Registration Dossier
Registration Dossier
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EC number: 230-386-8 | CAS number: 7085-19-0
Endpoint summary
Administrative data
Description of key information
7 -Week Range Finding Study: Kirsch et al. (1986).
Under the conditions of the study, the no effect level is between 50 and 400 ppm..
4-Week Range Finding Study: Schilling (1990a).
Under the conditions of the study the "no adverse effect level" for male and female mice is between 900 ppm (~ 220 mg/kg body weight) and 2 700 ppm (~ 890 mg/kg body weight).
4-Week Range Finding Study: Schilling (1990b).
Under the conditions of the sudy the "no adverse effect level" for male and female mice could not be determined but was estimated to be < 2 700 ppm.
Sub-Chronic Repeated Dose Toxicity: Kirsch et al. (1985).
Under the conditions of this study the no-adverse-effect-level (NOAEL) is between 50 ppm and 150 ppm from the pathological point of view.
Sub-Chronic Repeated Dose Toxicity: Reinart (1977).
Under the conditions of the study the no-effect level was 200 ppm.
Sub-Chronic Repeated Dose Toxicity: Rondot et al. (1978).
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.
Chronic Repeated Dose Toxicity: Kuehborth (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. Therefore 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).
Repeated Dose Toxicity: Oral 13 week. Reuzel (1979)
Under the conditions of this study, the NOAEL was 4 mg/kg bw/day.
Range-Finding Study: Reuzel et al. (1977)
Under the conditions of this study, no noticeable differences in the effects observed by oral administration of the test material either in the diet or by capsule were seen.
Short-Term Repeated Dose Toxicity Via the Dermal Route: Allan et al. (1993)
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.
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:
- 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 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 3 and 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:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 September 1977 to 20 December 1977
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- GLP compliance:
- no
- Remarks:
- The performance of this study pre-dates the inception of GLP
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: about 4-5 months old
- Weight at study initiation: females: 6.3-6.4 kg, males: 7.6-8.1 kg
- Housing: The dogs were divided into 3 test groups of 4 males and 4 females and one control group of 4 males and 4 females each. They were individually housed in indoor cages. The dog and
cage number were indicated on each cage.
- Diet: All dogs received daily a weighed portion of food in an amount of 35 g/kg body weight during the first two weeks, and 40 g/kg body weight during the remainder of the test period. The dogs were fed a basal diet of the following percentage composition: instant wheat product 25, beet pulp 5,
instant oat product 25, vitamin B preparation 0.2, fish meal 10, brewer's yeast 3, meat scraps 10, vitamin AD3EK preparation 0.3, skimmed milk powder 5, NaCl + trace minerals 0.5, soya bean oil meal 8, soya bean oil 5, grass meal 3. Analyses are made for nutrients and contaminants.
- Water: unfluorised tap water was always available
- Acclimation period: Two weeks. Animals received an oral anthelmintic treatment (piperazine adipate, 200 mg/kg) twice during the quarantine period. - Route of administration:
- oral: feed
- Vehicle:
- other: molasses/water
- Details on oral exposure:
- DIET PREPARATION
- The test material was administered by admixture of the test material in the diet. To prepare the test diets firstly a premix was made by mixing 100 g test material in 2000 g basal diet in a Stephan cutter for 2 minutes. The test diets were prepared by diluting appropriate quantities of the premix with basal diet and a molasses/water mixture (1:1). Homogeneity of the diets was achieved by mixing for two minutes in a Lodige mixer. The test diets were mixed in the sequence of increasing concentrations of the test material in the diet.
- Rate of preparation of diet: The diets were freshly prepared at the initiation of the study and then at approximately biweekly intervals.
- Storage temperature of food: The diets were stored at room temperature and were identified by their group number.
- The control group received the basal diet without the test material. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - The actual levels of the test material in the diets were determined by analyses of the batches of each of the diets prepared in weeks 1, 6 and 12.
- The analyses were made after a storage period of at most 2 weeks.
- The concentrations of the test material recovered in the diet at the different stages were well in line with the nominal concentrations. - Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- Daily (continuous in diet)
- Dose / conc.:
- 4 mg/kg bw/day (nominal)
- Dose / conc.:
- 16 mg/kg bw/day (nominal)
- Dose / conc.:
- 64 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Four animals per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The dose-levels were selected on the basis of the results of a previous range-finding study, in which levels of up to 32 mg/kg body weight/day did not induce any noticeable untoward effect.
- Positive control:
- No
- Observations and examinations performed and frequency:
- OBSERVATIONS FOR SIGNS OF TOXICITY: Yes
- Health, condition and behaviour of all dogs were checked daily. Special attention was paid to the buccal mucosa and eyelids. All signs of reaction to treatment were recorded.
BODY WEIGHT: Yes
- The weight of each dog was recorded twice weekly for one week pre-dosing and for the first four weeks on test, and then weekly. The weighing’s were generally done when the animals had food available.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The quantity of food consumed by each dog was recorded daily and the mean weekly intake per dog was calculated.
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- At the beginning and at weeks 7 and 13 the eyes of all dogs were examined by indirect ophthalmoscopy.
HAEMATOLOGY: Yes
- Each dog was examined at the beginning and at week 6 and 12. Examinations included:
1.Haemoglobin concentrations - by cyanmethemoglobin method of Van Kampen and Zijlstra (Clin. Chim. Acta 6 (1961) 538-544).
2.Packed cell volume - as microhaematocrit.
3.Erythrocyte count - by Coulter Counter.
4.Leucocyte count - by Coulter Counter.
5. Differential leucocyte count - by direct visual count of smear after May-Griinwald-Giemsa staining.
6.Platelet count - by Coulter Counter.
7. Blood clotting time - using Normotest reagents (Nyegard and Co., Oslo, Norway).
CLINICAL CHEMISTRY: Yes
- Each dog was examined at the beginning and at week 7 and 13. Examinations included:
1. GPT - by Reitman and Frankel (Am. J. Clin. Path. 28 (1957) 56-63).
2. GOT - by Reitman and Frankel.
3. AP - by Bessey, Lowry and Brock (J. Biol. Chem. 164 (1946) 321-329).
4. Total protein - by biuret reaction.
5. Albumin - by Doumas, et al. (Clin. Chim. Acta 31 (1971) 87-96).
6. Serum electrophoresis - by agar gel electrophoretic method on microscopic slides of Wieme.
7. Urea nitrogen - by the automated phenazone/diacetyl monoxime technique of Ceriotti and Spandrio (Clin. Chim. Acta 11 (1965) 519-522).
8. Fasting plasma glucose - by Technicon Auto-Analyzer, method N-9a.
9. Total bilirubin - according to the method of Nederlands Normalisatie Instituut.
10. Serum electrolytes Na+ and K+ - by Paschen and Fuchs (Clin. Chim. Acta 35 (1971) 401-408).
11. OCT - by Ohshita (Clin. Chim. Acta 67 (1976) 145-152).
12. Creatinine - by Jaffe-reaction (In: Gorter en De Graaff, Klinische Diagnostiek).
URINALYSIS: Yes
- Each dog was examined at the beginning and at week 6 and 12. Individual urine samples were collected by catheterisation after overnight deprivation of water and food and examined for:
1. Specific gravity - by refractometer.
2. Electrolytes Na+ and K+ - method of Paschen and Fuchs (Clin. Chim. Acta 35 (1971) 401-408). The following semi-quantitative tests were made:
1. pH - Labstix
2. Protein - Labstix
3. Sugar - Labstix
4. Occult blood - Labstix
5. Ketones - Labstix
6. Bilirubin - Urobilistix
7. Microscopy of the sediment - after centrifugation at 3,000 rpm for 3 minutes. Deposits were examined for: erythrocytes, leucocytes, epithelial cells, crystals, casts, bacteria, sperm cells, and worm eggs. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- On completion of 91 days of treatment, all surviving dogs were anaesthetised by intravenous administration of Nembutal and exsanguinated by cutting the carotid artery. The dogs were subjected to a thorough autopsy.
- The following organs from all animals were dissected free of fat and weighed: liver, heart, adrenals, kidneys, brain, pituitary, spleen, lungs, testicles, thymus, thyroid, ovaries and prostate.
- Samples of the following tissues from all dogs were preserved in a 4 per cent neutral, phosphate-buffered formaldehyde solution: liver, kidneys, spleen, jejunum, ileum, caecum, thymus, heart, brain, lung, thyroid, adrenals, pituitary, testicles, ovaries, prostate, salivary glands (3), trachea, diaphragm, gall bladder, buccal mucosa, lymph nodes(cervical, popliteal and mesenteric), oesophagus, stomach, duodenum, colon, urinary bladder, ureter, uterus, nervus ischiadicus, spinal cord, pancreas, tongue, skin, anal sac, circumanal glands, thoracid aorta, abdominal aorta, skeletal muscle, mammary gland, bone marrow, bone, eye with optic nerve, other gross changes. Bone marrow smears were prepared and fixed in methanol. All tissues preserved, except the tongue, skin, anal sac, circumanal glands, teachea popliteal and bones, were processed for wax embedding and 5 μm sections, stained by hematoxylin and eosin, and examined microscopically. - Other examinations:
- FAECAL BLOOD CONTENT
- The faeces samples of all dogs were examined for the presence of occult blood during 4 consecutive days pre-dosing and daily during four consecutive days at week 1, 4, 8 and 12 of treatment.
ORGAN FUNCTION TESTS
- A liver-function test (bromosulphophthalein method) was carried out upon all dogs of the top-dose and control group at week 13. Blood samples were taken at one and at thirty minutes after intravenous injection of 12.5 mg BSP/kg body weight. The retention after 30 minutes was calculated from the extinction values at one and at 30 minutes.
- A kidney-function test was conducted upon all dogs at week 13. The phenolsulphophthaleine (PSP) method was used, After a 16 hour period without food 1 mg PSP/kg body weight was intravenously injected. Next a blood sample was collected after 60 minutes and the concentration of PSP in the plasma was measured. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - A few small ulcera of the buccal mucosa were observed in the male dog 7-364 (high-dose group) after 10 days of treatment. A few days later, the dog became ill, still showing ulcera, accompanied
by a swollen mandibular lymph node. The food intake decreased markedly and slight fever occurred. Therefore, 225,000 IU penicillin and 375 mg streptomycin were administered for five consecutive days. The dog recovered within a week but the food intake continued to be too low. In week 7 of the study the same dog had a moderate purulent conjunctivitis. The next few weeks the dog became thin, showed a mucous discharge from eye and nose, and pale to slightly yellow conjunctivae and buccal mucosae. To prevent the dog from severe emaciation the test material was omitted from the diet from week 10 and onward. Within two weeks the dog had clinically recovered.
- Reddening of the gingivae was observed in female dog 7.303 and in male dog 7.301 both of the top-dose group in week 2 only.
- None of the other dogs showed any abnormalities in health condition. Behaviour was not affected in any of the dogs. - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Mean body weights were distinctly lower in dogs of the top-dose group than in the controls during the major part of the study.
- A similar phenomenon, though to a lesser degree, was observed in dogs of the mid-dose group. One dog of this group gained more weight than did any of the dogs used in the present study. Therefore, the resulting differences between the mean values of mid-dose dogs and the controls were relatively small. Body weights of males of the low-dose group were also slightly lower than those of the controls. This was attributed to the relatively low initial weights of this group, since the weight gain figures were comparable with those of the controls. Weight gain in females of the low-dose group was very similar to that of control females. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- During the first two weeks of the test period all dogs received 35 g/food/kg body weight/day. This quantity appeared to be not sufficient for the dogs. Therefore the quantity was increased to 40 g food/kg body weight/day for the remainder part of the test period. All dogs consumed the daily portions of food given, except for male dog 7.364 of the top-dose group.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A small corneal ulcer was seen in male dog 7.368 of the top dose group at the end of the study. Since corneal ulcers do not occur frequently in dogs of this colony, it is possible that the ulcer was induced by the test material.
No treatment-related changes were observed in the other dogs. - Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The ingestion of 64 mg/kg body weight/day test material resulted in clear decreases in haemoglobin content, packed cell volume and red blood cell count at week 6 and 13. Moreover, the number of lymphocytes was slightly diminished, while the number of neutrophils was elevated. In dogs fed 16 mg/kg body weight/day the haemoglobin content, packed cell volume and red blood cell count were generally also lower than in the controls, though the differences were not always statistically significant. The increased number of thrombocytes at week 6 in dogs fed 16 mg/kg body weight/day was an isolated finding and was considered to be unrelated to treatment.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Dogs of the top-dose group showed a slight, though consistent, increase in serum urea values. Total protein and albumin values of this group were slightly, though statistically significantly lower
than those of the controls at week 6. The alkaline phosphatase activities of this group were generally lower than those of the controls, though male dog 7.3.64 showed increased values at week 6
and 13. Since increases, rather than decreases are indicative of adverse effects, the decrease of the alkaline phosphatase activities were not considered to be of toxicological importance. Dogs of the mid-dose group showed significantly lower bilirubin values compared with the controls at week 6 only. Since there was no dose-response relationship, no toxicological significance was attached to this finding. - Endocrine findings:
- not examined
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At week 13, the pH values of the urine of dogs given 16 or 64 mg/kg body weight/day were generally slightly higher than those of the controls. All values however were within the normal range. The remainder of the semi-quantitative tests did not reveal indications of treatment-related differences between test and control dogs. The incidence of cells or of other constituents in urine sediment was similar in all groups. The statistically significant increase of the Na content at the low intake level at week 6, and of the K content at the intermediate intake level at week 12 were isolated findings, and considered unrelated to treatment.
- 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 organ weights were generally similar in the different groups. When, however, expressed relative to body weight, the values of the heart, kidneys, liver, brain and lungs turned out to be statistically significantly higher in dogs fed 64 mg/kg body weight/day than in controls. Slightly higher values of relative liver, brain and lung weights were observed also in the mid-dose group, but the differences with the controls were not statistically significant. Dogs fed the test material showed lower mean thymus weights than the controls. The differences with the controls were however, not statistically significant.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At gross examination male dog 7.364 revealed several changes. The kidneys and gall bladder appeared to be enlarged. The bronchial lymph nodes of this dog were enlarged and haemorrhagic. Moreover slightly brown discoloured adipose tissue was found in the mesentery and pericardium of this dog. A similar discoloration of the adipose tissue was found in the mesentery of two females (viz. 7.325 and 7.392) of the top-dose group.
Many dogs had intestinal parasites, most probably Toxocara canis.
In six female dogs a haemorrhagic mucosa of the urinary bladder was found, but this finding was attributed to catheterisation of the bladder.
Other minor gross lesions were about equally distributed between control and test animals or were isolated findings. These findings were, therefore, considered to be of no-toxicological significance. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No changes were observed in the brown discoloured fat tissues of the pericardium and/or mesentery.
Slight differences in incidence of mononuclear inflammatory cell infiltrates in the liver, were observed between males of control- and top-dose groups. These differences, however, are not considered to represent a toxic effect since they were only slight and since, moreover, similar differences in incidence are quite common in the colony of dogs used.
A focal area of necrosis surrounded by neutrophilic leucocytes slight periportal fibrosis and a focal sub-acute pericarditis were observed only in high-dose animals. Each of the changes, however, occurred only in one dog. Since, moreover, these alterations are rather common in control dogs used in other toxicity studies, they are considered to be fortuitous findings unrelated to treatment.
Some atrophic glomeruli were seen in the kidneys of two dogs of both the top- and mid-dose group. This change, however, is a very common finding in beagles of this colony; moreover, the incidence in both groups was the same. Therefore, this change was not ascribed to the test material.
Slightly increased deposits of brown pigment were found in a lymph node of one male and one female of the top-dose group. Brown pigment dipositions, in various degrees, are rather frequently observed in lymph-nodes of our dogs. It is therefore highly unlikely that the deposition seen in the present study has any toxicological significance.
The other pathological changes observed were about equally distributed amongst the control group and the different test groups or they occurred only in a single animal. Therefore, none of these abnormalities, which are common findings in beagle dogs of the TNO-colony, are ascribed to the feeding of the test material. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- KIDNEY AND LIVER FUNCTION TESTS
- Specific gravity values of urine were similar in all groups. These values were measured in urine collected from dogs which had constant access to drinking water. No specific gravity values are available of urine obtained after an 18-hour period of water deprivation, because it appeared impossible to collect any urine after such a period. Phenol red retention was statistically significantly higher in dogs fed 64 mg/kg body weight/day than in controls. In the same group the bromosulphophthalein retention was also significantly higher than in the controls. However, a large part of this increase in BSP retention was caused by an extremely high value of male dog 7.364. The BSP retention values of the other males of this group were similar to the control values. These values of the females, however, were slightly higher than those of the controls.
FAECAL BLOOD
- During the pre-treatment period several dogs in each of the groups had once or a few times minimal quantities of occult blood in the faeces. After the initiation of the treatment period only one control dog and one of the intermediate dose group showed only once a minimal quantity of occult blood in the faeces. - Details on results:
- The present study revealed changes in dogs of the high-dose group, which are attributed to treatment.
The distinct depression in body weight gain in dogs fed 64 mg/kg bw/day test material was not accompanied by a reduced food intake, which indicates an unfavourable effect on food efficiency. Therefore, the depression of weight gain was considered to be a deleterious effect of the test material.
The decrease in haemoglobin content, packed cell volume and red blood cell count in dogs of the high-dose group were ascribed to treatment, since these chances occurred both after 6 and 13 weeks. Moreover, indications of similar changes were observed also, though less pronounced, in dogs of the mid-dose group.
A major organ affected was the kidney of dogs of the top-dose group. This appeared from an impaired kidney function, elevated serum urea values and increases in relative kidney weights. Microscopically, however, no treatment-related changes were observed. Although microscopy of the liver likewise failed to reveal changes attributable to treatment, the results of the BSP function test and the relative liver weight suggested an effect of the test material in the high-dose group.
The increases of the relative weights of the brain and lungs in the top-dose group were probably unrelated to treatment, because there were no indications of treatment-related histopathological changes in these organs. Moreover, it has been shown that in rats increases in the relative weight of the brain and lungs may occur as a result of depression in body weight gain (Feron et al., Fd. Cosmet. Toxicol. 11 (1973) 85 - 94). Since the body weights of top-dose dogs were depressed a similar relationship may be responsible for the increased relative weights of the brain and lungs observed in these dogs. The increased relative weight of the heart in dogs of the top-dose group is considered to be of doubtful toxicological significance since it was not associated with morphological changes in this organ.
Clinical symptoms were restricted to a few dogs of the top-dose group and were severe in one dog. The rapid improvement of the dog after withdrawing the test material from the diet was considered an indication that the test material had induced the symptoms. Corneal ulcers, as observed in one dog of this group may have been induced also by the test material since they are rarely if ever observed in dogs of the colony used. The reddening of the gingivae observed in two other dogs might be due to the irritating properties of the test material on mucous membranes. It is very doubtful whether the brown discoloured adipose tissue in three dogs of the top-dose group is of any toxicological significance, since the phenomenon was not accompanied by any microscopical changes in this tissue.
Although the thymus weights, both absolute and relative, were generally lower in the test groups than in the controls no statistically significant differences occurred amongst the groups. Since similar low thymus weights were found also in the control group and moreover, no noticeable microscopical changes were observed in this organ, it does not seem justified to attach any toxicological significance to the relatively low weights in the test groups.
Dogs fed 16 mg/kg body weight test material only showed relatively low weight gain and slight, and transitory decreases in red blood cells and packed cell volume. Since these changes occurred more markedly in the top-dose group their occurrence in the mid-dose group cannot be disregarded.
On the basis of these results it seems justified to conclude that 4 mg/kg body weight test material was the no-effect level in the present study. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 4 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
- gross pathology
- haematology
- ophthalmological examination
- organ weights and organ / body weight ratios
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 64 mg/kg bw/day (nominal)
- System:
- urinary
- Organ:
- kidney
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Under the conditions of this study, the NOAEL was 4 mg/kg bw/day.
- Executive summary:
A sub-chronic (90-day) feeding study with the test material was performed in beagle dogs fed diets containing the test material at levels which provided intakes of 0, 4, 16 or 64 mg/kg body weight/day. The study was similar in design to OECD 409.
Health, condition and behaviour were generally not affected by the test material. One dog of the top-dose group showed ulcera in the buccal mucosa, a purulent conjunctivitis, pale mucous membranes and a poor condition. Gain in body weight was markedly decreased in dogs of the top-dose group and slightly decreased in dogs of the intermediate-dose group. Food consumption was decreased only in one dog of the top-dose group. Haemoglobin content, packed cell volume and red blood cell counts were decreased in the top-dose group. Rather low value occurred in the intermediate-dose group. In the top-dose group there were moderate increases in serum urea values. Phenol-red, and BSP-retention were increased in the top-dose group. The pH of the urine was generally slightly higher in dogs of the two highest dose groups than in controls. On ophthalmoscopical examination no treatment-related changes were observed. A purulent conjunctivitis was seen in one dog of the top-dose group.
The relative weights of the heart, kidneys, and liver were increased in the top-dose group. On gross examination brown discoloured fat tissue in pericard and/or mesenterium was observed in three dogs of the top-dose group. Microscopic examination was essentially negative.
Under the conditions of this study, the NOAEL was 4 mg/kg bw/day.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 14 July 1988 to 15 August 1988
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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: Age when supplied: 40 days old. Age when administration period started: 49 days old.
- Weight at study initiation: Weights when administration period started: male animals: 24 (22 - 25) g; female animals: 19 (18 - 21) g.
- Housing: During the study period the mice were housed singly in type I Makrolon cages with wire mesh tops (floor area about 200 cm^2). The cages with the test animals were arranged on the racks in such a manner that uniform experimental conditions (fresh air/waste air/light) were guaranteed.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation: Nine days
DETAILS OF FOOD AND WATER QUALITY
- Analysis of feed: The feed used in the study was assayed for contaminants. On the basis of the duration of use and the analytical results the feed was found to be suitable. The copper values were found to have dropped in individual cases; they were, however, acceptable since they were within the range of error expected with this method of determination. In the feed batch 97/88 higher total nitrosamine levels (DMN; DEN; NPIP; NMORPH) were found: 0.04 mg/kg (proposed maximum limit: 0.01 mg/kg). In respect to the administration period and the slight increase in the total nitrosamine levels as well as the absence of any specific finding in all groups (including the control group), these amounts of nitrosamines were assessed as being of no biological relevance and have no impairment of the object of the study.
- Analysis of drinking water: The drinking water was regularly assayed both by the municipal authorities of Frankenthal and by the Department of Water Chemistry and Technical Services of BASF Aktiengesellschaft. In view of the aim and duration of the study there are no special requirements exceeding the specifications of the drinking water.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Fully air conditioned room.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 a.m. - 6.00 p.m. and 12 hours darkness from 6.00 p.m. - 6.00 a.m.) - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- At the end of the acclimatisation period, the test material was added to the ground feed in different amounts depending on the test group.
- The test material preparations were prepared once for the 4-week study period. Each concentration was prepared separately. The test material was thoroughly mixed with a small amount of feed using a BOSCH mixer. This premix was then adjusted to the desired concentration with the appropriate amount of feed and mixed in a laboratory mixer of GEBR. LODIGE for about 10 minutes. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analysis of test material
- The test material was characterised and the homogeneity was verified before the study began.
The stability of the test material was analysed after the termination of all studies with the test material.
Analysis of test material preparations
- To verify the correct concentration of the test material preparations samples were taken of each concentration at the start of the study and sent for analysis. To prove the homogenous distribution of the test material in the diet samples of the lowest and highest dose were sent to the analytical laboratory
- Methods: The content of test material in the feed was determined by HPLC.
ANALYSES
- Analysis of the test material: The homogeneity of the test material has been verified analytically. Results of the stability analysis are not available.
- Analysis of the test material preparations: The stability of the test material in the diet has been demonstrated for a period of 33 days before the start of the study. The results obtained verified the correctness of the concentrations and the homogeneous distribution of the test material in the diet. - Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- Continuous (in diet).
- Dose / conc.:
- 0 ppm
- Remarks:
- Test group 0: Control
- Dose / conc.:
- 100 ppm
- Remarks:
- Test group 1
- Dose / conc.:
- 300 ppm
- Remarks:
- Test group 2
- Dose / conc.:
- 900 ppm
- Remarks:
- Test group 3
- Dose / conc.:
- 2 700 ppm
- Remarks:
- Test group 4
- No. of animals per sex per dose:
- Five per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: In literature the LD50 value in mice was reported as 300 mg/kg body weight by intraperitoneal application. Due to this finding and experimental experience with a similar substance the dose levels of 100, 300, 900 and 2 700 ppm were chosen.
- Animal assignment rationale: 1 day prior to the start of the study, the male and female mice were assigned to the test groups on the basis of their weights. The list of randomization instructions was generated by a computer. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Every day.
- The animals were inspected for evident signs of toxicity each day. Once a week an additional exact clinical examination was carried out for each animal.
- A check for dead or moribund animals was carried out twice a day (Mondays to Fridays) or once a day (Saturdays, Sundays and on public holidays).
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
- All animals were weighed once a week, on each occasion on the same day.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: During the administration period feed consumption was determined weekly for the course of 7 days.
- Compound intake: The mean amount of test substance consumed each day (in mg) per kg body weight was calculated using the following formula:
[FC x D / BWx]
Where:
FC = mean daily feed consumption (in g) in one week of the study (from day x-7 to day x)
D = dose in ppm
BWx = mean body weight (in g) on day x of the study
HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus and after decapitation. The blood samplings and the subsequent analysis of the blood and serum samples were carried out in a randomised sequence.
- The following examinations were carried out: Leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets.
- The differential blood count was evaluated visually. The data were transferred into the computer.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, blood sampling took place in the morning from fasted animals.
- How many animals: 5 animals per test group and sex.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus and after decapitation. The blood samplings and the subsequent analysis of the blood and serum samples were carried out in a randomised sequence.
- The following examinations were carried out:
Enzymes
- Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase.
Blood chemistry:
- Inorganic phosphate, calcium, urea, creatine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol. In deviation from the protocol the concentrations of the electrolytes (sodium, potassium and chloride) could not be determined due to the low amount of serum.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, blood sampling took place in the morning from fasted animals.
- How many animals: 5 animals per test group and sex. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- The animals were sacrificed by decapitation under CO2 anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The weight of liver, kidneys and testes from all animals sacrificed at scheduled dates was determined. For determination of relative weights, the terminal body weight measured in the animal house was taken.
- The following organs from all animals were fixed in 4 % formaldehyde solution: All gross lesions, spleen, testes, heart, kidneys, liver, adrenal glands.
HISTOPATHOLOGY: Yes
- After fixation, the histotechnical processing was performed. - Statistics:
- The statistical evaluation of the data was carried out on the computing systems.
Clinical examinations: For the statistical evaluation of the study, means and standard deviation of the variables feed consumption, body weight and intake of test material were calculated for the animals of each test group, and printed out together with the individual values in the form of tables (in case of test material intake only the means were shown in the tables). The statistical significance of the clinical data (body weight) was checked using the KRUSKAL-WALLIS ANOVA test and the MANN-WHTNEY-U-test.
Clinical chemistry and haematology: Mean and standard deviation were calculated for each test group and tabulated together with the individual values. Except of the differential blood count, a non-parametric one-way analysis of variance is done via the Kruskal-Wallis- h-test. If the resulting p-value is equal or less than 0.05 a pairwise comparison of each dose group with the control group was carried out. This comparison is done using the Mann-Whitney-U-test for the hypotheses of equal medians. If the results of this test are significant, p-markers (* for p ≤ 0.02, *** for p ≤ 0.002) were printed together with the group mean in the tables.
Pathology: 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).
A non-parametric one-way analysis of variance was done via the KRUSKAL-WALLIS-h-test. If the resulting p-value was less than 0.05 a pairwise comparison
of each dose group with the control group was carried out. This comparison was done using the WILCOXON-U-test for the hypotheses of equal means. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No signs attributable to the administration of the test material were observed throughout the study period.
- Mortality:
- no mortality observed
- Description (incidence):
- During the entire administration period no animal died prematurely.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- In all male and female animals of dose groups 1 - 4 in comparison to the control groups no influence on the body weight gain could be detected during the administration period.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Throughout the course of the study, the male and female animals of test groups 1 - 4 (100, 300, 900 and 2 700 ppm) compared with those of the control groups showed no test material-related adverse effect on the feed consumption.
- Food efficiency:
- not specified
- 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):
- See 'Clinical biochemistry findings'.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - Alkaline phosphatase and cholesterol: The alkaline phosphatase activities and the cholesterol concentrations in the serum of the males and females of test group 4 (2 700 ppm) were significantly increased at the end of the 4-week administration period. These changes in the peripheral blood are attributable to the test material administered, and are probably due to a hepatic dysfunction.
- Platelets: The slight decrease in the platelet count seen in the females of group 4 (2 700 ppm) was also test material-induced as corresponding findings were also observed in both sexes of the supplementary study) with higher doses. Pathogenetically, however, the finding of a thrombocytopenia is hard to allocate to a certain clinical syndrome because there are no concomitant symptoms.
- Other examinations: The other examinations exhibited no changes which are causally related to the test material administration. A few significant deviations from the control group figures were found in isolated cases in the parameters Haemoglobin, inorganic phosphate and albumin. On the basis of the plausibility criteria described in detail below they are not regarded as being related to the test material administered.
Plausibility criteria:
- Haemoglobin: The values lie within the range of biological variation; effect not relevant and similar effect absent in both sexes; not very plausible.
- Inorganic phosphate: The values lie within the range of biological variation; effect not relevant, random increase or decrease in the control value; effect not relevant and similar effect absent in both sexes; not very plausible.
- Albumin: The values lie within the range of biological variation; effect not relevant and similar effect absent in both sexes; not very plausible. - Endocrine findings:
- not examined
- 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):
- Oral administration of the test material as a dose of 2 700 ppm results in a statistically significant increase in the liver weight (absolute and relative values) in male and female mice. This change is to be regarded as a consequence of administration of the test material.
No relationship to the test material can be seen from the statistically significantly increased kidney weights (relative values: dose groups 1 - 3 in females and dose group 2 in males; absolute values: dose groups 2 and 3 in females; no statistically significant change in kidney weight in dose group 4). - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Light-microscopy reveals that the increased liver weights in dose group 4 are correlated to a centrally pronounced hepatocellular hypertrophy, as is also similarly observed and described at the same dose in a separate study. Further changes, such as hyalin degeneration of individual liver cells in 2 male and 4 female animals, focal and multifocal necroses respectively in one male and 2 females, and isolated mitoses in the liver cells of 2 males, are findings which are also observed at the same dose in a separate study. In dose group 3 light-microscopy of the liver reveals only incidental and spontaneous changes.
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not examined
- Details on results:
- Oral administration of the test material over a period of 4 weeks results in a statistically significant increase in the liver weights in male and female mice in dose
group 4 (2 700 ppm).
Light-microscopy reveals that the increase in liver weight is correlated with a central hepatocyte hypertrophy, as is also similarly observed at the same dose and with the same administration method in a second range-finding study. According to the results of electron microscopy in the second study, the essential etiology of the hepatocyte hypertrophy is considered to be peroxisome proliferation. In dose group 3 (900 ppm) no test material-related liver changes or other organ changes are found which are attributable to administration of the test material.
From the point of view of pathology, and taking into consideration the results of study the second-range-finding study, a "no adverse effect level" is to be expected between 900 and 2 700 ppm. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 900 - < 2 700 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the study the "no adverse effect level" for male and female mice is between 900 ppm (~ 220 mg/kg body weight) and 2 700 ppm (~ 890 mg/kg body weight).
- Executive summary:
The short-term repeated dose toxicity of the test material was assessed according to OECD Test Guideline 407 and in compliance with GLP.
The test material was administered to 40 B6C3F1 mice (20 males and 20 females, 15 males and 15 females respectively) via the diet over a period of 4 weeks. A group of untreated animals (5 per sex) was used for a control group.
In each case 5 animals per sex received the test material in doses of 100, 300, 900 and 2 700 ppm.
Feed consumption and body weight were determined each week. The animals state of health was checked daily, and once a week they were subjected to an additional exact clinical examination.
At the end of the study a clinicochemical and haematological examination was carried out.
It can be stated that the dietary administration of the test material to mice for a period of 4 weeks led to the following findings:
2 700 ppm (about 780 mg/kg body weight):
- 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 a central hypertrophy of the hepatocytes in both sexes900 ppm (about 220 mg/kg body weight), 300 ppm (about 70 mg/kg body weight) and 100 ppm (about 25 mg/kg body weight):
- no test material-induced changesThe evaluation of the results indicate that the "no adverse effect level" for male and female mice is between 900 ppm (about 220 mg/kg body weight) and 2 700 ppm (about 780 mg/kg body weight).
However, the assessment of the results shows that no clear toxic effect was detected that meets the EPA "MTD-criteria", i.e. body weight reduction of about 10 % or distinct organ toxicity among others which is necessary for the dose level selection for the carcinogenicity study. In order to determine the target organs and to facilitate the dose level selection for the carcinogenicity study a further 4-week study in mice with the dose levels of 2 700, 4 500 and 7 000 ppm will be carried out.
Under the conditions of the study the "no adverse effect level" for male and female mice is between 900 ppm (~ 220 mg/kg body weight) and 2 700 ppm (~ 890 mg/kg body weight).
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 08 November 1988 to 07 December 1988
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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: Age when supplied: 44 days old. Age when administration period started: 49 days old.
- Weight at study initiation: Weights when administration period started: male animals: 26 (24 - 27) g; female animals: 20 (20 - 21) g.
- Housing: During the study period the mice were housed singly in type I Makrolon cages with wire mesh tops. The cages with the test animals were arranged on the racks in such a manner that uniform experimental conditions (fresh air/waste air/light) were guaranteed.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation: 5 days
DETAILS OF FOOD AND WATER QUALITY
- Analysis of feed: The feed used in the study was assayed for contaminants. On the basis of the duration of use and the analytical results the feed was found to be suitable. The copper values were found to have dropped in individual cases; 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 was regularly assayed both by the municipal authorities of Frankenthal and by the Department of Water Chemistry and Technical Services of BASF Aktiengesellschaft. In view of the aim and duration of the study there are no special requirements exceeding the specifications of the drinking water.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Fully air conditioned room.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 a.m. - 6.00 p.m. and 12 hours darkness from 6.00 p.m. - 6.00 a.m.) - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- At the end of the acclimatisation period, the test material was added to the ground feed in different amounts depending on the test group.
- The test material preparations were prepared once for the 4-week study period. Each concentration was prepared separately. The test material was thoroughly mixed with a small amount of feed using a BOSCH mixer. This premix was then adjusted to the desired concentration with the appropriate amount of feed and mixed in a laboratory mixer of GEBR. LODIGE for about 10 minutes. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test material was characterised and the homogeneity was verified before the test began.
The stability of the test material was analysed after the termination of all studies.
Before the start of the study the stability of the test material in the diet had been demonstrated for a period of 33 days. The homogeneity of the mixtures was verified in the former study. To verify the correct concentration of the test material preparations samples were taken of each concentration at the start of the study and sent for analysis.
The content of the test material in the feed was determined by HPLC.
The analysis of the test material showed a content of active ingredient of 92.7 %.
The homogeneity of the test material has been verified analytically.
Analysis of the test material preparations: The stability of the test material in the diet has been demonstrated for a period of 33 days before the start of the study. The results obtained verified the correctness of the concentrations. The homogenous distribution of the test material in the diet has already been verified in - Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- Continuous (in diet).
- Dose / conc.:
- 0 ppm
- Remarks:
- Control
- Dose / conc.:
- 2 700 ppm
- Dose / conc.:
- 4 500 ppm
- Dose / conc.:
- 7 000 ppm
- No. of animals per sex per dose:
- Five per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: In literature the LD50 value in mice was reported as 300 mg/kg by intraperitoneal application. In the previous 4-week 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 a central hypertrophy of the hepatocytes in both sexes.
100, 300 and 900 ppm:
- No test material induced changes.
Due to this the dose levels of 2 700, 4 500 and 7 000 ppm were chosen.
- Animal assignment rationale: 1 day prior to the start of the study, the male and female mice were assigned to the test groups on the basis of their weights. The list of randomisation instructions was generated by a computer. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Every day.
- The animals were inspected for evident signs of toxicity each day. Once a week an additional exact clinical examination was carried out for each animal.
- A check for dead or moribund animals was carried out twice a day (Mondays to Fridays) or once a day (Saturdays, Sundays and on public holidays).
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
- All animals were weighed once a week, on each occasion on the same day.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: During the administration period feed consumption was determined weekly for the course of 7 days.
- Compound intake: The mean amount of test material consumed each day (in mg) per kg body weight was calculated using the following formula:
[FC x D / BWx]
Where:
FC = mean daily feed consumption (in g) in one week of the study (from day x-7 to day x)
D = dose in ppm
BWx = mean body weight (in g) on day x of the study
HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus and after decapitation. The blood samplings and the subsequent analysis of the blood and serum samples were carried out in a randomised sequence.
- The following examinations were carried out: Leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets.
- The differential blood count was evaluated visually. The data were transferred into the computer.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, blood sampling took place in the morning from fasted animals.
- How many animals: All surviving animals per test group and sex.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus and after decapitation. The blood samplings and the subsequent analysis of the blood and serum samples were carried out in a randomised sequence.
- The following examinations were carried out:
Enzymes:
- Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase.
Blood chemistry:
- Sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, blood sampling took place in the morning from fasted animals.
- How many animals: All surviving animals per test group and sex. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- At the end of the 4-week administration period all surviving animals were sacrificed after a fasting period (withdrawal of feed for abot 20 h).
- The animals were sacrificed by decapitation under CO2 anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The weight of liver, kidneys, testes and adrenal glandsfrom all animals sacrificed at scheduled dates was determined. For determination of relative weights, the terminal body weight measured in the animal house was taken.
- The following organs from all animals were fixed in 4 % formaldehyde solution: All gross lesions, spleen, testes, heart, kidneys, liver, adrenal glands.
- Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after death and assessed by gross pathology.
HISTOPATHOLOGY: Yes
- After fixation, the histotechnical processing was performed.
- After fixation, the histopathological processing, the examination by light microscopy and the evaluation of findings were performed (see table 'Histopathological processing.
- Further examinations were done as follows: Liver samples from animals No. 5 (control) and No. 20 (dose group 3) were embedded in epoxy resin. Semithin sectioning was followed and ultramicrotomy was done. The ultrathin sections were evaluated using a Hitachi H 600 Electron microscope. Characteristic lesions were photographed. - Optional endpoint(s):
- Electron microscopic investigation to demonstrate the cause of the prominent hypertrophy of hepatocytes ultrastructurally, since the etiology is unsolved by light microscopy.
- Statistics:
- The statistical evaluation of the data was carried out on the computing systems.
Clinical examinations: For the statistical evaluation of the study, means and standard deviation of the variables feed consumption, body weight and intake of test material were calculated for the animals of each test group, and printed out together with the individual values in the form of tables (in case of test material intake only the means were shown in the tables). The statistical significance of the clinical data (body weight) was checked using the KRUSKAL-WALLIS ANOVA test and the MANN-WHTNEY-U-test.
Clinical chemistry and haematology: Mean and standard deviation were calculated for each test group and tabulated together with the individual values. Except of the differential blood count, a non-parametric one-way analysis of variance is done via the Kruskal-Wallis- h-test. If the resulting p-value is equal or less than 0.05 a pairwise comparison of each dose group with the control group was carried out. This comparison is done using the Mann-Whitney-U-test for the hypotheses of equal medians. If the results of this test are significant, p-markers (* for p ≤ 0.02, *** for p ≤ 0.002) were printed together with the group mean in the tables.
Pathology: 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).
A non-parametric one-way analysis of variance was done via the KRUSKAL-WALLIS-h-test. If the resulting p-value was less than 0.05 a pairwise comparison
of each dose group with the control group was carried out. This comparison was done using the WILCOXON-U-test for the hypotheses of equal means. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- At the end of the study all male animals and one female animal of group 3 (7 000 ppm) showed a reduced general state. On the day of its death, one female animal of group 3 (7 000 ppm) showed a strong decrease in body weight and its general state was reduced. All other animals stayed uninfluenced by the test material administered.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- During the administration period one female animal of group 3 (7 000 ppm) died on day 20 of the study.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Both sexes of all 3 dose groups showed clearly dose-dependent influences of the body weight gain at any time of the study period. Particularly the body weights of group 3 (7 000 ppm) were influenced and had compared to the control, significantly decreased in both sexes from
day 7 onwards (27 % females and 32 % males at the end of the study).
Body weight gain was also significantly affected in both sexes of groups 1 and 2 (2 700 ppm and 4 500 ppm). The body weights of group 2 were at the end of the study 11 % (male) and 13 % (female), and in group 1, 6 % (male) and 5 % (female) lower than the corresponding control values. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- In all dose groups of both sexes, compared to the control, the feed consumption values had highly fluctuated (females) and had clearly increased, being up to 124 % in the males. However, these values, especially the ones found for the males, are not at all reflected in the body weight gain. They are attributable to the spilling of feed by the animals.
- 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):
- See 'Clinical biochemistry findings'.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase:
4-week oral administration of the test material resulted, both in the male and in the female mice in test groups 1 to 3 (2 700, 4 500 and 7 000 ppm), in a dose-related increase in alkaline phosphatase activity. Moreover, significantly increased alanine aminotransferase activities were detectable in the serum of the male and female animals in test group 3 (7 000 ppm). In addition, in the males in test group 3 (7 000 ppm) the aspartate aminotransferase activities and in the females in test group 2 (4 500 ppm) the alanine aminotransferase activities were significantly increased compared to the levels in the control group.
A particularly increase in enzyme activities was noted in the animals in test group 3 (7 000 ppm). In this dose group the alanine aminotransferase and alkaline phosphatase activities were increased 5- to 10-fold compared with the control value. The reason for the increased enzyme activities in the periphery was probably a hepatic dysfunction. The extreme increase in activities in the 7 000 ppm group furthermore points to massive liver cell damage in animals receiving
this dose.
Leukocytes, lymphocytes and platelets:
In the serum of the male and female mice in test group 3 (7 000 ppm) the leukocyte levels were decreased at the end of the administration period, however, the decrease in white blood cells in the females was expressed only as a trend. The reason for the reduced leukocyte count in the peripheral blood is a decrease in the lymphocytes in both sexes receiving the highest dose. Also undoubtedly of test material-related origin is the reduced platelet count found in both sexes in the 7 000 ppm group and also in the females in test group 2 (4 500 ppm).
The changes in the distribution of the white blood cells and platelets cannot be assigned to a specific pathological state since concomitant symptoms, which could explain these findings, are absent. In particular, no morphological variations were found in the differential blood count of either sex.
Glucose, albumin and globulins:
At the end of the study increased albumin levels and decreased globulin concentrations were detected in the serum of the males in test group 3 (7 000 ppm). Repeated administration of the test material to female animals led to a dose-dependent increase in the albumin concentration and to a decrease in the glucose level which, however, was expressed only as a trend. In deviation to the previous range finding study an increase of the albumin level was detected in the females of test group 1 (2 700 ppm).
The decrease in the glucose level in the females and possibly also the decrease in the globulin levels in the male mice were probably of nutritional origin and are presumably causally connected to the reduced body weight change in the animals in test group 3 (7 000 ppm).On the other hand, the reason for the increase in the albumin levels in both sexes is unclear. Since an absolute increase in the serum albumin is physiologically impossible and, moreover, there are no indications of a relative increase in the albumin concentration as a result of a reduction in the plasma volume (for example, decreased haematocrit levels).
Other examinations:
The other examinations exhibited no changes which are causally related to the test material administration. A few significant deviations from the control group figures were found in isolated cases in the parameters Mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, alanine aminotransferase, calcium, total protein, albumin, globulins and triglycerides. On the basis of the plausibility criteria described in detail below they are not regarded as being related to the test material administered.
Plausibility criteria:
- Mean corpuscular haemoglobin: The values lie within the range of biological variation;
effect not relevant, Random increase or decrease in the control value; effect not relevant, No monotonic trend present (absence of dose response relationship; effect not very plausible and Similar effect absent in both sexes; not very plausible.
- Mean corpuscular haemoglobin concentration: The values lie within the range of biological variation; effect not relevant, Random increase or decrease in the control value; effect not relevant, No monotonic trend present (absence of dose response relationship; effect not very plausible and Similar effect absent in both sexes; not very plausible.
- Alanine aminotransferase: The changes have no pathognomic relevance, The values lie within the range of biological variation; effect not relevant and No monotonic trend present (absence of dose-response relationship); effect not very plausible.
- Calcium: The values lie within the range of biological variation; effect not relevant, No monotonic trend present (absence of dose-response relationship); effect not very plausible, Similar effect absent in both sexes; not very plausible.
- Total protein: The values lie within the range of biological variation; effect not relevant, Random increase or decrease in the control value; effect not relevant, No monotonic trend present (absence of dose response relationship; effect not very plausible and Similar effect absent in both sexes; not very plausible.
- Albumin: The values lie within the range of biological variation; effect not relevant, Contrary change in both sexes; effect not very plausible, Random increase or decrease in the control value; effect not relevant, No monotonic trend present (absence of dose response relationship; effect not very plausible and Similar effect absent in both sexes; not very plausible.
- Globulins: The values lie within the range of biological variation; effect not relevant, No monotonic trend present (absence of dose-response relationship); effect not very plausible, Similar effect absent in both sexes; not very plausible.
- Triglycerides: The values lie within the range of biological variation; effect not relevant, Random increase or decrease in the control value; effect not relevant, No monotonic trend present (absence of dose response relationship; effect not very plausible and Similar effect absent in both sexes; not very plausible.
From the viewpoint of clinical chemistry and haematology the no effect level, under the test conditions given in this study, is for the males and females below 2 700 ppm. - Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Description (incidence and severity):
- The principal changes in organ weights were statistically significant increases in the liver weights (absolute and relative values) of the male and female animals in test groups 1 and 2 and the males in test group 3. Similar increases in liver weight were also observed in the females in test group 3, but no statistical calculation was carried out, because the group was too small. The decrease in body weight and the increase in liver weight were considered as being dose-dependent effects of the test material.
Further weight changes related to the absolute kidney weights in the males (decreases in test group 3), the relative testes weights (increase in test groups 2 and 3) and the relative kidney weights of the females (increase in test group 1). In addition, the adrenal weights showed slightly significant changes in the females (decrease in absolute values in test group 1, increase in the relative values in test group 2). The absolute and relative adrenal weights of the males in test group 3 were increased, no correlating findings being observed by light-microscopy. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Macroscopic examination did not reveal any test material-related findings.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Light microscopy showed that the essential changes were to be found in the liver: In test group 1 both the male and female animals exhibited a centrally pronounced hepatocyte hypertrophy. A central hepatocyte hypertrophy was also evident in the males in test group 2 (4 animals), while all the females in this test group and one male had developed a diffuse hypertrophy. A diffuse hypertrophy of the hepatocytes was found in all of the female and male animals in group 3.. In addition, as regards the hypertrophic hepatocytes, comparison with the control animals revealed a granular cytoplasm structure. Moreover, haematoxylin-eosin staining of a paraffin section showed that a cytoplasmic eosinophilia had occurred in test groups 2 and 3, which was distinctly pronounced compared to the control. Signs of hyalin degeneration of liver cells were observed to an isolated extent (1st degree) in two males and to a slight extent (2nd degree) in all the females in dose group 1.
In dose group 2, one male showed signs of a hyalin degeneration to an isolated extent (1st degree) and two females to a slight extent (2nd degree), and all the animals in dose group 3 to a slight to moderate extent (2nd to 3rd degrees).
Focal and multifocal necroses were found in one animal each in groups 1, 2 and 3 of the males and in group 2 of the females. They were interpreted as spontaneous changes. Histological examination further revealed mitotic figures in liver cell nuclei, these being observed in all animals in dose group 3, in one male and 4 females in dose group 2, and in 2 females in dose group 1. Mitotic figures were found in dose group 3, in some animals, with a clearly higher incidence than in dose groups 2 or 1 (females). No mitotic figures were found in the control.
All other histological findings were regarded as being spontaneous or incidental. - Histopathological findings: neoplastic:
- not specified
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Electron microscopic investigation:
The liver specimens originated from material which had originally been intended only for paraffin histological examinations. The necropsy conditions and fixation were planned correspondingly. This led to individual cell compartments showing post-mortem damage which could not be differentiated reliably from degenerative damage possibly having occurred during life.
Nevertheless, the following assessment attempts to describe changes in individual cell compartments, to formulate an opinion and to bring out differences in comparison with hepatocytes of the control animal, even though the evaluation and diagnosis of the substructure was impaired by necropsy and fixation conditions which were unfavorable in this respect.
- Cell nuclei: The nuclei showed chromatin clumping and in some cases chromatin margination with loss of chromatin from other nucleoplasm localisations. These images could come within the framework of a necrobiotic process or be connected to a post-mortem autolysis. Since these non-specific changes found both in the treated animal and in the control animal, these findings were interpreted as signs of incipient, autolytic cell processes which had occurred following the necropsy. However, it was not possible to reliably distinguish between these and nucleic changes which had taken place intra vitam and had their roots in a degeneration. Compared to the control, the hepatocytes of the animal from dose group 3 exhibited nuclei having increased irregularities of the nuclear membrane. However, true invaginations were not yet visible. In addition, isolated lipid-like droplets (intranuclear lipid inclusions?) were observed in the nucleoplasm of the nuclei in this animal whereas, however, lipid droplets in the cytoplasm, as appear regularly in the hepatocytes of the control were not detectable.
- Mitochondria: Most mitochondria in the hepatocytes of the animal from dose group 3 showed hyperplasia of their cristae. Also noticeable were structural changes, as manifested in the formation of lamellar cristae (stacks) oriented longitudinally to the axis of the mitochondria. At the same time, a tubular or vesicular character of the cristae could not be ruled out, it being difficult to make a reliable assessment on the available material. The mitochondria in the hepatocytes of the control animal showed a partly flocculent and moderately osmiophilic matrix in which cristae appeared, isolated or arranged loosely.
The limiting double membrane of the mitochondria in the control animal and the dose group 3 animal was very well developed and was partially lined with granular endoplasmic reticulum.
Isolated cup-shaped or ring-shaped mitochondria also appeared in the hepatocytes of the control and the group 3 animal. Their occurrence is known for "normal" hepatic tissue.
- Smooth endoplasmatic reticulum (SER): Smooth endoplasmic reticulum was found to a varying extent in each hepatocyte. Between the control animal and the animal from dose group 3 there were no essential differences indicative of an increase or decrease in this cell compartment. Furthermore, no degenerative changes which could be reliably distinguished from fixation-related artefacts were observed.
- Rough endoplasmatic reticulum (RER): In the hepatocytes of the control animal RER appeared in the form of prominent, parallel bundles of relatively long tubular sections; parts of the RER were additionally associated with mitochondria, which were completely or partly lined with RER. In contrast to this, parallel tubular bundels of RER were not found in the hepatocytes of the group 3 animal, but only suggestions of a parallel arrangement. Overall, compared to the control, the RER appeared to only a limited extent, often as short, solitary segments, but also partly in association with mitochondria. These RER findings pointed to a reduction of this cell compartment in the cytoplasm of the hepatocytes of the group 3 animal. There were no indications of degranulation and vesiculation. The occurrence of many shorter segments of the RER in the liver cell may be regarded as a sign of fragmentation of the RER, as can occur in the context of degenerative cytoplasm processes.
- Lysosomal activity: The lysosomal activity appeared to be distinctly increased in the liver cells of the group 3 animal. This was seen especially in the increased occurrence of for the most part peribiliary, secondary lysosomes, as a rule autophagosomes in addition to other cytoplasm fractions, mitochondria were also visible in the autophagic vacuoles of the lysosomal system.
Increased incidence of autophagosomes is an expression of increased degenerative processes in the cytoplasm and their elimination from the cell by lysosomal digestion or filtering out.
- Glycogen: Glycogen, an important cytoplasmic inclusion of the liver cell, generally appeared in the form of glycogen rosettes to varying extents in hepatocytes. As a result of unsuitable fixation conditions, hardly any glycogen inclusions were found either in the control or in the animal receiving the highest dose. Thus a comparative assessment of the glycogen content could not be carried out.
- Lipids: Intracytoplasmic lipid storage was observed in the form of medium-sized, non-membrane-bound lipid vacuoles in many hepatocytes in the control, these lipid vacuoles appearing in a loosely-packed form in the hepatocyte cytoplasm. No lipid vacuoles of the same kind were detected in the cytoplasm of the group 3 hepatocytes. This pointed to a decreased lipid storage in the group 3 hepatocytes. Lipid-like droplets were visible only in a few cell nuclei in the hepatocytes of the highest dose group as pseudoinclusions/ inclusions
- Peroxisomes: Peroxisomes appeared to a greatly increased extent in the cytoplasm of the group 3 hepatocytes. The accumulation of peroxisomes was of the same quality both in centrilobular as well as in perilobular cytoplasm areas. The organelles limited by a single membrane exhibited a moderately osmiophilic matrix, in parts flocculent, in parts more homogeneous. In many peroxisomes there were crystalloid inclusion bodies (nucleoids) of varying size and polymorphic structure, partly thread-like, partly lamellar. The outer membrane of the peroxisomes showed marked irregularities in the form of invaginations of small membrane sections, giving these organelles a pleomorphic character. Some small groups of peroxisomes lay in direct contact alongside each other and displayed small, jigsaw-like connections. Short, agranular tubular sections in direct contact with the outer membrane of the proxisomes appeared irregularly. Peroxisomes in the hepatocyte cytoplasm of the control animal were found to only a limited extent.
In summary, the main finding on examination of the hepatocytes from the liver of a group 3 animal was massive accumulation of peroxisomes in the cytoplasm, this being interpreted as a result of peroxisome proliferation. Structural changes in the mitochondria were also evident, as manifested by lamellar and longitudinally organized cristae and a cristae proliferation. The mitochondrial changes could be interpreted as a sign of increased metabolic activity triggered by an increase in the metabolic stress of the cell. This was still an adaptative process. Mitochondrial changes of clearly degenerative causes were not found in the material investigated. However, the marked increase in lysosomal activity in many group 3 hepatocytes, the reduction and fragmentation of the RER and the irregularities of the nuclear membrane were findings which were connected to non-specific, degenerative cell processes. The hepatocyte hypertrophy found on light-microscopic examination was regarded, from a structural point of view, as being caused mainly by the extensive peroxisome accumulations in the cell cytoplasm. - Dose descriptor:
- NOAEL
- Remarks on result:
- not determinable
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the sudy the "no adverse effect level" for male and female mice could not be determined but was estimated to be < 2 700 ppm.
- Executive summary:
The repeated dose toxicity of the test material was assessed according to OECD Test Guideline 407 and in compliance with GLP.
The test material was administered to 30 86C3F1/Cr1Br mice (15 males and 15 females) via the diet over a period of 4 weeks. A group of untreated animals (5 per sex) was used for a control group. In each case 5 animals per sex received the test material in doses of 2 700, 4 500 and 7 000 ppm (test groups 1 - 3). Feed consumption and body weight were determined each week. The animals state of health was checked daily, and once a week they were subjected to an additional exact clinical examination. At the end of the study a clinicochemical and haematological examination was carried out. It can be stated that the dietary administration of the test material to mice for a period of 4 weeks led to the following findings:
7 000 ppm (about 4 110 mg/kg body weight):
- 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 amino-transferase 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, degenerative liver cell changes 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 organisatoric structure (only one male was examined by electron microscopy).
4 500 ppm (about 1 950 g/kg body weight):
- 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, diffuse hypertrophy of liver cells, degenerative liver cell changes and cytoplasmatic eosinophilia as sign of proliferation of the peroxisomes in both sexes.
2 100 ppm (about 890 mg/kg body weight):
- 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, central hypertrophy of liver cells and degenerative liver cell changes in both sexes 2.
The electron microscopic investigation was performed in one male of the control group and in one male of the 7 000 ppm group to demonstrate the cause of the prominent hypertrophy of hepatocytes ultrastructurally. For this purpose the condition was not ideal due to the unfavourable necropsy and fixation conditions. But nevertheless the examinations showed signs of massive proliferation of peroxisomes in the hepatocytes as predominant finding.
In summary, it can be stated that the "no adverse effect level" for male and female mice is below 2 700 ppm (~ 890 mg/kg body weight) and in respect to the former study (Project No.: 50S0047/83075) it is between 900 ppm (~ 220 mg/kg body weight) and 2 700 ppm (~ 890 mg/kg body weight).
- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- The study was used as a range-finder for a sub-chronic toxicity study.
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 17 March 1977 to 14 April 1977
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- The toxicity of the test material was examined in a four-week study with pure-bred beagle dogs as the experimental animals. The study was used as a range-finder for a sub-chronic toxicity study.
- GLP compliance:
- no
- Remarks:
- This study pre-dates the inception of GLP
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: About 3 months old.
- Weight at study initiation: 3 to 5 kg
- Housing: They were individually housed in metabolism cages. The dog and group number were marked on each cage.
- Diet: 50 g food/kg body weight/day. The dogs were fed a basal diet of the following percentage composition: Instant wheat product 25 %, beet pulp 5 %, instant oat product 25 %, B-vitamin preparation 0.2 %, fish meal 10 %, brewer's yeast 3 %, meat scraps 10 %, vitamin AD3EK-preparation 0.3 %, skimmed milk powder 5 %, NaCl+ trace minerals 0.5 %, soyabean-oil meal 8 %, soya bean-oil 5 %, grass meal 3 %. The diets are freshly prepared at the initiation of the study.
- Water: Unfluoridised tap water
- Acclimation period: Two weeks. Animals received an oral anthelmintic treatment (piperazine adipate, 200 mg/kg) twice during the quarantine period.
DETAILS OF FOOD AND WATER QUALITY:
- The nutrient composition and the levels of various contaminants was determined periodically in batches of basal diet. - Route of administration:
- other: Oral in diet or oral capsule.
- Vehicle:
- soya oil
- Remarks:
- (capsule administration)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
BY ADMIXTURE IN THE DIET
- The test material was administered to groups of 1 male and 1 female at dietary levels of 8, 20 or 32 mg/kg body weight. Firstly, a premix of 26.0 g of the teat material in 1274 g basal diet was prepared by blending in a Kenwood mixer for 2 minutes. From this premix the high-dose diet (32 mg/kg body weight) was obtained by diluting 640 g premix with 19 360 g basal diet. The medium-dose diet (20 mg/kg body weight) was obtained by mixing 400 g premix with 19 600 g basal diet, whilst the low-dose diet (8 mg/kg body weight) was obtained by mixing 160 g premix with 19 840 g basal diet. Homogeneity of the test diets was achieved by mixing for 2 minutes in a Lodige mixer.
- The test diets were stored at ambient temperature and were identified by their group number. One control group (one male and one female) received the basal diet without supplement of the test material.
- The stability of the test material in the diets was examined by analysis soon after diet preparation and after one and four weeks of storage.
BY CAPSULE
- Appropriate quantities of the test material were weighed out in capsules no. 00 (Ely Lilly and Cy, Indianapolis, USA) on a Sauter milligram balance, and thereafter the capsules were filled with soya bean oil, and sealed air-tight with a warm concentrated solution of gelatin. The capsules were freshly prepared daily with amounts of the test material based upon the last body weight measurement. Each total daily amount teat material needed for one day to obtain the same intake of the teat material aa the dogs of the corresponding groups receiving the test material by dietary administration. The dogs received their capsule each day, one hour after feeding, 7 days a week for a period of 4 weeks.
- The control dogs received an equal amount of soya bean oil/kg body weight/day. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- -The stability of the test material in the diets was examined by analysis soon after diet preparation and after one and four weeks of storage.
- Quantities of the test material recovered in the respective diet samples taken after 7 or 28 days of storage were lower than in food samples taken immediately after preparing. These differences, however, do not necessarily imply that loss of test material occurred during storage. Since the test material is a stable compound and since, moreover, there are practically no differences between the test material contents after 7 and 28 days, it is likely that the apparent losses are the result of analytical errors (10 - 15 %). - Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 8 mg/kg bw/day (nominal)
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Dose / conc.:
- 32 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- One male and one female
- Control animals:
- yes, concurrent vehicle
- yes, plain diet
- Details on study design:
- - Dose selection rationale: Doses were selected to examine the toxicity of the test material and decide on the doses to be used in the sub-chronic toxicity study.
- Positive control:
- No
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- General appearance, condition, health and behaviour of all dogs were checked daily. Special attention was paid to the buccal mucosa and eyelids.
BODY WEIGHT: Yes
- The weight of each dog was recorded daily for one week pre-dosing and for the first week on test and thereafter twice weekly. The weighing’s were generally done when the animals had food available.
FOOD CONSUMPTION: Yes
- The quantity of food consumed by each dog was recorded daily and the mean weekly intake per dog was calculated.
OPHTHALMOSCOPIC EXAMINATION: Yes
- At the beginning and at days 25 - 27 the eyes of all dogs were examined by indirect ophthalmoscopy.
HAEMATOLOGY: Yes
- Each dog was examined at the beginning and at days 25 - 27. Examinations included:
1. Haemoglobin concentration - by cyanmethemoglobin method of Van Kampen and Zijlstra (Clin. Chim. Acta 6 (1961) 538-544).
2. Packed cell volume - as microhaematocrit.
3. Erythrocyte count - by Coulter Counter.
4. Leucocyte count - by Coulter Counter.
5. Differential leucocyte count - by direct visual count of smear after May-Grunwald-Giemsa staining.
6. Platelet count – by Coulter Counter.
7. Blood clotting time - using Normotest reagents (Byegard and Co., Oslo, Norway).
CLINICAL CHEMISTRY: Yes
- Each dog was examined at the beginning and at days 25 - 27. Examinations included:
1. SGPT - by Reitman and Frankel (Am. J. Clin. Path. 28 (1957) 56-63).
2. SGOT - by Reitman and Frankel.
3. SAP - by Bessey, Lowry and Brock (J. Biol. Chem. 164, (1946) 321-329).
4. Total serum protein - biuret reaction.
5. Serum albumin - De Leeuw-Israel, Arp-Neefjes and Hollander (Exptl. Geront.3 (1967) 255-260).
6. Serum electrophoresis- by agar gel electrophoretic method on microscopic slides of Wieme.
7. BUN- by the automated phenazone/diacetyl monoxime technique of Ceriotti and Spandrio (Clin. Chim. Acta 11 (1965) 519-522).
8. Fasting plasma glucose - by Technicon Auto-Analyzer, method N-9a.
9. Bilirubin - Ishida and Nobuoka (Clin. Chim. Acta .19 (1968) 249-255).
10. Serum electrolytes Na+ and K+ - Paschen and Fuchs ( Clin. Chim. Acta 35, (1971) 401-408).
11. OCT - Kontinnen (Clin. Chim. Acta 18 (1967) 147-150).
URINALYSIS: Yes
- Each dog was examined at the beginning and at days 25 - 27. Individual urine samples were collected after overnight deprivation of water and food and examined for:
1. Specific gravity - by refractometer.
2 Electrolytes Na+ and K+ - method of Paachen and Fuchs (Clin. Chim. Acta 35 (1971) 401-408).
The following semi-quantitative tests were made:
1) pH
2) Protein
3) Sugar
4) Occult blood
5) Ketones
6) Microscopy of the sediment - after centrifuging at 3 000 rpm for 3 minutes. Deposits were examined for: Erythrocytes, leucocytes, epithelial cells, crystals, casts, bacteria, sperm cells, and worm eggs. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- On completion of 28 days of treatment, all surviving dogs were anaesthetised by intravenous administration of Nembutal® and exsanguinated by cutting the carotid artery. The dogs were subjected to a thorough autopsy. The following organs from all animals were dissected free of fat and weighed: Liver, kidneys, spleen, thymus, heart, brain, lung, thyroid, adrenals, pituitary’s, testicles, ovaries, and prostate.
- Samples of the following tissues from all dogs were preserved in a 4 % neutral, phosphate buffered formaldehyde solution: Liver, jejunum, kidneys, spleen, ileum, caecum, thymus, heart, brain, lung, thyroid, adrenals, pituitary, testicles, ovaries, prostate, salivary glands (3), trachea, diaphragm, gall bladder, buccal mucosa, lymph nodes (cervical, popliteal, mesenteric), oesophagus, stomach, duodenum, colon, urinary bladder, ureter, uterus, nervus ischiadicus, spinal cord, pancreas, tongue, skin, anal sac, circumanal glands, thoracic aorta, abdominal aorta, skeletal muscle, mammary gland, bone marrow, bone and eye with optic nerve.
- Bone marrow smears were prepared and fixed in methanol.
HISTOPATHOLOGY: Yes
- Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. Microscopic examination was restricted to the following tissues: Liver, kidneys, stomach, buccal mucosa, bone marrow, spleen, testes and thymus. - Other examinations:
- FAECAL BLOOD CONTENT
- During 7 consecutive days pre-dosing and daily during the first and fourth weeks of treatment the faeces sample of all dogs were examined for the presence of occult blood. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No abnormalities in behaviour, health, condition or general appearance were observed in any of the groups. Although special attention was paid to changes of the buccal mucosa and eyelids no grossly visible abnormalities were seen.
- Mortality:
- no mortality observed
- Description (incidence):
- No mortality occurred.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- All dogs gained weight during the study. No obvious differences were observed in bodyweight gain amongst the groups.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- Ophthalmoscopy did not reveal any changes in the eyes.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At week four, relatively high numbers of thrombocytes were observed in all test groups. It may be mentioned that this parameter shows a wide variation in the dogs used (range at our institute 129 - 511/mm^3). Since moreover the increases were not dose-related no toxicological significance is attached to this finding. There were no indications that the other haematological parameters investigated were affected by the test material.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no indications that the biochemical blood values were influenced by the ingestion of the test material. SAP values were generally very high, at both stages for dogs of this age, but no explanation can be given for this finding.
- Endocrine findings:
- not examined
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Na+ and K+ concentrations in the urine samples showed very wide individual variation both between different stages and between individual dogs. In view of this variation no toxicological importance is attached to the apparent tendency of the mean K+ concentrations to decrease at increasing level of treatment. At week 4, the Na+ concentrations in urine of the test dogs were higher than in urine of the controls. These differences were, however, not dose-related, and moreover the N+ concentrations were very low in urine of the control group. Therefore no toxicological significance was attached to this finding.
- Control dog D7-4 showed an extremely high specific gravity of the urine at week 4, which increased considerably the mean value of the group. At the same stage, urine from three of the four dogs of the top-dose group was found to be rather low in specific gravity.
- The large amounts of crystals and bacteria found at week 0 are attributed to the relatively long period during which samples were collected. - 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 relative kidney weights tended to increase with increasing intake levels of the test material. Both the relative and the absolute weights of the pituitary gland were decreased in the top-dose group. The weights of the remaining organs shoved a normal variation.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - At autopsy no gross abnormalities attributable to the test material were observed. Careful examination of the buccal mucosa and gingivae showed no changes, bleedings or ulcerations in any of the dogs.
- In the following dogs gross lesions were observed:
male, no. D7-13 (low-dose group) - greyish-white nodules in the lungs (visceral larva migrans);
male, no. D7-12 (low-dose group) - pale kidneys;
male, no. D7-11 (high-dose group) - greyish-white nodules in the lungs (visceral larva migrans);
female, no. 7-21 (control group) - a few petechia in the mucosa of the urinary bladder;
female, no. 7-16 (low-dose group) - pale kidneys;
female, no. 7-15 (high-dose group) - pale kidneys. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Microscopic examination of the liver, kidneys, spleen, stomach, testicles, buccal mucosa, thymus and bone marrow did not reveal abnormalities that could be related to treatment.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- FAECES
- Minimal quantities of occult blood were occasionally found in all groups during the last week of the quarantine period and during the first week of the treatment period. The quantities and frequencies however, were about equally distributed between the different groups. During the last week of the treatment period no occult blood was observed in any of the groups. - Details on results:
- The administration of the test material to young beagle dogs either by capsule or by inclusion in the diet did not induce any major abnormalities. Some slight changes observed cannot be easily interpreted as a result of the small number of dogs used.
Since there were no obvious differences in the effects induced by the two ways of administering the test material, the mean results of the dogs in the same group have been combined.
The increase in the relative weight of the kidneys was noticeable only in dogs of the top-dose group. In this group all individual kidney weights were above the highest weight in the control group. The specific gravity of urine samples of the dogs of this group were relatively low. These findings point to possible renal damage, but no treatment-related microscopical renal changes were detected.
Likewise, no microscopical changes were found in the pituitary gland of the top-dose animals, although the weights of the organ were relatively low. The latter finding might therefore be without toxicological significance.
The results obtained failed to reveal any noticeable differences in the effects observed by oral administration of the test material either in the diet or by capsule. - Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Conclusions:
- Under the conditions of this study, no noticeable differences in the effects observed by oral administration of the test material either in the diet or by capsule were seen.
- Executive summary:
The toxicity of the test material was examined in a four-week study with pure-bred beagle dogs as the experimental animals. The study was used as a range-finder for a sub-chronic toxicity study.
Four groups (one control group and three test groups) each consisting of two males and two females were used. The groups received respectively 0, 8, 20 or 32 mg/kg test material body weight/day. The test material was administered in two different ways, one male and one female of each group received the test material by admixture in the diet, the other male and female by capsules. All dogs were provided with 50 g diet/kg body weight/day. The study included clinical, haematological, biochemical and pathological observations.
No mortality occurred. General appearance, health, condition or behaviour were not affected by treatment. Opthalmoscopic examination did not reveal changes of the eyes or of their adnexa which could be related to treatment. There was no noticeable effect of the test material on body weights. Haematological and biochemical blood values were comparable in all groups. Urine composition did not indicate any influence of the test material on kidney-function. Incidence and quantity of occult blood in faeces were not influenced by the ingestion of the test material. The relative weight of the kidney was increased, that of the pituitary was decreased in the top-dose group. Gross and microscopic examination was essentially negative.
Under the conditions of this study, no noticeable differences in the effects observed by oral administration of the test material either in the diet or by capsule were seen.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- April to July 1983
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age on the day they were supplied: 29 days
- Age at study initiation: 42 days
- Weight 6 days after they had been supplied: Male animals: 121.9 (112 - 141) g; female animals: 109.1 (101 - 119) g.
- Weight at study initiation: Male animals: 171.5 (157 - 195) g; female animals: 135.3 (124 - 155) g.
- Housing: During the study period the rats were housed in DK III type stainless steel wire mesh cages. Male or female rats of one test group were split up into groups of five. The animals of a group of five were placed in 2 cages identified with the same number. 3 rats were put in one cage and 2 in the other. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (fresh air/ventilation/light) were ensured.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At the age of 35 days, a 7-day adaptation period began during which the animals received ground feed and water ad libitum.
DETAILS OF FOOD AND WATER QUALITY:
- The feed used in the study was assayed for contaminants. On the basis of duration of use and the analytical findings the feed was found to be suitable.
- The drinking water is regularly assayed for contaminants. In view of the aim and duration of the study there are no special requirements exceeding the specification of drinking water.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 - 18.00 o'clock and 12 hours darkness from 18.00 - 6.00 o'clock). - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- After the adaptation period, various amounts of test material, depending on the test group, were added to the diet.
- The mixtures of test material and feed were prepared freshly once a week after their stability had been verified for this period before the beginning of the study. The weighed test material was mixed thoroughly with a small amount of the feed in a beaker using a spatula. Subsequently a premix was prepared in a mixer (MX 32) of BRAUN. This premix was then adjusted to the concentrations desired with the appropriate amounts of feed and mixed in a laboratory mixer of GEBR. LODIGE for about 10 minutes. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analyses of the test material were carried out before the beginning of the study in several analytical laboratories. The content of the active ingredients and impurities was determined.
The correctness of the weighings after 6 and 12 weeks of the study was verified by examining a sample of each concentration analytically. The homogeneity and the stability of the test substance material were determined analytically before the beginning of the study.
The samples were extracted, and the test material concentrations were fractioned by thin layer chromatography and then directly quantitatively determined by U-remission-spectrophotometry.
Results
- The characteristics of the test material (content of active ingredient and impurities) were determined before the beginning of the study. The analyses revealed an degree of purity of 92.7 %. The stability of the test substance was verified.
- Analysis of the test material formulations: Corresponding to the variability of the analytical method (coefficient of variation 1 .8 - 3.8 %) the analyses of the test material formulations carried out provided evidence of the correctness of the actual concentration of the test material in the specific samples. Taking into account the above mentioned variability of the method the stability of the test material formulations over a period of 31 days and the homogeneity of the test material in the test material formulation was verified analytically. - Duration of treatment / exposure:
- 3 months
- Frequency of treatment:
- Continuous (in diet)
- Dose / conc.:
- 0 ppm
- Remarks:
- Test group 0: Control
- Dose / conc.:
- 50 ppm
- Remarks:
- Test group 1
- Dose / conc.:
- 150 ppm
- Remarks:
- Test group 2
- Dose / conc.:
- 450 ppm
- Remarks:
- Test group 3
- No. of animals per sex per dose:
- 15 per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
The object of the study was to establish, by administering the test material orally to rats for 3 months, a dose with marginally toxic effects that can be used as the highest dose in an oncogenicity study over a period of 2 years. The choice of the dose was based on feeding studies with MCPA and the test material as well as with 2,4 DP.
These studies showed that after three months of the administration of 400 ppm test material a reduction of the body weights and an increase in the relative kidney weights occurred. In this investigation the "no effect level" was 50 ppm.
The administration of 400 ppm test material over a period of 7 months resulted in anemic symptoms and an increase in the relative liver and kidney weights. The increase of relative kidney weight was also observed at a dose of 100 ppm.
The most striking finding after the application of 500 ppm 2,4 DP over a period of 3 months was the increase of the relative kidney weights. At a dose level of 100 ppm the increased kidney weights could not be observed.
In accordance with the results of the studies mentioned above, 450 ppm was fixed as the highest dose, 150 ppm as the medium one, and 50 ppm as the lowest dose for the present study.
- Rationale for animal assignment: The rats were split up into the test groups strictly at random. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed each day for any evident signs of toxicity. Each time that the animals were weighed they were also inspected and palpated.
- A check was made for any dead or moribund animals twice each day (Mondays to Fridays) or once each day (Saturdays, Sundays and on holidays).
BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed weekly (Tuesdays). 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:
- In order to determine the amount of food consumed, the contents of the two food bowls of a group of give were weighed together and subtracted from the amount offered. During the adaptation and administration periods, the food consumption was determined weekly.
- The mean amount of test material intake consumed daily (in mg) per kg body weight was calculated exemplary for the 1st, 4th, 8th and 12th test week according to the following formula:
[FC x D] / [(BWx + BWx + 7)/2]
Where:
FC = mean daily food consumption (in g) during one test week (from day x to day x + 7)
D = dose in ppm
BWx = mean body weight at test day x (in g)
BWx + 7 = mean body weight at test day x + 7 (in g)
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At the beginning of the study and at the end of the administration period, the eyes of all the animals were examined with a focusable hand slit lamp for any changes to the refracting media. If changes to the refracting media were suspected or existed, photographs were taken with a KOWA camera.
- Dose groups that were examined: All animals.
BLOOD AND PLASMA EXAMINATION
- The blood required was taken from the orbital venous plexus in an amount of 1.2 - 1.5 mL. In each case, blood sampling took place in the morning between 07.00 and 12.00 o'clock.
- Blood samplings and the subsequent analysis of the blood and plasma samples without the differential-blood-count and reticulocytes were carried out in randomised sequence 43 and 85 days after the beginning of administration (period of administration; blood samplings 1 and 2). The clinicochemical and haematological examinations ware carried out in 10 animals per test group and sex during the period of administration.
HAEMATOLOGY: Yes
- The following parameters were determined using a particle counter: Haemoglobin, erythrocyte count, haematocrit, mean haemoglobin content per erythrocyte, mean cell volume, mean corpuscular haemoglobin concentration, platelet count, leukocyte count.
The reticulocytes were evaulated visually.
CLINICAL CHEMISTRY: Yes
- In this study the following parameters were determined: Total bilirubin, creatinine, urea, sodium, potassium, total protein, glucose, inorganic phosphate, calcium, chloride, cholesterol, albumin.
PLASMA/SERUM HORMONES/LIPIDS: Yes
- Enzymes: In this study the activity of the following enzymes was recorded: Glutamic pyruvic transaminase, alkaline phosphatase, glutamic oxalacetic transaminase.
- Clotting analysis: The following parameter was determined: Hepato Quick Test.
URINALYSIS: Yes
- Time schedule for collection of urine: Individual urine was collected overnight from 10 animals per test group and sex 38 and 80 days after the beginning of treatment (administration period, urine collections 1 and 2).
- The following examinations were carried out: pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, sediment microscopy. With the exception of the sediment examination, all urine constituents were determined semiquantitatively with test strips and a reflecting photometer. The sediment was evaluated microscopically. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- At the end of the study the animals were anaesthetised with CO2 after a fasting period (withdrawal of food and water) of about 16 hours and exsanguinated by decapitation, then necropsied and assessed by gross pathology.
- From all animals body weight (determination in exsanguinated animals) and the weight of liver, kidneys, adrenals, testes/ovaries, and brain was determined.
HISTOPATHOLOGY: Yes
- Of all animals the organs listed were fixed in toto or in representative parts depending on their size in 4 % formaldehyde solution: Heart - aorta - trachea - lungs - esophagus - stomach - duodenum - jejunum - ileum - cecum - colon - rectum - liver - pancreas - spleen - thymus - mesenteric lymph node - sternum with sternal marrow - kidneys - urinary bladder - testes - ovaries - uterus - pituitary - adrenals - thyroids (with parathyroids) - brain - peripheral nerve - skin - all gross lesions (to include a border of apparently normal tissue) - tongue - and possible target organs.
- After fixation processing, the examination by light microscopy and the evaluation of findings were performed using the SNOP-Code (Systematized Nomenclature of Pathology) for histopathological findings. - Statistics:
- Clinical examinations and pathology:
- Mean and standard deviation were calculated for the statistical evaluation of the study for the variables food intake, body weight and body weight change of the animals of each test group; mean, and standard deviation were calculated for the statistical evaluation of the variables (exsanguinated body weight, absolute and relative organ weights) of the animals in each test group.
- The statistical evaluation was carried out using at-test generalised by WILLIAMS for the simultaneous comparison of several dose groups with a control group for the parameters body weight change, absolute and relative organ weights.
Clinical chemistry and haematology:
- Blood and plasma examinations: After statistical correction (NALIMOV criterion), mean and standard error were calculated and tabulated together with the individual values. For the purpose of testing the significance, the individual dose groups were compared with the control group using the t test.
- Urinalysis: The assessment whether certain pronounced characteristics are different in the control and test groups was carried out by means of the chi^2 test in appropriate two-by-two tables taking the Yates correction for continuity into account. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinically evident signs of toxicity that could be attributed to the administration of the test material were found throughout the administration period.
- Mortality:
- no mortality observed
- Description (incidence):
- No animal died during the period of the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- In none of the test groups (male and female rats) was the body weight gain adversely affected throughout the course of the study.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No differences were evident between the control animals of both sexes and the animals of test group 1 - 3 as regards food consumption.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No impairment of the refracting media was revealed by the ophthalmoscopy carried out with the hand slit lamp either before the administration of the test material or after 12 weeks of the study. Nor did the examinations carried out in cases of doubt with the KOWA camera show any changes to the refracting media or the ocular fundus that had been induced by the test material .
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In many cases the significant deviations from the values of the control group cannot be considered to be induced by the test material. Plausibility criteria have been introduced in order to avoid a detailed assessment and discussion of each individual statistically significant deviation for each parameter. The purpose of these criteria is to give all the reasons why the deviations observed are of no relevance with reference to the administration of the test material or why any relevance is improbable. All the parameters for which a test material-induced change is even merely suspected are assessed below.
Creatinine:
At the end of the 3-month administration period increased creatinine values were recorded in the plasma of the female animals in test group 3 (450 ppm), these being no doubt attributable to the administration of the test material . The cause of the increase in creatinine is probably a renal impairment. Whereas the other clinicochemical and urinalytical examinations did not reveal any findings that could support such clinical symptoms, the gross pathological findings showed indications of a a test material - related effect to the kidney of the test material in the form of increased kidney weights
Glucose:
Reduced glucose concentrations were found in the plasma of the male animals in test group 3 (450 ppm) after blood sampling 2 (3 months after the beginning of administration). The question whether this finding is induced by the test material or is of an incidental nature cannot be answered, since a reduction in the glucose values was observed in only one sex and only after one blood sampling. Moreover, it is difficult to assign the isolated finding of a drop in glucose to a specific syndrome.
Other parameters:
To sum up, it can be said that the repeated administration of 450 ppm test material caused an increase in the creatinine concentration in the plasma of the female animals, which is presumably attributable to a renal impairment. In the case of the male animals, reduced glucose values were recorded after the administration of 450 ppm test material which cannot be explained pathogenetically.
The administration of 50 ppm and 150 ppm test material did not lead to any changes, whose causes can be related to the administration of the substance. - Endocrine findings:
- not examined
- 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):
- The main findings in this study were significantly increased absolute and relative kidney weight as well in the male rats (dose groups 2 and 3) as in the female (absolute weight in dose group 3 only and dose groups 2 and 3 in the relative kidney weight). These findings are interpreted as test material related ones although no alterations could be detected histopathologically that could be related to the increase of the kidney weights.
All other findings are incidental or single findings of spontaneous origin that - from the obtained findings - are not relatable to the application of the test test material. - Gross pathological findings:
- effects observed, treatment-related
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not examined
- Details on results:
- On the basis of the clinicochemical and haematological results, the no adverse effect level both for male and for female rats should be in a range of between 150 ppm and 450 ppm.
After the oral administration of 50 ppm, 150 ppm and 450 ppm respectively the no-adverse-effect-level is between 50 ppm and 150 ppm from the pathological point of view. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 50 - <= 150 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- gross pathology
- Dose descriptor:
- NOAEL
- Effect level:
- >= 150 - <= 400 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical biochemistry
- haematology
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of this study the no-adverse-effect-level (NOAEL) is between 50 ppm and 150 ppm from the pathological point of view.
- Executive summary:
The sub-chronic repeated dose toxicity of the test material was assessed according to OECD Test Guideline 408 and in compliance with GLP.
The test material was administered to 90 Wistar rats (45 male, 45 female) via their diet for 3 months. For comparison, a group of untreated animals (15 per sex) was used as control. The doses were 0, 50, 150, 450 ppm.
Food consumption and body weight were determined once a week; the rats' state of health was checked each day. At the beginning of the study and toward the end of the administration period all the animals were subjected to ophthalmoscopic examinations. Two clinicochemical and haematological examinations as well as 2 urinalyses were carried out. All animals were subjected to a gross-pathological examination. Finally an extensive histopathological examination was carried out.
The following findings were obtained and assessed or discussed in relation to the test material.
450 ppm group:
- Haematological and clinicochemical findings: Increase in the creatinine values in the plasma of female rats.
- Organ weights: Increase of absolute and relative kidney weights in the animals of both sexes.150 ppm group:
- Organ weights: Increase of absolute and relative kidney weights in the male animals, increase of relative kidney weights in the female animals.
50 ppm group:
- No changes to which a material-induced relevance can be attributed.
The findings that occured in the animals of the 450 ppm group indicate a renal impairment in the male und female animals. An increase of the absolute and relative kidney weights could also be shown in the 150 ppm group.
Under the conditions of this study the no-adverse-effect-level (NOAEL) is between 50 ppm and 150 ppm from the pathological point of view.
- Endpoint:
- chronic toxicity: oral
- Remarks:
- Combined repeated dose and carcinogenicity
- Type of information:
- experimental study
- Adequacy of study:
- key 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. Therefore 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 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. Therefore 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
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.
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 |
Ophthalmoscopic Changes
Animal No. and Sex |
Mg/kg bw/day |
Changes Observed |
At Week(s) |
7.321 M |
Control |
Slight reddening of the conjunctivae |
1 |
7.314 M |
Control |
Idem |
7 |
7.382 F |
Control |
Idem |
1 |
7.304 F |
Control |
Idem |
7 and 13 |
7.386 M |
4 |
Pale conjunctivae |
7 |
7.309 F |
4 |
Hypertrophy of the membrana nictitans |
1, 7 and 13 |
7.349 F |
16 |
Vascular injection in the cornea |
1 |
7.364 M |
64 |
Pale conjunctivae |
7 |
7.368 M |
64 |
Small corneal ulcer |
13 |
7.392 F |
64 |
Slight reddening of the conjunctivae |
7 |
Mean Body Weights and Mean Weight Gain
Mg/kg bw/day |
Body Weight (in kg) at Week |
Weight Gain (kg) |
||||||
0 |
2 |
4 |
6 |
8 |
10 |
13 |
1-13 |
|
Males |
||||||||
0 |
8.1 |
8.6 |
9.4 |
10.2 |
10.9 |
12.0 |
12.9 |
4.8 |
4 |
7.6 |
8.0 |
8.9 |
9.6 |
10.3 |
11.1 |
11.8 |
4.2 |
16 |
7.8 |
8.3 |
8.9 |
9.6 |
10.3 |
10.8 |
11.8 |
4.0 |
64 |
8.0 |
7.8 |
8.1 |
8.6 |
8.6 |
9.0 |
9.9 |
1.9 |
Females |
||||||||
0 |
6.4 |
6.6 |
7.1 |
7.5 |
8.1 |
8.4 |
9.2 |
2.8 |
4 |
6.3 |
6.3 |
6.7 |
7.2 |
7.8 |
8.1 |
8.9 |
2.6 |
16 |
6.3 |
6.2 |
6.5 |
7.0 |
7.4 |
7.5 |
8.3 |
2.0 |
64 |
6.4 |
6.2 |
6.4 |
6.7 |
7.1 |
7.4 |
8.1 |
1.7 |
Concentrations of Test Material Recovered in the Diets
Group (mg/kg bw/day) |
Concentration of Test Material (in ppm) in Diet Samples Taken After Storage at Day |
Nominal Concentration (ppm) at Day |
|||
0 |
7 |
28 |
1 (2)) |
43 and 84 (3)) |
|
4 |
101 (89) |
97 (95) |
95 (93) |
113 |
102 |
16 |
437 (96) |
411 (104) |
370 (93) |
454 |
397 |
64 |
1840 (101) |
1537 (97) |
1602 (101) |
1814 |
1587 |
() in brackets the percentages recovered
2) based on a food intake of 35 g/kg body weight/day
3) based on a food intake of 40 g/kg body weight/day
Mean Haematological Indices
Mg/kg bw/day |
Clotting Time (sec.) |
Hb (m mol/L) |
Packed Cell Volume (L/L) |
RBC ((x10^12 /)L) |
WBC ((x10^9 /)L) |
Thrombocytes ((x10^9 /)L) |
Differential Count (%) |
||||
Lymph |
Neutr |
Mono |
Eos |
Bas |
|||||||
Week 0 |
|||||||||||
0 |
26 |
8.1 |
0.411 |
5.0 |
13.2 |
335 |
44 |
53 |
1 |
2 |
0 |
4 |
26 |
8.1 |
0.406 |
5.1 |
14.8 |
281 |
40 |
54 |
2 |
5 |
0 |
16 |
26 |
8.3 |
0.411 |
5.1 |
15.1 |
323 |
41 |
55 |
1 |
3 |
0 |
64 |
26 |
8.0 |
0.397 |
5.0 |
13.1 |
415 |
41 |
56 |
1 |
2 |
0 |
Week 6 |
|||||||||||
0 |
30 |
8.7 |
0.436 |
5.6 |
12.4 |
274 |
37 |
53 |
2 |
8 |
0 |
4 |
28 |
8.6 |
0.424 |
5.5 |
12.7 |
323 |
38 |
55 |
2 |
5 |
0 |
16 |
27 |
8.0 |
0.404* |
5.1* |
12.8 |
368* |
33 |
60 |
7 |
1 |
0 |
64 |
29 |
6.7*** |
0.330*** |
4.2*** |
14.7 |
324 |
25* |
66* |
1 |
9 |
0 |
Week 13 |
|||||||||||
0 |
25 |
9.3 |
0.456 |
6.2 |
13.9 |
270 |
39 |
55 |
1 |
4 |
0 |
4 |
25 |
9.3 |
0.444 |
6.2 |
14.0 |
258 |
40 |
55 |
1 |
4 |
0 |
16 |
24 |
9.0 |
0.436 |
5.9 |
16.1 |
292 |
41 |
56 |
0 |
3 |
0 |
64 |
24 |
7.8** |
0.371*** |
5.1** |
14.6 |
329 |
32 |
63 |
1 |
4 |
0 |
Hb = haemoglobin content
RBC = red blood cell count
WBC = white blood cell count
* P<0.05; ** P<0.01; *** P<0.001 according to Wilcoxon test
Mean Biochemical Parameters
Mg/kg bw/day |
Glucose (m mol/L) |
Urea (m mol/L) |
GOT (U/L) |
GPT (U/L) |
AP (U/L) |
TP (g/L) |
Albumin (g/L) |
Week 0 |
|||||||
0 |
7.0 |
2.2 |
3.9 |
4.0 |
59 |
60 |
32 |
4 |
6.9 |
2.6 |
4.1 |
4.6 |
57 |
57** |
31 |
16 |
6.8 |
2.8 |
4.0 |
4.1 |
54 |
59 |
32 |
64 |
7.0 |
2.6 |
3.6 |
3.3 |
53 |
59 |
32 |
Week 6 |
|||||||
0 |
6.2 |
3.4 |
6.4 |
8.5 |
46 |
54 |
29 |
4 |
6.5 |
4.0 |
6.1 |
8.0 |
46 |
54 |
27 |
16 |
6.4 |
3.8 |
5.6 |
7.7 |
39 |
53 |
26 |
64 |
5.9 |
5.4** |
5.0 |
6.6 |
33*** |
49** |
23** |
Week 13 |
|||||||
0 |
6.3 |
4.5 |
6.9 |
8.1 |
43 |
60 |
32 |
4 |
6.5 |
4.5 |
6.5 |
7.8 |
42 |
62 |
33 |
16 |
6.4 |
4.5 |
5.3 |
6.2 |
39 |
60 |
32 |
64 |
5.0 |
5.6* |
5.5 |
8.8 |
35 |
56 |
31 |
Urea = urea nitrogen
GOT = glutamic-oxalacetic transaminase
GPT = glutamic-pyruvic transaminase
AP = alkaline phosphatase
TP = total protein
* P < 0. 05; ** P <0.01; *** P < 0.001 according to Wilcoxon
Mean Biochemical Parameters Continued
Mg/kg bw/day |
Na in Plasma (mg/100 mL) |
K in Plasma (mg/100 mL) |
Billirubin (Umol/L) |
OCT (U/L) |
Creatinine (Umol/L) |
Week 0 |
|||||
0 |
350 |
16.3 |
1.6 |
6.7 |
59 |
4 |
351 |
16.2 |
1.7 |
6.9 |
56 |
16 |
350 |
15.7 |
1.8 |
6.4 |
57 |
64 |
352 |
16.3 |
1.8 |
6.5 |
57 |
Week 6 |
|||||
0 |
351 |
16.9 |
1.6 |
4.8 |
64 |
4 |
327 |
16.2 |
1.4 |
4.8 |
69 |
16 |
337 |
16.3 |
1.2* |
4.6 |
60 |
64 |
328 |
16.0 |
1.6 |
4.4 |
59 |
Week 13 |
|||||
0 |
372 |
18.0 |
1.3 |
7.9 |
71 |
4 |
371 |
17.3 |
1.2 |
8.2 |
69 |
16 |
378 |
18.0 |
1.3 |
7.5 |
64 |
64 |
386 |
18.8 |
1.5 |
8.0 |
67 |
OCT = ornithine carbamoyl transferase
*P<0.05 , according to Wilcoxon test
Mean Percentages of Globulins
Mg/kg bw/day |
Week 0 |
Week 6 |
Week 13 |
||||||
α |
β |
γ |
α |
β |
γ |
α |
β |
γ |
|
0 |
49 |
31 |
20 |
51 |
33 |
16 |
51 |
32 |
17 |
4 |
51 |
29 |
20 |
51 |
34 |
15 |
53 |
31 |
17 |
16 |
51 |
29 |
20 |
51 |
34 |
15 |
53 |
32 |
15 |
64 |
49 |
31 |
20 |
53 |
32 |
15 |
53 |
31 |
16 |
Kidney and Liver Function Tests
Mg/kg bw/day |
Specific Gravity at the End of Week |
Phenol Red (µg/100 mL) |
BSP Retention (%) |
||
0 |
6 |
13 |
12 |
12 |
|
0 |
1.0258 |
1.0412 |
1.0263 |
41 |
3.0 |
4 |
1.0248 |
1.0355 |
1.0257 |
38 |
- |
16 |
1.0284 |
1.0403 |
1.0293 |
44 |
- |
64 |
1.0324 |
1.0352 |
1.0238 |
74** |
6.2* |
BSP = bromosulphophthalein
* P < 0.05; ** P < 0.01 according to Wilcoxon test
Mean Na and K Content in Urine
Mg/kg bw/day |
Na |
K |
Na |
K |
Na |
K |
In Urine (mg %) |
||||||
Week 0 |
Week 6 |
Week 12 |
||||
0 |
247 |
227 |
432 |
502 |
253 |
209 |
4 |
267 |
244 |
614* |
612 |
337 |
203 |
16 |
398 |
227 |
545 |
556 |
303 |
328* |
64 |
405 |
246 |
532 |
529 |
238 |
241 |
* p < 0.05 according to Wilcoxon test
Mean Relative Organ Weights (in g/100 g body weight)
Mg/kg bw/day |
Heart |
Kidneys |
Liver |
Spleen |
Brain |
Testes |
Ovaries |
Pituitary |
Thyroid |
Adrenals |
Lungs |
Thymus |
Prostate |
0 |
0.80 |
0.49 |
3.52 |
0.22 |
0.69 |
0.14 |
0.0077 |
0.00065 |
0.0084 |
0.0109 |
0.87 |
0.18 |
0.039 |
4 |
0.82 |
0.49 |
3.52 |
0.21 |
0.67 |
0.15 |
0.0077 |
0.00069 |
0.0088 |
0.0106 |
0.85 |
0.15 |
0.020 |
16 |
0.81 |
0.48 |
3.72 |
0.20 |
0.78 |
0.14 |
0.0100 |
0.00067 |
0.0091 |
0.0120 |
0.95 |
0.14 |
0.032 |
64 |
0.94* |
0.59** |
4.40*** |
0.24 |
0.87* |
0.14 |
0.0091 |
0.00076 |
0.0095 |
0.0113 |
1.01* |
0.12 |
0.033 |
* P < 0.05; ** P < 0.01; *** P < 0.001 according to Wilcoxon test
Concentrations of Test Material Recovered in the Diets
Group |
Concentration of Test Material (in ppm) Recovered in Diet Samples Taken After Storage at Day |
Intended Concentration (ppm) |
||
0 |
7 |
28 |
||
Low Dose |
159 |
127 |
138 |
160 |
Mid Dose |
371 |
340 |
350 |
400 |
High Dose |
658 |
562 |
552 |
640 |
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
- 1.1 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
- System:
- urinary
- Organ:
- 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:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- 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.
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- Read-across to structurally similar supporting substance.
- Reason / purpose for cross-reference:
- read-across source
- 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
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- A single study conducted in an analogue read across substance conducted according to standardised guidelines and in compliance with GLP.
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:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- 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.
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- Read-across to structurally similar supporting substance.
- Reason / purpose for cross-reference:
- read-across source
- 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
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
Additional information
7 -Week Range Finding Study: Kirsch et al. (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 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.
4-Week Range Finding Study: Schilling (1990a).
The short-term repeated dose toxicity of the test material was assessed according to OECD Test Guideline 407 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 material was administered to 40 B6C3F1 mice (20 males and 20 females, 15 males and 15 females respectively) via the diet over a period of 4 weeks. A group of untreated animals (5 per sex) was used for a control group.
In each case 5 animals per sex received the test material in doses of 100, 300, 900 and 2 700 ppm.
Feed consumption and body weight were determined each week. The animals state of health was checked daily, and once a week they were subjected to an additional exact clinical examination.
At the end of the study a clinicochemical and haematological examination was carried out.
It can be stated that the dietary administration of the test material to mice for a period of 4 weeks led to the following findings:
2 700 ppm (about 780 mg/kg body weight):
- 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 a central hypertrophy of the hepatocytes in both sexes
900 ppm (about 220 mg/kg body weight), 300 ppm (about 70 mg/kg body weight) and 100 ppm (about 25 mg/kg body weight):
- no test material-induced changes
The evaluation of the results indicate that the "no adverse effect level" for male and female mice is between 900 ppm (about 220 mg/kg body weight) and 2 700 ppm (about 780 mg/kg body weight).
However, the assessment of the results shows that no clear toxic effect was detected that meets the EPA "MTD-criteria", i.e. body weight reduction of about 10 % or distinct organ toxicity among others which is necessary for the dose level selection for the carcinogenicity study. In order to determine the target organs and to facilitate the dose level selection for the carcinogenicity study a further 4-week study in mice with the dose levels of 2 700, 4 500 and 7 000 ppm will be carried out.
Under the conditions of the study the "no adverse effect level" for male and female mice is between 900 ppm (~ 220 mg/kg body weight) and 2 700 ppm (~ 890 mg/kg body weight).
4-Week Range Finding Study: Schilling (1990b).
The repeated dose toxicity of the test material was assessed according to OECD Test Guideline 407 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 material was administered to 30 86C3F1/Cr1Br mice (15 males and 15 females) via the diet over a period of 4 weeks. A group of untreated animals (5 per sex) was used for a control group. In each case 5 animals per sex received the test material in doses of 2 700, 4 500 and 7 000 ppm (test groups 1 - 3). Feed consumption and body weight were determined each week. The animals state of health was checked daily, and once a week they were subjected to an additional exact clinical examination. At the end of the study a clinicochemical and haematological examination was carried out. It can be stated that the dietary administration of the test material to mice for a period of 4 weeks led to the following findings:
7 000 ppm (about 4 110 mg/kg body weight):
- 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 amino-transferase 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, degenerative liver cell changes 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 organisatoric structure (only one male was examined by electron microscopy).
4 500 ppm (about 1 950 g/kg body weight):
- 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, diffuse hypertrophy of liver cells, degenerative liver cell changes and cytoplasmatic eosinophilia as sign of proliferation of the peroxisomes in both sexes.
2 100 ppm (about 890 mg/kg body weight):
- 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, central hypertrophy of liver cells and degenerative liver cell changes in both sexes 2.
The electron microscopic investigation was performed in one male of the control group and in one male of the 7 000 ppm group to demonstrate the cause of the prominent hypertrophy of hepatocytes ultrastructurally. For this purpose the condition was not ideal due to the unfavourable necropsy and fixation conditions. But nevertheless the examinations showed signs of massive proliferation of peroxisomes in the hepatocytes as predominant finding.
In summary, it can be stated that the "no adverse effect level" for male and female mice is below 2 700 ppm (~ 890 mg/kg body weight) and in respect to the former study (Project No.: 50S0047/83075) it is between 900 ppm (~ 220 mg/kg body weight) and 2 700 ppm (~ 890 mg/kg body weight).
Sub-Chronic Repeated Dose Toxicity: Kirsch et al. (1985).
The sub-chronic repeated dose toxicity of the test material was assessed according to OECD Test Guideline 408 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 material was administered to 90 Wistar rats (45 male, 45 female) via their diet for 3 months. For comparison, a group of untreated animals (15 per sex) was used as control. The doses were 0, 50, 150, 450 ppm.
Food consumption and body weight were determined once a week; the rats' state of health was checked each day. At the beginning of the study and toward the end of the administration period all the animals were subjected to ophthalmoscopic examinations. Two clinicochemical and haematological examinations as well as 2 urinalyses were carried out. All animals were subjected to a gross-pathological examination. Finally an extensive histopathological examination was carried out.
The following findings were obtained and assessed or discussed in relation to the test material.
450 ppm group:
- Haematological and clinicochemical findings: Increase in the creatinine values in the plasma of female rats.
- Organ weights: Increase of absolute and relative kidney weights in the animals of both sexes.
150 ppm group:
- Organ weights: Increase of absolute and relative kidney weights in the male animals, increase of relative kidney weights in the female animals.
50 ppm group:
- No changes to which a material-induced relevance can be attributed.
The findings that occured in the animals of the 450 ppm group indicate a renal impairment in the male und female animals. An increase of the absolute and relative kidney weights could also be shown in the 150 ppm group.
Under the conditions of this study the no-adverse-effect-level (NOAEL) is between 50 ppm and 150 ppm from the pathological point of view.
Sub-Chronic Repeated Dose Toxicity: Reinart (1977).
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.
Sub-Chronic Repeated Dose Toxicity: Rondot et al. (1978).
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.
Chronic Repeated Dose Toxicity: Kuehborth (1988).
The 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. Therefore 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).
4 -Week Range-Finder: Reuzel et al. (1977)
The toxicity of the test material was examined in a four-week study with pure-bred beagle dogs as the experimental animals. The study was used as a range-finder for a sub-chronic toxicity study. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Four groups (one control group and three test groups) each consisting of two males and two females were used. The groups received respectively 0, 8, 20 or 32 mg/kg test material body weight/day. The test material was administered in two different ways, one male and one female of each group received the test material by admixture in the diet, the other male and female by capsules. All dogs were provided with 50 g diet/kg body weight/day. The study included clinical, haematological, biochemical and pathological observations.
No mortality occurred. General appearance, health, condition or behaviour were not affected by treatment. Opthalmoscopic examination did not reveal changes of the eyes or of their adnexa which could be related to treatment. There was no noticeable effect of the test material on body weights. Haematological and biochemical blood values were comparable in all groups. Urine composition did not indicate any influence of the test material on kidney-function. Incidence and quantity of occult blood in faeces were not influenced by the ingestion of the test material. The relative weight of the kidney was increased, that of the pituitary was decreased in the top-dose group. Gross and microscopic examination was essentially negative.
Under the conditions of this study, no noticeable differences in the effects observed by oral administration of the test material either in the diet or by capsule were seen.
Repeated dose toxicity: oral 13 week. Reuzel (1979)
A sub-chronic (90-day) feeding study with the test material was performed in beagle dogs fed diets containing the test material at levels which provided intakes of 0, 4, 16 or 64 mg/kg body weight/day. The study was similar in design to OECD 409. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Health, condition and behaviour were generally not affected by the test material. One dog of the top-dose group showed ulcera in the buccal mucosa, a purulent conjunctivitis, pale mucous membranes and a poor condition. Gain in body weight was markedly decreased in dogs of the top-dose group and slightly decreased in dogs of the intermediate-dose group. Food consumption was decreased only in one dog of the top-dose group. Haemoglobin content, packed cell volume and red blood cell counts were decreased in the top-dose group. Rather low value occurred in the intermediate-dose group. In the top-dose group there were moderate increases in serum urea values. Phenol-red, and BSP-retention were increased in the top-dose group. The pH of the urine was generally slightly higher in dogs of the two highest dose groups than in controls. On ophthalmoscopical examination no treatment-related changes were observed. A purulent conjunctivitis was seen in one dog of the top-dose group.
The relative weights of the heart, kidneys, and liver were increased in the top-dose group. On gross examination brown discoloured fat tissue in pericard and/or mesenterium was observed in three dogs of the top-dose group. Microscopic examination was essentially negative.
Under the conditions of this study, the NOAEL was 4 mg/kg bw/day.
Short-Term Repeated Dose Toxicity Via the 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 2 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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