicantly increased creatinine and urea values, which, however, after blood sampling 2 (43 days after the beginning of administration) only appeared as a trend. However, the administration of 400 ppm test material as racemate did not cause any changes to the above-mentioned parameters in the female animals. The male animals of this test group (400 ppm racemate), however, were found to have a slight increase in the urea concentration in the plasma after blood sampling. The cause of the increased creatinine and urea values is probably the marginal renal insufficiency induced both by the racemate and by the isomer, although the different reactions of the two sexes to the two test materials must be assessed as incidental. The reduction in the calcium concentration detected at the end of the administration period (blood sampling 2) in the female animals of teat group 2 (400 ppm racemate) is probably also attributable to renal insufficiency.
Other parameters: On the basis of the plausibility criteria the other significant changes can be denied any test material induced relevance.
In summary, it can be said that the 6-week administration of 400 ppm racemate and isomer to rats via their feed led to the following clinico-chemical changes.
- Reduction of the cholesterol level in the female (racemate and isomer) and male rats (isomer).
- Reduction of the calcium concentration in the female animals (racemate).
- Increase in the creatinine values in the female animals (isomer) and in the urea concentration in the male (racemate) and female rats (isomer). The cause of these findings is probably a marginal renal insufficiency.
The administration of 50 ppm test material did not cause any changes that could be related to the administration of the test material.
No differences could be established between the racemate and the isomer regarding their toxicological profile.
Thus, in terms of clinical chemistry, the no adverse effect level should be in a range between 50 ppm and 400 ppm for both sexes and for both test materials. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The administration of the test material as racemate in concentrations of 50 ppm and 400 ppm and in the isomer in concentrations of 50 ppm and 400 ppm for 7 weeks in the diet caused a slight, significant increase of the kidney weights both in the male and in the female animals of group 4 (isomer, 400 ppm) in comparison with the control.
The gross-pathological and histopathological examinations revealed neither changes that were able to explain this weight increase nor any other test material-induced alterations.
On account of their distribution in the control and test groups all the other findings that were obtained are to be regarded not as test material-induced but as spontaneous. - Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The minimum to moderate fatty infiltration of the hepatocytes observed in all the groups exhibited an accentuated distribution pattern in the periphery in most cases; this suggests an alimentary origin. Similarly, the focal interstitial nephritis, and the calcified casts in the tubules of the cortical-medullary margin of the kidney found particularly in the female animals must be assessed as spontaneous changes on account of their distribution in the control and test groups.
The same applies to the testicular atrophy, the haemorrhagic erosions in the stomach, the focal dystrophic calcification of the adrenal, the cystically dilated sinus in the adrenal medulla and the single fiber necrosis in the heart, each of which was observed only in one animal. On the basis of the results obtained in the present study it can be said that a statistically significant increase in the mean kidney weight was found in both sexes after the 7-week administration of the isomer of the test material in the diet to male and female rats in a concentration of 400 ppm. A morphological equivalent for this weight increase could not be verified by light microscopy. Nor were there any morphological differences between the animals to which the test material was administered as racemate or the isomer in the diet. - Histopathological findings: neoplastic:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 50 other: ppm (corresponding to 4.4 - 4.8 mg/kg bw and day)
- Sex:
- male/female
- Basis for effect level:
- other: Increased kidney weights (only females) and clinicochemical effects (cholesterol and calcium reduced, urea and creatinine increased) in next higher dose group (400 ppm) for both racemic Mecoprop and Mecoprop-P (R-isomer)
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the study, the no effect level is between 50 and 400 ppm for both the racemate and the isomer.
- Executive summary:
The repeated dose toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 407.
During the study, the test material was administered as the racemate and as an isomer to Wistar rats over a period of 49 days via their diet. 100 rats (50 males and 50 females) in 5 groups each containing 10 animals per sex were used in the study. The racemate was administered to the animals of test groups 1 and 2 and the isomer to the animals of test groups 3 and 4. For comparison a joint control group (10 male and 10 female rats) was used. The dose levels in the feed were 50 ppm (test groups 1 and 3) and 400 ppm (test groups 2 and 4) in the isomer and as racemate. The rats' food consumption and body weight were determined weekly; the state of health of the animals was checked each day. The duration of the study of 28 days was extended by 21 days since results that were difficult to interpret were obtained in determining the creatinine level at the 1st blood sampling on the 23rd day of administration, and therefore a 2nd blood sampling was carried out on the 43rd day of administration. All the animals were subjected to a gross-pathological examination. This was followed by a histopathological examination.
The following findings were obtained and assessed or discussed in relation to the test materials.
400 ppm group, racemate: Clinico-chemical and haematological findings included a drop in the cholesterol level in the female animals; increased urea concentration in the male rats; reduction of the calcium concentration in the female animals.
400 ppm group, isomer: Clinico-chemical and haematological findings included a drop in the cholesterol level in the male and female animals; increase in the values for urea and creatinine in the female rats.
400 ppm group, isomer: Increase in the absolute kidney weights in the male and female rats.
The administration of 50 ppm of the substance as racemate and isomer did not cause any test material-induced changes.
On the basis of the results of the clinico-chemical examinations – reduction in the calcium values in the female rats and increase in the urea value in the male rats of the 400 ppm racemate group as well as increase in the creatinine and urea values in the female rats of the 400 ppm isomer group - there is an indication of a test material-induced marginal renal insufficiency in the case of both test materials. The increase in kidney weight (400 ppm, isomer) can also be ascribed to this, although no histopathologial changes were found that could explain the weight increases.
The similar reduction in the cholesterol level - particularly pronounced in the female animals - after the administration of 400 ppm is a further indication that there is no difference between the toxic effect of the racemate and the isomer.
Under the conditions of the study, the no effect level is between 50 and 400 ppm for both the racemate and the isomer.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 12 March 1991 to 25 June 1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-1 (90-Day Oral Toxicity)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 41-day old animals on receipt. At the start of the administration period the animals were 51 days old.
- Weight at study initiation: Male animals: 26 (24 - 27) g; female animals: 21 (19 - 22) g.
- Housing: The mice were housed singly in Makrolon® cages, type MI with wire cover.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: 10-day acclimatisation period.
DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical as well as for microbiological contaminants.
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory.
The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light from 06.00 - 18.00 h, 12 hours dark from 18.00 - 6.00 h. - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: The test material was weighed out and thorougly mixed with a small amount of food in a beaker. The premix was subsequently prepared in a BOSCH household mixer. A corresponding amount of food was then added to the premix to obtain the desired concentration, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Investigations to characterise the test material in respect of purity and the homogeneity were carried out at the start of the study. The stability of the test material over the test period was verified analytically.
The stability of the test material in the food for a period of 33 days was demonstrated on a comparable batch.
The homogeneity of the test material in the food was checked on 6 samples of the high and low dose group at the start of the study. In order to ascertain that the middle concentration in the food was correct, one sample of this concentration was also sent to the analytical laboratory at the start of the study. At the end of the study sample from all three doses were checked in the analytical laboratory.
The content of the test material was determined by HPLC.
The homogeneity and concentration control analyses at the start and the concentration control analyses at the end of the study revealed values in an acceptable range of ± 10 % of the theoretical concentration. - Duration of treatment / exposure:
- 3 months
- Frequency of treatment:
- Fed in diet, continuously.
- Dose / conc.:
- 100 ppm
- Dose / conc.:
- 1 000 ppm
- Dose / conc.:
- 2 500 ppm
- No. of animals per sex per dose:
- 10 per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The doses were chosen on the basis of previous investigations with the racemic form of the test material. In literature the LD50 value in mice was reported as 300 mg/kg by intraperitoneal application. In a 4-week feeding study mice received the test material in the dose levels of 100, 300, 900 and 2 700 ppm via the diet. The following findings were obtained and assessed or discussed as possibly being test material-related:
2 700 ppm: Increase in alkaline phosphatase and cholesterol in both sexes; decrease in platelets in the females; increase in absolute and relative liver weights as well as central hypertrophy of the hepatocytes in both sexes.
100, 300 and 900 ppm: No test material-induced changes.
Due to these findings a supplementary 4-week range-finding feeding study was carried out with doses of 7 000, 4 500 and 2 700 ppm to determine the target organs and to find the possible MTD for the long-term study. The following possibly test material-related findings were obtained:
7 000 ppm: Retarded body weight gain with decreased body weights in both sexes at the end of the study (27 % in females and 32 % in males); reduced general state in both sexes; death of 1/5 females; increase in alkaline phosphatase, alanine aminotransferase and albumin in both sexes; increase in aspartate aminotransferase in the males; decrease in leukocytes, lymphocytes and platelets in both sexes; decrease in globulins (males) and glucose (females); increased absolute and relative liver weights in both sexes and adrenal weights in males; diffuse hypertrophy of hepatocytes and degenerative liver cell changes; damages and cytoplasmatic eosinophilia of the hepatocytes as sign of the proliferation of the peroxisomes (both sexes), massive proliferation of the peroxisomes in the hepatocytes and additionally hyperplasia of the mitochondric crista as well as changes in their structure.
4 500 ppm: Retarded body weight gain with decreased body weights in both sexes at the end of the study (13 % in females and 11 % in males); increase in alkaline phosphatase in both sexes; increase in alanine aminotransferase and albumin in females; decrease in the platelets (females); increased absolute and relative liver weights as well as diffuse hypertrophy of liver cells with cytoplasmatic eosinophilia as sign of proliferation of peroxisomes (in both sexes).
2 700 ppm: Minimal retarded body weight gain with slight decreased body weights in both sexes at the end of the study (5 % in females and 6 % in males); increase in alkaline phosphatase in both sexes; increase in albumin (females); increased absolute and relative liver weights and central hypertrophy of liver cells in both sexes
In the following 3-month feeding study, dose levels of 600, 1 200, 2 400 and 4 500 ppm were chosen. The following possibly test material-related findings were obtained (as no histological examinations were carried out, only the results of macroscopy are available):
4 500 ppm: Retarded body weight gain in both sexes with reduced body weights at the end of the study of about 10 % in the males and about 19 % in the females; reduced haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration in both sexes as well as reduced red blood cells and haematocrit in females and mean corpuscular volume in males; increased alkaline phosphatase in both sexes and alanine aminotransferase in females; increased creatinine in both sexes and glucose in males as well as total bilirubin, cholesterol and urea in females; decreased total protein and globulins in males and triglycerides in females; liver enlargement and discolouration and in 4/10 animals liver foci in both sexes; absolute and relative increased liver weights in both sexes.
2 400 ppm: Reduced feed consumption (max. 20 %) in the females; decreased haemoglobin, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration in both sexes; increased alkaline phosphatase in both sexes and glucose in the males as well as creatinine and cholesterol in the females; decreased total protein and globulin in the males and triglycerides in the females; liver discolouration in both sexes and in 1/10 male and 6/10 females enlargement as well as liver foci in 1/10 males; absolute and relative increased liver weights in both sexes.
1 200 ppm: Reduced feed consumption (max. 9 and 18 %) in males and females; decreased hemoglobin and triglycerides in the females; increased cholesterol in the females.
600 ppm: Reduced feed consumption (max. 13 %) in the females; decreased triglycerides in the females.
On the basis of the results of the above studies 100, 1 000 and 2 500 ppm were chosen as doses for the present investigations.
- Rationale for animal assignment: Before the start of the administration period the animals were distributed according to weight among the individual test groups separated by sex. The randomisation list was drawn up by a computer.
- Fasting period before blood sampling for clinical biochemistry: 16 - 20 hours. - Positive control:
- None
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made for dead or moribund animals twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and public holidays).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The general state of health of the animals was checked twice a day (Monday to Friday) and once a day (Saturdays, Sundays and public holidays). Additionally, the animals were examined in detail and palpated once a week.
In order to prepare tables of the clinical symptoms observed the data were entered off-line into the computer systems; according to the particular health status of the individual animals, sometimes several different clinical signs were documented during one week.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was determined before the start of the administration period in order to randomise the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as the body weight change.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The food consumption over a period of 7 days was determined weekly and calculated as mean food consumption in grams per animal per day.
The mean amount of daily ingested test material (in mg/kg bw) was calculated taking into account food consumption, dosage and body weight data. The parameter test material intake was determined using the following formula:
test material intake: (FC x D) / BWx
Where:
FC = Mean daily food consumption (in g) from day x-7 to day x
D = Dosage in ppm
BWx = Body weight on day x of the study (in g)
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No. Blood was taken from the retroorbital venous plexus and after decapitation in the morning from fasted, not anesthetised or anesthetised animals respectively.
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex.
- Parameters checked: The following parameters were determined in blood with EDTA-K 3 as anticoagulant using a particle counter (S Plus model, by Coulter, Krefeld, FRG): Leukocytes, erythrocytes, heamoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets. The differential blood count was evaluated visually.
CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Parameters checked: An automatic analyser was used to examine the clinicochemical parameters. The cyanide-insensitive palmitoyl-CoA-oxidation in liver homogenate was recorded with an automatic enzyme analyser. The following parameters were determined:
1. Enzymes: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase, cyanide-insensitive palmitoyl-CoA-oxidation in liver homogenate (only in the animals of the controls and high dose groups).
2. Blood chemistry: Sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes. The animals were anesthetised with CO2 and decapitated. The exsanguinated animals were necropsied and assessed for gross pathology. From all animals sacrificed at scheduled dates, the following weights were determined: Exsanguinated animals (body weight), Liver, kidneys, adrenal glands, testes.
Subsequently the following organs and tissues of all animals were fixed in 4 % formaldehyde solution: All gross lesions, brain, pituitary gland, thyroid glands (with parathyroid glands), thymus, trachea, lungs, heart, liver, gallbladder, spleen, kidneys, adrenal glands, pancreas, testes, ovaries, uterus/vagina, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, mesenteric and mandibular lymph nodes, sciatic nerve, sternum with marrow, salivary glands (mandibular and sublingual glands), femur with knee joint and bone marrow, skin, female mammary gland, skeletal muscle, eyes, spinal cord (cervical, thoracic, lumbar cord), aorta, extraorbital lacrimal glands, accessory genital organs (prostate, seminal vesicle, coagulation gland).
HISTOPATHOLOGY: Yes. After the tissues have been fixed, histopathological processing, the examination by light microscopy and the evaluation of findings were performed. - Statistics:
- The statistical evaluation and calculation of the data was carried out on the computer systems of the Department of Toxicology of BASF Aktiengesellschaft.
For the statistical evaluation of the study, means and standard deviations were calculated for the variables food consumption, body weight, body weight change and substance intake for the animals in each test group.
Statistical significance of the clinical data (body weight, body weight change) were determined by analysis of variance (ANOVA) followed by a Dunnett's test.
Clinical chemistry and haematology: Means and standard deviations have been calculated for each test group and tabulated together with the individual values. In order to test if the results of the individual dose groups differed statistically significantly from the results of the control group, the means for the dose groups, excepting the differential blood count, were compared with those for the control group using the analysis of variance (ANOVA) and Dunnett's test or Student's t-test. Significances resulting from the statistical comparison have been indicated in the tables on means.
Mean and standard deviation were calculated for the statistical evaluation of the study for the variables of body weight and of absolute and relative organ weights (related to body weight) of the animals in each test group and
tabulated together with the individual values (absolute and relative organ weights). The statistical evaluation was carried out using the DUNNETT's test. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No abnormal clinical were observed throughout the study period.
- Mortality:
- no mortality observed
- Description (incidence):
- No animal died intercurrently during the test period.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weight:
The body weight of the male animals of test group 3 was reduced statistically significantly from day 63 onwards, resulting in reduced values of about 10 % at the end of the study.
In male animals of test group 2, the body weight was reduced statistically significantly only on day 91 (about 8 %). In males of test group 1, on day 42 the body weight was increased, this was, however, assessed as being incidental and thus no test material-related effect was observed in this group.
In females of test groups 3 and 2, body weight was reduced statistically significantly on day 91 (9 %), in test group 3 additionally on day 63. In test group 1 the body weights were slightly lower than in the control group, there was, however no statistical significance.
Body weight change:
In males of test group 3, body weight change was reduced statistically significantly from day 56 onwards.
In males of test group 2, statistically significantly reduced values were seen on days 7, 35 and 91.
In test group 1 no statistically significant changes were seen in comparison to the control group.
In females of test group 3, body weight change was reduced statistically significantly from day 49 onwards (with the exception of day 56).
In females of test group 2, statistically significantly reduced values were seen on days 28, 49, 63, 70, 84 and 91.
In test group 1, body weight was reduced statistically significantly on days 63 and 91, only.
However, as the control values for females were unusually high in this study (historical control data), it seems very unlikely, that the slightly reduced values of body weight or body weight change in test group 1 were a test material-related effect. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- During the study there was spilling of food by the mice in all groups. On account of the housing of the animals (Makrolon cage with bedding), it was not possible to determine the exact food consumption. The recorded values for food consumption were therefore only considered to be estimates of the food consumption rather than the true amounts of food consumed by the animals.
The values of food consumption were reduced in male animals of test groups 1 and 2 and in female animals of test groups 1, 2 and 3. However, as already mentioned above, these values might be misleading and probably do not reflect the real food consumption of the animals. Moreover, as there is no dose-response relationship and as the values of the control groups were unexpectedly high, this finding was assessed as being incidental and not test material-related.
The amount of test material intake was calculated based upon the weekly determined food consumption and body weight. These values serve only for orientation sense, because of spilling of food by the mice, only an approximate food consumption value can be determined to be used in the calculation of the test material intake. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- After 3-months of test material administration a slight decrease in haemoglobin and mean corpuscular haemoglobin was found in the peripheral blood of the high-dose males. These changes are probably test material-related and might be due to a haemotoxic effect of the test material at a dose level of 2 500 ppm. The other haematological examinations revealed no test material-related changes.
There are some statistically significant inter-group differences in the results of the haematological examinations. These deviations are marginal, inconsistent, sporadic or lacking dosage-relationship. Accordingly, these changes are considered to be of no toxicological significance. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significantly increased urea concentrations were detected in the serum of both sexes of test groups 2 and 3 (1 000 ppm and 2 500 ppm); in the females of test group 1 (100 ppm) the urea level was also statistically significantly increased. Furthermore, in the serum of the mid- and high-dose males a statistically significant, dose-dependent increase in creatinine was found. These changes are related to the test material administered and indicate an impairment of kidney function in both sexes.
A dose-dependent decrease in triglyceride concentrations was noted in the male animals of test groups 2 and 3; in the serum of the female treatment-groups (100 ppm, 1 000 ppm and 2 500 ppm) statistically significantly decreased triglyceride levels were found also. Moreover, statistically significantly increased cholesterol concentrations were observed in the serum of both sexes of the highest dose group. The changes in the triglyceride and cholesterol levels in the serum of both sexes are considered to be treatment-related and might be due to a disturbance of lipid metabolism probably caused by the induction of hepatic peroxisome-associated enzymes.
In addition, a statistically significant increase in glucose and a decrease in globulins were detected in the serum of the high-dose males at the end of the administration period. These changes are also attributed to the test material. However, it is difficult to assign these findings to a specific action pattern of the test compound.
There are some statistically significant inter-group differences in the results of the clinico-chemical examinations. These deviations are marginal, inconsistent, sporadic or lacking dosage-relationship. Accordingly, these changes are considered to be of no toxicological significance. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The administration of the test material led to a treatment-related statistically significant reduction of the terminal body weight in males and females treated with 2 500 ppm.
In females, the terminal body weight was reduced in animals treated with 100 ppm and 1 000 ppm also, showing only a tendency for a dose-resonse relationship. But the statistical significance was only slight (p ≤ 0.05), and there was no clinically observed body weight reduction prior to day 91 in females treated with 100 ppm or 1 000 ppm. Furthermore, it is noticeable that the terminal body weight of the control females was unusually high, when compared with the historical control data. Therefore, a test material-related effect seems unlikely.
For the decrease of the absolute kidney weights in top dose males, a test material-induced effect cannot be ruled out, though it seems very unlikely, because there were no histopathological findings that could explain the weight changes. In females, the relative kidney weights were increased in all treated animals. Because there was no dose-response relationship, and there were no histopathological findings, that may explain reasonably the weight changes, a test material-related effect seems unlikely.
For the decrease of the absolute weights of the adrenal glands in top dose females, there were no histopathological correlates. Furthermore, the relative weights of the adrenal glands is most likely related to the reduction of the terminal body weight in top dose females. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The target organs for the test material were liver and kidneys.
In the liver, the cytoplasm of the hepatocytes stained eosinophilic in all males and females treated with 2 500 ppm. The eosinophilia of hepatocytes is induced by the test material and might cause the macroscopically diagnosed dark-brown discolouration and might result in increased liver weights.
In the kidney, the cytoplasm of single tubular epithelial cells was eosinophilic in all males and females treated with 2 500 ppm. The eosinophilia in tubular cells is induced by the test material.
Both the light microscopically visible eosinophilia of hepatocytes as well as the eosinophilia of single tubular epithelial cells are known as possible correlates of proliferation of peroxisomes. The decrease of lipid storage in mid and top dose males and females is interpreted as a result of increased lipid metabolism, possibly in connection with peroxisome proliferation. The reduction of terminal body weight in both sexes may explain this effect further.
All other macroscopically or histopathologically diagnosed findings are considered to have developed spontaneously. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All other histopathologically diagnosed findings are considered to have developed spontaneously.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All other histopathologically diagnosed findings are considered to have developed spontaneously.
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Enzymes: At the end of the administration period the cyanide-insensitive palmitoyl-CoA-oxidation in the liver of both sexes of the highest dose group (2 500 ppm) was increased in the males and females more than 10- and 15-times, respectively, when compared with the control. The marked induction of the oxidation of palmitoyl-CoA in the presence of cyanide is assessed as being test material-related and is probably caused by a proliferation of peroxisomes in the hepatocytes.
In addition to the induction of the peroxisomal marker cyanide-insensitive palmitoyl-CoA-oxidation in the liver increased alkaline phosphatase activities were found in the serum of both sexes of the highest dose group (2 500 ppm). Moreover, statistically significantly increased alanine-aminotransferase activities were seen in the serum of the high-dose females. The increase in alkaline phosphatase and alanine aminotransferase activities in the peripheral blood is also considered to be treatment-related and is presumably associated with a disturbed liver function. - Dose descriptor:
- NOAEL
- Effect level:
- 100 other: ppm (corresponding to 20 mg/kg bw and day)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Toxicity on liver and kidneys at next higher dose level (1000 ppm)
- Dose descriptor:
- LOAEL
- Effect level:
- 100 other: ppm (corresponding to 20 mg/kg bw and day)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Toxicity on liver and kidneys was also evident in this dose group (100 ppm)
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the study, the no observed adverse effect level in this study is for male animals 100 ppm and for female animals slightly below 100 ppm.
- Executive summary:
The repeated dose toxicity of the test material was assessed via the oral route in a sub-chronic study according to OECD Test Guideline 408 and in compliance with GLP.
The test material was administered to 10 male and 10 female B6C3Fl mice per group in the diet for 3 months at doses of 0 ppm (test group 0), 100 ppm (test group 1), 1 000 ppm (test group 2), and 2 500 ppm (test group 3). Food consumption and body weight were determined once a week. A check of the general state of health of the mice as well as a check for dead or moribund animals was done twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and public holidays). Furthermore the animals were additionally examined in detail and palpated once a week. At the end of the study, blood samplings were performed. All animals were subjected to gross-pathological assessment, followed by histopathological examination.
The findings below were obtained and assessed to be related to the test material administration.
Test group 3 (2 500 ppm; about 740 and 930 mg/kg body weight for males and females, respectively):
Clinical examinations: Statistically significantly reduced body weight (about 10 % in males and 9 % in females on day 91); statistically significantly reduced body weight change in both sexes; clinical chemistry and haematology; increase in alkaline phosphatase, cyanide-insensitive palmitoyl-CoA-oxidation in liver homogenate, urea and cholesterol in both sexes; decrease in triglycerides in both sexes; increase in creatinine and glucose in the males; increase in alanine aminotransferase in the females; decrease in haemoglobin, mean corpuscular haemoglobin and globulins in the males.
Pathology: Statistically significant reduction of the terminal body weight in both sexes; statistically significant increase of the absolute and relative liver weight in both sexes; macroscopically diagnosed dark-brown discolouration of the liver in both sexes; in the liver: Eosinophilic cytoplasm of hepatocytes in both sexes; in the kidneys: Eosinophilic cytoplasm of tubular epithelial cells in both sexes; decrease of lipid storage in the liver of both sexes.
Test group 2 (1 000 ppm; about 220 and 330 mg/kg body weight for males and females, respectively):
Clinical examinations: Statistically significantly reduced body weight (about 8 % in males and 9 % in females on day 91); statistically significantly reduced body weight change in both sexes.
Clinical chemistry and haematology: Increase in urea in both sexes decrease in triglycerides in both sexes; increase in creatinine in the males.
Pathology: In the liver: Eosinophilic cytoplasm of hepatocytes in one female; decrease of lipid storage in the liver of both sexes.
Test group 1 (100 ppm; about 20 and 30 mg/kg body weight, respectively):
Clinical examinations: No test material-related findings.
Clinical chemistry and haematology: Increase in urea in the females; decrease in triglycerides in the females.
Pathology: No test material-related findings.
Both clinical chemistry and pathology indicate that the liver and the kidneys are the target organs. Effects on these organs were clearly seen in test group 3 (2 500 ppm) and test group 2 (1 000 ppm). In test group 1 (100 ppm) slight changes of clinical-chemical parameters were seen in females, but no morphological correlate was obtained in the pathological examinations.
Cyanide-insensitive palmitoyl-CoA-oxidation in liver homogenate was determined only at the highest dose level and in the control group as an indicator for peroxisomal proliferation. The determination resulted in a 10- to 15-fold increase in the treatment group. The values were not determined in the other dose groups as it was not intended to obtain a "no observed effect level" for this parameter. Both the increased values of cyanide-insensitive palmitoyl-CoA-oxidation and the eosinophilic cytoplasm of the hepatocytes indicate a peroxisomal proliferation in the liver. The eosinophilic cytoplasm of tubular epithelial cells suggest that peroxisomal proliferation also occurs in the kidneys.
Under the conditions of the study, the no observed adverse effect level in this study is for male animals 100 ppm and for female animals slightly below 100 ppm
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 12 April 1977 to 13 July 1977
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- , no satellite group for follow-up observation, some minor deviations with respect to the examination of clinical chemical parameters
- GLP compliance:
- no
- Remarks:
- study pre-dates GLP
- Limit test:
- no
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
A stability test confirmed the stability of the substances in the feed (200 and 3 200 ppm) for over one month. - Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- OFA, SPF
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: Average weight for males 188.7 (167-208) g; average weight for females 145.6 (131-167) g
- Housing: In groups of five in Makrolon cages 37.5 x 23.5 x 16 cm.
- Diet: Ad libitum. The food is replaced once a week.
- Water: Ad libitum. Water-bottles are changed and filled once a week.
- Acclimation period: 5 days
DETAILS OF FOOD AND WATER QUALITY:
The food was sterilised before use.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1.5 °C
- Humidity (%): 50 ± 10 %
- Air changes (per hr): The air was completely changed 10 times per hour.
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: The compounds were incorporated into the basic diet bi-weekly. The bi-weekly preparation of blends was chosen subsequent to a stability test which showed that the concentration did not change with time (the stability tests were carried out for both compound blends at 200 and 3 200 ppm stored for over one month at room temperature before assay). During the experiment a sample of the various blends obtained during the 1st, 3rd and 5th preparations (i.e. begin of 1st, 2nd and 3rd month of treatment) were sent to Rhone-Poulenc for control of concentration levels.
- Mixing appropriate amounts with:
Racemate: The blend for the high dose level group (group 3, 3 200 ppm) was obtained by dissolving 68.8 g of compound (a purity of 93% equals 64 g of pure compound) in 500 mL of acetone, and mixing, for 30 minutes, the solution with 20 kg of basic diet. The blend for the medium dose level group (group 2, 800 ppm) was obtained by mixing 5 kg of the above blend with 15 kg of basic diet. The blend at 200 ppm (group 1) was obtained by mixing 4 kg of the above blend (800 ppm) with 12 kg of basic diet.
Isomer: The same procedure was applied as for the racemate. The blend at 3 200 ppm (group 8) was obtained by dissolving 96 g of compound in 750 mL of acetone. The solution was slowly incorporated to 30 kg of basic diet and mixed for 30 minutes. Blends for groups 7, 6, 5 and 4 were obtained by successive half dilutions (15 kg of preceding blend + 15 kg basic diet). Basic diets administered to untreated control groups (CA and CB) were subjected to the same procedure, i.e. 500 mL acetone were added to 20 kg basic diet and mixed.
- Storage temperature of food: Room temperature.
VEHICLE
- Justification for use and choice of vehicle: The two test materials were shown to be practically not soluble in water but were both soluble in acetone. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Generally the variations in comparison to theoretical concentrations did not exceed 5 %.
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Continuously in diet.
- Dose / conc.:
- 200 ppm
- Remarks:
- Racemate
- Dose / conc.:
- 800 ppm
- Remarks:
- Racemate
- Dose / conc.:
- 3 200 ppm
- Remarks:
- Racemate
- Dose / conc.:
- 200 ppm
- Remarks:
- Isomer
- Dose / conc.:
- 400 ppm
- Remarks:
- Isomer
- Dose / conc.:
- 800 ppm
- Remarks:
- Isomer
- Dose / conc.:
- 1 600 ppm
- Remarks:
- Isomer
- Dose / conc.:
- 3 200 ppm
- Remarks:
- Isomer
- No. of animals per sex per dose:
- 15 per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- Objective of the study: Comparative assessment of repeated dose toxicity of the racemic test material and the R-isomer.
- Observations and examinations performed and frequency:
- BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly
WATER CONSUMPTION: Yes
- Time schedule for examinations: Twice a week
CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: Daily
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 4 times (week 0, 5, 9 and 13: All animals/sex and group).
HAEMATOLOGY:
- Time schedule for collection of blood: 3 times (week 4, 8 and 12)
- Anaesthetic used for blood collection: Yes. Incision of the tip of the tail under light ether anaestesia, carried out between 8.30 and 9.00 h.
- Animals fasted: Yes. The food hopper was removed between 16.00 and 17.00 h the day before blood sampling and replaced afterwards.
- How many animals: 10 animals/sex and group, except 200 ppm groups; week 12, 10 animals/sex and group.
- Parameters checked: Mean cell volume, Erythrocyte count, White blood cell count, Haemoglobin, Haematocrit, Hean corpuscular haemoglobin, Differential white blood cell count, Reticulocytes%.
Bone marrow smears: At autopsy on 5 animals/sex and group.
Mean corpuscular haemoglobin concentration (MCH) is calculated by: (Hb concentration (g/100 mL) of the animal considered x Mean erythrocyte count (/mm³) of control group) / Erythrocyte count (/mm³) of the animal considered x Mean Hb concentration (g/100 mL of control group)
Erythrocyte (RBC) and leucocyte (WBC) counts: Carried out with a Coulter Counter.
Differential Leucocyte Count: Determined microscopically after May-Grunwald-Giemsa staining.
Haemoglobin concentration (HB): Carried out colorimetrically in Drabkin's solution and compared with a standard.
Haematocrit (H): Measured by centrifugation of a blood sample rendered incoagulable in a capillary tube. The following equation is then used: (Length of erythrocyte deposit / total length) x 100
Mean cell volume (HCV): Measured on a COULTRONICS along with blood cell counts.
Reticulocyte Count (RET): Direct visual count under microscope, after staining the preparation with a Cresyl Blue solution. The number of reticulocytes is expressed in a ratio to 1.000 erythrocytes.
Bone marrow smears: Microscopical determination after May-Griinwald-Giemsa staining.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3 times (week 4, 8 and 12).
- Animals fasted: Yes. The food hopper was removed between 16.00 and 17.00 h the day before blood sampling and replaced afterwards.
- How many animals: 5 animals/sex and group except 200 ppm groups; week 12: 10 animals/sex and group.
- Parameters checked. Blood collected from incision of the tip of the tail under light ether anaestesia, carried out between 8.30 and 9.00 h.
Parameters examined: Sodium, Potassium, Chloride, Glucose, Urea, SGOT - SGPT, Alkaline phosphatase, Total proteins, Calcium, Phosphorus, Cholesterol, Bilirubin.
Electrophoresis of proteins: Once (week 12: 5 animals/sex).
Methods used:
Sodium (Na+) and Potassium (K+): Flame spectrophotometry.
Chloride (Cl-): The chloride ions react with a solution of mercuric nitrite and the thiocyanate ions liberated give a red-coloured complex with ferric ions, measured at 480 nm.
Calcium (Ca): Determination by methylthymol. Magnesium interference is prevented by the presence of 8-hydroxyquinoline.
Phosphorous (P): After the formation of a phospho-molybdate there is a reduction by p-methylaminophenol to form molybden blue.
Glucose (GLUC): ULTROLAB LKB determination. Glucose oxidase method with the production of the chromogen 4 amino-phenazole.
Urea (BUN): ULTROLAB LKB determination. By urease action urea is transformed yielding ammonium carbonate which gives a blue colouration in the presence of phenol and hypochlorite in an alkaline medium.
Bilirubin (BIL): ULTROLAB LKB determination. With 24-dichloraniline diazote a coloured azotic compound is formed in the presence of bilirubin. The determination is carried out after substraction of a serum blank.
Cholesterol (CHOL): ULTROLAB LKB determination. After hydrolysis of cholesterol esterified by cholesterolesterase, the free cholesterol is oxidised by cholesterol oxydase. Hydrogen peroxide which is fanned gives formaldehyde in the presence of methanol and catalase. The aldehyde is assayed by the formation of a yellow derivative from dehydrolutidin.
Total proteins (PROT): ULTROLAB LKB determination. Biuret method. In an alkaline medium proteins fonn a complex chelate with copper salts.
Serum alkaline phosphatase (SAP): ULTROLAB LKB determination. Paranitrophenylphosphate is split by alkaline phosphatase to give paranitrophenol. The speed of its appearance is proportional to the activity of SAP and the speed is analysed by the LKB 8 600.
Serum glutamic-oxaloacetic transaminase (SGOT): Optimised technique. SGOT catalyses the reaction α-ketoglutarate + asparate → L glutamate + oxaloacetate. The oxaloacetate is reduced in the presence of NADH+ which is eliminate at 340 nn. The determination is made at 37 °C using an LKB 8600.
Normal values for the rat are: 120 - 180 mU/mL at 37 °C.
Serum glutamic-pyruvic transaminase (SGPT): Optimised technique. SGPT catalyses the reaction α-ketoglutamate + alanine → glutamate + pyruvate. The pyruvate is reduced in the presence of NADH+ which is eliminated at 340 nm. Determinations are made at 37 °C using an LKB 8600.
Normal values for the rat are: 35 - 70 mU/mL 37 °C.
Electrophoresis (week 12 only): The proteinogramme represents the migration of serum proteins in a uniform electric field, on a cellulose acetate strip in a buffer solution. The buffer is a solution of sodium veronal TRIS, pH 8.8 having an ionic strength of 0.05 M. The migration is carried out at 190 volts for 2 hours. The different fractions are stained with a solution of Amidoschwarz, and measured with a densitometer coupled to an integrator.
URINALYSIS: Yes
- Time schedule for collection of urine: 3 times (week 4, 8 and 12).
- Metabolism cages used for collection of urine: Yes. Rats are given orally 20 mL/kg of water (8 to 10 mL per animal) before being placed individually in metabolic cage. All the urine voided is then collected.
- Animals fasted: Yes. Animals were deprived of food for 16 to 18 hours prior to urine collection.
- Parameters examined: pH, Glucose, Ketone bodies, Urobilin, Proteins, Blood, Specific gravity, Microscopy of sediment.
Urinalysis is carried out as follows:
- Specific gravity: Measured by means of a refractometer TS Meter.
- Proteins, blood, glucose, ketone bodies, bilirubin: Determined by means of test strips (Combur 6 Test). protein is checked by reaction with nitric acid.
- Microscopy of sediments: Determined after centrifugation of urine samples. Evaluation of erythrocytes, leucocytes, epithelial cells, cell casts, crystals and yeast.
The presence of protein, blood, glucose, ketone bodies, bilirubin is estimated from 0 to ++++, as follows :
0: No trace
+ " Traces" - "slight traces"
++ Clearly defined presence
+++ Large quantity
++++ Very large quantity
The presence of sediments is quantitatively estimated as follows:
+ 0 to 20 units per field
++ 20 to 50 units per field
+++ > 50 units per field - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Decapitation under ether anaesthesia
Organ weights (all survivors): Heart, Liver, Spleen, Kidneys, Adrenals, Gonads, Brain, Thyroid, Pituitary, Prostate, Thymus, Uterus.
HISTOPATHOLOGY: Yes (15 males and 14 females/groups 0 ppm and 3 200 ppm isomer, 15 animals/sex and group for 3 200 ppm racemate, 10 animals/sex and group for 800 ppm racemate and for 400, 800 and 1 600 ppm isomer)
Brain, Trachea, Tongue, Duodenum, Ileum, Pancreas, Mesenteric lymph node, Pituitary, Striated muscle, Aorta, Lung, Oesophagus, Caecum, Colon, Thymus, Kidneys, Thyroid, Skin, Heart, Salivary glands, Stomach, Jejunum, Liver, Spleen, Bone, Uterus, Mammary gland, Testes, Bladder, Adrenals, Sciatic nerve, Ovaries, Prostate, Seminal vesicles.
Histopathological examinations were carried out on six treated and the two untreated control groups. The lesions are tabulated in such a way that their frequency can be compared between treated and control groups for each compound. - Statistics:
- In addition to individual values obtained the tables of results provide for each parameter examined and for each group the mean and the standard deviation.
The results obtained in the treated groups were followed by the value for the Student "t" test.
This test indicates if the difference between the mean of treated and control groups is significantly different from zero. The difference is determined by subtraction: Treated group - control group. A positive "t" test indicates therefore an increase in comparison to controls while a negative test indicates a decrease.
The calculation of the Student test was done systematically between the treated and both control groups (Control A and Control B). The results of the tests compared to control group A are always indicated. Results of the tests in comparison to control group B are indicated only if a significant difference at 95 % has been observed in comparison to at least one of the treated groups. It is important to have both "t" values available in order to draw a conclusion, should the two tests not agree. Haematological, clinical chemical data and organ weights were also compared by the "t" test between groups which had received the same concentration of either compound in the diet i.e. groups 2 and 6 on the one hand and 3 and 8 on the other. - Clinical signs:
- not specified
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Control group A: One female died in month 1 under anaesthesia at blood sampling.
Control group B: One female died in month 2 under anaesthesia at blood sampling.
Racemate:
800 ppm: One female died 3 days after blood sampling in month 3and was replaced by another female.
Isomer:
400 ppm: One male died in month 3 killed at blood sampling and was replaced by another male.
800 ppm: One female died at blood sampling in month 3.
3 200 ppm: One female died at blood sampling in month 1 and was necropsied. One female died at blood sampling in month 3 and was not replaced. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 200 ppm: The growth rate of the treated groups (1: Racemate; 4: Isomer) was comparable to that of the untreated control groups. The few changes observed (group 1, males week 2; group 4, males week 4 and females, week 12) were only significant in comparison to untreated control group A and were not confirmed the following weeks.
400 ppm: Group 5 (racemate): A few variations were noted only in comparison to the untreated control group A, mainly positive in male rats and more pronounced and always negative in the females, which lost 5 to 8 % in comparison to untreated control group A. These differences were only significant in females in comparison to both control groups at weeks 11 and 12.
800 ppm: The differences were more pronounced but only significant in comparison to both control groups for the females of group 2 (racemate) at weeks 11 and 12. The differences were systematically negative for male and female animals of group 2 (racemate) and negative from week onwards for the females of group 6 (isomer); on the other hand, for the males of group 6, the differences were negative from week 0 to 3 then positive (or close to 0) from week 4 onwards.
1 600 ppm (group 7 isomer) and 3 200 ppm (groups 3, racemate and 8, isomer): From week 1 onwards, a marked depression in the mean weight of males and females was noted. The decrease ranged from 5 to 7 % for males and from 7 to 16 % for females at 1 600 ppm (group 7 isomer) in comparison to control group A. Decreases in growth rate (30 % in the females and 35 % in males), which were comparable between each other were observed at the concentration of 3 200 ppm for both compounds. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Food consumption was measured weekly. Spilling of food, presumably on account of its taste, made the accurate estimation of consumption for groups 3 and 8 difficult.
From the graphs it appeared that, for some treated groups (males of groups 2 and 3; males and females of groups 7 and 8) the consumption had increased in comparison to the controls. This is not the case because all these groups spilled food to some degree, while the other groups spilled and consumed less food with no significant difference between groups.
Three peaks can be noted for males and females in all groups at weeks 5, 9 and 13. This overall increase in food consumption can be explained by the fact that at these periods animals were deprived of food for 16 to 18 hours prior to blood sampling and urine collection. This was subsequently followed by an increased food consumption because of a greater appetite.
Compound intake: The mean compound intake over 13 weeks, expressed in mg/kg body weight/day for the different groups was as follows
- Racemate:
Group 1 (200 ppm): 16.5 mg/kg bw/day (males); 18.2 mg/kg bw/day (females)
Group 2 (800 ppm): 67.9 mg/kg bw/day (males); 75.9 mg/kg bw/day (females)
Group 3 (3200 ppm): 390.8 mg/kg bw/day (males); 398.7 mg/kg bw/day (females)
- R-isomer:
Group 4 (200 ppm): 15.6 mg/kg bw/day (males); 18.4 mg/kg bw/day (females)
Group 5 (400 ppm): 31.9 mg/kg bw/day (males); 37.8 mg/kg bw/day (females)
Group 6 (800 ppm): 67.6 mg/kg bw/day (males); 75.8 mg/kg bw/day (females)
Group 7 (1600 ppm): 146.4 mg/kg bw/day (males); 170.1 mg/kg bw/day (females)
Group 8 (3200 ppm): 403.2 mg/kg bw/day (males); 403.5 mg/kg bw/day (females) - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Water consumption was measured twice a week per cage of 5 animals and was expressed in mL of water intake/animal/day. No inter-group differences were apparent.
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No ocular lesions which could be attributed to the administration of the compounds were observed at the examinations at weeks 0, 5, 9 and 13. The only abnormality noted was in male 214 (group 2), at week 0 who showed small pigmentary spots on the cornea of the right eye. At weeks 5, 9 and 13 this was absent. Slit-lamp examination did not reveal any opacifications.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A certain number of slight but statistically significant changes were observed of which the most noticeable are described below:
Week 4: Decrease in haemoglobin concentration in males and females of groups 2 and 3 (racemate), in males of groups 5 and 8, and in females of group 7 (isomer). The changes concerning red blood cell counts (decrease in females of group 7) and white blood cell counts (decrease in females of group 3 and increase in females of group 7) are of little significance.
Week 8: Decrease in red blood cell count in females of group 2, in males and females of group 3 (racemate) and group 8 (isomer). Decrease in haemoglobin concentration in males and females of group 3, the males of group 7 and males and females of group 8. Decrease in white blood cell count of males of groups 3 and 8.
Week 12: No major change was observed at the lowest concentrations (groups 1 and 2, 4, 5 and 6, except for the latter a slight leucopenia in males). At the highest concentrations - group 7 (isomer -1 600 ppm): a slight decrease in haemoglobin concentration in females and in red blood cell counts in males accompanied by an increase in polynuclear count. The same observations were made for groups 3 and 8 (3 200 ppm), i.e. decrease in haemoglobin concentration in males and females of both groups; decrease in red blood cell count in males. For group 8 only, a decrease in white blood cell count and an increase in polynuclear count was noted in males.
The comparison by 't" test of groups 2 and 6 (800 ppm) and groups 3 and 8 (3 200 ppm) showed at week 12 a decrease in white blood cell count in the males of group 6 in comparison to group 2 (t = 4.22), the same decrease in white blood cell count in males of group 8 in comparison to group 3 (t = 3.87) but with an increase in polynuclear count in males of group 8 compared to group 3.
In conclusion, a treatment-related effect was observed on red blood cell counts and haemoglobin concentration, mainly in groups 3 (racemate at 3 200 ppm), 7 and 8 (isomer at 1 600 and 3 200 ppm) and was probably related to a general toxic effect.
Bone marrow smears: Examination of bone marrow smears which were done on 5 males and 5 females of each group revealed no change. Granulocytic and erythrocytic series were normal.
The administration of the racemate and the isomer induced a slight normochromic anaemia at the highest concentration used (3 200 ppm). - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A number of values from all groups showed statistically significant differences from control values but in no instances did these fall outside the normal ranges of values for this laboratory nor were any consistent trends seen. They were therefore not considered to be treatment related.
Glucose: A slight increase in blood glucose levels was seen in males in groups 3, 6 and 8 at 4 weeks when compared with both control groups. This was also seen at 12 weeks in groups 7 and 8 (both sexes).
Blood urea nitrogen: Slight but statistically significant increases in B.U.N. levels when compared with both control groups were seen at 4 weeks in group 3 females and group 8 males (for the former, the mean value was 0.57 g/L); at 8 weeks in group 3 males and group 8 females (for the latter, the mean value was 0.55 g/L); at 12 weeks in group 2 females, group 3 males and females, groups 5 and 6 females, and groups 7 and 8 males and females. Only in group 8 males did the mean value exceed the normal upper value for this laboratory of 0.50 g/L (the mean value in this group being 0.52 g/L).
Cholesterol: There were slight but statistically significant decreases in cholesterol levels, when compared with both control groups at 4 weeks in groups 3 and 7 males and group 8 males and females; at 8 weeks in group 2 females and groups 3 and 8 males and at 12 weeks in groups 3, 7 and 8 males.
Bilirubin: A statistically significant decrease in biliburin, when compared with both control groups was seen at week 8 in group 2 females. However, this change was slight and transient and in view of the absence of any trend elsewhere it was not considered to be treatment related.
Serum alkaline phosphatase: Significant increases in S.A.P. levels, when compared with both control groups were seen at 4 weeks in group 3 males and females; at 8 weeks in group 3 males and females, group 7 males and group 8 males and females; at 12 weeks in group 3 males and females, group 7 males and group 8 males and females.
SGOT: Only a statistically significant reduction, when compared with both control groups was seen at 12 weeks in group 8 females.
SGPT: Statistically significant increases were seen, when compared with both control groups at 4 weeks in group 3 males; at 8 weeks in group 3 females and group 8 males and females; at 12 weeks in group 3 males and group 7 and 8 males and females (in group 3 females the elevated mean value was mainly due to the female 320 which showed a particularly high level).
Serum proteins and protein electrophoresis: Slight but statistically significant reductions in total protein levels, when compared with both control groups were seen at 8 weeks in groups 6, 7 and 8 males; at 12 weeks in groups 3 and 7 males.
At 12 weeks, electrophoresis showed: Increased albumin levels with an elevated A/G ratio in group 2 females, group 3 males and females, group 6 females and group 8 males and females; decreased β globulins in group 3 males; decreased α² globulins in group 3 males, group 7 males and females and group 8 males; decreased α³ globulins in group 8 males.
The comparison by "t" test of groups 2 and 6 (800 ppm) and groups 3 and 8 (3 200 ppm) showed only that mean glucose levels of males and females in groups 6 and 8 were significantly higher than those of males and females in groups 2 and 3 respectively.
The changes seen in blood biochemical values indicate an effect on liver function in group 3 (racemate, 3 200 ppm) and in groups 7 and 8 (isomer, 1 600 ppm and 3 200 ppm respectively). However, these changes were generally slight. - Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Urinalysis was carried out at weeks 4, 8 and 12. Some changes were noted, both in control and treated animals, for example traces of protein. This is a common finding in rats.
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No behavioural changes which could be attributed to the treatment with the test materials were observed.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased kidney weights were noted in group 1 (males and females) and in groups 2, 4, 5 and 6 (males) but the values remained within the normal range for this laboratory.
There was a dose-related increase in liver weights in females which is probably related to the elevation in SAP, and SGPT values.
The apparent decrease in all organ weights, with the exception of the liver, at the highest concentration of racemate and R-isomer was proportional to the reduction in growth rate and body weight and is considered to be a result of the toxic effect of the compounds at this concentration. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At week 13 of treatment all animals were sacrificed and necropsied. The major changes observed at the post-mortem examination were the following:
Control group A:
Male: Yellowish colour of one liver lobe.
Female: Hyaline cyst on the right ovary.
Female: Sub-cutaneous mass on the right costal flank.
Control Group B:
Male: Haemoarrhagic lungs
Female: Hyaline cyst on one kidney.
Female: Small necrotic foci on one liver lobe.
Female: Haemorrhagic spot on one liver lobe.
Group 1:
Male: Atrophic thyroid
Male: Hydrometria.
Group 3:
Male: Very pale adrenals and hyperotrophic right cerebral hemisphere.
Male: Very pale adrenals and atrophic thymus.
Male: Right gonad appeared small and soft.
Female: Very pale adrenals.
Group 5:
Male: Small “diverticle” on the spleen.
Male: Small whitish mass on the surface of the kidney.
Female: Atrophic mammary glands.
Female: Colon adherent to the uterus. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Histopathological examinations were carried out on six treated and the two untreated control groups. The lesions are tabulated in such a way that their frequency can be compared between treated and control groups for each compound. None of the lesions observed is related to the administration of the compounds.
A technical problem with the preparation of the slides did not permit the histological examination of the eyes. - Histopathological findings: neoplastic:
- no effects observed
- Dose descriptor:
- NOEL
- Effect level:
- 200 other: ppm (corresponding to 16 mg/kg bw and day)
- Sex:
- male/female
- Basis for effect level:
- other: Effects on liver and kidney (increased organ weight in combination with altered biochemical parameters) in next higher dose group for either racemic Mecoprop or Mecoprop-P (R-isomer)
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the study the no-effect level was 200 ppm for both the racemate and the isomer.
- Executive summary:
The repeated dose toxicity of the test material in the rat were assessed in a study which was conducted according to a method siilar to that which is outlined in the standardised guideline, OECD 408.
During the study, weanling rats of Sprague-Dawley origin (strain 0FA, SPF, IFFA-CREDO) were given continuously for 3 months a diet containing either the racemate at levels of 200, 800 and 3 200 ppm, or the isomer at levels of 200, 400, 800, 1 600 and 3 200 ppm, to determine the potential toxicity of these two compounds. Two untreated control groups received the powdered diet only.
Clinical symptoms: No adverse symptoms or behavioural changes, which could be attributed to the ingestion of the compounds, were observed during the study.
Mortality: There was no compound-related mortality. Some animals died accidentally during the bleeding procedure from the effects of ether anaesthesia.
Growth rate: During the course of the study no significant differences between the two untreated control groups were noted. With compound the racemate at 800 ppm, a slight reduction in weight gain became apparent in females. With the isomer a reduction in body weight gain was evident at 1 600 ppm in both sexes. At the highest concentration tested (3 200 ppm), the depression of the growth curve became pronounced for both compounds and in both sexes.
Food consumption: This remained in general the same for the treated and untreated groups. Waste of food was noted in groups 3 (the racemate at 3 200 ppm) and 8 (the isomer at 3 200 ppm) and this made it impossible to record accurately the food consumption.
In both sexes of all groups at weeks 5, 9 and 13, three "peaks" in food consumption were observed. This transient increase in food consumption may have been caused by starvation at these periods for 16 to 18 hours, prior to blood and urine collections, with subsequently increased appetite.
Haematology: Both compounds produced slight and sporadic decreases in white blood cells at the highest concentrations and mainly in the male groups. Decreases in haemoglobin concentrations and red blood cell counts, more often the former, were noted at the two highest concentrations tested and with both compounds. This was especially noticeable at the 3 200 ppm level in both sexes at 8 and 12 weeks.
Clinical chemistry: Increases, both in frequency and degree, of urea, SAP and SGPT values, were noted. Considering values above 0.50 g/L urea as the upper limit of normality, an increased frequency of values above this limit became evident at concentrations of 800 ppm of the racemate and 400 ppm of the isomer. These changes are considered to be due to a toxic action of the compounds as the frequency of abnormal SAP and SGPT activities increased with the higher concentrations, with the duration of treatment and they were observed in both sexes. Increased albumin levels together with increases in SAP and SGPT activities (and liver weight increase in females) indicate that the liver is the target organ. A fall in cholesterol levels below 0.80 g/L was apparent and this was more marked in frequency and degree, particularly in males, with time of administration and concentrations.
Organ weights: Increased kidney weights were noted in group I (males and females) and in groups 2, 4, 5 and 6 (males) but the values remained within the normal range for this laboratory.
There was a dose-related increase in liver weights in females which is probably related to the elevation in SAP, and SGPT values. The apparent decrease in all organ weights, with the exception of the liver, at the highest concentration of the racemate and the isomer was proportional to the reduction in growth rate and body weight and is considered to be a result of the toxic effect of the compounds at this concentration. No lesions were observed in livers or kidneys, nor in any of the other organs examined.
Since concentrations between 800 and 200 ppm for the racemate and between 400 and 200 ppm for the isomer have not been studied, the no-effect dose level for both compounds in this study was 200 ppm.
The changes in body weights and/or in the clinical laboratory findings at 800 and 3 200 ppm for the racemate and at 400 to 3 200 ppm for the isomer together with the increase in liver weight at 3 200 ppm for both compounds can be considered to be treatment related. The biochemical changes indicate a disturbance of liver function not accompanied with histopathological change. The haematological findings, whilst related to treatment are considered to be a non-specific toxic response.
Under the conditions of the study the no-effect level was 200 ppm for both the racemate and the isomer.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 06 April 1978 to 03 July 1978
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents); study was focussed on histopathological examination of the eyes supplementary to Reinert, 1977
- Deviations:
- yes
- Remarks:
- , no guideline study as such; supplemental investigations for previous study
- GLP compliance:
- no
- Remarks:
- study pre-dates GLP
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- OFA, SPF
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 4 - 5 weeks old at start of trial.
- Weight at study initiation: 100 - 120 g at start of trial.
- Housing: 5 animals per cage in Makrolon cages (37.5 x 23.5 x 16 cm) placed on racks which were rotated weekly.
- Diet: Ad libitum
- Water: Ad libitum. Water bottles were changed and filled once a week.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22° C ± 1.5 °C
- Humidity (%): 50 ± 10 %
- Air changes (per hr): The air was completely changed 10 times per hour.
- Photoperiod (hrs dark / hrs light): Artificial lighting was provided from 0700 to 1900 H.
- Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: The compounds were incorporated into the basic diet bi-weekly. The bi-weekly preparation of blends was chosen subsequent to a stability test carried out by RHONE-POULENC for the preceding experiment. The test showed that the concentration did not change with time (the stability test was carried out for both compound blends at 200 and 3 200 ppm stored for over one month at room temperature).
- Mixing appropriate amounts with:
Racemate: The blend for the high dose level group (group III - 3 200 ppm) was obtained by dissolving 51.6 g of compound (a purity of 93 % equals 48 g of pure compound) in 375 mL of acetone, and mixing at length the solution with 15 kg of basic diet. The blend at 800 ppm (group II) was obtained by mixing 4 kg of the above blend (3 200 ppm) with 8 kg of basic diet.
Isomer: The same procedure was applied. The blend at 3 200 ppm (group VIII) was obtained by dissolving 64 g of compound in 500 mL of acetone. The solution was slowly incorporated to 204 g of basic diet. Blends for groups VII and VI were obtained by half dilutions (10 kg of preceding blend + 10 kg of basic diet).
VEHICLE
- Justification for use and choice of vehicle: The two test materials were shown to be practically not soluble in water but were both soluble in acetone. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- During this experiment a sample of the various blends obtained during the 1st, 2nd and 3rd preparations (i.e. begin of 1st, 2nd and 3rd fortnights of treatment) was sent to RHONE-POULENC for control of concentration levels.
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Continuous for 13 weeks.
- Dose / conc.:
- 800 ppm
- Remarks:
- Racemate
- Dose / conc.:
- 3 200 ppm
- Remarks:
- Racemate
- Dose / conc.:
- 800 ppm
- Remarks:
- Isomer
- Dose / conc.:
- 1 600 ppm
- Remarks:
- Isomer
- Dose / conc.:
- 3 200 ppm
- Remarks:
- Isomer
- No. of animals per sex per dose:
- 10 per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- As histopathological examination of the eyes due to technical problems were not conducted in a previously conducted study (IFREB 1977), this study was undertaken. Therefore only the following parameters were registered: Body weight (weekly), food consumption (weekly), clinical observations (daily), ophthalmoscopic examinations (week 0 and 13), and histopathological examinations of the eyes.
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Week 0 and week 12.
- Dose groups that were examined: All animals in all groups. Cornea, lens and fundus were examined. Both eyes were examined with a Heine mini-set 1974 F ophthalmoscope.
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes. The eyes of all animals were examined in week 13 after sacrifice by decapitation under ether anaesthesia. Eyes were fixed in Bouin, and embedded in paraffin-polywax. 5-microns thick sections were cut and stained with trichrome hemalum-phloxin-saffron (FPS) then mounted between slide and coverslip in afcolene medium. - Statistics:
- All numerical data obtained were compiled and analysed statistically as follows:
MEAN, STANDARD DEVIATION, STUDENT 't' TEST: In addition to individual values, for each group, the mean and the standard deviation were calculated. The results obtained in the treated groups were followed by the value for the Student 't' test. This test indicates if the difference between the mean of treated and control groups is significantly different from zero. The difference is determined by substraction: treated group - control group. A positive 't' test indicates therefore an increase in comparison to controls while a negative test indicates a decrease. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Two isolated cases of diarrhoea occurred: In male 368 (group III, racemate, 3 200 ppm) during the first week, and in male 261 (group II, racemate, 800 ppm) during the 9th week of treatment.
- Mortality:
- no mortality observed
- Description (incidence):
- There was no mortality during the course of the study.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 800 ppm, racemate and isomer: Throughout the study the growth of male rats treated with either compound remained close to that of control animals while, in females, body weights decreased slightly, but significantly, from week 3 onwards.
1600 ppm, isomer: In comparison to control animals body weights lowered significantly in males from week 1 and in females from week 3 onwards.
3200 ppm, racemate and isomer: A very significant reduction in body weights was observed in animals of both sexes from week 1 onwards. The marked body weight decrease of male 368 had no apparent cause. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption was measured weekly and remained, in the whole, for both compounds, comparable to that of the control group. The slight increase in food consumption in treated animals might be attributed to spilling of food, presumably on account of its taste. Results can be found in Table 1.
The weekly mean food consumption and mean body weights allowed calculation of the mean compound intake per group. Results can be found in Table 2. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- Ophthalmological examination carried out at weeks 0 and 12 revealed no lesion which could be attributed to treatment.
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No major behavioural change was observed.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Histological examination of the eyes of Sprague-Dawley rats treated orally for 3 months with either the racemate or the isomer revealed no lesion.
- Histopathological findings: neoplastic:
- not examined
- Dose descriptor:
- other: No ocular lesions and no treatment related effects were found at any dose level at the histopathological examination of the eyes.
- Remarks on result:
- not measured/tested
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the study no behavioural change which could be attributed to the ingestion of the test materials was observed. A dose-related reduction of the growth rate was however noted. No ocular lesion was observed. The histological examination of the eyes of all animals at the end of the study revealed no treatment-related lesion.
- Executive summary:
A technical problem with the preparation of the slides did not permit the histological examination of the eyes in a previous study. Consequently, an additional 13-week study, subject of this report, was undertaken for the evaluation of eye lesions.
The racemate was administered at concentrations of 800 and 3 200 ppm and the isomer at concentrations of 800, 1 600 and 3 200 ppm. Both compounds were administered orally and continuously, mixed with the diet, for 3 months to groups of 10 males and 10 females. The following examinations were performed during the course of the study: Ophthalmology at months 0 and 3; weekly recording of body weights and food consumption and histology of the eyes at the end of treatment.
As in the preceding study, no behavioural change which could be attributed to the ingestion of the test materials was observed. A dose-related reduction of the growth rate was however noted. No ocular lesion was observed. The histological examination of the eyes of all animals at the end of the study revealed no treatment-related lesion.
- Endpoint:
- chronic toxicity: oral
- Remarks:
- Combined repeated dose and carcinogenicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 16 May 1984 to 12 June 1986
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other:
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Version / remarks:
- (1981)
- Deviations:
- yes
- Remarks:
- , use of two satellite groups of 10 and 15 rats/sex at each dose level for 12 and 24 months, respectively, whereas testing guideline of 1981 stipulates only one high dose satellite group of 20 animals/sex - but no conflict to testing guideline of 2009.
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Chbb = THOM (SPF)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 42 days
- Weight at study initiation:
Main groups: Males 167 g (141 g - 192 g); females 136 g (120 g - 156 g)
Satellite group I: Males 164 (14-183); females 135 g (111 g - 149 g)
- Housing: During the test, the rats were housed singly in type DK III stainless steel wire cages, floor area about 900 cm^2 (Becker & CO, Castrop-Rauxel, Germany).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 14 days acclimatisation period during which they received ground diet and water ad libitum and were accustomed to the environmental conditions.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity
- Air changes (per hr): No data (completely air conditioned rooms).
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06:00 - 18:00 h, 12 hours dark from 18:00 - 6:00 h). - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Remarks:
- Diet pre-mix used
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: To prepare the test material preparations, the test material was weighed out depending on the dose group, and thoroughly mixed with a small amount of the feed in a beaker, using a spatula. The premix was subsequently prepared, initially in a BRAUN mixer (Mx 32) and further in the study also in a BOSCH household mixer. This premix was then adjusted to the required concentration by adding the appropriate amount of feed and was mixed in a laboratory mixer supplied by GEBR. LÖDIGE, for about 10 minutes.
DIET PREPARATION
- Rate of preparation of diet: The feed/substance mix was freshly prepared at intervals of not more than 24 days. The stability of the test material in the feed was verified for a period of 33 days.
- Mixing appropriate amounts with: Kliba rats/mice/hamsters maintenance diet. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test material in the maintenance diet was analytically investigated before the study began. To verify homogeneity of the test material preparations, samples were sent to the Analytical Laboratory before the start of the study and than again in the initial phase of the administration period after the mixing procedure had been optimised (additional premix).
Furthermore, samples of each one of the doses were sent to the analytical laboratory at the beginning of the study and thereafter at 3-monthly intervals.
The content of the test material in the feed/test material mixes was determined by means of HPLC. - Duration of treatment / exposure:
- 24 months
- Frequency of treatment:
- Continuously in diet
- Dose / conc.:
- 20 ppm
- Remarks:
- Nominal in diet
- Dose / conc.:
- 100 ppm
- Remarks:
- Nominal in diet
- Dose / conc.:
- 400 ppm
- Remarks:
- Nominal in diet
- No. of animals per sex per dose:
- Main group: 50 per sex per dose.
Satellite group I: 10 per sex per dose level was dosed for 12 months.
Satellite group II: 15 per sex per dose level was dosed for 24 months. - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The selection of the doses was based on the results of a 3-month feeding study with the test material in rats. In this study, dosages of 450, 150 and 50 ppm were used. The following test material-related findings were obtained:
450 ppm: Increase in the creatinine values in the plasma of the female animals; increase in the absolute and relative kidney weights in the male animals.
150 ppm: Increase in the absolute and relative kidney weights in the males; increase in the relative kidney weights in the females.
50 ppm: No changes to be attributable to the test material administration.
On the basis of the above findings, the following dose levels were fixed for the current study on a potential carcinogenic and chronic-toxic effect:
20 ppm as a definite no effect level; 100 ppm was chosen as the intermediate dose and 400 ppm as the highest dose.
Marginal toxic effects on administration of 400 ppm could probably be expected, however, without any adverse effect on the normal lifespan.
- Rationale for animal assignment: 12 days prior to the start of administration (day 0), the male and female rats were allocated to the test groups on the basis of their weights. The list of randomisation instructions was generated by a computer.
- Rationale for selecting satellite groups:
Main group: Determination of the body weight and feed consumption up to 24 months, urinalysis at the end of the study, subsequently necropsy.
Satelite group I: Determination of the body weight and feed consumption up to 12 months, urinalyses, hormone analyses (T3/T4), interim sacrifice after 12 months.
Satelite group II: Clinico-chemical and haematological examinations, necropsy after 24 months.
- Post-exposure recovery period in satellite groups: No. - Positive control:
- None
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: The state of health was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week. A check for mortality was made twice daily (Mondays to Fridays) and once daily (Saturdays, Sundays and public holidays).
BODY WEIGHT: Yes
- Time schedule for examinations: The animals of the main groups and satellite group I were weighed once a week, including week 14 of the administration; subsequently, body weight was determined at 4-weekly intervals. The body weights were determined on the same day of the week each time. There was an additional determination of the amount of feed consumed and of the body weight, which took place at the end of the study outside the 4-weekly cycle (satellite group I and main groups).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: For animals of main group and satellite group I once a week up to 14 weeks and thereafter once a month until the end of the study. The mean amount of daily ingested test material (in mg) per kg body weight was determined at the same intervals as was feed consumption.
To determine the amount of feed consumed, the feed box with contents was weighed and the value obtained was subtracted from the initial value.
The feed consumption of the main groups and satellite group I was weighed once a week, including week 14 of administration, for the course of the preceding week; subsequently it was determined at 4-weekly intervals.
The mean amount of daily ingested test material (in mg) per kg body weight was determined at the same intervals as was feed consumption.
The values given represent a group mean calculated from the amounts of test material ingested by each individual animal, and were determined using the formula:
(FC x D) / BWx
Where:
FC = Mean daily feed consumption (in g) within one week of the study (from day x-7 to day x)
D = Dose in ppm
BWx = Mean bodyweight (in g) on day x of the study
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of the animals in the main groups (control, and highest dose group) were examined for changes to the refracting media using a focusable hand-held slit lamp before the start of the study and after about 6 and 12 months. Further eye examinations were carried out after about 18 months and at the end of the study in each of the first 10 of the surviving animals of both sexes in the control and highest dose group, using a hand-held slit lamp. If changes to the refracting media had either been expected or existed, photographs would have been taken where necessary, using a KOWA camera.
During examinations of the eyes before the start of the study and after about 12 months of the study, the fundus of 10 male and 10 female rats of the control and highest dose
HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus of the non-fasted animals in the mornings. The blood samplings and the subsequent analysis of the blood and plasma samples - except for the differential blood counts and the reticulocytes - were carried out in randomised sequence 6 days before the test material was administered (acclimatisation period; blood sampling 0) and about 26, 52, 78, and 104 weeks after the start of the administration (administration period; blood samplings 1, 2, 3, and 4). The lists of randomisation instructions were generated with a random number generator on a computer.
The haematological examinations were carried out before and during the administration period in 15 animals per test group and sex (satellite group II). For blood sampling 4, the animals that died in satellite group II were supplemented to 15 animals per test group and sex by adding main group animals. For the hormone analyses, blood was taken after 52 weeks from 10 animals per group and sex of satellite group I.
- Parameters checked: Haemoglobin, erythrocytes, haematocrit, mean haemoglobin content per erythrocyte, mean cell volume, mean corpuscular haemoglobin concentration, platelets, leukocytes, differential blood count, reticulocytes, prothrombin time.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus of the non-fasted animals in the mornings. The blood samplings and the subsequent analysis of the blood and plasma samples - except for the differential blood counts and the reticulocytes - were carried out in randomised sequence 6 days before the test material was administered (acclimatisation period; blood sampling 0) and about 26, 52, 78, and 104 weeks after the start of the administration (administration period; blood samplings 1, 2, 3, and 4). The lists of randomisation instructions were generated with a random number generator on a computer.
The clinico-chemical examinations were carried out before and during the administration period in 15 animals per test group and sex (satellite group II). For blood sampling 4, the animals that died in satellite group II were supplemented to 15 animals per test group and sex by adding main group animals. For the hormone analyses, blood was taken after 52 weeks from 10 animals per group and sex of satellite group I.
- Parameters checked: Total bilirubin, creatinine, urea, sodium, potassium, glucose, inorganic phosphate, calcium, chloride, triglycerides, total cholesterol, total protein, albumin, globulins, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, alkaline phosphatase, triiodothyronine (T 3), thyroxine (T 4).
URINALYSIS: Yes
- Time schedule for collection of urine: The urine of each individual animal was collected overnight from the satellite group I animals about 26 and 52 weeks after the administration began (administration period: Urine collections 1 and 2) and from 10 animals per group of the main group animals about 104 weeks after the administration began (administration period; urine colleection 3).
- Parameters checked: pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, sediment.
Except sediment microscopy, all the urine constituents were determined semi-quantitatively with test strips and a reflection photometer. The sediment was evaluated under a microscope. - Sacrifice and pathology:
- The rats that survived in satellite group I were killed at the end of the 12-month administration period and the surviving main and satellite groups II rats at the end of the 24-month administration period after a fasting period (Satellite group I; withdrawal of feed and water, Satellite group II and main group withdrawal of feed).
The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks.
GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes - Statistics:
- Clinical examinations: For the statistical evaluation of the test, means and standard deviation were calculated for the variables feed consumption, body weight and test material intake for the animals in each test group. Statistical significance of the clinical data (body weight) was determined by analysis of variance (ANOVA) followed by a Dunnett's test.
Blood and plasma examinations: Following statistical adjustment (NALIMOV criterion), the means and standard errors were calculated and have been printed out in the tables together with the individual figures.
For judging the significance, the t test (with the exception of the differential blood count) was used to compare the individual dose groups both with the control group and with their corresponding intitial values (blood sampling 0).
Urinalyses
The assessment as to whether specific features are pronounced to various extents in the control and test groups was carried out by the chi^2 test in corresponding two-by-two contingency tables. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The three doses (20, 100 and 400 ppm) administered as addition to the diet did not lead to any disturbances the general state of health of any one of the animals participating in the test. As the duration of the test increased, findings such as decubitus in the tarsal region or alopecia, etc., became more frequent in the animals used. The changes are spontaneous ones, also regularly to be found in untreated older rats.
- Mortality:
- no mortality observed
- Description (incidence):
- The mortality of the male and female animals was not influenced by the administration of the test material.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The body weight of the male and female animals of all doses in the main groups as well as that of the females in satellite group I was comparable to that of the control animals.
There was a significant reduction in body weight between days 154 and 364 in the satellite group I male rats dosed 20 and 100 ppm; a dose-response relationship was not seen, and hence the reduction was assessed as being incidental in nature. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- The amount of feed consumed daily by the male and female animals of all dose groups (20, 100, and 400 ppm) in the main groups and satellite groups I during the specific duration of the study did not differ substantially when compared to the untreated control rats.
The amounts of test material taken up (mg/kg body weight) each day by the animals in the specific dose groups corresponded to the dose factor chosen. The mean amounts (in mg/kg body weight) of test material taken up in the course of the study are:
20 ppm main group males: 1.1 mg/kg bodyweight.
20 ppm satellite group 1 males: 1.3 mg/kg bodyweight.
20 ppm main group females: 1.4 mg/kg bodyweight.
20 ppm satellite group 1 females: 1.6 mg/kg bodyweight.
100 ppm main group males: 5.5 mg/kg bodyweight.
100 ppm satellite group 1 males: 6.5 mg/kg bodyweight.
100 ppm main group females: 6.9 mg/kg bodyweight.
100 ppm satellite group 1 females: 7.9 mg/kg bodyweight.
400 ppm main group males: 22.2 mg/kg bodyweight.
400 ppm satellite group 1 males: 26.1 mg/kg bodyweight.
400 ppm main group females: 27.9 mg/kg bodyweight.
400 ppm satellite group 1 females: 31.8 mg/kg bodyweight. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The ophthalmological examinations carried out with of a hand-held slit lamp before the start of the test and subsequently every 6 months showed no test material-induced impairment of the refracting media.
At no time was there any difference between the treated and untreated rats in terms of frequency distribution of the corneal findings in the form of remainders of the pupillary membrane or isolated corneal stipplings. Incidentally, these changes are frequently to be found in untreated Wistar rats. The examination with a KOWA camera carried out before the start of the test and after about 12 months showed no changes in the eye fundus. - Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- From the haematology point of view, the 24-month administration of the test material to male and female rats in doses of 20, 100, and 400 ppm did not cause any changes that could be ascribed to the test material administered.
The significant differences from the control group are not regarded as being related to the test material. Plausibility criteria have been introduced in order to avoid a detailed assessment and discussion of each individual statistically significant difference for each parameter.
The purpose of these criteria is to give all the reasons why there is no relation to administration of the test material, or why such a relation is improbable. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- From the clinical chemistry point of view, the 24-month administration of the test material to male and female rats in doses of 20, 100, and 400 ppm did not cause any changes that could be ascribed to the test material administered.
The significant differences from the control group are not regarded as being related to the test material. Plausibility criteria have been introduced in order to avoid a detailed assessment and discussion of each individual statistically significant difference for each parameter.
The purpose of these criteria is to give all the reasons why there is no relation to administration of the test material, or why such a relation is improbable. - Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- 400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.
100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.
20 ppm: No changes whatsoever that could be associated with the test material administered.
The 24-month administration of the test material therefore showed a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups, while the kidney weights of the female animals remained uninfluenced. - Gross pathological findings:
- not specified
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
- Dose descriptor:
- NOAEL
- Effect level:
- 20 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- organ weights and organ / body weight ratios
- Dose descriptor:
- NOAEL
- Effect level:
- 400 ppm
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: No adverse effects observed at highest dose level tested.
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the study a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups was observed, while the kidney weights of the female animals remained uninfluenced. Thereore the NOAEL for male rats was 20 ppm (equivalent to 1.1 mg/kg bodyweight/ day) and for female rats was 400 ppm (equivalent to 27.9 mg/kg bodyweight/ day).
- Executive summary:
The carcinogenicity and chronic repeated dose toxicity of the test material was assessed according to OECD Test Guideline 453 and in compliance with GLP.
Three different doses (20, 100 and 400 ppm) of the test material were administered with the diet to 450 Wistar rats (225 males and 225 females) for 24 months.
Each dose group was split up into a main group (50 animals per sex) and into the satellite groups I and II comprising 10 and 15 animals per sex respectively.
A group of untreated animals (75 males and 75 females) was maintained in parallel for comparison. It was likewise split into a main group and satellite groups I and II. The feed consumption and body weight of the animals in the main group and of satellite group I was determined once a week up to 14 weeks and thereafter once a month until the end of the study. The state of health of all animals was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week.
At the beginning of the study and then about every 6 months, animals of the main groups (control and highest dose) were examined ophthalmologically.
Blood samples for haematological and clinico-chemical examinations were taken 5 times altogether from the animals of the satellite group II (at the last blood sampling, dead animals in this satellite group were supplemented by animals of the main groups).
Urinalyses were carried out on the animals of satellite group I two times in the first year, and on 10 animals of each main group about 104 weeks after the beginning of the administration period.
The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks.
All animals used ware assessed by gross pathology. This was followed by a comprehensive histopathological examination.
The following findings were obtained and assessed or discussed to be test material-related:
400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.
100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.
20 ppm: No changes whatsoever that could be associated with the test material administered.
Under the conditions of the study a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups was observed, while the kidney weights of the female animals remained uninfluenced. Thereore the NOAEL for male rats was 20 ppm (equivalent to 1.1 mg/kg bodyweight/ day) and for female rats was 400 ppm (equivalent to 27.9 mg/kg bodyweight/ day).
Referenceopen allclose all
Intake of Test Substance
The mean daily test material intake in mg/kg body weight/day (mg/kg bw/d) over the entire study period is shown in the following table:
Test Group |
Concentration in the Diet (ppm) |
Mean Daily Test Substance Intake (mg/kg bw/d) |
|
Males |
Females |
||
1 |
100 |
6.15 |
7.2 |
2 |
400 |
24.5 |
29.1 |
3 |
800 |
50.7 |
58.7 |
Clinical Chemistry
Cyanide-insensitive palmitoyl-CoA-oxidation activity (PALCOA) values in liver and kidney of male and female rats are given in the following table:
|
Dose (ppm) |
PALOCA (mU/mg protein) |
||||
0 |
100 |
400 |
800 |
|||
Liver |
Males |
Mean |
6.51 |
6.62 |
7.54 |
9.12* |
SD |
0.82 |
0.83 |
0.93 |
0.93 |
||
Females |
Mean |
6.62 |
6.63 |
7.53* |
9.77* |
|
SD |
0.81 |
0.57 |
0.28 |
1.17 |
||
Kidney |
Males |
Mean |
2.27 |
4.03 |
5.88* |
6.39* |
SD |
0.81 |
0.84 |
0.50 |
0.67 |
||
Females |
Mean |
0.64 |
2.17 |
3.73 |
3.60 |
|
SD |
0.10 |
0.51 |
0.63 |
0.70 |
||
Kruskal Wallis + Wilcoxon-test; * = p ≤0.05; SD = standard deviation;
Number of animals/group = 4 except in the control group of the female animals of kidney examination (N = 3). The PALCOA-value of the control animal No. 17 was measured as 0.00 mU/mg protein and the integrity of this result cannot be assured. Therefore, this value was eliminated from the statistical evaluation. Due to the low number of control animals, the increases in kidney PALCOA activity in the treated females, although biologically meaningful, were not statistically significant.
Absolute Organ Weights
Compared with the control, the following deviations were present:
Males |
Test Groups |
||
1 |
2 |
3 |
|
Terminal body weight |
- 3 % |
+ 2 % |
0 % |
Liver |
+ 1 % |
+ 4 % |
+ 2 % |
Kidneys |
+ 16 % |
+ 16 % |
+ 15 % |
Females |
Test Groups |
||
1 |
2 |
3 |
|
Terminal body weight |
0 % |
-2 % |
-1 % |
Liver |
-6 % |
0 % |
+1 % |
Kidneys |
-1 % |
+1 % |
+4 % |
In all treated males, the absolute kidneys weights were increased (up to 16 % above control). Although there was no dose-response relationship, this might be related to treatment.
Relative Organ Weights
Compared with the control, the following deviations were present:
Males |
Test Groups |
||
1 |
2 |
3 |
|
Liver |
+ 3 % |
+ 2 % |
+ 2 % |
Kidneys |
+ 19 % |
+ 14 % |
+ 16 % |
Females |
Test Groups |
||
1 |
2 |
3 |
|
Liver |
-6 % |
+1 % |
+1 % |
Kidneys |
-1 % |
+2 % |
+4 % |
In all treated males, the relative kidneys weights were increased (up to 19 % above control). Although there was no dose-response relationship, this might be related to treatment.
Body weight of male rats during treatment over 14 days at the dose levels of 125, 250, 500, 1 000, 1 500, and 2 200 ppm test material in feed during 14 days (corresponding to about 10, 20, 40, 80, 120, and 180 mg/kg bw).
10 mg/kg bw |
||||||
Animal No. |
201 |
202 |
203 |
204 |
Mean |
SD |
Body weight d 0 (g) |
220.4 |
200.6 |
196.8 |
222.2 |
210.0 |
13.16 |
Body weight d 7 (g) |
260.9 |
239.4 |
224.2 |
255.9 |
245.1 |
16.69 |
Body weight d 14 (g) |
303.1 |
271.4 |
253.3 |
291.3 |
279.78 |
21.97 |
20 mg/kg bw |
||||||
Animal No. |
205 |
206 |
207 |
208 |
Mean |
SD |
Body weight d 0 (g) |
202.0 |
195.5 |
215.3 |
209.2 |
205.5 |
8.60 |
Body weight d 7 (g) |
241.4 |
224.0 |
252.2 |
245.3 |
240.73 |
12.01 |
Body weight d 14 (g) |
272.5 |
255.3 |
289.9 |
276.9 |
273.65 |
14.29 |
40 mg/kg bw |
||||||
Animal No. |
209 |
210 |
211 |
212 |
Mean |
SD |
Body weight d 0 (g) |
205.6 |
186.6 |
178.1 |
197.0 |
191.83 |
12.0 |
Body weight d 7 (g) |
233.7 |
208.3 |
190.1 |
231.4 |
215.88 |
20.66 |
Body weight d 14 (g) |
256.0 |
232.4 |
204.0 |
259.8 |
238.05 |
25.73 |
80 mg/kg bw |
||||||
Animal No. |
213 |
214 |
215 |
216 |
Mean |
SD |
Body weight d 0 (g) |
201.7 |
193.1 |
207.6 |
193.3 |
198.93 |
7.04 |
Body weight d 7 (g) |
229.6 |
215.0 |
230.3 |
224.2 |
224.78 |
7.06 |
Body weight d 14 (g) |
260.1 |
235.3 |
260.1 |
248.5 |
251.0 |
11.81 |
120 mg/kg bw |
||||||
Animal No. |
217 |
218 |
219 |
220 |
Mean |
SD |
Body weight d 0 (g) |
182.5 |
197.9 |
205.6 |
216.7 |
200.68 |
14.37 |
Body weight d 7 (g) |
203.4 |
213.4 |
224.7 |
238.7 |
220.05 |
15.18 |
Body weight d 14 (g) |
229.1 |
241.8 |
246.3 |
276.3 |
248.38 |
19.99 |
180 mg/kg bw |
||||||
Animal No. |
221 |
222 |
223 |
224 |
Mean |
SD |
Body weight d 0 (g) |
206.2 |
202.8 |
192.0 |
196.2 |
199.3 |
6.4 |
Body weight d 7 (g) |
238.5 |
219.6 |
205.3 |
191.1 |
213.63 |
20.26 |
Body weight d 14 (g) |
258.3 |
223.6 |
224.3 |
214.7 |
230.23 |
19.22 |
Dose due to feed intake of male rats during treatment with 125, 250, 500, 1 000, 1 500, and 2200 ppm test material in feed during 14 days (corresponding to about 10, 20, 40, 80, 120, and 180 mg/kg bw).
10 mg/kg bw |
||||||
Animal No. |
201 |
202 |
203 |
204 |
Mean |
SD |
Dose d 0 – 7 (mg/kg bw/day) |
13.05 |
12.79 |
13.03 |
12.66 |
12.88 |
0.19 |
Dose d 8 – 14 (mg/kg bw/day) |
12.70 |
11.75 |
12.09 |
13.46 |
12.50 |
0.75 |
20 mg/kg bw |
||||||
Animal No. |
205 |
206 |
207 |
208 |
Mean |
SD |
Dose d 0 – 7 (mg/kg bw/day) |
24.33 |
24.03 |
25.29 |
25.90 |
24.89 |
0.86 |
Dose d 8 – 14 (mg/kg bw/day) |
22.10 |
23.40 |
23.95 |
23.86 |
23.33 |
0.85 |
40 mg/kg bw |
||||||
Animal No. |
209 |
210 |
211 |
212 |
Mean |
SD |
Dose d 0 – 7 (mg/kg bw/day) |
46.70 |
49.38 |
48.54 |
51.92 |
49.14 |
2.17 |
Dose d 8 – 14 (mg/kg bw/day) |
42.74 |
47.13 |
52.34 |
47.38 |
47.40 |
3.92 |
80 mg/kg bw |
||||||
Animal No. |
213 |
214 |
215 |
216 |
Mean |
SD |
Dose d 0 – 7 (mg/kg bw/day) |
88.11 |
95.91 |
88.67 |
90.54 |
90.81 |
3.56 |
Dose d 8 – 14 (mg/kg bw/day) |
83.67 |
83.37 |
86.52 |
86.86 |
85.11 |
1.84 |
120 mg/kg bw |
||||||
Animal No. |
217 |
218 |
219 |
220 |
Mean |
SD |
Dose d 0 – 7 (mg/kg bw/day) |
128.38 |
121.08 |
117.43 |
122.15 |
122.26 |
4.55 |
Dose d 8 – 14 (mg/kg bw/day) |
127.04 |
165.52 |
128.39 |
138.14 |
139.77 |
17.86 |
180 mg/kg bw |
||||||
Animal No. |
221 |
222 |
223 |
224 |
Mean |
SD |
Dose d 0 – 7 (mg/kg bw/day) |
196.47 |
191.82 |
189.85 |
150.61 |
182.19 |
21.23 |
Dose d 8 – 14 (mg/kg bw/day) |
202.31 |
188.49 |
205.72 |
196.10 |
198.16 |
7.57 |
The Mean Body Weight Change (in kg and %) for the Male Dogs on Study Day 49 and at the End of the Administration Period
Test Group (Dose) Male Dogs |
Increase |
|||
Kg |
% |
|||
Study Day 49 |
Study Day 364 |
Study Day 49 |
Study Day 364 |
|
0 (0 ppm) |
0.4 |
2.0 |
4 |
19 |
1 (60 ppm) |
0.8 |
1.9 |
8 |
18 |
2 (180 ppm) |
0.7 |
2.6 |
7 |
25 |
3 (600 ppm) |
0.1 |
1.7 |
1 |
16 |
The Approximate, Mean Daily Test Material Intake (in mg/kg/body weight) During the Administration Period
|
Test Group (Dose) |
||
1 (60 ppm) |
2 (180 ppm) |
3 (600 ppm) |
|
Male animals |
1.8 |
5.2 |
18.3 |
Female animals |
2.0 |
5.7 |
19.0 |
All animals |
1.9 |
5.4 |
18.7 |
Intake of Test Material
The mean daily test material intake in mg/kg body weight/day (mg/kg bw/d) over the entire study period is shown in the following table:
Test Group |
Concentration in Diet (ppm) |
Mean Daily Test Material Intake (mg/kg bw/d) |
|
Males |
Females |
||
1 |
250 |
57.9 |
87.0 |
2 |
750 |
165.0 |
251.4 |
3 |
2500 |
681.0 |
955.9 |
Clinical Chemistry
Cyanide-insensitive palmitoyl-CoA-oxidation activity (PALCOA) values in liver and kidney of male and female mice are given in the following table:
|
|
Dose (ppm) |
PALCOA (mU/mg protein) |
|||
0 |
250 |
750 |
2500 |
|||
Liver |
Males |
Mean |
5.12 |
5.22 |
6.91 |
57.15* |
SD |
0.89 |
0.57 |
1.86 |
3.47 |
||
Females |
Mean |
4.23 |
4.64 |
11.05* |
57.86* |
|
SD |
2.44 |
0.95 |
0.73 |
5.56 |
||
Kidney |
Males |
Mean |
4.23 |
9.85* |
12.94* |
18.29* |
SD |
0.81 |
0.53 |
0.81 |
1.42 |
||
Females |
Mean |
1.08 |
5.99* |
10.54* |
15.56* |
|
SD |
0.51 |
0.89 |
1.45 |
0.76 |
||
Kruskal Wallis+ Wilcoxon-test; * = p s 0.05; SD = standard deviation;
Number of animals/group = 4
Absolute Organ Weights
Compared with the control, the following deviations were present:
|
Tests Groups |
||
1 |
2 |
3 |
|
Males |
|||
Terminal body weight |
+ 2 % |
- 1 % |
- 7 % |
Liver |
+ 9 % |
- 1 % |
+ 56 % |
Kidneys |
+ 12 % |
- 2 % |
- 13 % |
Females |
|||
Terminal body weight |
+ 3 % |
+ 1 % |
- 9 % |
Liver |
+ 4 % |
+ 7 % |
+ 83 % |
Kidneys |
+ 9 % |
+ 9 % |
- 8 % |
Relative Organ Weights
Compared with the control, the following deviations were present:
|
Tests Groups |
||
1 |
2 |
3 |
|
Males |
|||
Liver |
+ 7 % |
0 |
+ 68 % |
Kidneys |
+ 10 % |
0 |
- 6 % |
Females |
|||
Liver |
+ 2 % |
+ 5 % |
+ 101 % |
Kidneys |
+ 8 % |
+ 8 % |
0 |
Concentration Control Analysis and Analysis of Homogeneity
Sample No. |
Nominal Concentration [mg/kg] |
Analytical Concentration [mg/kg] |
% of Nominal Concentration |
||||
Sample I |
Sample II |
Sample III |
|
Mean |
SD |
||
2 |
0 |
n.d. |
n.d. |
n.d. |
- |
- |
- |
3 |
250 |
243 |
244 |
244 |
97.6 |
96.1 |
4.8 |
4 |
250 |
250 |
250 |
250 |
100.0 |
||
5 |
250 |
225 |
228 |
227 |
90.8 |
||
6 |
750 |
713 |
719 |
716 |
95.5 |
- |
- |
7 |
2500 |
2454 |
2454 |
2454 |
98.2 |
96.9 |
1.3 |
8 |
2500 |
2426 |
2421 |
2424 |
97.0 |
||
9 |
2500 |
2394 |
2385 |
2390 |
95.6 |
||
n.d.: Not detectable
Semiquantitative Assessment of Peroxisomes in Hepatocytes Using PMP70-Immunohistochemistry.
Males |
|
Animal No. |
Intracytoplasmic Peroxisomes |
1 |
(+) |
2 |
(+) |
3 |
(+) |
4 |
(+) |
13 |
++ |
14 |
++ |
15 |
++ |
16 |
++ |
Females |
|
17 |
(+) |
18 |
(+) |
19 |
(+) |
20 |
(+) |
29 |
++(+) |
30 |
++(+) |
31 |
++(+) |
32 |
++(+) |
Quantitative Assessment of Peroxisomes in Hepatocytes Using PMP70-Immunohistochemistry
Dose (ppm) |
|
Area % |
Count of Pos. Area |
Males |
|||
0 |
M |
0.56 |
173 |
SD |
0.16 |
36 |
|
n |
4 |
4 |
|
250 |
M |
1.31 |
375 |
SD |
0.74 |
187 |
|
n |
4 |
4 |
|
750 |
M |
2.38* |
602* |
SD |
0.09 |
99 |
|
n |
4 |
4 |
|
2500 |
M |
3.34* |
857* |
SD |
0.59 |
136 |
|
n |
4 |
4 |
|
Females |
|||
0 |
M |
1.16 |
346 |
SD |
0.14 |
50 |
|
n |
4 |
4 |
|
250 |
M |
1.90* |
523* |
SD |
0.24 |
61 |
|
n |
4 |
4 |
|
750 |
M |
2.48* |
668* |
SD |
0.86 |
171 |
|
n |
4 |
4 |
|
2500 |
M |
4.23* |
987* |
SD |
0.61 |
80 |
|
n |
4 |
4 |
|
*: p < = 0.05
**: p < = 0.01: Wilcoxon-test (one-sided)
Many of the indicated significant differences from the figures in the control group cannot be regarded as induced by the test material. Plausibility criteria have been introduced in order to avoid a detailed assessment and discussion of each statistically significant difference for each parameter. The purpose of these criteria is to give all the reasons why there is no relation to the administration of the test material or why any relation is improbable.
All the parameters for which a change is even merely suspected to be related to the test material were assessed.
The plausibility criteria are:
- The changes have no pathognomic relevance;
- The values lie within the range of biological variation, effect not relevant;
- Contrary change in both sexes, not very plausible;
- Similar effect absent for parameters which have a similar dependence on one another, not very plausible;
- Reciprocal effect absent for parameters which have a reciprocal dependence on one another, not very plausible;
- Random increase or decrease in the control value, effect not relevant.
- No monotonic trend present (absence of dose-response relationship), not very plausible;
- Similar effect absent in both sexes, not very plausible;
- Trend within the test group, effect not relevant;
- First change in the observation period, not very plausible.
Observations after 3 months oral administration of test material to mice
Dose (ppm) sex |
0 m/f |
100 m/f |
1000 m/f |
2500 m/f |
| mean dose, mg/kg bw/day | -/- | 20/30 | 220/330 | 740/930 |
| body weight, g (3 mo) | 33.6/28.4 | 32.2/26.1 | 30.9*/25.8* | 30.4**/25.8* |
| Organ weights: | ||||
| liver weight, g | 1.21/1.11 | 1.15/0.99 | 1.21/1.13 | 1.63**/1.66** |
| kidneys weight, g | 0.51/0.37 | 0.50/0.36 | 0.48/0.40 | 0.42**/0.35 |
| adrenal glands weight, g | 6.2/11 | 7.1/10.2 | 5.8/10.1 | 6.4/8.8* |
| Clinical chemistry/ haematology: | ||||
| alkaline phosphatase | -/- | -/- | -/- | ↑/↑ |
| cyan.in.pal.-CoA-ox.1 | -/- | n.d. | n.d. | ↑/↑ |
| urea | -/- | -/↑ | ↑/↑ | ↑/↑ |
| cholesterol | -/- | -/- | -/- | ↑/↑ |
| triglycerides | -/- | -/↓ | ↓/↓ | ↓/↓ |
| creatinine | -/- | -/- | ↑/- | ↑/- |
| glucose | -/- | -/- | -/- | ↑/- |
| alanine aminotransferase | -/- | -/- | -/- | -/↑ |
| haemoglobin | -/- | -/- | -/- | ↓/- |
| mean corpuscular haemogl. | -/- | -/- | -/- | ↓/- |
| globulins | -/- | -/- | -/- | ↓/- |
* p < 0.05 ** p < 0.01 Dunnet-test (two sided) n.d.: not determined
1cyanide-insensitive palmitoyl-CoA-oxidation in liver homogenate
Historical Control Data: Female Body Weight in Mice (B6C3F1).
Project Number |
Duration |
Absolute Weights in Gram (SD*) |
187029-OXI |
06.87 - 09.87 |
23.79 (3.19) |
85123-DCC |
08.87 - 11. 87 |
22.4 (1.38) |
87057-CCC |
10.87 - 01. 88 |
23. 49 (2.28) |
87078-OXI |
12.87 - 03.88 |
22.30 (1.92) |
87085-2-EH 01. |
01.88 - 04.88 |
21.11 (1.5) |
88013-CDA |
03.88 - 06.88 |
24.98 (2.94) |
89013-PIX |
06.89 - 09.89 |
22.49 (1.67) |
89055-2,4-DP |
02.90 - 05.90 |
23.16 (3.09) |
83079-MCPP |
02.90 - 05.90 |
24.51 (1.28) |
90036-METI |
09.90 - 12.90 |
21. 97 (1.35) |
90038-INDO |
10.90 - 01.91 |
19.53 (1.45) |
91001-2,4 DP |
02.91 - 05.91 |
19.98 (1.42) |
187029-OXI |
06.87 - 09.87 |
23.79 (3.19) |
91002-MCPP |
03.91 - 06.91 |
24.09 (2.92) |
n = 13 |
|
M = 22.48 SD = 1.96 |
* SD=Standard deviation
Test Material Concentrations
Group |
Theoretical Concentration (ppm) |
Control 1st Preparation |
Control 2nd Preparation (ppm) |
Control 5th Preparation (ppm) |
|
Racemate |
1 |
200 |
185 (-7.5 %) |
200 (0 %) |
210 (+5 %) |
2 |
800 |
760 (-5 %) |
840 (+5 %) |
840 (+5 %) |
|
3 |
3200 |
3200 (0 %) |
3300 (+3 %) |
3250 (+16 %) |
|
Isomer |
4 |
200 |
200 (0 %) |
200 (0 %) |
190 (-5 %) |
5 |
400 |
405 (+1.3 %) |
400 (0 %) |
385 (-3.8 %) |
|
6 |
800 |
815 (+1.9 %) |
800 (0 %) |
815 (+ 1.9 %) |
|
7 |
1600 |
1650 (+3 %) |
1550 (-3 %) |
1650 (+ 3 %) |
|
8 |
3200 |
3250 (+1.6 %) |
3200 (0 %) |
3300 (+3 %) |
|
Parentheses: % of variation.
Bodyweight (g)
Group |
Day of Test |
||||||||||||||
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
||
Control group A males |
m |
116 |
167 |
212 |
246 |
273 |
289 |
304 |
325 |
334 |
342 |
349 |
361 |
370 |
376 |
s |
5 |
8 |
11 |
12 |
13 |
16 |
17 |
19 |
18 |
20 |
20 |
21 |
22 |
23 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Control group A females |
m |
111 |
145 |
165 |
186 |
206 |
212 |
219 |
23 |
238 |
242 |
247 |
257 |
260 |
258 |
s |
3 |
7 |
9 |
14 |
11 |
17 |
19 |
16 |
20 |
18 |
22 |
19 |
19 |
21 |
|
n |
15 |
15 |
15 |
15 |
15 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
Control group B males |
m |
117 |
167 |
203 |
243 |
279 |
294 |
315 |
333 |
346 |
351 |
358 |
369 |
382 |
384 |
s |
6 |
8 |
9 |
9 |
11 |
11 |
12 |
12 |
11 |
12 |
14 |
13 |
15 |
16 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Control group B females |
m |
109 |
143 |
159 |
186 |
199 |
211 |
218 |
227 |
233 |
235 |
240 |
251 |
255 |
251 |
s |
4 |
6 |
10 |
11 |
11 |
10 |
14 |
13 |
13 |
13 |
14 |
16 |
16 |
14 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
14 |
14 |
14 |
14 |
|
Group 1 males (racemate: 200 ppm) |
m |
115 |
162 |
204 |
244 |
276 |
293 |
306 |
326 |
338 |
343 |
349 |
357 |
371 |
377 |
s |
5 |
8 |
9 |
12 |
15 |
16 |
17 |
19 |
20 |
21 |
21 |
21 |
21 |
21 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 1 females (racemate: 200 ppm) |
m |
110 |
142 |
164 |
186 |
204 |
211 |
218 |
230 |
236 |
245 |
245 |
248 |
249 |
251 |
s |
5 |
6 |
7 |
10 |
11 |
13 |
9 |
12 |
14 |
14 |
14 |
16 |
16 |
15 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 2 males (racemate: 800 ppm) |
m |
116 |
161 |
202 |
241 |
259 |
285 |
300 |
322 |
334 |
337 |
343 |
353 |
366 |
373 |
s |
6 |
8 |
10 |
12 |
15 |
15 |
18 |
19 |
19 |
20 |
21 |
22 |
24 |
27 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 2 females (racemate: 800 ppm) |
m |
108 |
139 |
156 |
177 |
196 |
201 |
208 |
221 |
228 |
226 |
229 |
233* |
235* |
242 |
s |
5 |
7 |
6 |
9 |
12 |
9 |
12 |
14 |
15 |
12 |
15 |
15 |
17 |
15 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
|
Group 3 males (racemate: 3200 ppm) |
m |
115 |
150* |
173* |
190* |
198* |
199* |
197* |
210* |
214* |
223* |
224* |
226* |
236* |
242* |
s |
6 |
9 |
13 |
15 |
15 |
18 |
19 |
18 |
18 |
19 |
19 |
18 |
19 |
21 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 3 females (racemate: 3200 ppm) |
m |
110 |
127* |
137* |
146* |
154* |
152* |
155* |
160* |
167* |
168* |
169* |
173* |
176* |
186* |
s |
6 |
7 |
9 |
9 |
12 |
15 |
13 |
13 |
15 |
15 |
15 |
16 |
17 |
20 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 4 males (isomer: 200 ppm) |
m |
117 |
168 |
208 |
252 |
282 |
299 |
316 |
334 |
346 |
352 |
358 |
366 |
380 |
382 |
s |
4 |
7 |
7 |
8 |
8 |
12 |
16 |
15 |
16 |
17 |
18 |
19 |
19 |
21 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 4 females (isomer: 200 ppm) |
m |
110 |
142 |
161 |
183 |
202 |
213 |
218 |
227 |
235 |
237 |
240 |
247 |
247 |
251 |
s |
4 |
6 |
7 |
7 |
10 |
11 |
11 |
11 |
10 |
10 |
11 |
13 |
12 |
12 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 5 males (isomer: 400 ppm) |
m |
117 |
165 |
203 |
249 |
281 |
295 |
315 |
336 |
352 |
352 |
360 |
371 |
384 |
388 |
s |
6 |
7 |
8 |
9 |
11 |
12 |
12 |
14 |
14 |
14 |
14 |
15 |
17 |
18 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 5 females (isomer: 400 ppm) |
m |
108 |
139 |
163 |
178 |
194 |
204 |
211 |
222 |
223 |
229 |
231 |
237 |
239* |
244 |
s |
4 |
5 |
8 |
8 |
10 |
13 |
10 |
12 |
13 |
12 |
17 |
14 |
14 |
16 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 6 males (isomer: 800 ppm) |
m |
115 |
163 |
197* |
243 |
273 |
289 |
306 |
629 |
342 |
345 |
352 |
360 |
374 |
378 |
s |
6 |
8 |
8 |
9 |
9 |
9 |
12 |
13 |
14 |
13 |
15 |
17 |
17 |
18 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 6 females (isomer: 800 ppm) |
m |
111 |
141 |
153 |
179 |
195 |
205 |
213 |
224 |
231 |
229 |
231 |
238 |
240 |
240 |
s |
4 |
6 |
8 |
12 |
12 |
12 |
15 |
15 |
15 |
15 |
15 |
18 |
22 |
21 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
|
Group 7 males (isomer: 1600 ppm) |
m |
118 |
159* |
189* |
234* |
261* |
273* |
290 |
308* |
321 |
322* |
327* |
333* |
347* |
348* |
s |
4 |
5 |
6 |
9 |
10 |
13 |
12 |
15 |
15 |
18 |
18 |
18 |
18 |
22 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 7 females (isomer: 1600 ppm) |
m |
112 |
135* |
146* |
168* |
185* |
190* |
195* |
203* |
210* |
209* |
209* |
215* |
218* |
221* |
s |
5 |
7 |
7 |
9 |
11 |
9 |
12 |
12 |
13 |
10 |
11 |
12 |
13 |
11 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 8 males (isomer: 3600 ppm) |
m |
115 |
143* |
157* |
183* |
197* |
196* |
204* |
214* |
220* |
217* |
218* |
225* |
238* |
241* |
s |
6 |
9 |
12 |
14 |
18 |
17 |
23 |
22 |
20 |
22 |
23 |
24 |
24 |
24 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
Group 8 females (isomer: 3600 ppm) |
m |
112 |
126* |
133* |
148* |
156* |
162* |
164* |
170* |
178* |
175* |
175* |
183* |
186* |
192* |
s |
6 |
6 |
9 |
9 |
9 |
7 |
8 |
7 |
8 |
11 |
12 |
12 |
10 |
14 |
|
n |
15 |
15 |
15 |
15 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
13 |
|
* Significantly different from CA and CB groups (99 % and over).
Organ Weights – Absolute Values
Group |
Wght. (g) |
Brain (g) |
Pit. (mg) |
Thyr. (mg) |
Thym. (g) |
Heart. (g) |
Liver. (g) |
Spleen (g) |
Kidn.‡ (g) |
Adren.‡ (mg) |
Gonad‡ (g) |
Prost. (g) |
Uter. (g) |
|
Control group A males |
m |
379 |
1.820 |
10 |
15 |
0.290 |
1.130 |
12.914 |
0.769 |
2.282 |
43 |
3.505 |
0.715 |
|
s |
23 |
0.095 |
2 |
2 |
0.063 |
0.089 |
1.154 |
0.096 |
0.139 |
4 |
0.202 |
0.116 |
|
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
15 |
15 |
|
|
Control group A females |
m |
258 |
1.750 |
12 |
15 |
0.251 |
0.860 |
8.133 |
0.648 |
1.514 |
55 |
0.139 |
|
0.406 |
s |
21 |
0.086 |
2 |
2 |
0.063 |
0.065 |
0.914 |
0.062 |
0.12 |
4 |
0.025 |
|
0.082 |
|
n |
14 |
13 |
14 |
14 |
14 |
14 |
14 |
13 |
14 |
14 |
14 |
|
14 |
|
Control group B males |
m |
384 |
1.818 |
11 |
13 |
0.334 |
1.094 |
12.217 |
0.802 |
2.227 |
43 |
3.383 |
0.774 |
|
s |
16 |
0.078 |
1 |
2 |
0.051 |
0.053 |
0.698 |
0.073 |
0.174 |
5 |
0.139 |
0.158 |
|
|
n |
15 |
15 |
14 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
Control group B females |
m |
251 |
1.717 |
13 |
14 |
0.237 |
0.831 |
7.558 |
0.530 |
1.476 |
53 |
0.134 |
|
0.429 |
s |
14 |
0.069 |
2 |
2 |
0.045 |
0.051 |
0.403 |
0.076 |
0.166 |
4 |
0.019 |
|
0.106 |
|
n |
14 |
14 |
13 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
14 |
|
Group 1 males (racemate: 200 ppm) |
m |
377 |
1.815 |
10 |
15 |
0.253 |
1.090 |
12.776 |
0.802 |
2.476* |
44 |
3.490 |
0.765 |
|
s |
21 |
0.049 |
1 |
3 |
0.075 |
0.072 |
1.009 |
0.090 |
0.214 |
3 |
0.203 |
0.098 |
|
|
n |
15 |
5 |
5 |
5 |
5 |
14 |
5 |
5 |
5 |
5 |
5 |
5 |
|
|
Group 1 females (racemate: 200 ppm) |
m |
251 |
1.751 |
13 |
14 |
0.244 |
0.809 |
7.814 |
0.658 |
1.641* |
54 |
0.135 |
|
0.415 |
s |
15 |
0.075 |
2 |
2 |
0.053 |
0.061 |
0.723 |
0.080 |
0.125 |
7 |
0.019 |
|
0.092 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
15 |
|
15 |
|
Group 2 males (racemate: 800 ppm) |
m |
373 |
1.789 |
11 |
14 |
0.295 |
1.078 |
12.720 |
0.745 |
2.521* |
42 |
3.523 |
0.754 |
|
s |
27 |
0.088 |
1 |
2 |
0.051 |
0.088 |
1.342 |
0.081 |
0.225 |
6 |
0.156 |
0.195 |
|
|
n |
15 |
15 |
15 |
15 |
15 |
14 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
Group 2 females (racemate: 800 ppm) |
m |
242 |
1.719 |
12 |
14 |
0.226 |
0.529 |
8.470 |
0.624 |
1.574 |
54 |
0.123 |
|
0.428 |
s |
15 |
0.071 |
2 |
3 |
0.045 |
0.054 |
0.952 |
0.080 |
0.135 |
8 |
0.019 |
|
0.121 |
|
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
14 |
|
Group 3 males (racemate: 3200 ppm) |
m |
242* |
1.746 |
7* |
10* |
0.148* |
0.838* |
12.797 |
0.591* |
1.631* |
33* |
3.194 |
0.295* |
|
s |
21 |
0.122 |
1 |
3 |
0.043 |
0.093 |
1.401 |
0.067 |
0.134 |
4 |
0.242 |
0.031 |
|
|
n |
15 |
15 |
15 |
15 |
15 |
14 |
15 |
15 |
15 |
14 |
14 |
15 |
|
|
Group 3 females (racemate: 3200 ppm) |
m |
186* |
1.626* |
9* |
12 |
0.182* |
0.667* |
9.452* |
0.542 |
1.267* |
40* |
0.078* |
|
0.255* |
s |
20 |
0.054 |
2 |
2 |
0.046 |
0.060 |
1.369 |
0.107 |
0.134 |
5 |
0.020 |
|
0.086 |
|
n |
15 |
15 |
15 |
15 |
14 |
15 |
15 |
15 |
15 |
14 |
15 |
|
15 |
|
Group 4 males (isomer: 200 ppm) |
m |
328 |
1.835 |
11 |
14 |
0.304 |
1.095 |
12.859 |
0..820 |
2.473* |
44 |
3.525 |
0.739 |
|
s |
21 |
0.065 |
1 |
3 |
0.068 |
0.070 |
1.116 |
0.083 |
0.139 |
8 |
0.178 |
0.136 |
|
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
Group 4 females (isomer: 200 ppm) |
m |
251 |
1.727 |
13 |
14 |
0.219 |
0.808 |
7.900 |
0.619 |
1.555 |
52 |
0.118 |
|
0.428 |
s |
12 |
0.065 |
3 |
3 |
0.049 |
0.047 |
0.373 |
0.073 |
0.021 |
5 |
0.013 |
|
0.148 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
|
15 |
|
Group 5 males (isomer: 400 ppm) |
m |
388 |
1.848 |
11 |
15 |
0.316 |
1.139 |
13.055 |
0.816 |
2.497* |
43 |
3.584 |
0.745 |
|
s |
18 |
0.072 |
1 |
2 |
0.044 |
0.053 |
1.550 |
0.093 |
0.167 |
4 |
0.188 |
0.157 |
|
|
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
|
|
Group 5 females (isomer: 400 ppm) |
m |
244 |
1.750 |
13 |
15 |
0.242 |
0.799 |
7.979 |
0.617 |
1.580 |
55 |
0.131 |
|
0.431 |
s |
16 |
0.061 |
2 |
3 |
0.051 |
0.049 |
1.035 |
0.096 |
0.124 |
7 |
0.033 |
|
0.125 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
14 |
|
14 |
|
Group 6 males (isomer: 800 ppm) |
m |
378 |
1.816 |
11 |
15 |
0.290 |
1.060 |
12.503 |
0.741 |
2.489* |
44 |
3.377 |
0.728 |
|
s |
18 |
0.064 |
2 |
3 |
0.040 |
0.083 |
0.972 |
0.052 |
0.153 |
5 |
0.854 |
0.157 |
|
|
n |
15 |
15 |
15 |
15 |
14 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
Group 6 females (isomer: 800 ppm) |
m |
240 |
1.738 |
12 |
13 |
0.215 |
0.791 |
8.391 |
0.614 |
1.574 |
55 |
0.118 |
|
0.383 |
s |
21 |
0.052 |
1 |
2 |
0.054 |
0.069 |
1.178 |
0.032 |
0.142 |
7 |
0.020 |
|
0.069 |
|
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
14 |
|
Group 7 males (isomer: 1600 ppm) |
m |
348* |
1.781 |
10 |
14 |
0.240 |
1.064 |
13.873 |
0.777 |
2.282 |
41 |
3.368 |
0.665 |
|
s |
22 |
0.119 |
1 |
2 |
0.041 |
0.085 |
1.472 |
0.100 |
0.221 |
3 |
0.287 |
0.132 |
|
|
n |
15 |
15 |
15 |
15 |
15 |
14 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
Group 7 females (isomer: 1600 ppm) |
m |
221* |
1.729 |
12 |
13 |
0.220 |
0.767* |
9.120* |
0.638 |
1.487 |
50 |
0.109* |
|
0.475 |
s |
11 |
0.047 |
2 |
1 |
0.046 |
0.037 |
0.926 |
0.093 |
0.120 |
6 |
0.024 |
|
0.098 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
15 |
|
Group 8 males (isomer: 3600 ppm) |
m |
241* |
1.655* |
8* |
11 |
0.148* |
0.867* |
12.899 |
0.584* |
1.625* |
36* |
3.13* |
0.288* |
|
s |
24 |
0.110 |
2 |
3 |
0.037 |
0.098 |
1.122 |
0.068 |
0.169 |
5 |
0.216 |
0.090 |
|
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
14 |
|
|
Group 8 females (isomer: 3600 ppm) |
m |
192* |
1.623* |
8* |
12* |
0.191* |
0.710* |
9.937* |
0.556 |
1.352 |
44* |
0.081* |
|
0.279* |
s |
14 |
0.055 |
2 |
2 |
0.035 |
0.055 |
1.035 |
0.079 |
0.106 |
8 |
0.013 |
|
0.105 |
|
n |
13 |
13 |
13 |
13 |
12 |
13 |
13 |
13 |
13 |
13 |
13 |
|
13 |
|
* Values are significantly different from Ca and CB groups (99 % and over).
‡ Mean values of individual weights.
Daily Mean Food Consumption Per Animal
Group |
Control (g) |
II Racemate (g) |
III Racemate (g) |
VI Isomer (g) |
VII Isomer (g) |
VIII Isomer (g) |
M |
27.0 |
26.7 |
28.0 |
28.3 |
28.2 |
28.5 |
F |
27.3 |
27.8 |
28.1 |
27.8 |
28.1 |
28.5 |
Compound Intake
Group |
Males |
Females |
II 800 ppm racemate |
81.7 |
121.1 |
III 3200 ppm racemate |
452.5 |
537.1 |
VI 800 ppm isomer |
84.1 |
117.8 |
VII 1600 ppm isomer |
178.1 |
239.9 |
VIII 3200 ppm isomer |
429.5 |
539.0 |
Treatment related changes in organ weights:
0 ppm (m/f) |
20 ppm (m/f) |
100 ppm (m/f) | 400 ppm (m/f) | |
Dose, mg/ kg bw/day* |
-/- | 1.1/1.4 | 5.5/6.9 | 22.2/27.9 |
kidney weight, g |
3.80/2.69 | 3.83/2.77 | 4.07#/1.97 | 4.31##/2.82 |
relative kidney weight, g |
0.60/0.76 | 0.61/0.74 | 0.62/0.76 | 0.66##/0.77 |
*: calculated for the main group # and ##: 5% and 1% significance level (Dunnett's test)
Analyses
- Analysis of the test material: The active ingredient content of the test material was determined before the start of the study, after about 19 months, and at the end of the study.
The active ingredient content was 92.7 % before the beginning of the study. The stability of the test material was verified after 19 months by the analytical results and at the end of the study HPLC yielded 91.9 % and the gas chromatographic method yielded a content of 94.9 %. In accordance with the study protocol, after one year the test material samples were sent for reanalysis; however, no results were passed on. This is of subordinate significance, since, as it had been expected, the stability of the test material was confirmed by analysis at the end of the test.
The homogeneity of the test material was confirmed analytically before the start of the test.
The technical active ingredient was subjected. to a storage test, which after 2 years of storage at room temperature, at 30, 40, and 50 °C showed that its degree of purity had not declined.
Analysis of the test material preparations: In the analysis of the samples drawn before the start of the study, the homogeneity of the test material preparations could not be unambiguously verified; therefore, new samples were analysed. The results of these investigations confirm the homogeneous distribution of the test material in the vehicle.
The stability of the test material in the maintenance diet over 33 days was demonstrated at the start of study.
- Analysis of feed: The feed was regarded as suitable on the basis of the duration of use and the results of the tests for contaminants. The guideline for maximum tolerable contaminants was the Proposed Guidelines of EPA of May 9, 1979, Fed. Reg. Vol. 44, No. 91, p. 27354.
Only the copper values were found to have dropped; they were however acceptable since they were within the range of error expected with this method of determination.
- Analysis of drinking water: The drinking water used was regarded as suitable on the basis of the results obtained.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 5.4 mg/kg bw/day
- Study duration:
- chronic
- Species:
- dog
- System:
- hepatobiliary
- Organ:
- adrenal glands
- kidney
- 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
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 September 1992 to 05 March 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Version / remarks:
- 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA Pesticide Assessment Guidelines, Subdivision F. Hazard Evaluation: Human and Domestic Animals 82-2 Repeated dose dermal toxicity study.
- Version / remarks:
- Revised Edition, November 1984
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on species / strain selection:
- The rabbit was chosen as the test species as it has been shown to be a suitable model for this type of study and is the species recommended in the test guidelines.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10 to 12 weeks old on arrival.
- Weight at study initiation: 2.0 to 2.8 kg on arrival.
- Fasting period before study: Food and water were only withdrawn overnight prior to collection of samples.
- Housing: All rabbits were caged individually in metal cages with perforated floors.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: An eight-day acclimatisation period was allowed between delivery of the animals and start of treatment. The health status of all animals was monitored, by daily observation throughout the acclimatisation period, to ensure that the rabbits selected for final assignment to the study were satisfactory.
DETAILS OF FOOD AND WATER QUALITY: The batches of diet used for the study had been analysed for nutrients, possible contaminants and microorganisms. Results of the routine physical and chemical examination of drinking water at source, as conducted usually weekly by the supplier, are made available to Huntingdon Research Centre Ltd., as quarterly summaries. There was no information available to the Study Director to indicate that any non-nutrient substance likely to influence the effect of the test material could reasonably have been expected to be present in the diet or the drinking water.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature was generally controlled in the range 15 to 21 °C, however on Day 11, minimum to maximum temperature readings of 17 to 30.5 °C were recorded as a result of animal building maintenance work.
- Humidity (%): Relative humidity was generally controlled in the region of 36 to 62 % R.H., however on Day 11, minimum to maximum humidity readings of 32 to 62 % R.H. were recorded as a result of animal building maintenance work.
- Air changes (per hr): Air exchange was maintained at approximately 19 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to provide 12 hours artificial light (0700 - 1900 hours) in each 24-hour period.
- Type of coverage:
- occlusive
- Vehicle:
- unchanged (no vehicle)
- Remarks:
- (the test material was moistened with distilled water to ensure good contact with the skin)
- Details on exposure:
- RANGE-FINDING STUDY
A group comprising one male and one female rabbit of the New Zealand White strain was treated by dermal application of the test material at a dosage of 1 000 mg/kg/day. The treatment area was a shaven region of skin measuring 12 x 8 cm (approximately 10% of the total body surface) and the test material was moistened with distilled water at a constant volume of 3 mL/rabbit/day. After dosing, the treatment site was covered by an impervious bandage for approximately six hours. Following the exposure period, the dressings were removed and the treated skin was washed with warm water (30 - 40 °C) and then gently blotted dry.
MAIN STUDY
- Two days before the start of treatment each animal was weighed and forty rabbits were randomly allocated to four groups, each consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.
- Prior to dosing on Day 1 (24 September 1992), a final health status examination revealed one of the rabbits allocated to Group 2, believed to be a female, to be in fact a very immature male. This animal was replaced before study start with an appropriate spare rabbit.
- Immediately after dosing, a male rabbit in Group 2 started to convulse violently. The animal was sacrificed immediately on humane grounds - its condition was attributed to shock brought on by the bandaging procedure (a very infrequent but recorded response in rabbits). Post mortem findings revealed congested lungs as the only notable finding. This animal was immediately replaced by an appropriate spare rabbit. Neither actions were considered to have affected the integrity of the study and the weights of replacement rabbits were similar to those of the original animals.
Following the commencement of treatment, spare animals were removed from the study. No further investigations were performed on these animals.
TEST SITE
- Area of exposure: The treatment area was a shaven region of skin measuring 12 x 8 cm on the back of each rabbit.
- % coverage: Approximating to 10 % of the total body surface.
- Type of wrap if used: The treatment site was covered with an impervious bandage consisting of gauze covered with 'Elastoplast' elastic adhesive dressing backed with impervious 'Sleek' plaster.
- Time intervals for shavings or clipplings: The hair was clipped from the dorsal region of each rabbit. Hair clipping was carried out approximately twenty-four hours prior to the first application of the test material. Hair clipping was repeated as necessary during the experimental period. The skin sites were not abraded.
REMOVAL OF TEST SUBSTANCE
- Washing: The treated skin was washed with warm water (30 - 40 °C) and gently blotted dry.
- Time after start of exposure: The test material remained on the back of each rabbit for approximately six hours each day after which time the dressings were removed.
TEST MATERIAL
- Amount(s) applied: The appropriate amount of test material (equating to the correct dosage) was weighed out daily for each animal and spread evenly over the prepared skin of the rabbits.
- Constant volume or concentration used: Yes. Each animal received a constant dosage based upon its most recently recorded bodyweight. The test material was moistened with distilled water at a constant volume/dosage (0.1, 0.3 and 3 mL/rabbit/day for Groups 2, 3 and 4 respectively) to ensure good contact with the skin. - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- Animals were treated for either twenty-one (males) or twenty-two (females) consecutive days. Treatment of animals not scheduled for sacrifice on Day 22 was continued up to 24 hours prior to sacrifice.
- Frequency of treatment:
- Animals were treated once daily.
- Dose / conc.:
- 10 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- Five per sex per dose
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale:
A high dosage of 1 000 mg/kg/day was selected on the basis of available toxicity information, in particular, rabbit acute dermal toxicity data [LD50 (rabbit) > 4 000 mg/kg bodyweight] and the results of a preliminary study.
- Fasting period before blood sampling for clinical biochemistry: Yes, food and water were withdrawn overnight prior to collection of samples. - Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each day and again in the late afternoon to look for dead or moribund animals.
DERMAL IRRITATION: Yes
- Time schedule for examinations: Local irritation was recorded immediately prior to the first daily application of the test material and subsequently daily. Local dermal reactions (erythema and oedema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system as follows:
Erythema and eschar formation
0 = No erythema
1 = Slight erythema
2 = Well-defined erythema
3 = Moderate erythema
4 = Severe erythema (beet redness) to slight eschar formation (injuries in depth)
Oedema
0 = No oedema
1 = Slight oedema
2 = Well-defined oedema (area well-defined by definite raising)
3 = Moderate oedema (raised approximately 1 mm)
4 = Severe oedema (raised more than 1 mm and extending beyond the area of exposure)
Dermal changes other than erythema and oedema were recorded separately.
BODY WEIGHT: Yes
- Time schedule for examinations: All rabbits were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.
FOOD CONSUMPTION:
- Food consumption: The quantity of food consumed by each rabbit was measured at weekly intervals throughout the study.
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was withdrawn from the median artery of the ear of all rabbits prior to termination. Male and female samples were collected on consecutive days (Days 20 and 21 respectively).
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- How many animals: All rabbits
- Parameters checked: The following parameters were analysed with an Ortho ELT-1500 Analyser, using standard Ortho methodology: Packed cell volume (PCV), Haemoglobin (Hb), Red blood cell count (RBC).
Absolute indices:
Mean corpuscular haemoglobin concentration (MCHC), Calculated: Hb (g/dL) x 100 / PCV (%)
Mean corpuscular volume (MCV), Calculated: PCV (%) x 10 / RBC (x10^6/mm³)
Mean corpuscular haemoglobin (MCH), Calculated: Hb (g/dL x 10 / RBC (x10^6/mm³)
Platelet count (Plts) and Total white blood cell count (WBC)
The following estimations were measured using the appropriate methodology:
Thrombotest (1T) - Method of Owren, P.A. (Lancet, 1959, ii, 754), Differential white blood cell count (Diff) - namely: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M).
The percentage distribution of each cell type was determined by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells. Percentage values were then converted to absolute values by computer inevitably involving a "rounding off'' in a proportion of the results. Hence, the measured total WBC may differ slightly from the calculated total for the differential count.
Additional blood film slides were prepared and examined for morphological abnormalities: Polychromasia, Hypochromasia, Anisocytosis, Rouleaux formation, Separate film report (generated for additional abnormalities).
The collected blood samples were divided into tubes containing EDTA anticoagulant for haematological investigations and citrate anticoagulant for coagulation tests.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was withdrawn from the median artery of the ear of all rabbits prior to termination. Male and female samples were collected on consecutive days (Days 20 and 21 respectively).
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- How many animals: All rabbits
- Parameters checked: The following parameters were analysed with an Hitachi 737 Clinical Chemistry Analyser:
Glucose - using BCL Test Kit (hexokinase mediated), Total protein, Albumin (Alb), Globulin (Glob), Calculated: Total protein (g/dl) minus Alb (g/dl), Albumin/Globulin ratio (A/G), Calculated from Total protein and Albumin concentrations, Urea nitrogen (Urea Nitr), Creatinine, Alkaline phosphatase (AP) - reaction temperature 30 °C, Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase (ALT)' - using BCL Test Kit, reaction temperature 30 °C, Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase (AST)' - using BCL Test Kit, reaction temperature 30 °C, Total bilirubin (Bilirubin), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl), Inorganic phosphorus (P), Cholesterol (Chol).
The collected blood samples were divided into tubes containing heparin anticoagulant for biochemical tests.
URINALYSIS: Yes.
- Time schedule for collection of urine: Overnight urine samples were collected for all rabbits prior to termination. As no overnight urine samples were obtained for various rabbits from all four groups, resampling occurred on the night prior to sacrifice.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- Parameters checked: The following estimations were measured using the appropriate methodology:
Volume (Vol), pH - using pH meter, Specific Gravity (SG) - using Atago UR-1 Refractometer, sample compared with water (nominal value of 1 000)
Protein - using Roche Cobas Centrifugal Analyser, utilising modified method of Macart, M. and Gerbaut, L. (Clin. Chim. Acta, 1984, 141, 77)
The following tests were also performed using qualitative indicators of analyte concentration:
Total reducing substances (TRS), Glucose (Gluc), Ketones (Ket), Bile pigments (Bile Pigs), Urobilinogen (Uro-bi), Haem pigments (Haem Pigs - positive or negative finding only).
Microscopic examination of urine samples was not performed as the turbidity of rabbit urine collected overnight prevents accurate assessment of cellular deposits.
The colour and appearance of samples was not recorded in the raw data. This deviation from protocol was not considered to affect the validity of the results as any abnormalities would have been noted.
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- All animals were randomly killed on Day 22 (males) or Day 23 (females) by means of an intravenous overdose of pentobarbitone sodium and a complete gross necropsy undertaken. This necropsy included observations of all external surfaces, orifices and cranial cavity, the external and cut surfaces of brain, all viscera and glands, and the carcass. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.
GROSS PATHOLOGY: Yes.
The following organs from each animal were weighed as soon after dissection as possible to avoid drying: Adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes (with epididymides), thyroid (with parathyroid).
The macroscopic appearance of the tissues of all rabbits was recorded and samples of the following tissues were preserved in 10% buffered formalin: Adrenals, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), gall bladder, heart, ileum, jejunum, kidneys*, larynx, liver*, lungs*, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin (treated and untreated)*, spleen, sternum (for bone and marrow section), stomach, testes (including epididymides), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina, any macroscopically abnormal tissues*.
* Tissues required for histopathological examination for rabbits from Groups 1 and 4.
HISTOPATHOLOGY: Yes
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (m.p. 56 °C); sections were cut at 4 μm and stained with haematoxylin and eosin.
Microscopic examination of prepared slides (from tissues indicated under Macroscopic pathology) was carried out for all rabbits of Group 1 (control group) and Group 4 (high dosage group) killed on Days 22 (males) or 23 (females).
Examinations were extended to include the spleens from all female rabbits following an apparent decrease in the weight of this organ for females from the intermediate and high dosage groups.
Slides of treated skin from all rabbits of Group 2 and 3 (low and intermediate dosage groups respectively) were also prepared as a contingency to cover possible changes at the high dosage. - Statistics:
- All statistical analyses were carried out separately for males and females.
The following sequence of statistical tests was used for bodyweight gains, weekly food consumption, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75 %), the proportion of values different from the mode was analysed by Fisher's exact test followed by Mantel's test for a trend in proportions. Otherwise:
(ii) Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level, a logarithmic transformation was tried to see if a more stable variance structure could have been obtained.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a oneway analysis of variance was carried out followed by Williams' test for a dose-related response.
(iv) If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Organ weight analysis was initially carried out using analysis of variance as outlined above on absolute organ weights. Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10 % level of significance.
Significant differences between control animals and those treated with the test material have been expressed at the 5 % (* P < 0.05) or 1 % (** P < 0.01) level. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment-related clinical signs.
Notable clinical findings were limited to transient cases of changes in faeces production (loose faeces, soft faeces and few faeces) and a thin-looking appearance in rabbits from all four groups. These changes are commonly seen in laboratory rabbits and at the frequency observed were not considered to be important. - Dermal irritation:
- effects observed, treatment-related
- Description (incidence and severity):
- Slight erythema was observed as early as Day 2 among rabbits treated at 1 000 mg/kg/day with reactions progressing to well-defined erythema with slight or well-defined oedema in eight animals by Day 8; slight erythema only was recorded in the remaining two males. Thereafter, these responses were generally maintained to the end of the study although well-defined erythema with moderate oedema was recorded for one male between Days 10 and 14 and for one female between Days 10 and 16. Additional reactions comprised cases of beige staining of the treatment site (not enough to prevent assessment of erythema) from as early as Day 3 and lasting to 19 days plus treatment site dryness and desquamation (sloughing) of the stratum corneum generally recorded during all three weeks and lasting up to 18 days in the majority of rabbits. Less common cases of cracking and/or hardening of the treatment site, lasting up to 8 days during the middle part of the study, were recorded for two males and up to two females; one further male also showed spots on the treatment site on Days 17 and 18.
Although slightly less marked than above, irritation reactions for rabbits treated at 100 mg/kg/day ranged from slight erythema with or without slight oedema from as early as Day 2 or 3 to well-defined erythema with slight or well-defined oedema in seven animals by Day 8; slight erythema only was recorded in the remaining three females. Thereafter, these reactions were maintained, generally to the end of the study, although a slight degree of amelioration was recorded during Week 3 for three males. Additional reactions comprised beige staining of the treatment site during Weeks 2 and 3 and lasting up to 11 days for three males and three females plus sloughing from as early as Day 6 and lasting up to 15 days for all rabbits.
Reactions were limited for rabbits treated at 10 mg/kg/day to cases of slight erythema as early as Day 3 in seven animals and lasting up to 20 days along with slight oedema in one male for 8 days. The remaining three rabbits showed no response throughout the study. Additional reactions comprised sloughing for one male and one female during the middle part of the study.
No dermal reactions were recorded for control rabbits. - Mortality:
- no mortality observed
- Description (incidence):
- There were no mortalities.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Intergroup variation in actual bodyweights and bodyweight gains revealed no disturbances that were considered to be related to treatment with the test material. Analysis of individual values shows that mean differences from controls can be attributed to natural variation in these parameters.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Intergroup variation in food consumption revealed no disturbances that were considered to be related to treatment with the test material. Analysis of individual values shows that mean differences from controls can be attributed to natural variation in these parameters.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant increases in monocyte counts for males treated at 1 000 mg/kg/day and in basophil values for females receiving 100 and 1 000 mg/kg/day in comparison with controls (P < 0.01 and P < 0.05 respectively) were small in nature and can be attributed to chance.
Intergroup variation in the remaining parameters measured revealed no disturbances that were considered to be related to treatment with the test material.
Analysis of blood slides revealed a low occurrence of slight polychromasia and/or slight anisocytosis (9/40 incidences) among study animals. This finding is not uncommon among young laboratory rabbits and was not considered to be important. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant (P < 0.05) decrease in urea nitrogen levels was recorded for all female rabbits treated with the test material in comparison with the controls. Cholesterol values were also statistically (P < 0.05) lower for female rabbits treated at 100 or 1 000 mg/kg/day. These differences in urea nitrogen and cholesterol were not dosage-related and, as wide variation is commonly seen in laboratory rabbits, statistical significance was considered to have arisen by chance.
There were no other statistically significant differences from the controls for remaining biochemical parameters measured. - Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Results revealed a statistically significant increase in protein levels for males treated at 1 000 mg/kg/day in comparison with controls (P < 0.05). Reduced urine volume (P < 0.05 or P < 0.01) was also recorded for all females treated with the test material. Both these changes can be attributed to chance with the latter finding arising as a result of a large urine output from two control females.
Intergroup variation in the remaining parameters measured revealed no disturbances that were considered to be related to treatment with the test material.
No overnight samples were obtained for 4/40 rabbits. Some success in obtaining a specimen (2/4 rabbits) occurred following repeat overnight sampling for these animals on the night prior to sacrifice. Results were essentially similar to those recorded at first analysis. - Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significantly (P < 0.05) lower than control absolute spleen weights were recorded for female rabbits treated at 100 or 1 000 mg/kg/day. This finding was not dosage-related and following histopathological examination was considered to be of no toxicological importance.
There were no other significant differences in organ weights between control and treated groups. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The macroscopic examination performed at termination revealed no changes that were attributable to treatment with the test material.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related changes were found in the treated skin samples. These were as follows:
Minimal diffuse acanthosis was found in the treated skin samples of three males and three females treated at 1 000 mg/kg/day and also in two females treated at 10 mg/kg/day. This change was not present in rabbits treated at 10 mg/kg/day.
Spleens from all rabbits showed minimal or moderate congestion with the majority of control animals showing a moderate degree. These findings were considered to be of no toxicological importance.
All other findings were considered to be incidental in origin and of no toxicological significance. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- RANGE-FINDING STUDY
Clinical signs
Soft faeces were recorded for the male rabbit on Day 1 and for the female on Days 1, 2 and 7 to termination; the latter animal also had diarrhoea on Days 5 and 6. No other clinical findings were seen throughout the study.
Dermal response
Irritation reactions progressed from slight erythema for both rabbits with slight oedema in the male on Day 3 to well-defined erythema with slight or well-defined oedema by the end of the study. These reactions were accompanied to termination by dryness and desquamation (sloughing) of the stratum corneum from Day 5 or 6 and beige staining on the dose site from Day 7; the latter observation did not prevent assessment of dermal scoring.
Bodyweight and food consumption
Satisfactory bodyweight gains and food consumption were recorded for both rabbits throughout the study.
Terminal procedures
Post mortem revealed no left kidney and an apparently enlarged gall bladder for the male as the only macroscopic abnormalities. Liver and spleen weights for the latter animal appeared slightly low while the right kidney weight appeared slightly raised; liver, spleen and kidney weights for the female were all unremarkable.
The results of this investigation with the test material indicated that 1 000 mg/kg/day was a suitable high dosage choice for the twenty-one day study in the rabbit. The proposed dosages for the latter study were 0, 10, 100 and 1 000 mg/kg/day. - Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No systemic effects observed at the highest dose tested.
- Critical effects observed:
- no
- Conclusions:
- Under the conditions of the study it was considered that 1 000 mg/kg/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.
- Executive summary:
The repeated dose toxicity of the test material via the dermal route was assessed according to OECD Test Guideline 410 and in compliance with GLP.
This study was performed to assess the cutaneous and systemic toxicity of the test material to the rabbit. The test material was administered by dermal application once daily, to groups of five male and five female rabbits for twenty-one (males) or twenty-two (females) consecutive days, at fixed dosages of 10, 100 and 1 000 mg/kg/day. The test material was moistened sufficiently with distilled water to ensure good contact with the skin. A further group of rabbits (five males and five females) was held as a concurrent control receiving distilled water alone. Bodyweights, food consumption and clinical observations were recorded during the study. Blood samples for clinical investigations were taken on Day 20 (males) or Day 21 (females) and animals were killed and examined macroscopically on Day 22 (males) or Day 23 (females). Histological examination of specified tissues was then initiated.
The following comments in relation to real or possible treatment-related findings are made in summary:
Among rabbits treated at 1 000 mg/kg/day, slight erythema was observed as early as Day 2 with reactions progressing to well-defined erythema with slight or well-defined oedema in eight animals by Day 8; slight erythema only was recorded in the remaining two males. Thereafter, these responses were generally maintained to the end of the study although well-defined erythema with moderate oedema was recorded for one male and one female between Days 10 and 16. Additional reactions comprised cases of beige staining of the treatment site (not enough to prevent assessment of erythema) from as early as Day 3 and lasting to 19 days plus treatment site dryness and desquamation (sloughing) of the stratum corneum generally recorded during all three weeks and lasting up to 18 days in the majority of rabbits. Less common cases of cracking and/or hardening of the treatment site, lasting up to 8 days during the middle part of the study, were recorded for two males and up to two females; one further male also showed spots on the treatment site on Days 17 and 18.
Among rabbits treated at 100 mg/kg/day, irritation reactions ranged from slight erythema with or without slight oedema from as early as Day 2 or 3 to well-defined erythema with slight or well-defined oedema in seven animals by Day 8; slight erythema only (developing to well-defined erythema) was recorded in the remaining three females. Thereafter, these reactions were maintained, generally to the end of the study, although a slight degree of amelioration was recorded during Week 3 for three males. Additional reactions comprised beige staining of the treatment site during Weeks 2 and 3 and lasting up to 11 days for three males and three females plus sloughing from as early as Day 6 and lasting up to 15 days for all rabbits.
Among rabbits treated at 10 mg/kg/day, reactions comprised slight erythema as early as Day 3 in seven animals and lasting up to 20 days along with slight oedema in one male for 8 days. The remaining three rabbits showed no response throughout the study. Additional reactions comprised sloughing for one male and one female during the middle part of the study.
Microscopic pathology: Minimal diffuse acanthosis was recorded in the treated skin samples of 3/5 males and 3/5 females at 1 000 mg/kg/day and 2/5 females at 100 mg/kg/day.
Remaining parameters, namely clinical signs, bodyweight, food consumption, biochemistry, haematology, urinalysis, organ weights and macroscopic pathology, were not affected by the dermal application of the test material at 10, 100 or 1 000 mg/kg/day.
Under the conditions of the study it was considered that 1 000 mg/kg/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
Repeated dose toxicity: dermal - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 September 1992 to 05 March 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Version / remarks:
- 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA Pesticide Assessment Guidelines, Subdivision F. Hazard Evaluation: Human and Domestic Animals 82-2 Repeated dose dermal toxicity study.
- Version / remarks:
- Revised Edition, November 1984
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on species / strain selection:
- The rabbit was chosen as the test species as it has been shown to be a suitable model for this type of study and is the species recommended in the test guidelines.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10 to 12 weeks old on arrival.
- Weight at study initiation: 2.0 to 2.8 kg on arrival.
- Fasting period before study: Food and water were only withdrawn overnight prior to collection of samples.
- Housing: All rabbits were caged individually in metal cages with perforated floors.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: An eight-day acclimatisation period was allowed between delivery of the animals and start of treatment. The health status of all animals was monitored, by daily observation throughout the acclimatisation period, to ensure that the rabbits selected for final assignment to the study were satisfactory.
DETAILS OF FOOD AND WATER QUALITY: The batches of diet used for the study had been analysed for nutrients, possible contaminants and microorganisms. Results of the routine physical and chemical examination of drinking water at source, as conducted usually weekly by the supplier, are made available to Huntingdon Research Centre Ltd., as quarterly summaries. There was no information available to the Study Director to indicate that any non-nutrient substance likely to influence the effect of the test material could reasonably have been expected to be present in the diet or the drinking water.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature was generally controlled in the range 15 to 21 °C, however on Day 11, minimum to maximum temperature readings of 17 to 30.5 °C were recorded as a result of animal building maintenance work.
- Humidity (%): Relative humidity was generally controlled in the region of 36 to 62 % R.H., however on Day 11, minimum to maximum humidity readings of 32 to 62 % R.H. were recorded as a result of animal building maintenance work.
- Air changes (per hr): Air exchange was maintained at approximately 19 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to provide 12 hours artificial light (0700 - 1900 hours) in each 24-hour period.
- Type of coverage:
- occlusive
- Vehicle:
- unchanged (no vehicle)
- Remarks:
- (the test material was moistened with distilled water to ensure good contact with the skin)
- Details on exposure:
- RANGE-FINDING STUDY
A group comprising one male and one female rabbit of the New Zealand White strain was treated by dermal application of the test material at a dosage of 1 000 mg/kg/day. The treatment area was a shaven region of skin measuring 12 x 8 cm (approximately 10% of the total body surface) and the test material was moistened with distilled water at a constant volume of 3 mL/rabbit/day. After dosing, the treatment site was covered by an impervious bandage for approximately six hours. Following the exposure period, the dressings were removed and the treated skin was washed with warm water (30 - 40 °C) and then gently blotted dry.
MAIN STUDY
- Two days before the start of treatment each animal was weighed and forty rabbits were randomly allocated to four groups, each consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.
- Prior to dosing on Day 1 (24 September 1992), a final health status examination revealed one of the rabbits allocated to Group 2, believed to be a female, to be in fact a very immature male. This animal was replaced before study start with an appropriate spare rabbit.
- Immediately after dosing, a male rabbit in Group 2 started to convulse violently. The animal was sacrificed immediately on humane grounds - its condition was attributed to shock brought on by the bandaging procedure (a very infrequent but recorded response in rabbits). Post mortem findings revealed congested lungs as the only notable finding. This animal was immediately replaced by an appropriate spare rabbit. Neither actions were considered to have affected the integrity of the study and the weights of replacement rabbits were similar to those of the original animals.
Following the commencement of treatment, spare animals were removed from the study. No further investigations were performed on these animals.
TEST SITE
- Area of exposure: The treatment area was a shaven region of skin measuring 12 x 8 cm on the back of each rabbit.
- % coverage: Approximating to 10 % of the total body surface.
- Type of wrap if used: The treatment site was covered with an impervious bandage consisting of gauze covered with 'Elastoplast' elastic adhesive dressing backed with impervious 'Sleek' plaster.
- Time intervals for shavings or clipplings: The hair was clipped from the dorsal region of each rabbit. Hair clipping was carried out approximately twenty-four hours prior to the first application of the test material. Hair clipping was repeated as necessary during the experimental period. The skin sites were not abraded.
REMOVAL OF TEST SUBSTANCE
- Washing: The treated skin was washed with warm water (30 - 40 °C) and gently blotted dry.
- Time after start of exposure: The test material remained on the back of each rabbit for approximately six hours each day after which time the dressings were removed.
TEST MATERIAL
- Amount(s) applied: The appropriate amount of test material (equating to the correct dosage) was weighed out daily for each animal and spread evenly over the prepared skin of the rabbits.
- Constant volume or concentration used: Yes. Each animal received a constant dosage based upon its most recently recorded bodyweight. The test material was moistened with distilled water at a constant volume/dosage (0.1, 0.3 and 3 mL/rabbit/day for Groups 2, 3 and 4 respectively) to ensure good contact with the skin. - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- Animals were treated for either twenty-one (males) or twenty-two (females) consecutive days. Treatment of animals not scheduled for sacrifice on Day 22 was continued up to 24 hours prior to sacrifice.
- Frequency of treatment:
- Animals were treated once daily.
- Dose / conc.:
- 10 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- Five per sex per dose
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale:
A high dosage of 1 000 mg/kg/day was selected on the basis of available toxicity information, in particular, rabbit acute dermal toxicity data [LD50 (rabbit) > 4 000 mg/kg bodyweight] and the results of a preliminary study.
- Fasting period before blood sampling for clinical biochemistry: Yes, food and water were withdrawn overnight prior to collection of samples. - Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each day and again in the late afternoon to look for dead or moribund animals.
DERMAL IRRITATION: Yes
- Time schedule for examinations: Local irritation was recorded immediately prior to the first daily application of the test material and subsequently daily. Local dermal reactions (erythema and oedema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system as follows:
Erythema and eschar formation
0 = No erythema
1 = Slight erythema
2 = Well-defined erythema
3 = Moderate erythema
4 = Severe erythema (beet redness) to slight eschar formation (injuries in depth)
Oedema
0 = No oedema
1 = Slight oedema
2 = Well-defined oedema (area well-defined by definite raising)
3 = Moderate oedema (raised approximately 1 mm)
4 = Severe oedema (raised more than 1 mm and extending beyond the area of exposure)
Dermal changes other than erythema and oedema were recorded separately.
BODY WEIGHT: Yes
- Time schedule for examinations: All rabbits were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.
FOOD CONSUMPTION:
- Food consumption: The quantity of food consumed by each rabbit was measured at weekly intervals throughout the study.
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was withdrawn from the median artery of the ear of all rabbits prior to termination. Male and female samples were collected on consecutive days (Days 20 and 21 respectively).
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- How many animals: All rabbits
- Parameters checked: The following parameters were analysed with an Ortho ELT-1500 Analyser, using standard Ortho methodology: Packed cell volume (PCV), Haemoglobin (Hb), Red blood cell count (RBC).
Absolute indices:
Mean corpuscular haemoglobin concentration (MCHC), Calculated: Hb (g/dL) x 100 / PCV (%)
Mean corpuscular volume (MCV), Calculated: PCV (%) x 10 / RBC (x10^6/mm³)
Mean corpuscular haemoglobin (MCH), Calculated: Hb (g/dL x 10 / RBC (x10^6/mm³)
Platelet count (Plts) and Total white blood cell count (WBC)
The following estimations were measured using the appropriate methodology:
Thrombotest (1T) - Method of Owren, P.A. (Lancet, 1959, ii, 754), Differential white blood cell count (Diff) - namely: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M).
The percentage distribution of each cell type was determined by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells. Percentage values were then converted to absolute values by computer inevitably involving a "rounding off'' in a proportion of the results. Hence, the measured total WBC may differ slightly from the calculated total for the differential count.
Additional blood film slides were prepared and examined for morphological abnormalities: Polychromasia, Hypochromasia, Anisocytosis, Rouleaux formation, Separate film report (generated for additional abnormalities).
The collected blood samples were divided into tubes containing EDTA anticoagulant for haematological investigations and citrate anticoagulant for coagulation tests.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was withdrawn from the median artery of the ear of all rabbits prior to termination. Male and female samples were collected on consecutive days (Days 20 and 21 respectively).
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- How many animals: All rabbits
- Parameters checked: The following parameters were analysed with an Hitachi 737 Clinical Chemistry Analyser:
Glucose - using BCL Test Kit (hexokinase mediated), Total protein, Albumin (Alb), Globulin (Glob), Calculated: Total protein (g/dl) minus Alb (g/dl), Albumin/Globulin ratio (A/G), Calculated from Total protein and Albumin concentrations, Urea nitrogen (Urea Nitr), Creatinine, Alkaline phosphatase (AP) - reaction temperature 30 °C, Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase (ALT)' - using BCL Test Kit, reaction temperature 30 °C, Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase (AST)' - using BCL Test Kit, reaction temperature 30 °C, Total bilirubin (Bilirubin), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl), Inorganic phosphorus (P), Cholesterol (Chol).
The collected blood samples were divided into tubes containing heparin anticoagulant for biochemical tests.
URINALYSIS: Yes.
- Time schedule for collection of urine: Overnight urine samples were collected for all rabbits prior to termination. As no overnight urine samples were obtained for various rabbits from all four groups, resampling occurred on the night prior to sacrifice.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, food and water were withdrawn overnight prior to collection of samples.
- Parameters checked: The following estimations were measured using the appropriate methodology:
Volume (Vol), pH - using pH meter, Specific Gravity (SG) - using Atago UR-1 Refractometer, sample compared with water (nominal value of 1 000)
Protein - using Roche Cobas Centrifugal Analyser, utilising modified method of Macart, M. and Gerbaut, L. (Clin. Chim. Acta, 1984, 141, 77)
The following tests were also performed using qualitative indicators of analyte concentration:
Total reducing substances (TRS), Glucose (Gluc), Ketones (Ket), Bile pigments (Bile Pigs), Urobilinogen (Uro-bi), Haem pigments (Haem Pigs - positive or negative finding only).
Microscopic examination of urine samples was not performed as the turbidity of rabbit urine collected overnight prevents accurate assessment of cellular deposits.
The colour and appearance of samples was not recorded in the raw data. This deviation from protocol was not considered to affect the validity of the results as any abnormalities would have been noted.
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- All animals were randomly killed on Day 22 (males) or Day 23 (females) by means of an intravenous overdose of pentobarbitone sodium and a complete gross necropsy undertaken. This necropsy included observations of all external surfaces, orifices and cranial cavity, the external and cut surfaces of brain, all viscera and glands, and the carcass. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.
GROSS PATHOLOGY: Yes.
The following organs from each animal were weighed as soon after dissection as possible to avoid drying: Adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes (with epididymides), thyroid (with parathyroid).
The macroscopic appearance of the tissues of all rabbits was recorded and samples of the following tissues were preserved in 10% buffered formalin: Adrenals, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), gall bladder, heart, ileum, jejunum, kidneys*, larynx, liver*, lungs*, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin (treated and untreated)*, spleen, sternum (for bone and marrow section), stomach, testes (including epididymides), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina, any macroscopically abnormal tissues*.
* Tissues required for histopathological examination for rabbits from Groups 1 and 4.
HISTOPATHOLOGY: Yes
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (m.p. 56 °C); sections were cut at 4 μm and stained with haematoxylin and eosin.
Microscopic examination of prepared slides (from tissues indicated under Macroscopic pathology) was carried out for all rabbits of Group 1 (control group) and Group 4 (high dosage group) killed on Days 22 (males) or 23 (females).
Examinations were extended to include the spleens from all female rabbits following an apparent decrease in the weight of this organ for females from the intermediate and high dosage groups.
Slides of treated skin from all rabbits of Group 2 and 3 (low and intermediate dosage groups respectively) were also prepared as a contingency to cover possible changes at the high dosage. - Statistics:
- All statistical analyses were carried out separately for males and females.
The following sequence of statistical tests was used for bodyweight gains, weekly food consumption, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75 %), the proportion of values different from the mode was analysed by Fisher's exact test followed by Mantel's test for a trend in proportions. Otherwise:
(ii) Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level, a logarithmic transformation was tried to see if a more stable variance structure could have been obtained.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a oneway analysis of variance was carried out followed by Williams' test for a dose-related response.
(iv) If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Organ weight analysis was initially carried out using analysis of variance as outlined above on absolute organ weights. Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10 % level of significance.
Significant differences between control animals and those treated with the test material have been expressed at the 5 % (* P < 0.05) or 1 % (** P < 0.01) level. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment-related clinical signs.
Notable clinical findings were limited to transient cases of changes in faeces production (loose faeces, soft faeces and few faeces) and a thin-looking appearance in rabbits from all four groups. These changes are commonly seen in laboratory rabbits and at the frequency observed were not considered to be important. - Dermal irritation:
- effects observed, treatment-related
- Description (incidence and severity):
- Slight erythema was observed as early as Day 2 among rabbits treated at 1 000 mg/kg/day with reactions progressing to well-defined erythema with slight or well-defined oedema in eight animals by Day 8; slight erythema only was recorded in the remaining two males. Thereafter, these responses were generally maintained to the end of the study although well-defined erythema with moderate oedema was recorded for one male between Days 10 and 14 and for one female between Days 10 and 16. Additional reactions comprised cases of beige staining of the treatment site (not enough to prevent assessment of erythema) from as early as Day 3 and lasting to 19 days plus treatment site dryness and desquamation (sloughing) of the stratum corneum generally recorded during all three weeks and lasting up to 18 days in the majority of rabbits. Less common cases of cracking and/or hardening of the treatment site, lasting up to 8 days during the middle part of the study, were recorded for two males and up to two females; one further male also showed spots on the treatment site on Days 17 and 18.
Although slightly less marked than above, irritation reactions for rabbits treated at 100 mg/kg/day ranged from slight erythema with or without slight oedema from as early as Day 2 or 3 to well-defined erythema with slight or well-defined oedema in seven animals by Day 8; slight erythema only was recorded in the remaining three females. Thereafter, these reactions were maintained, generally to the end of the study, although a slight degree of amelioration was recorded during Week 3 for three males. Additional reactions comprised beige staining of the treatment site during Weeks 2 and 3 and lasting up to 11 days for three males and three females plus sloughing from as early as Day 6 and lasting up to 15 days for all rabbits.
Reactions were limited for rabbits treated at 10 mg/kg/day to cases of slight erythema as early as Day 3 in seven animals and lasting up to 20 days along with slight oedema in one male for 8 days. The remaining three rabbits showed no response throughout the study. Additional reactions comprised sloughing for one male and one female during the middle part of the study.
No dermal reactions were recorded for control rabbits. - Mortality:
- no mortality observed
- Description (incidence):
- There were no mortalities.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Intergroup variation in actual bodyweights and bodyweight gains revealed no disturbances that were considered to be related to treatment with the test material. Analysis of individual values shows that mean differences from controls can be attributed to natural variation in these parameters.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Intergroup variation in food consumption revealed no disturbances that were considered to be related to treatment with the test material. Analysis of individual values shows that mean differences from controls can be attributed to natural variation in these parameters.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant increases in monocyte counts for males treated at 1 000 mg/kg/day and in basophil values for females receiving 100 and 1 000 mg/kg/day in comparison with controls (P < 0.01 and P < 0.05 respectively) were small in nature and can be attributed to chance.
Intergroup variation in the remaining parameters measured revealed no disturbances that were considered to be related to treatment with the test material.
Analysis of blood slides revealed a low occurrence of slight polychromasia and/or slight anisocytosis (9/40 incidences) among study animals. This finding is not uncommon among young laboratory rabbits and was not considered to be important. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant (P < 0.05) decrease in urea nitrogen levels was recorded for all female rabbits treated with the test material in comparison with the controls. Cholesterol values were also statistically (P < 0.05) lower for female rabbits treated at 100 or 1 000 mg/kg/day. These differences in urea nitrogen and cholesterol were not dosage-related and, as wide variation is commonly seen in laboratory rabbits, statistical significance was considered to have arisen by chance.
There were no other statistically significant differences from the controls for remaining biochemical parameters measured. - Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Results revealed a statistically significant increase in protein levels for males treated at 1 000 mg/kg/day in comparison with controls (P < 0.05). Reduced urine volume (P < 0.05 or P < 0.01) was also recorded for all females treated with the test material. Both these changes can be attributed to chance with the latter finding arising as a result of a large urine output from two control females.
Intergroup variation in the remaining parameters measured revealed no disturbances that were considered to be related to treatment with the test material.
No overnight samples were obtained for 4/40 rabbits. Some success in obtaining a specimen (2/4 rabbits) occurred following repeat overnight sampling for these animals on the night prior to sacrifice. Results were essentially similar to those recorded at first analysis. - Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significantly (P < 0.05) lower than control absolute spleen weights were recorded for female rabbits treated at 100 or 1 000 mg/kg/day. This finding was not dosage-related and following histopathological examination was considered to be of no toxicological importance.
There were no other significant differences in organ weights between control and treated groups. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The macroscopic examination performed at termination revealed no changes that were attributable to treatment with the test material.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related changes were found in the treated skin samples. These were as follows:
Minimal diffuse acanthosis was found in the treated skin samples of three males and three females treated at 1 000 mg/kg/day and also in two females treated at 10 mg/kg/day. This change was not present in rabbits treated at 10 mg/kg/day.
Spleens from all rabbits showed minimal or moderate congestion with the majority of control animals showing a moderate degree. These findings were considered to be of no toxicological importance.
All other findings were considered to be incidental in origin and of no toxicological significance. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- RANGE-FINDING STUDY
Clinical signs
Soft faeces were recorded for the male rabbit on Day 1 and for the female on Days 1, 2 and 7 to termination; the latter animal also had diarrhoea on Days 5 and 6. No other clinical findings were seen throughout the study.
Dermal response
Irritation reactions progressed from slight erythema for both rabbits with slight oedema in the male on Day 3 to well-defined erythema with slight or well-defined oedema by the end of the study. These reactions were accompanied to termination by dryness and desquamation (sloughing) of the stratum corneum from Day 5 or 6 and beige staining on the dose site from Day 7; the latter observation did not prevent assessment of dermal scoring.
Bodyweight and food consumption
Satisfactory bodyweight gains and food consumption were recorded for both rabbits throughout the study.
Terminal procedures
Post mortem revealed no left kidney and an apparently enlarged gall bladder for the male as the only macroscopic abnormalities. Liver and spleen weights for the latter animal appeared slightly low while the right kidney weight appeared slightly raised; liver, spleen and kidney weights for the female were all unremarkable.
The results of this investigation with the test material indicated that 1 000 mg/kg/day was a suitable high dosage choice for the twenty-one day study in the rabbit. The proposed dosages for the latter study were 0, 10, 100 and 1 000 mg/kg/day. - Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No systemic effects observed at the highest dose tested.
- Critical effects observed:
- no
- Conclusions:
- Under the conditions of the study it was considered that 1 000 mg/kg/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.
- Executive summary:
The repeated dose toxicity of the test material via the dermal route was assessed according to OECD Test Guideline 410 and in compliance with GLP.
This study was performed to assess the cutaneous and systemic toxicity of the test material to the rabbit. The test material was administered by dermal application once daily, to groups of five male and five female rabbits for twenty-one (males) or twenty-two (females) consecutive days, at fixed dosages of 10, 100 and 1 000 mg/kg/day. The test material was moistened sufficiently with distilled water to ensure good contact with the skin. A further group of rabbits (five males and five females) was held as a concurrent control receiving distilled water alone. Bodyweights, food consumption and clinical observations were recorded during the study. Blood samples for clinical investigations were taken on Day 20 (males) or Day 21 (females) and animals were killed and examined macroscopically on Day 22 (males) or Day 23 (females). Histological examination of specified tissues was then initiated.
The following comments in relation to real or possible treatment-related findings are made in summary:
Among rabbits treated at 1 000 mg/kg/day, slight erythema was observed as early as Day 2 with reactions progressing to well-defined erythema with slight or well-defined oedema in eight animals by Day 8; slight erythema only was recorded in the remaining two males. Thereafter, these responses were generally maintained to the end of the study although well-defined erythema with moderate oedema was recorded for one male and one female between Days 10 and 16. Additional reactions comprised cases of beige staining of the treatment site (not enough to prevent assessment of erythema) from as early as Day 3 and lasting to 19 days plus treatment site dryness and desquamation (sloughing) of the stratum corneum generally recorded during all three weeks and lasting up to 18 days in the majority of rabbits. Less common cases of cracking and/or hardening of the treatment site, lasting up to 8 days during the middle part of the study, were recorded for two males and up to two females; one further male also showed spots on the treatment site on Days 17 and 18.
Among rabbits treated at 100 mg/kg/day, irritation reactions ranged from slight erythema with or without slight oedema from as early as Day 2 or 3 to well-defined erythema with slight or well-defined oedema in seven animals by Day 8; slight erythema only (developing to well-defined erythema) was recorded in the remaining three females. Thereafter, these reactions were maintained, generally to the end of the study, although a slight degree of amelioration was recorded during Week 3 for three males. Additional reactions comprised beige staining of the treatment site during Weeks 2 and 3 and lasting up to 11 days for three males and three females plus sloughing from as early as Day 6 and lasting up to 15 days for all rabbits.
Among rabbits treated at 10 mg/kg/day, reactions comprised slight erythema as early as Day 3 in seven animals and lasting up to 20 days along with slight oedema in one male for 8 days. The remaining three rabbits showed no response throughout the study. Additional reactions comprised sloughing for one male and one female during the middle part of the study.
Microscopic pathology: Minimal diffuse acanthosis was recorded in the treated skin samples of 3/5 males and 3/5 females at 1 000 mg/kg/day and 2/5 females at 100 mg/kg/day.
Remaining parameters, namely clinical signs, bodyweight, food consumption, biochemistry, haematology, urinalysis, organ weights and macroscopic pathology, were not affected by the dermal application of the test material at 10, 100 or 1 000 mg/kg/day.
Under the conditions of the study it was considered that 1 000 mg/kg/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.
Reference
Additional information
Short-Term Repeated Dose Toxicity, Oral Route
Mellert (2003a)
The repeated dose toxicity of the test material was assessed via the oral route and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was administered to groups of 4 male and 4 female Wistar rats at dietary concentrations of 0, 100, 400 and 800 ppm for 4 weeks. Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. At the end of the administration period, cyanide-insensitive palmitoyl-CoA-oxidation activity (PALCOA) was examined in liver and kidney as an indicator for peroxisome proliferation.
Increased PALCOA values were seen in the liver of the high dose animals of both sexes as well as in the kidney of the high and mid dose males and of all treated females.
The PALCOA-value of one control animal was measured as 0.00 mU/mg protein. The integrity of this result cannot be assured. Therefore, this value was eliminated from the statistical evaluation. Due to the low number of control animals, the increases in kidney PALCOA activity in the treated females, although biologically meaningful, were not statistically significant.
The absolute and relative weights of the kidneys were increased in all treated males; this, however, is not considered to be an adverse effect but rather an adaptation to increased functional demand.
Under the conditions of the study there are clear indications of peroxisome proliferation in both liver and kidney at the 400 and 800 ppm inclusion rates. At 100 ppm there was a clear effect in the kidney, but not the liver, for both sexes, this is important as it reinforces the kidney as the primary organ affected, and shows that there was already significant proliferation at the high dose in the 2 year study.
Beimborn & Leibold (2003)
The repeated dose toxicity of the test material was assessed as part of an in vivo toxicokinextics study according to OECD Test Guideline 417 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
4 male rats were fed non-radiolabelled test material for 14 days at dietary concentrations of 125, 250, 500, 1 000, 1 500, and 2 200 ppm and the daily equivalent by a single gavage dose of radiolabelled test material at day 15.
As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1500 and 2 200 ppm.
In the first week of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding week, i.e. the week before radioactive dosing, feed doses were closer to target at all dose levels.
No test-material related clinical findings were observed.
Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1 500 and 2 200 ppm. No test-material related clinical findings were observed.
Beimborn & Leibold (2004)
The repeated dose toxicity of the test material was assessed as part of an in vivo toxicokinextics study according to OECD Test Guideline 417 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Four male rats were fed non-radiolabelled test material for 60 days at dietary concentrations of 190, 380, 570, 760, and 950 ppm and the daily equivalent by a single gavage dose of radiolabelled test material at day 61.
As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 570, 760, and 950 ppm.
During the first month of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding month, i.e. the month before radioactive dosing, feed doses were closer to target at all dose levels.
No test material related clinical findings were observed.
Under the conditions of the study the body weight changes were slightly decreased in groups dosed with 570, 760 and 950 ppm. No test-material related clinical findings were observed.
Mellert (2003b)
The aim of the study was to obtain additional information on the toxicological profile of the test material, especially with regard to peroxisome proliferation. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was administered to groups of 4 male and 4 female B6C3F1 mice at dietary concentrations of 0, 250, 750 and 2 500 ppm for 4 weeks. Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. At the end of the administration period, cyanide-insensitive palmitoyl-CoA-oxidation (PALCOA) was examined in liver and kidney as an indicator for peroxisome proliferation.
Increased PALCOA values were seen in the liver of the high dose males and high and mid dose females as well as in the kidney of all treated male and female animals.
The absolute and relative liver weights were increased in high dose males and females; this, however, is not considered to be an adverse effect but rather an adaptation to increased functional demand.
Under the conditions of the study the results are clearly indicative of peroxisome proliferation in both liver and kidney.
Kirsch (1986)
The repeated dose toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 407. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
During the study, the test material was administered as the racemate and as an isomer to Wistar rats over a period of 49 days via their diet. 100 rats (50 males and 50 females) in 5 groups each containing 10 animals per sex were used in the study. The racemate was administered to the animals of test groups 1 and 2 and the isomer to the animals of test groups 3 and 4. For comparison a joint control group (10 male and 10 female rats) was used. The dose levels in the feedwere50 ppm (test groups 1 and 3) and 400 ppm (test groups 2 and 4) in the isomer and as racemate. The rats' food consumption and body weight were determined weekly; the state of health of the animals was checked each day. The duration of the study of 28 days was extended by 21 days since results that were difficult to interpret were obtained in determining the creatinine level at the 1st blood sampling on the 23rd day of administration, and therefore a 2nd blood sampling was carried out on the 43rd day of administration. All the animals were subjected to a gross-pathological examination. This was followed by a histopathological examination.
The following findings were obtained and assessed or discussed in relation to the test materials.
400 ppm group, racemate: Clinico-chemical and haematological findings included a drop in the cholesterol level in the female animals; increased urea concentration in the male rats; reduction of the calcium concentration in the female animals.
400 ppm group, isomer: Clinico-chemical and haematological findings included a drop in the cholesterol level in the male and female animals; increase in the values for urea and creatinine in the female rats.
400 ppm group, isomer: Increase in the absolute kidneyweightsin the male and female rats.
The administration of 50 ppm of the substance as racemate and isomer did not cause any test material-induced changes.
On the basis of the results of the clinico-chemical examinations – reduction in the calcium values in the female rats and increase in the urea value in the male rats of the 400 ppm racemate group as well as increase in the creatinine and urea values in the female rats of the 400 ppm isomer group - there is an indication of a test material-induced marginal renal insufficiency in the case of both test materials. The increase in kidney weight (400 ppm, isomer) can also be ascribed to this, although no histopathologial changes were found that could explain the weight increases.
The similar reduction in the cholesterol level - particularly pronounced in the female animals - after the administration of 400 ppm is a further indication that there is no difference between the toxic effect of the racemate and the isomer.
Under the conditions of the study, the no effect level is between 50 and 400 ppm for both the racemate and the isomer.
Sub-Chronic Repeated Dose Toxicity, Oral Route
Mellert et al. (1995)
The sub-chronic toxicity of the test material following repeated dose administration of the test material via the oral route was assessed according to OECD Test Guideline 408 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was administered to groups of 15 male and 15 female Wistar rats for 3 months at dietary concentrations of 0 ppm, 75 ppm, 500 ppm, 2 500 ppm (males only) and 3 000 ppm (females only). Based upon the results of a range finding study, different high concentrations were selected for males and females.
Food consumption and body weight were determined at least once a week. Water consumption was determined once a week from day 21 onwards. A check of the general state of health of the rats was made at least daily. Furthermore, the animals were thoroughly examined and palpated once a week. In addition to these general clinical examinations, functional observational batteries and measurements of motor activity were carried out in 10 animals per group and sex before the start of the administration period, and in the 4th, 8th and 13th week of the administration period. Before the commencement and at the end of the administration ophthalmological examinations were carried out in 10 males and 10 females of control and high dose groups. Haematological and clinic-chemical examinations as well as urinalyses were carried out at the end of the administration period in 10 animals per sex and dose. Five animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations. All other animals were subjected to gross-pathological assessment, followed by histopathological examinations.
The following findings were obtained and assessed as being related to the test substance administration:
3 000 ppm (females; about 240 mg/kg bw/day) and 2 500 ppm (males; about 189 mg/kg bw/day):
- Reduced food consumption in both sexes.
- Increased water consumption in both sexes.
- Decreased body weights, resulting in reduced values of about 18 % (males) and 14 % (females) on day 91.
- Decreased body weight change, resulting in reduced values of about 28 % (males) and 29 % (females) on day 91.
- Reduced food efficiency in males and females.
- Decrease in red blood cells, haemoglobin, haematocrit, calcium, total protein, globulins, triglycerides and cholesterol in both sexes.
- Decrease in chloride in females increase in alkaline phosphatase and urea in both sexes.
- Increase in serum creatinine and transitional epithelial cells in the urine of males.
- Increase in alanine aminotransferase in females.
- Increase of absolute and relative liver weights in both sexes alterations of hepatocytes with cytoplasmic eosinophilia and granular cytoplasm in both sexes.
- Centrolobular hypertrophy of hepatocytes in males (2/10).
- Presence of fine lipid vacuoles (lipid storage) in the adrenal cortex of 8/10 males and 10/10 females.
500 ppm (about 38 mg/kg bw/day):
- Increased water consumption in females.
- Decrease in red blood cells, haemoglobin, haematocrit, triglycerides and cholesterol in males.
75 ppm (about 6 mg/kg bw/day):
- No substance-related findings.
In conclusion, the oral administration of the test material led to signs of toxicity at dietary concentrations of 3 000 ppm (females), 2 500 ppm (males) and 500 ppm (both sexes). Target organs were liver and adrenals. Toxicity was further characterised by an anaemic process and slight functional impairment of kidneys. Lipids were also lowered in either sex. No signs of neurotoxicity were detected.
Under the conditions of this study the no effect level for neurotoxicity was therefore above 3 000 ppm in females and above 2500 ppm in males. The NOAEL for systemic toxicity was 75 ppm (about 6 mg/kg bw/day).
Rondot (1998)
A technical problem with the preparation of the slides did not permit the histological examination of the eyes in a previous study. Consequently, an additional 13-week study, subject of this report, was undertaken for the evaluation of eye lesions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The racemate was administered at concentrations of 800 and 3 200 ppm and the isomer at concentrations of 800, 1 600 and 3 200 ppm. Both compounds were administered orally and continuously, mixed with the diet, for 3 months to groups of 10 males and 10 females. The following examinations were performed during the course of the study: Ophthalmology at months 0 and 3; weekly recording of body weights and food consumption and histology of the eyes at the end of treatment.
As in the preceding study, no behavioural change which could be attributed to the ingestion of the test materials was observed. A dose-related reduction of the growth rate was however noted. No ocular lesion was observed. The histological examination of the eyes of all animals at the end of the study revealed no treatment-related lesion.
Reinert (1998)
The repeated dose toxicity of the test material in the rat were assessed in a study which was conducted according to a method siilar to that which is outlined in the standardised guideline, OECD 408. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
During the study, weanling rats of Sprague-Dawley origin (strain 0FA, SPF, IFFA-CREDO) were given continuously for 3 months a diet containing either the racemate at levels of 200, 800 and 3 200 ppm, or the isomer at levels of 200, 400, 800, 1 600 and 3 200 ppm, to determine the potential toxicity of these two compounds. Two untreated control groups received the powdered diet only.
Clinical symptoms: No adverse symptoms or behavioural changes, which could be attributed to the ingestion of the compounds, were observed during the study.
Mortality: There was no compound-related mortality. Some animals died accidentally during the bleeding procedure from the effects of ether anaesthesia.
Growth rate: During the course of the study no significant differences between the two untreated control groups were noted. With compound the racemate at 800 ppm, a slight reduction in weight gain became apparent in females. With the isomer a reduction in body weight gain was evident at 1 600 ppm in both sexes. At the highest concentration tested (3 200 ppm), the depression of the growth curve became pronounced for both compounds and in both sexes.
Food consumption: This remained in general the same for the treated and untreated groups. Waste of food was noted in groups 3 (the racemate at 3 200 ppm) and 8 (the isomer at 3 200 ppm) and this made it impossible to record accurately the food consumption.
In both sexes of all groups at weeks 5, 9 and 13, three "peaks" in food consumption were observed. This transient increase in food consumption may have been caused by starvation at these periods for 16 to 18 hours, prior to blood and urine collections, with subsequently increased appetite.
Haematology: Both compounds produced slight and sporadic decreases in white blood cells at the highest concentrations and mainly in the male groups. Decreases in haemoglobin concentrations and red blood cell counts, more often the former, were noted at the two highest concentrations tested and with both compounds. This was especially noticeable at the 3 200 ppm level in both sexes at 8 and 12 weeks.
Clinical chemistry: Increases, both in frequency and degree, of urea, SAP and SGPT values, were noted. Considering values above 0.50 g/L urea as the upper limit of normality, an increased frequency of values above this limit became evident at concentrations of 800 ppm of the racemate and 400 ppm of the isomer. These changes are considered to be due to a toxic action of the compounds as the frequency of abnormal SAP and SGPT activities increased with the higher concentrations, with the duration of treatment and they were observed in both sexes. Increased albumin levels together with increases in SAP and SGPT activities (and liver weight increase in females) indicate that the liver is the target organ. A fall in cholesterol levels below 0.80 g/L was apparent and this was more marked in frequency and degree, particularly in males, with time of administration and concentrations.
Organ weights: Increased kidney weights were noted in group I (males and females) and in groups 2, 4, 5 and 6 (males) but the values remained within the normal range for this laboratory.
There was a dose-related increase in liver weights in females which is probably related to the elevation in SAP, and SGPT values. The apparent decrease in all organ weights, with the exception of the liver, at the highest concentration of the racemate and the isomer was proportional to the reduction in growth rate and body weight and is considered to be a result of the toxic effect of the compounds at this concentration. No lesions were observed in livers or kidneys, nor in any of the other organs examined.
Since concentrations between 800 and 200 ppm for the racemate and between 400 and 200 ppm for the isomer have not been studied, the no-effect dose level for both compounds in this study was 200 ppm.
The changes in body weights and/or in the clinical laboratory findings at 800 and 3 200 ppm for the racemate and at 400 to 3 200 ppm for the isomer together with the increase in liver weight at 3 200 ppm for both compounds can be considered to be treatment related. The biochemical changes indicate a disturbance of liver function not accompanied with histopathological change. The haematological findings, whilst related to treatment are considered to be a non-specific toxic response.
Under the conditions of the study the no-effect level was 200 ppm for both the racemate and the isomer.
Mellert (1993)
The repeated dose toxicity of the test material was assessed via the oral route in a sub-chronic study according to OECD Test Guideline 408 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was administered to 10 male and 10 female B6C3Fl mice per group in the diet for 3 months at doses of 0 ppm (test group 0), 100 ppm (test group 1), 1 000 ppm (test group 2), and 2 500 ppm (test group 3). Food consumption and body weight were determined once a week. A check of the general state of health of the mice as well as a check for dead or moribund animals was done twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and public holidays). Furthermore the animals were additionally examined in detail and palpated once a week. At the end of the study, blood samplings were performed. All animals were subjected to gross-pathological assessment, followed by histopathological examination.
The findings below were obtained and assessed to be related to the test material administration.
Test group 3 (2 500 ppm; about 740 and 930 mg/kg body weight for males and females, respectively):
Clinical examinations:Statistically significantly reduced body weight (about 10 % in males and 9 % in females on day 91); statistically significantly reduced body weight change in both sexes; clinical chemistry and haematology; increase in alkaline phosphatase, cyanide-insensitive palmitoyl-CoA-oxidation in liver homogenate, urea and cholesterol in both sexes; decrease in triglycerides in both sexes; increase in creatinine and glucose in the males; increase in alanine aminotransferase in the females; decrease in haemoglobin, mean corpuscular haemoglobin and globulins in the males.
Pathology:Statistically significant reduction of the terminal body weight in both sexes;statistically significant increase of the absolute and relative liver weight in both sexes;macroscopically diagnosed dark-brown discolouration of the liver in both sexes;in the liver: Eosinophilic cytoplasm of hepatocytes in both sexes;in the kidneys: Eosinophilic cytoplasm of tubular epithelial cells in both sexes; decrease of lipid storage in the liver of both sexes.
Test group 2 (1 000 ppm; about 220 and 330 mg/kg body weight for males and females, respectively):
Clinical examinations: Statistically significantly reduced body weight (about 8 % in males and 9 % in females on day 91); statistically significantly reduced body weight change in both sexes.
Clinical chemistry and haematology: Increase in urea in both sexes decrease in triglycerides in both sexes; increase in creatinine in the males.
Pathology:In the liver: Eosinophilic cytoplasm of hepatocytes in one female; decrease of lipid storage in the liver of both sexes.
Test group 1 (100 ppm; about 20 and 30 mg/kg body weight, respectively):
Clinical examinations: No test material-related findings.
Clinical chemistry and haematology:Increase in urea in the females; decrease in triglycerides in the females.
Pathology: No test material-related findings.
Both clinical chemistry and pathology indicate that the liver and the kidneys are the target organs. Effects on these organs were clearly seen in test group 3 (2 500 ppm) and test group 2 (1 000 ppm). In test group 1 (100 ppm) slight changes of clinical-chemical parameters were seen in females, but no morphological correlate was obtained in the pathological examinations.
Cyanide-insensitive palmitoyl-CoA-oxidation in liver homogenate was determined only at the highest dose level and in the control group as an indicator for peroxisomal proliferation. The determination resulted in a 10- to 15-fold increase in the treatment group. The values were not determined in the other dose groups as it was not intended to obtain a "no observed effect level" for this parameter. Both the increased values of cyanide-insensitive palmitoyl-CoA-oxidation and the eosinophilic cytoplasm of the hepatocytes indicate a peroxisomal proliferation in the liver. The eosinophilic cytoplasm of tubular epithelial cells suggest that peroxisomal proliferation also occurs in the kidneys.
Under the conditions of thestudy, the no observed adverse effect level in this study is for male animals 100 ppm and for female animals slightly below 100 ppm
Chronic Repeated Dose Toxicity, Oral Route
Bachmann et al. (1997)
The chronic toxicity from oral administration of the test material was assessed according to OECD Test Guideline 542 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
The test materialwas administered to groups of 5 male and 5 female purebred Beagle dogs in the diet at concentrations of 0, 60, 180 and 600 ppm over a period of about 12 months. Food consumption of the animals was determined daily and their body weight once a week; the dogs' state of health was checked each day. Clinical chemistry and haematological examinations as well as urinalyses were carried out once before and three times during the administration period. Ophthalmological examinations were carried out7days before the beginning of the administration period and on study day 363. All animals were subjected to complete gross and histopathological examination.
The following findings were obtained and assessed as being test material-related:
600 ppm (approx. 19 mg/kg bw/day):
- Slight but not statistically significant influence on body weight gain in the males (especially from study days 14 - 49 mean body weight change was stagnant and/or retarded; thereafter the animals gained weight but still in a reduced manner);
- Decrease in haemoglobin and haematocrit in the males;
- Decrease in calcium and inorganic phosphate in the females.
180 ppm (approx. 5 mg/kg bw/day):
- No substance-induced changes.
60 ppm (approx. 2 mg/kg bw/day):
- No substance-induced changes.
Under the conditions of the study it can be stated thatthe test material administered with the diet to Beagle dogs in a dose of 600 ppm led to retarded body weight change and to slight decreases in haemoglobin concentrations and haematocrit values in the peripheral blood in the males and to slight decreases in inorganic phosphate and calcium in the females.
No toxicologically relevant findings occurred in the male and female dogs receiving 180 ppm and 60 ppm of the test material.
The no observed adverse affect level (NOAEL) for male and female Beagle dogs under the chosen test conditions is 180 ppm (approx. 5 mg/kg body weight/day).
Kuehborth et al. (1988)
The carcinogenicity and chronic repeated dose toxicity of the test material was assessed according to OECD Test Guideline 453 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Three different doses (20, 100 and 400 ppm) of the test material were administered with the diet to 450 Wistar rats (225 males and 225 females) for 24 months.
Each dose group was split up into a main group (50 animals per sex) and into the satellite groups I and II comprising 10 and 15 animals per sex respectively.
A group of untreated animals (75 males and 75 females) was maintained in parallel for comparison. It was likewise split into a main group and satellite groups I and II. The feed consumption and body weight of the animals in the main group and of satellite group I was determined once a week up to 14 weeks and thereafter once a month until the end of the study. The state of health of all animals was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week.
At the beginning of the study and then about every 6 months, animals of the main groups (control and highest dose) were examined ophthalmologically.
Blood samples for haematological and clinico-chemical examinations were taken 5 times altogether from the animals of the satellite group II (at the last blood sampling, dead animals in this satellite group were supplemented by animals of the main groups).
Urinalyses were carried out on the animals of satellite group I two times in the first year, and on 10 animals of each main group about 104 weeks after the beginning of the administration period.
The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks.
All animals used ware assessed by gross pathology. This was followed by a comprehensive histopathological examination.
The following findings were obtained and assessed or discussed to be test material-related:
400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.
100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.
20 ppm: No changes whatsoever that could be associated with the test material administered.
Under the conditions of the study a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups was observed, while the kidney weights of the female animals remained uninfluenced. Thereore the NOAEL for male rats was 20 ppm (equivalent to 1.1 mg/kg bodyweight/ day) and for female rats was 400 ppm (equivalent to 27.9 mg/kg bodyweight/ day).
Short-Term Repeated Dose Toxicity, Dermal Route
Allan et al. (1993)
The repeated dose toxicity of the test material via the dermal route was assessed according to OECD Test Guideline 410 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
This study was performed to assess the cutaneous and systemic toxicity of the test material to the rabbit. The test material was administered by dermal application once daily, to groups of five male and five female rabbits for twenty-one (males) or twenty-two (females) consecutive days, at fixed dosages of 10, 100 and 1 000 mg/kg/day. The test material was moistened sufficiently with distilled water to ensure good contact with the skin. A further group of rabbits (five males and five females) was held as a concurrent control receiving distilled water alone. Bodyweights, food consumption and clinical observations were recorded during the study. Blood samples for clinical investigations were taken on Day 20 (males) or Day 21 (females) and animals were killed and examined macroscopically on Day 22 (males) or Day 23 (females). Histological examination of specified tissues was then initiated.
The following comments in relation to real or possible treatment-related findings are made in summary:
Among rabbits treated at 1 000 mg/kg/day, slight erythema was observed as early as Day 2 with reactions progressing to well-defined erythema with slight or well-defined oedema in eight animals by Day 8; slight erythema only was recorded in the remaining two males. Thereafter, these responses were generally maintained to the end of the study although well-defined erythema with moderate oedema was recorded for one male and one female between Days 10 and 16. Additional reactions comprised cases of beige staining of the treatment site (not enough to prevent assessment of erythema) from as early as Day 3 and lasting to 19 days plus treatment site dryness and desquamation (sloughing) of the stratum corneum generally recorded during all three weeks and lasting up to 18 days in the majority of rabbits. Less common cases of cracking and/or hardening of the treatment site, lasting up to 8 days during the middle part of the study, were recorded for two males and up to two females; one further male also showed spots on the treatment site on Days 17 and 18.
Among rabbits treated at 100 mg/kg/day, irritation reactions ranged from slight erythema with or without slight oedema from as early as Day 2 or 3 to well-defined erythema with slight or well-defined oedema in seven animals by Day 8; slight erythema only (developing to well-defined erythema) was recorded in the remaining three females. Thereafter, these reactions were maintained, generally to the end of the study, although a slight degree of amelioration was recorded during Week 3 for three males. Additional reactions comprised beige staining of the treatment site during Weeks 2 and 3 and lasting up to 11 days for three males and three females plus sloughing from as early as Day 6 and lasting up to 15 days for all rabbits.
Among rabbits treated at 10 mg/kg/day, reactions comprised slight erythema as early as Day 3 in seven animals and lasting up to 20 days along with slight oedema in one male for 8 days. The remaining three rabbits showed no response throughout the study. Additional reactions comprised sloughing for one male and one female during the middle part of the study.
Microscopic pathology: Minimal diffuse acanthosis was recorded in the treated skin samples of 3/5 males and 3/5 females at 1 000 mg/kg/day and 2/5 females at 100 mg/kg/day.
Remaining parameters, namely clinical signs, bodyweight, food consumption, biochemistry, haematology, urinalysis, organ weights and macroscopic pathology, were not affected by the dermal application of the test material at 10, 100 or 1 000 mg/kg/day.
Under the conditions of the study it was considered that 1 000 mg/kg/day represents a NOAEL in the rabbit in terms of systemic toxicity while a no effect level for dermal irritation was not established.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the material does not require classification with respect to specific target organ toxicity following repeated dose application.
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