Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Description of key information
Chronic study; oral (diet), mouse (CD-1; 50/sex/dose), OECD TG 453, GLP;
NOAEL(males) = 2 mg a.i./kg bw/day, LOAEL(females) = 4 mg a.i./kg
bw/day; no NOAEL identified for females
Subchronic study; oral (gavage), rat (Sprague Dawley, Crl: CD(SD)BR;
10/sex/dose), EPA OPP 82-1, GLP; NO(A)EL = 2.5 mg a.i./kg bw/day
Subchronic study; oral (diet), mouse (CD-1; 10/sex/dose), OECD TG 453, GLP (dose-range finder for Combined chronic toxicity/carcinogenicity study); NOAEL = 12 mg a.i./kg bw/day
Subchronic study; oral (gavage), dog (Beagle; 4/sex/dose), OECD TG 409,
GLP; NOAEL = 5 mg a.i./kg bw/day
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:
- chronic toxicity: oral
- Remarks:
- combined repeated dose and carcinogenicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
- Version / remarks:
- 1988
- Deviations:
- yes
- Remarks:
- urinalysis is missing.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Version / remarks:
- adopted May 12, 1981
- Deviations:
- yes
- Remarks:
- urinalysis is missing.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- mouse
- Strain:
- other: CD®-1/Crl:CD1(lcr)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 37–42 days
- Weight at study initiation: 15.9–23.3 g (male) / 14.8–21.5 g (females)
- Housing: individually in MAKROLON cages (type I)
- Diet: commercial ssniff® R/M-H V1530 diet, ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
Test concentration, homogeneity and stability of the test substance in the diet were analysed several times during the study period. All diets were freshly prepared once a week and stored at ambient temperature. - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- Groups 1 (m+f), 2 (m+f), 3 (m): 78 weeks
In the following groups administration was discontinued from the test week stated below as a mortality rate of 70 % was reached:
Group 3 (f): 75 weeks
Group 4 (m): 67 weeks
Group 4 (f): 66 weeks - Frequency of treatment:
- Daily
- Dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- Remarks:
- Chronic study (males)
corresponding to 2 mg a.i./kg bw/day - Dose / conc.:
- 30 mg/kg bw/day (actual dose received)
- Remarks:
- Chronic study (males)
corresponding to 6 mg a.i./kg bw/day - Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Chronic study (males): reduced in several steps to 35 mg/kg bw/day due to high mortality; corresponding to 20 (7) mg a.i./kg bw/day
- Dose / conc.:
- 20 mg/kg bw/day (actual dose received)
- Remarks:
- Chronic study (females)
corresponding to 4 mg a.i./kg bw/day - Dose / conc.:
- 60 mg/kg bw/day (actual dose received)
- Remarks:
- Chronic study (females)
corresponding to 12 mg a.i./kg bw/day - Dose / conc.:
- 200 mg/kg bw/day (actual dose received)
- Remarks:
- Chronic study (females): reduced in several steps to 65 mg/kg bw/day due to high mortality; corresponding to 40 (14) mg a.i./kg bw/day
- Dose / conc.:
- 200 mg/kg bw/day (actual dose received)
- Remarks:
- Interim pathology group (females): reduced in several steps to 75 mg/kg bw/day due to high mortality; corresponding to 40 (15) mg a.i./kg bw/day
Basis: - Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Interim pathology group (males): reduced in several steps to 65 mg/kg bw/day due to high mortality; corresponding to 20 (13) mg a.i./kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- Carcinogenicity study: 50
Interim pathology group: Control group: 10 males and 10 females / High dose group: 20 males and 20 females - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The dose levels were selected in agreement with the Sponsor based on the results of the 13-week dose-range finding study in mice
- Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 13 weeks, at week 15 and thereafter at 2-week intervals
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
weekly for the first 13 weeks, at week 15 and thereafter at 2-week intervals
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily, by visual appraisal
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in 10 animals/sex/group prior to dosing, in test week 52 and at study termination
HAEMATOLOGY: Yes
- Time schedule for collection of blood: In test week 13, 26, 52 and at study termination
- Anaesthetic used for blood collection: ether
- Animals fasted: yes
- How many animals: The first 20 surviving animals/sex/group of the main study
- Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, leukocyte count, platelet count, relative and absolute differential blood count including neutrophilic, eosinophilic
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In test week 26, 52 and at study termination
- Animals fasted: Yes
- How many animals: The first 10 surviving animals/sex/group of the main study
- Parameters: Sodium, potassium, calcium, chloride, glucose, total cholesterol, blood urea, total bilirubin, creatinine, total protein and albumin concentration, globulin, alanine aminotransferase, aspartate amino-transferase, alkaline phosphatase, lactate dehydrogenase
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
From:
All surviving animals of the satellite (at interim sacrifice) and of the main study and all animals that died prematurely
Organs:
Adrenal, aorta abdominalis, bone, bone marrow, brain, caecum, clitorial gland, coagulating gland, epididymis, extraorbital lachrymal gland, eye with optic nerve and Harderian gland, gall bladder, heart, large and small intestine, kidney and ureter, macroscopically visible lesions, liver, lungs, lymph nodes, mammary gland, muscle, nasal cavity with nasopharynx, sciatic nerve, oesophagus, ovary, pancreas, pituitary, preputial gland, prostate, salivary glands, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testicle, thymus, thyroid, tissue masses or tumours, tongue, trachea, urinary bladder, uterus, vagina, Zymbal’s gland
Organ weights: Yes
From:
All satellite animals (at interim sacrifice) and of the first 10 surviving animals of main study/sex/group (at terminal sacrifice)
Organs:
Liver, kidneys, adrenals, testes, epididymides, ovaries, brain
HISTOPATHOLOGY: Yes
From:
All control and high dose animals and all animals that died prematurely
Organs:
Adrenal, aorta abdominalis, bone, bone marrow, brain, caecum, clitorial gland, coagulating gland, epididymis, extraorbital lachrymal gland, eye with optic nerve and Harderian gland, gall bladder, heart, large and small intestine, kidney and ureter, macroscopically visible lesions, liver, lungs, lymph nodes, mammary gland, muscle, nasal cavity with nasopharynx, sciatic nerve, oesophagus, ovary, pancreas, pituitary, preputial gland, prostate, salivary glands, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testicle, thymus, thyroid, tissue masses or tumours, tongue, trachea, urinary bladder, uterus, vagina
Other:
All grossly visible tumours or lesions suspected of being tumours or tissues suspected to show non-neoplastic lesions in all groups occurring in any organ were examined microscopically. - Other examinations:
- Bone marrow evaluation (myeloid:erythroid ratio; M:E ratio) from control and high dose groups
Macroscopic investigations: beginning in test week 27 all animals of the main study were palpated once every week to record palpable masses
An additional pathological assessment was conducted to address possible systemic effects on the immune system. For the enhanced immune system evaluation, the following organs were evaluated based on the Enhanced Histopathology of the Immune System by Elmore (2012) and the details given by Elmore (2006): spleen, thymus, mesenteric lymph nodes, Peyer’s patches - jejunum (GALT – gut associated lymphoid tissue), and bone marrow (high dose and control animals only) (high dose and control animals only). Organs from animals affected by amyloidosis, autolysis, lymphoma and other tumors could not be evaluated. Furthermore, in some cases, the organs were not present on the slide. - Statistics:
- Main study:
Chi²-test: Bone marrow (α = 0.01);
Dunnett’s multiple t-test: Body weight, food consumption, haematology, clinical biochemistry, relative and absolute organ weights (α = 0.01);
Fisher’s exact test: Non-neoplastic and neoplastic lesions, mortality, mean survival and mean survival time (α = 0.01);
Guidelines for simple sensitive significance tests for carcinogenic effects in long-term animal experiments (Peto, R. et al., IARC Monograph Series, Supplement 2: 311–426, 1980): Tumours, tumour chronology (α = 0.01)
Interim pathology study:
Student’s t-test: Body weight, food consumption, relative and absolute organ weights (α = 0.01)
Fisher’s exact test: Histopathology (α = 0.01)
Enhanced immune system evaluation: From the M:E ratios obtained within the combined chronic toxicity and carcinogenicity study, the mean was calculated by group and sex for further evaluation. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Animals treated with the low dose of the test item showed no test item-related clinical signs. Treatment with the intermediate and high dose of test item caused increased incidences of local changes to eyes including swollen and/or reddened eyes/eyelids, as well as ptosis on several days during the course of the study. Other clinical findings (piloerection, reduced motility, pale parts of the body, reduced body temperature, irregular respiration, and abdominal/lateral position) were considered to be incidental and not treatment-related.
Interim pathology group:
Treatment with the high dose of test item caused increased incidences of local changes to eyes including swollen and/or reddened eyes/eyelids (males: 9/20, females: 9/20), as well as ptosis (males: 5/20, 2/20) on several days during the course of the study. Other clinical findings were not considered to be treatment-related. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- No treatment-related effects on mortality rate were observed in the low dose (16 %) and in the intermediate dose group of males (40 %) when compared to the male control animals (34 %).
The mortality rate of the low dosed females (40 %) was statistically significantly increased when compared to the control (8 %). However, the mortality rates are within the normal range of variation when compared with the historical control of female mice (6–42 %), and the mortality rate of the female control animals (8 %) in the study is unusually low. Therefore, the increase of premature deaths in the low dosed female group is not considered to be treatment related.
The mortality rates of the intermediate dosed females (74 %) and of the high dosed animals of both sexes (m + f: 74 %) were statistically significantly increased when compared with the control at the end of the study.
Interim pathology group:
One male and one female animal of the control group died prematurely. In the high dose group 9 of 20 males and 10 of 29 females died prematurely. The increased number of prematurely deceased high dosed animals is regarded to be treatment-related. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Animals treated with the low dose and males of the intermediate dose group showed no test item-related effects on body weight or body weight gain.
The group mean body weights of females of the intermediate dose and of both sexes of the high dosed groups were statistically significantly decreased over several test weeks. A ≥ 10% decrease of body weight was observed in the following study periods until administration of test item was terminated: In the intermediate dose group of female mice from test week 63 to 75 and in the high dose groups of male and female mice from test week 15 to 67 and test week 29 to 77, respectively.
At the end of the study period (test week 77) the total body weight gain was reduced in the female mice of the intermediate dose group and in the male and female animals of the high dose group when compared to the respective start values and to the control group.
The consistent effect on body weight and body weight gain in the intermediate (f) and high (m + f) dose groups throughout the treatment period is regarded as an adverse effect.
Interim pathology group:
Treatment with the high dose of test item led to a reduced body weight of males and females from approximately week 3 onwards. The total body weight gain (test week 0 to 51) was reduced in high dosed male (−44%) and female (−10%) mice when compared to controls. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- In both sub-studies (chronic study and interim pathology) no test item-related effect on food consumption was noted.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Test item-related effects on water consumption were not observed in any study group.
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- Examinations in test weeks 52 and 78 revealed no test item-related changes of the eyes and the optic region in any of the treated mice. Further, there was no indication of any impairment to auditory acuity.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No test item-related effect was observed in the low dosed animals of any sex.
In the intermediate and high dosed animals the following statistically significant changes of haematological parameters compared to the control were noted and are considered to be treatment related: Decrease of haemoglobin content and haematocrit value in both sexes; increase of leucocytes (in week 52), neutrophilic granulocytes and monocytes (in test week 52) in female mice only, which is indicative of inflammatory processes. Further evidence to support this view were microscopic findings such as muscle degeneration with cell reaction and large macrophages and/or mild lymphoid hyperplasia in the mesenteric lymph nodes in high dose animals of either sex.
All other findings are not considered to be test item related. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No test item-related effect was observed in the low dosed animals of both sexes.
In the intermediate dose group and the high dose group of both sexes statistically significantly increased activity of aspartate-aminotransferase (ASAT) and lactate-dehydrogenase (LDH) was observed in test weeks 26, 52 and /or 78/79. The elevated levels of both enzymes are considered to be test item-related.
All other findings are not considered to be test item-related. - 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):
- No test item-related effects were observed in the low and intermediate dosed animals of both sexes.
In high dosed females the absolute organ weight of the left kidney was statistically significantly decreased. In high dosed animals of both sexes reduction of the absolute kidney (left and right) and liver weights was observed. However, as the relative kidney and liver weights remained unchanged, accounting for the low body weight of high dose animals at necropsy, these effects are considered to be not treatment related.
Interim pathology group:
In males and females of the high dose groups the absolute kidney weights (left and right) were reduced and in high dose females the relative right kidney weight was statistically significantly decreased, and the absolute and relative liver weight was increased. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No test item-related effects were observed in the low dosed animals of both sexes.
The following macroscopic findings in the spleen and the urinary bladder of males and females of the intermediate and the high dose group were considered to be test item-related: reduced size of spleen, dilated, enlarged and tightly filled urinary bladder. The tightly filled urinary bladder observed during necropsy predominantly in animals of the intermediate and high dose group is regarded to be a secondary effect caused by possible muscle degeneration of the urogenital tract, resulting finally in dilatation of the urinary bladder as noted during the microscopic examination.
Other macroscopic findings are commonly found in mice of this strain and age and are therefore considered incidental or related to dissection or premature death.
Interim pathology group:
No test item related effects were observed in any of the high dosed treated animals of both sexes. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related systemic non-neoplastic lesions were noted in males and females of all dose level groups (10, 30 or 100/35 mg /kg bw/day (male animals) or 20, 60 or 200/65 mg/kg bw/day (female animals)) at terminal sacrifice (high dose group) and in the prematurely deceased animals (all dose groups):
- muscle degeneration with cellular reaction in the skeletal muscle (leg) of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the low dosed prematurely deceased females (20 mg /kg b.w./day (11/20 – 55%)) and the intermediate and high dosed prematurely deceased animals (30 or 100/35 mg (males) or 60 or 200/65 (females) mg /kg b.w./day);
- muscle degeneration with cellular reaction in the bone (sternum) of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- muscle degeneration with cellular reaction in the heart of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- large macrophages in the mesenteric lymph nodes of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- atrophy with cellular reduction in the spleen (in some cases correlating with macroscopic findings) of the high dosed females (200/65 mg/kg bw/day) at terminal sacrifice, and of the high dosed prematurely deceased males (100/35 mg/kg bw/day) and the intermediate and high dosed prematurely deceased females (60 or 200/65 mg/kg bw/day);
- dilatation of the urinary bladder (in some cases correlating with macroscopic findings) of the intermediate dosed males (30 mg/kg bw/day) and of the high dosed males and females (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the intermediate and
high dosed prematurely deceased males (30 or 100/35 mg/kg bw/day)
- vacuolisation of epithelial cells in the epididymides of the high dosed male (100/35 mg /kg bw/day) at terminal sacrifice, and of the intermediate and high dosed prematurely deceased males (30 or 100/35 mg /kg bw/day). The highest incidence and severity of these lesions were in general observed in the high dose group (animals treated with 100/35 (males) or 200/65 (females) mg/kg b.w./day). This effect is not considered as biologically relevant related to reproduction toxicity as the two-generation reproduction toxicity study did not show adverse effects related to reproduction toxicity. In addition, the dose-related vacuolisation of the epithelial cells lining the epididymides were observed in the presence of marked general systemic toxicity and reductions in body weight and body weight gain, which were considered adverse. Moreover, isolated epididymal vacuolisation is generally considered to be an age related effect in mice.
All other non-neoplastic lesions noted for various organs (partly statistically significant compared to the control group) are considered to be within the normal range of background pathology commonly observed in mice of this strain and age. The difference in the incidences of some findings in treated groups were regarded as random events or occurred without any dose-response relationship and are therefore considered to be unrelated to the treatment with the test item.
Interim pathology group:
Test item-related systemic non-neoplastic lesions were noted in males and females treated the high dose of 100/65 (males) or 200/75 (females) mg/kg bw/day at terminal sacrifice and in the prematurely deceased animals:
- muscle degeneration with cellular reaction in the skeletal muscle (leg) of males and females;
- muscle degeneration with cellular reaction in the heart of males and females;
- large macrophages in the mesenteric lymph nodes of males and females;
- large macrophages in the small intestine (duodenum, jejunum, ileum) of males and females;
- hyperplasia of the secretory cells of the bronchial epithelium in the lungs of males and females;
- vacuolisation of epithelial cells in the epididymides of the male animals. This effect is not considered as biologically relevant related to reproduction toxicity as the the two-generation reproduction toxicity study did not show adverse effects related to reproduction toxicity. In addition, the dose-related vacuolisation of the epithelial cells lining the epididymides were observed in the presence of marked general systemic toxicity and reductions in body weight and body weight gain, which were considered adverse. Moreover, isolated epididymal vacuolisation is generally considered to be an age related effect in mice.
The enhanced immune system evaluation (see also details in the table under 'Overall remarks, attachments') revealed that all changes noted in the 78-Week study (higher mean sums mainly for decreased mesenteric lymph node values in interim and terminal sacrifices, minor differences in the thymus, higher mean sums for decreased splenic values in the spleen at both interim and terminal sacrifices, and in Peyer's patches at the terminal sacrifice, minor decreased M:E ratio in both sexes at 200/65 mg/kg bw in the bone marrow at the interim sacrifice) are rather linked to age-related nonneoplastic or neoplastic lesions in CD-1 mice but not to treatment with the test item. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- Bone marrow examination: No effects were noted in the myeloid:erythroid ratio of high dosed males and females when compared with control.
Latency period of palpable masses: No effects were noted for high dosed males and females when compared with the control. - Details on results:
- CLINICAL SIGNS AND MORTALITY
Carcinogenicity study:
No treatment-related effects on mortality rate were observed in the low dose (16 %) and in the intermediate dose group of males (40 %) when compared to the male control animals (34 %).
The mortality rate of the low dosed females (40 %) was statistically significantly increased when compared to the control (8 %). However, the mortality rates are within the normal range of variation when compared with the historical control of female mice (6–42 %), and the mortality rate of the female control animals (8 %) in the study is unusually low. Therefore, the increase of premature deaths in the low dosed female group is not considered to be treatment related.
The mortality rates of the intermediate dosed females (74 %) and of the high dosed animals of both sexes (m + f: 74 %) were statistically significantly increased when compared with the control at the end of the study.
Animals treated with the low dose of the test item showed no test item-related clinical signs. Treatment with the intermediate and high dose of test item caused increased incidences of local changes to eyes including swollen and/or reddened eyes/eyelids, as well as ptosis on several days during the course of the study. Other clinical findings (piloerection, reduced motility, pale parts of the body, reduced body temperature, irregular respiration, and abdominal/lateral position) were considered to be incidental and not treatment-related.
Interim pathology group:
One male and one female animal of the control group died prematurely. In the high dose group 9 of 20 males and 10 of 29 females died prematurely. The increased number of prematurely deceased high dosed animals is regarded to be treatment-related.
Treatment with the high dose of test item caused increased incidences of local changes to eyes including swollen and/or reddened eyes/eyelids (males: 9/20, females: 9/20), as well as ptosis (males: 5/20, 2/20) on several days during the course of the study. Other clinical findings were not considered to be treatment-related.
BODY WEIGHT AND WEIGHT GAIN
Carcinogenicity study:
Animals treated with the low dose and males of the intermediate dose group showed no test item-related effects on body weight or body weight gain.
The group mean body weights of females of the intermediate dose and of both sexes of the high dosed groups were statistically significantly decreased over several test weeks. A ≥ 10% decrease of body weight was observed in the following study periods until administration of test item was terminated: In the intermediate dose group of female mice from test week 63 to 75 and in the high dose groups of male and female mice from test week 15 to 67 and test week 29 to 77, respectively.
At the end of the study period (test week 77) the total body weight gain was reduced in the female mice of the intermediate dose group and in the male and female animals of the high dose group when compared to the respective start values and to the control group.
The consistent effect on body weight and body weight gain in the intermediate (f) and high (m + f) dose groups throughout the treatment period is regarded as an adverse effect.
Interim pathology group:
Treatment with the high dose of test item led to a reduced body weight of males and females from approximately week 3 onwards. The total body weight gain (test week 0 to 51) was reduced in high dosed male (−44%) and female (−10%) mice when compared to controls.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In both sub-studies (carcinogenicity and interim pathology) no test item-related effect on food consumption was noted.
WATER CONSUMPTION AND COMPOUND INTAKE
Test item-related effects on water consumption were not observed in any study group.
OPHTHALMOSCOPIC EXAMINATION
Examinations in test weeks 52 and 78 revealed no test item-related changes of the eyes and the optic region in any of the treated mice. Further, there was no indication of any impairment to auditory acuity.
HAEMATOLOGY
No test item-related effect was observed in the low dosed animals of any sex.
In the intermediate and high dosed animals the following statistically significant changes of haematological parameters compared to the control were noted and are considered to be treatment related: Decrease of haemoglobin content and haematocrit value in both sexes; increase of leucocytes (in week 52), neutrophilic granulocytes and monocytes (in test week 52) in female mice only, which is indicative of inflammatory processes. Further evidence to support this view were microscopic findings such as muscle degeneration with cell reaction and large macrophages and/or mild lymphoid hyperplasia in the mesenteric lymph nodes in high dose animals of either sex.
All other findings are not considered to be test item related.
CLINICAL CHEMISTRY
No test item-related effect was observed in the low dosed animals of both sexes.
In the intermediate dose group and the high dose group of both sexes statistically significantly increased activity of aspartate-aminotransferase (ASAT) and lactate-dehydrogenase (LDH) was observed in test weeks 26, 52 and /or 78/79. The elevated levels of both enzymes are considered to be test item-related.
All other findings are not considered to be test item-related.
ORGAN WEIGHTS
Carcinogenicity study:
No test item-related effects were observed in the low and intermediate dosed animals of both sexes.
In high dosed females the absolute organ weight of the left kidney was statistically significantly decreased. In high dosed animals of both sexes reduction of the absolute kidney (left and right) and liver weights was observed. However, as the relative kidney and liver weights remained unchanged, accounting for the low body weight of high dose animals at necropsy, these effects are considered to be not treatment related.
Interim pathology group:
In males and females of the high dose groups the absolute kidney weights (left and right) were reduced and in high dose females the relative right kidney weight was statistically significantly decreased, and the absolute and relative liver weight was increased.
GROSS PATHOLOGY
Carcinogenicity study:
No test item-related effects were observed in the low dosed animals of both sexes.
The following macroscopic findings in the spleen and the urinary bladder of males and females of the intermediate and the high dose group were considered to be test item-related: reduced size of spleen, dilated, enlarged and tightly filled urinary bladder. The tightly filled urinary bladder observed during necropsy predominantly in animals of the intermediate and high dose group is regarded to be a secondary effect caused by possible muscle degeneration of the urogenital tract, resulting finally in dilatation of the urinary bladder as noted during the microscopic examination.
Other macroscopic findings are commonly found in mice of this strain and age and are therefore considered incidental or related to dissection or premature death.
Interim pathology group:
No test item related effects were observed in any of the high dosed treated animals of both sexes.
HISTOPATHOLOGY:
Carcinogenicity study:
Test item-related systemic non-neoplastic lesions were noted in males and females of all dose level groups (10, 30 or 100/35 mg /kg bw/day (male animals) or 20, 60 or 200/65 mg/kg bw/day (female animals)) at terminal sacrifice (high dose group) and in the prematurely deceased animals (all dose groups):
- muscle degeneration with cellular reaction in the skeletal muscle (leg) of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the low dosed prematurely deceased females (20 mg /kg b.w./day (11/20 – 55%)) and the intermediate and high dosed prematurely deceased animals (30 or 100/35 mg (males) or 60 or 200/65 (females) mg /kg b.w./day);
- muscle degeneration with cellular reaction in the bone (sternum) of the high
dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- muscle degeneration with cellular reaction in the heart of the high dosed
animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- large macrophages in the mesenteric lymph nodes of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- atrophy with cellular reduction in the spleen (in some cases correlating with macroscopic findings) of the high dosed females (200/65 mg/kg bw/day) at terminal sacrifice, and of the high dosed prematurely deceased males (100/35 mg/kg bw/day) and the intermediate and high dosed prematurely deceased females (60 or 200/65 mg/kg bw/day);
- dilatation of the urinary bladder (in some cases correlating with macroscopic findings) of the intermediate dosed males (30 mg/kg bw/day) and of the high dosed males and females (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the intermediate and
high dosed prematurely deceased males (30 or 100/35 mg/kg bw/day)
- vacuolisation of epithelial cells in the epididymides of the high dosed male (100/35 mg /kg bw/day) at terminal sacrifice, and of the intermediate and high dosed prematurely deceased males (30 or 100/35 mg /kg bw/day). The highest incidence and severity of these lesions were in general observed in the high dose group (animals treated with 100/35 (males) or 200/65 (females) mg/kg b.w./day). This effect is not considered as biologically relevant related to reproduction toxicity as the the two-generation reproduction toxicity study did not show adverse effects related to reproduction toxicity. In addition, the dose-related vacuolisation of the epithelial cells lining the epididymides were observed in the presence of marked general systemic toxicity and reductions in body weight and body weight gain, which were considered adverse. Moreover, isolated epididymal vacuolisation is generally considered to be an age related effect in mice.
All other non-neoplastic lesions noted for various organs (partly statistically significant compared to the control group) are considered to be within the normal range of background pathology commonly observed in mice of this strain and age. The difference in the incidences of some findings in treated groups were regarded as random events or occurred without any dose-response relationship and are therefore considered to be unrelated to the treatment with the test item.
Interim pathology group:
Test item-related systemic non-neoplastic lesions were noted in males and females treated the high dose of 100/65 (males) or 200/75 (females) mg/kg bw/day at terminal sacrifice and in the prematurely deceased animals:
- muscle degeneration with cellular reaction in the skeletal muscle (leg) of males and females;
- muscle degeneration with cellular reaction in the heart of males and females;
- large macrophages in the mesenteric lymph nodes of males and females;
- large macrophages in the small intestine (duodenum, jejunum, ileum) of males and females;
- hyperplasia of the secretory cells of the bronchial epithelium in the lungs of males and females;
- vacuolisation of epithelial cells in the epididymides of the male animals. This effect is not considered as biologically relevant related to reproduction toxicity as the the two-generation reproduction toxicity study did not show adverse effects related to reproduction toxicity. In addition, the dose-related vacuolisation of the epithelial cells lining the epididymides were observed in the presence of marked general systemic toxicity and reductions in body weight and body weight gain, which were considered adverse. Moreover, isolated epididymal vacuolisation is generally considered to be an age related effect in mice.
OTHER FINDINGS
Bone marrow examination: No effects were noted in the myeloid:erythroid ratio of high dosed males and females when compared with control.
Latency period of palpable masses: No effects were noted for high dosed males and females when compared with the control.
The enhanced immune system evaluation revealed that in animals at 200/65 mg/kg bw/day, there were higher mean sums mainly for decreased mesenteric lymph node values in interim and terminal sacrifices, i.e., differences were mainly due to minor atrophic changes. Macrophages in the mesenteric lymph nodes were laden with pigment or amyloid or foamy, and a differentiation was not considered to be possible. Inflammation could not be noted in the lymph nodes. Only minor differences were noted in the thymus and those were not considered of relevance. In the spleen at both interim and terminal sacrifices, and in Peyer’s patches at the terminal sacrifice, there were slightly higher mean sums for decreased splenic and Peyer’s patches values, i.e., differences were mainly due to minor atrophic changes. These however could not be attributed to treatment with the test item because there were no findings in the mesenteric lymph nodes and in intestinal lactea as noted in the available 13-Week study with mice. Most importantly, there were no inflammatory changes in the mesenteric lymph nodes. Findings in the bone marrow caused a minor decreased M:E (myeloid/erythroid) ratio in both sexes at 200/65 mg/kg bw/day, at the interim sacrifice only. It is deemed that all changes noted in the 78-Week study are rather age-related nonneoplastic or neoplastic lesions in CD-1 mice but not related to treatment with the test item. A general effect of the test substance on the immune system can be excluded. There was no accumulation or inflammation in mesenteric lymph nodes. Although the findings of foamy macrophage accumulation was a clearly notable finding in the mesenteric lymph nodes in mice at 60 and 200 mg/kg bw/day in the availble 13-week study in rats, it is therefore questionable if there was a direct relation between macrophage accumulation and inflammation in the 13-week study. The absence of inflammatory findings in mesenteric lymph nodes after 75 weeks at comparable doses is considered to be a strong indicator for adaptation. Results are also shown in the table under 'Overall remarks, attachments'. - Dose descriptor:
- NOAEL
- Effect level:
- 10 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- 20% aqueous solution, "product by process"
- Sex:
- male
- Basis for effect level:
- other: overall effects
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 2 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Sex:
- male
- Basis for effect level:
- other: overall effects
- Dose descriptor:
- LOAEL
- Effect level:
- 20 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- 20% aqueous solution, "product by process"
- Sex:
- female
- Basis for effect level:
- other: see 'Remark'
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 4 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Sex:
- female
- Basis for effect level:
- other: see 'Remark'
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 4 mg/kg bw/day (actual dose received)
- System:
- musculoskeletal system
- Organ:
- bone
- heart
- sternum
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The chronic NOAEL derived from the combined chronic toxicity and carcinogenicity study in mice is established at 2 mg a.i./kg bw/d in males and was not identified in females (histological change to muscle at 4 mg a.i./kg bw/d).
- Executive summary:
The chronic toxicity and carcinogenicity of the registration substance (20% a.i.) was tested in CD®-1 mice. Fifty mice per sex per group were fed the test item at low, intermediate and high dose levels for 78 weeks. A control group of 50 animals per sex received untreated diet. Due to increased mortality rate in the high dose group the initial high dose level was successively reduced to 35 mg/kg bw/d in males and 65 mg/kg bw/d in females. Towards the end of the study dosing was discontinued in the intermediate dosed females and high dosed males and females. A satellite group of 20 dosed animals per sex and 10 associated control animals per sex for chronic toxicity testing was included for 52 weeks. Observations and examinations were carried out. Treatment-related effects on survival were observed. The mortality rate of the intermediate dosed female mice treated with 60 mg/kg bw/day was statistically significantly increased from approximately test week 60 onwards. The male and female animals treated with the high dose of 100/35 (males) or 200/65 (females) mg/kg bw/day revealed an increased mortality and a corresponding decreased survival rate from approximately test weeks 38 (males) and 14 (females) onwards (statistically significant at p ≤ 0.01 from test weeks 51 (males) and 26 (females) onwards) (RMS Table 2). The time-point of the occurrence of statistical significance of the mortality rate was directly influenced by the low mortality rate of the control group. The severely increased mortality rates noted for these animals resulted in dose reductions in the high dose group during the course of the study and, subsequently, in the discontinuation of dosing of the high dosed animals from test weeks 68 (males) and 67 (females) onwards. Furthermore, the dosing of the intermediate dosed females was discontinued from test week 76 onwards.
Body weights were unaffected in the low dose animals and in intermediate dose males. The body weight of the intermediate dosed female mice (60 mg/kg bw/day) was below the body weight of the control group from approximately test week 41 onwards (up to 15%, p ≤ 0.01 in test weeks 41 and 49 to 73). Treatment with the high dose led to a reduced body weight of males and females from approximately test week 3 onwards in males (by 4% to 20%, on average 12%, p ≤ 0.01 in test weeks 3 to 67) and from test week 7 onwards in females (by 4% to 17%, on average 11%, p ≤ 0.01 in test weeks 7 to 17 and 21 to 77). The total body weight gain was accordingly reduced in the female animals of the intermediate dose group and in the male and female animals of the high dose group when compared to the respective start values and to the control group.
Local changes to eyes were seen in the intermediate and high dose animals, including swollen/reddened eyes and ptosis were seen. The observed effects to the eyes are known properties of the test item as it may cause serious damage to eyes (hazard identification amongst others: H314 'Causes severe skin burns and eye damage'). The local exposure of the eye to the test item during the food intake or grooming periods is very likely as the test item was administered via the diet.
Haematological examination revealed increased levels of leucocytes, neutrophilic granulocytes and monocytes in high dosed female mice, which are indicative of inflammatory processes. Clinical chemistry showed increased levels of aspartate-aminotransferase and lactate-dehydrogenase in treated animals. Increased absolute and relative liver weights were noted for the female animals.
The macroscopic inspection did not reveal any test item-related changes in the organs or tissues of the high dosed animals.
Histopathological examinations revealed the following treatment-related findings: degeneration with cellular reaction in skeletal muscle, bone and heart, large macrophages in mesenteric lymph nodes and the small intestine, vacuolisation of epithelial cells in the epididymides, hyperplasia of the secretory cells of the bronchial epithelium in the lungs as follows:
Non-neoplastic lesions-carcinogenicity study
Test item-related systemic non-neoplastic lesions were noted in males and females of all dose level groups (10, 30 or 100/35 mg /kg bw/day (male animals) or 20, 60 or 200/65 mg/kg bw/day (female animals)) at terminal sacrifice (high dose group) and in the prematurely deceased animals (all dose groups):
- muscle degeneration with cellular reaction in the skeletal muscle (leg) of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the low dosed prematurely deceased females (20 mg /kg b.w./day (11/20 – 55%)) and the intermediate and high dosed prematurely deceased animals (30 or 100/35 mg (males) or 60 or 200/65 (females) mg /kg b.w./day);
- muscle degeneration with cellular reaction in the bone (sternum) of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- muscle degeneration with cellular reaction in the heart of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- large macrophages in the mesenteric lymph nodes of the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the prematurely deceased males and females of all dose groups;
- atrophy with cellular reduction in the spleen (in some cases correlating with macroscopic findings) of the high dosed females (200/65 mg/kg bw/day) at terminal sacrifice, and of the high dosed prematurely deceased males (100/35 mg/kg bw/day) and the intermediate and high dosed prematurely deceased females (60 or 200/65 mg/kg bw/day);
- dilatation of the urinary bladder (in some cases correlating with macroscopic findings) of the intermediate dosed males (30 mg/kg bw/day) and of the high dosed males and females (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal sacrifice, and of the intermediate and high dosed prematurely deceased males (30 or 100/35 mg/kg bw/day).
- vacuolisation of epithelial cells in the epididymides of the high dosed male (100/35 mg /kg bw/day) at terminal sacrifice, and of the intermediate and high dosed prematurely deceased males (30 or 100/35 mg /kg bw/day). The highest incidence and severity of these lesions were in general observed in the high dose group (animals treated with 100/35 (males) or 200/65 (females) mg/kg b.w./day). This effect is not considered as biologically relevant related to reproduction toxicity as the the two-generation reproduction toxicity study did not show adverse effects related to reproduction toxicity. In addition, the dose-related vacuolisation of the epithelial cells lining the epididymides were observed in the presence of marked general systemic toxicity and reductions in body weight and body weight gain, which were considered adverse. Moreover, isolated epididymal vacuolisation is generally considered to be an age related effect in mice.
Non-neoplastic lesions-carcinogenicity study - Interim Pathology Study
Test item-related systemic non-neoplastic lesions were noted in males and females treated the high dose of 100/65 (males) or 200/75 (females) mg/kg bw/day at terminal sacrifice and in the prematurely deceased animals:
- muscle degeneration with cellular reaction in the skeletal muscle (leg) of males and females;
- muscle degeneration with cellular reaction in the heart of males and females;
- large macrophages in the mesenteric lymph nodes of males and females;
- large macrophages in the small intestine (duodenum, jejunum, ileum) of males and females;
- hyperplasia of the secretory cells of the bronchial epithelium in the lungs of males and females;
- vacuolisation of epithelial cells in the epididymides of the male animals. This effect is not considered as biologically relevant related to reproduction toxicity as the the two-generation reproduction toxicity study did not show adverse effects related to reproduction toxicity. In addition, the dose-related vacuolisation of the epithelial cells lining the epididymides were observed in the presence of marked general systemic toxicity and reductions in body weight and body weight gain, which were considered adverse. Moreover, isolated epididymal vacuolisation is generally considered to be an age related effect in mice.
There was no evidence of carcinogenicity.
Taken together, mice treated with 30 or 100/35 mg/kg bw/d test item (males) and 60 or 200/65 mg /kg bw/d test item (females) showed an increase of numbers of white blood cells and an increased incidence of large macrophages in mesenteric lymph nodes. Therefore, treatment of mice with high dose of test item is associated with indications of inflammatory processes.
Mice of both sexes treated with high dose of the test item and additional females treated with 60 mg/kg bw/d test item showed a statistically significant increase of levels of aspartate aminotransferase and lactate dehydrogenase and a dose dependent statistically significant increase of findings of muscle degeneration with cellular reaction in the heart, sternum and skeletal tissues.
In low dose mice of both sexes no statistically significant differences of biochemical parameters to the controls were observed. Any histopathological findings in the low dose mice that died prematurely (8/50 males and 20/50 females) were not statistically significant. However, in the prematurely deceased females treated with 20 mg/kg bw/d of test item an increased incidence of muscle degeneration in heart (7/20), sternum (3/20) and skeletal muscle (11/20) were observed. In low dose prematurely deceased males treated with 10 mg/kg bw/d test item only one single incidence of muscle degeneration in sternum (2/8) and heart (1/8) were observed. No treatment-related neoplastic lesions were observed and it is concluded that registration substance has no carcinogenic potential in mice.
The study reported above does not conform to the requirements of OECD 35 or OECD 116 in that excessive toxicity including mortality (>50%) occurred at both the intermediate dose (females) and high dose levels (males and females). This study is therefore not strictly acceptable as a negative carcinogenicity study.
As significant toxicity was seen in females at all dose levels, an overall study NOAEL for a chronic study could not be established. Thus, based on the observed findings in the interim pathology study (after one year of treatment) and in the carcinogenicity study an overall NOAEL for chronic toxicity of the registration substance (20% a.i.) can be established at 10 mg/kg bw/day for male mice corresponding to 2 mg a.i./kg bw/day. The LOAEL in females was 20 mg registration substance (20% a.i.)/kg bw/day corresponding to 4 mg a.i./kg bw/day.
An additional pathological assessment was conducted in 2022 to address possible systemic effects on the immune system. For the enhanced immune system evaluation, the following organs were evaluated: spleen, thymus, mesenteric lymph nodes, Peyer’s patches - jejunum (GALT – gut associated lymphoid tissue), and bone marrow (high dose and control animals only) (high dose and control animals only). The enhanced immune system evaluation revealed that all changes noted in the 78-Week study (higher mean sums mainly for decreased mesenteric lymph node values in interim and terminal sacrifices, minor differences in the thymus, higher mean sums for decreased splenic values in the spleen at both interim and terminal sacrifices, and in Peyer's patches at the terminal sacrifice, minor decreased myeloid:erythroid ratio in both sexes at 200/65 mg/kg bw in the bone marrow at the interim sacrifice) are rather linked to age-related non-neoplastic or neoplastic lesions in CD-1 mice but not related to treatment with the test item. From the enhanced immune system evaluation it is finally concluded that the changes observed in the different available repeated dose toxicity studies were limited to local reactions in the mesenteric lymph nodes as a consequence of an uptake and transfer of the test item by intestinal macrophage via lacteal lymph fluid. A general effect of the test substance on the immune system is excluded. Inflammatory lesions in the mesenteric lymph nodes were of minimal incidence and severity and were noted in the available 13-week study in mice at 60 and 200 mg/kg bw/day only. No such findings were noted in the current 78-Week study in mice. Even foamy macrophage agglomeration was not seen in mesenteric lymph nodes. Especially, there was no indication for inflammation in the lymph nodes after 78 weeks. In addition, in the available 90-Day study in dogs, no inflammation was noted. Therefore, it was concluded, that the few cases of minimal inflammatory lesions in mesenteric lymph nodes from mice of the 13-week study were not related to treatment with the test item. In addition, it was noted that macrophages agglomerated in the intestinal lactea, which is deemed to be the first step in the pathway of development of lesions. It is noteworthy, that no intestinal inflammation was encountered in mice after 78 weeks. Therefore, an adaptation was considerd to be very likely in mice.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- no hormone levels measured, no sensory reactivity test, no assessment of grip strength, no motor activity assessment and no functional observations were performed
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Version / remarks:
- May 12, 1981
- Deviations:
- yes
- Remarks:
- exposure time 90 days
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
- Version / remarks:
- 1988
- Deviations:
- yes
- Remarks:
- exposure time 90 days
- Principles of method if other than guideline:
- The study served as dose-range-finder for a combined chronic toxicity and carcinogenicity study.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Remarks:
- CRl:CD1 (ICR)
- Details on species / strain selection:
- The mouse is a commonly used rodent species for toxicity studies.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 38 - 44 days
- Weight at study initiation: Males: 22.3 - 30.1 g; Females: 19.4 - 27.4 g
- Fasting period before study: not applicable
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): not provided
- Photoperiod (hrs dark / hrs light): 12/12
- Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance mixture was freshly prepared once a week.
- Mixing appropriate amounts with (Type of food): commercial ssniff (R) R/M-H V1530 - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 91 days
- Frequency of treatment:
- daily
- Dose / conc.:
- 6 mg/kg bw/day (nominal)
- Remarks:
- corresponding to 1.2 mg active ingredient/kg bw/day (nominal)
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Remarks:
- corresponding to 4 mg active ingredient/kg bw/day (nominal)
- Dose / conc.:
- 60 mg/kg bw/day (nominal)
- Remarks:
- corresponding to 12 mg active ingredient/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Remarks:
- corresponding to 40 mg active ingredient/kg bw/day (nominal)
- Dose / conc.:
- 400 mg/kg bw/day (nominal)
- Remarks:
- corresponding to 80 mg active ingredient/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
based on available toxicity data
- Fasting period before blood sampling for clinical biochemistry: yes, overnight - Positive control:
- none
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations: behavioural changed, reaction to treatment or illness (skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor acitivity and behaviour patterns)
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: monitored by visual appraisal throughout the study
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: test week 13
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: haemoglogin content (HGB), erythrocytes (RBC), leucocytes (WBC), platelets (PCT), differential blood count (relative and absolute), haematocrit value (HCT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: albumin, globulin, albumin/globulin ration, bilirubin (total), cholesterol (total), glucose, protein (total), urea, calcium, chloride, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
OTHER: auditory acuity was checked with a simple noise test in all animals - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes: all superficial tissues were examined visually and by palpation; observation of brain, pituitary gland, cranial nerves, all subcutaneous tissues
, thoracic viscera, thymus, lymph nodes, heart, abdominal viscera, urinary bladder, gastro-intestinal tract as a whole, stomach, caecum, pleural surfaces, liver, kidneys gonads, adrenal glands, uterus, intra-abdominal lymph nodes, accessory reproductive organs
Following organ weights were recorded: adrenal gland, brain, epididymis, liver, testicle, kidney, ovary
HISTOPATHOLOGY: Yes: adrenal gland, aorta abdominalis, bone, bone marrow, brain, caecum, coagulating gland with seminal vesicles, epididymis, extraorbital lachrymal gland, eye with optic nerve and harderian gland, gall bladder, heart, large and small intestine, kidney and ureter, macroscopically visible lesions, liver, lungs, lymph nodes (cervial, mesenteric), mammary gland, muscle, nasal cavity with nasopharynx, sciatic nerve, oesophagus, ovary, pancreas, pituitary, preputial gland, prostate, salivary glands, skin, spinal cord, spleen, sternum, stomach, testicle, thymus, thyroid incl. parathyroids, tissue masses or tumours, tongue, trachea, urinary bladder, uterus, vagina, Zymbal's gland
An additional pathological assessment was conducted to address possible systemic effects on the immune system. For the enhanced immune system evaluation, the following organs were evaluated based on the Enhanced Histopathology of the Immune System by Elmore (2012) and the details given by Elmore (2006): spleen, mesenteric lymph nodes, Peyer’s patches - jejunum (GALT – gut associated lymphoid tissue) (high dose and control animals only). - Statistics:
- Body weight, food consumption, haematology, clinical biochemistry, relative and absolute organ weights: Dunnett’s multiple t-test
Histology: Fisher’s exact test - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- No test substance related changes of behaviour or external appearance were observed in mice treated with 6, 20, 60 or 200 mg/kg bw/day.
At 400 mg/kg bw/day the following was observed: One male revealed piloerection from test day 84 onwards, on female showed reduced motility and for another female piloerection was noted, both from test day 91 onwards. The male mouse which died showed reduced motility and an abdominal position from test day 90 - 92. These findings are considered test substance related. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- One male mouse died on test day 92 in the 400 mg/kg bw/day dose group.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No changes were observed in animals treated with 6, 20 or 60 mg/kg bw/day.
The body weight was reduced in male and female mice in the 200 and 400 mg/kg bw/day dose groups from test week 2 onwards by up to 26% and 35% in test weeks 13 and 12, respectively, for the male and up to 16% and 30% in test week 13 for the female animals compared to the control.
Correlating, the body weight gain of females and males was reduced. The body weight gain from start (test week 0) to the end of test week 13 of the male animals was -10% in the 200 mg/kg bw/day group and -17% in the 400 mg/kg bw/day dose group compared to 17% in the control group. The body weight gain of the females was -2% in the 200 mg/kg bw/day dose group and -18% in the 400 mg/kg bw/day group compared to 16% in the control group.
Body weight at autopsy was reduced by 30% for the males and by 18% for the females in the 200 mg/kg bw dose group an by 37% for the males and 31% for the females in the 400 mg/kg bw/day group compared to control group. This difference was statistically signficant.
All findings are considered to be test substance related. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- No changes were observed in animals treated with 6, 20 or 60 mg/kg bw/day.
From test week 1 onwards, the males and females treated with 200 or 400 mg/kg bw/day revealed a test item-related increase in food consumption with a maximum of 87% for the male and 56% for the female animals (400 mg/kg bw/day group) in test weeks 8 and 3, respectively, compared to the control.
Test item intake:
6 mg/kg bw/day group: mean ± SD: Males: 5.9 ± 1.2 mg/kg bw/day, Females: 6.2 ± 1.8 mg/kg bw/day
20 mg/kg bw/day group: mean ± SD: Males: 19.4 ± 3.4 mg/kg bw/day, Females: 20.7 ± 5.5 mg/kg bw/day
60 mg/kg bw/day group: mean ± SD: Males: 59.7 ± 11.2 mg/kg bw/day, Females: 63.5 ± 15.1 mg/kg bw/day
200 mg/kg bw/day group: mean ± SD: Males: 214.0 ± 66.4 mg/kg bw/day, Females: 216.9 ± 66.6 mg/kg bw/day
400 mg/kg bw/day group: mean ± SD: Males: 448.4 ± 143.0 mg/kg bw/day, Females: 444.6 ± 153.3 mg/kg bw/day - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 1/10 males treated with 200 mg/kg bw/day revealed the following findings for the left eye:
- conjunctivae: redness: diffuse, crimson red, individual vessels not easily discernible; chemosis: obvious swelling with partial eversion of lids; discharge: any amount different from normal;
- cornea: opacity: slight clouding
- lens: opacity: slight clouding and a pupillar size of 6 - 7 mm for the right eye.
Furthermore, 1/10 females treated with 200 mg/kg bw/day via the diet revealed the following changes in both eyes:
- iris: pupillar size: > 8 mm
- lens: opacity: complete opacity, obscuring underlaying structures.
These findings are considered to be incidental due to the low number of animals affected. - Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No changes were observed in animals treated with 6, 20 or 60 mg/kg bw/day.
Treatment with 200 mg/kg bw/day caused a statistically significant increase of leucocytes, platelets, neutrophilic granulocytes, lymphocytes and basophilic granulocytes in females. In the 400 mg/kg bw/day dose group an statistically significant increase was only observed in platelets and neutrophilic granulocytes in males and females. For leucocytes and basophilic granulocytes an increase in females was seen which was not statistically significant.
The increase of leucocytes, platelets, neutrophilic granulocytes, lymphocytes and basophilic granulocytes by female mice treated with 200 mg/kg bw/day (statistically significant at p <= 0.01) are considered to be test item related although dose dependency is missing.
Statistically significant differences (at p <= 0.01) for haemoglobin in males in 60, 200 and 400 mg/kg bw/day dose groups, for haematocrit in males of 60, 200 and 400 mg/kg bw/day and females of 400 mg/kg bw/day groups, as well as for monocytes in males of 20 mg/kg bw/day group, were only minor in comparison to control animals and are considered to be without any biological relevance.
No influence was noted for the number of erythrocytes, eosinophilic granulocytes and large unstained cells. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No changes were observed in animals treated with 6, 20 or 60 mg/kg bw/day.
Creatinine was statistically significant decreased in males and females in the 200 and 400 mg/kg bw/day dose groups. Urea was statistically significant increased in males and females in the 200 and 400 mg/kg bw/day dose groups except the increase in females in the 200 mg/kg bw/day group was not statistically significant. Potassium was increased in females in 200 and 400 mg/kg bw/day dose groups. Alanine aminotransferase was increase in males of the 200 and 400 mg/kg bw/day dose group and females of the 400 mg/kg bw/day group and decreased in females of the 200 mg/kg bw/day dose group. Alkaline phosphatase was increased in males of the 200 and 400 mg/kg bw/day dose group and infemales of the 400 mg/kg bw/day dose group. Aspartate aminotransferase was increased in males and females of the 200 and 400 mg/kg bw/day dose groups.
Statistically signficant differences in males and females for chloride and sodium in 200 and 400 mg/kg bw/day dose group were only minor in comparison to control animals and are considered to be without any biological relevance. No influence was noted for the plasma levels of albumin, globulin and the albumin/globulin ratio as well as for bilirubin, cholesterol, glucose, protein, calcium and lactate dehydrogenase. - 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):
- No changes were observed in animals treated with 6, 20 or 60 mg/kg bw/day.
Absolute weight of kidneys statistically significant reduced in males and females of the 400 mg/kg bw/day group. Reduction of the absolute kidney weight was also reduced in males and females of the 200 mg/kg bw/day group, however it was not statistically significant. The decreases of the absolute weights of the kidneys at 200 or 400 mg/kg bw/day are considered to be test item-related.
Treatment with 200 or 400 mg/kg bw/day via the diet caused an increase (statistically significant at p <= 0.01) in the relative brain, gonads, liver and kidney weights. These findings are considered to be due to the reduced body weight.
The statistically significant decreases (at p <= 0.01) of the absolute epididymis, gonads and brain weight of male and female animals compared to the control group are not considered to be test item-related because of the lacking dose dependence. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Macroscopic inspection at necropsy revealed test item-related changes in form of haemorrhagic foci and haemorrhagic deposits in the stomachs by one male and 3 females treated with 20 mg/kg bw/day, one male and 3 females treated with 60 mg/kg bw/day, 3 males treated with 200 mg/kg bw/day and 3 males and 3 females treated with 400 mg/kg bw/day. These findings are considered to be test item-related.
Further macroscopic lesions were noted in (parts of) the intestinal tract (blackbrown content), lungs (dark-red discoloured), lymph node (mesenteric) (enlarged), right ovary (cystic, filled with clear liquid), right preputial glands (hardened), seminal vesicle (reduced in size), right testis (reduced in size), eyes (reduced in size, corneal opacity), penis (protruded) and uterus (nodular thickened) in individual animals of the test item-treated groups. These changes are considered to be incidental findings due to the low number of animals affected. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- 2/10 male and female animals treated with 20 mg/kg bw/day, all males and 4/10 females treated with 60 mg/kg bw/day and 8/10 male and 7/10 female mice treated with 200 mg/kg bw/day via the diet showed a minimal to moderate dose dependent increase of macrophages in the mesenteric lymph nodes with vacuolization of the cytoplasm. In some mice purulent inflammatory reactions were noted. Furthermore, an abscess was noted in one male and female animal treated with 200 mg/kg bw/day. Two male mice revealed foci of neutrophilic granulocytes. These effects were considered test substance-related, but a patholgical re-evaluation was condcuted for further evaluation and a final conclusion on the nature of the effects observed. The enhanced immune system evaluation (see also Tables 5 - 7 under 'Any other information on results incl. tables') revealed higher mean sums for increased and decreased mesenteric lymph node values in males at 60 and 200 mg/kg bw/day. In females the mean sum was higher for increased mesenteric lymph node values at 200 mg/kg bw/day. Increased values were mainly due to few cases of granulocytic infiltrate and single cell necrosis/apoptosis. These findings were related to the accumulation of foamy macrophages in the mesenteric lymph nodes from both sexes at 60 and 200 mg/kg bw/day. In addition, these findings were associated with an accumulation of foamy macrophages in the mucosal lactea from jejunum and ileum in both sexes at 200 mg/kg bw/day, an unreported finding in the original study report of the 90-day oral toxicity study in mice. This accumulation was considered to be directly related to accumulation of foamy macrophages in the mesenteric lymph nodes.
There were no significant differences noted for the spleen and Peyer’s patches comparing the mean sums from controls with animals at 200 mg/kg bw/day. General effects on the immune system are excluded. All findings were local in mesenteric lymph nodes. They were considered to be due to increased storage of the test item or its metabolite(s) as a consequence of uptake by macrophages via lactea and lymphatic vessels. Inflammatory lesions in the mesenteric lymph nodes were of minimal incidence and severity and were noted at 60 and 200 mg/kg bw/day only. In addition, no such findings were noted in the available 78-week study in mice. Even foamy macrophage aglomeration was not seen in mesenteric lymph nodes. Especially, there was no indication for inflammation in the lymph nodes after 78 weeks. Also in the available 90-day study in dogs, no inflammation was noted. It was therefore concluded, that the few cases of minimal inflammatory lesions in mesenteric lymph nodes from mice of the 13-week study were not related to treatment with the test item. Instead, an adaptation was deemed to be very likely in mice.
Male and female mice treated with 200 mg/kg bw/day via the diet revealed a minimal to moderate increase of basophilic tubular cells with interstitial lymphocytic inflammatory reaction in the kidneys which is considered to be related to the administration of the test item. The renal changes were more pronounced in the kidneys of male animals and occurred in 7 of 10 male mice and in 3 of 10 female mice treated with 200 mg/kg bw/day. As these findings were limited to the 200 mg/kg bw/day group and no enhanced lesions were reported in the kidneys from animals of the other available repeated-dose toxicity studies, especially the 78-Week mouse study, the kidneys were not part of the enhanced immune system evaluation.
The lympho-histiocytic infiltrations in liver, kidney and heart of the control and test item-treated animals are considered to be spontaneous organ changes and thus are not test item-related.
The coincidental findings in a small number of control and test animals are considered to be spontaneous organ changes and are thus not test item-related. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- There was no indication of any impairment to auditory acuity.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 12 mg/kg bw/day (nominal)
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- food consumption and compound intake
- haematology
- Dose descriptor:
- NOAEL
- Effect level:
- 60 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- food consumption and compound intake
- haematology
- Key result
- Critical effects observed:
- no
- Conclusions:
- CD-1 mice were treated with the registration substance for 90 consecutive days via diet at nominal dose levels of 0, 1.2, 4, 12, 40 and 80 mg a.i./kg bw/day. Test item-related changes were observed starting at a dose of 40 mg a.i./kg bw/day, including decreased body weight and food consumption, changes in haematological and biochemical parameters, decreased kidney weights and effects on mesenteric lymph nodes. The NOAEL was 12 mg a.i./kg bw/day.
- Executive summary:
The aim of this 13-week dose-range-finding study was to obtain information on the toxicity of the registration substance administered via the diet to mice for 13 weeks, and to obtain information on the dose levels be selected for a carcinogenicity study in mice. One of 20 mice treated with 400 mg/kg bw/day died prematurely. In addition, marginal changes in behaviour were noted at 400 mg/kg bw/day. The body weight of the male and female animals treated with 200 or 400 mg/kg bw/day via the diet was reduced from test week 2 onwards. The body weight gain and body weight at autopsy were reduced accordingly. Treatment with 200 or 400 mg/kg bw/day caused changes in haematological and biochemical parameters (increase of leucocytes, platelets, neutrophilic granulocytes, lymphocytes and basophilic granulocytes, urea, aspartate aminotransferase and decrease in creatinine). No test item-related influences were noted during ophthalmological and auditory examination. Macroscopic inspection at necropsy revealed test item-related changes in form of haemorrhagic foci and haemorrhagic deposits in the stomachs of male and of female animals treated with 20, 60, 200 or 400 mg/kg bw/day. Additionally, the absolute weights of the kidneys were decreased at 200 and 400 mg/kg bw/day. The histopathological examination of male and female mice treated with 20, 60 or 200 mg/kg bw/day showed a dose dependent increase of macrophages in the mesenteric lymph nodes with vacuolization of the cytoplasm. In some mice purulent inflammatory reactions were noted. The animals treated with 200 mg/kg bw/day via the diet revealed a minimal to moderate increase of basophilic tubular cells with interstitial lymphocytic inflammatory reaction in the kidneys which is considered to be related to the administration of the test item. The mean actual intake of the test item via the diet was 5.9, 19.4, 59.7, 214.0 and 448.4 mg test substance/kg bw/day for the males and 6.2, 20.7, 63.5, 216.9 and 444.6 mg/kg bw/day for the females at the nominal dose levels of 6, 20, 60, 200 and 400 mg/kg bw/day.
Based on the above results dose levels were chosen for the combined chronic toxicity and carcinogenicity study. Based on the effects observed in the mesenteric lymph nodes starting from 20 mg/kg bw/day the NOAEL was determined to be 6 mg /kg bw/day which corresponds to 1.2 mg active ingredient/kg bw/day. However, an additional pathological assessment was conducted in 2022 to address possible systemic effects on the immune system. For the enhanced immune system evaluation, the following organs were evaluated: spleen, mesenteric lymph nodes, Peyer’s patches - jejunum (GALT – gut associated lymphoid tissue) (high dose and control animals only). The enhanced immune system evaluation revealed higher mean sums for increased and decreased mesenteric lymph node values in males at 60 and 200 mg/kg bw/day. In females, the mean sum was higher for increased mesenteric lymph node values at 200 mg/kg bw/day. Increased values were mainly due to few cases of granulocytic infiltrate and single cell necrosis/apoptosis. These findings were related to the accumulation of foamy macrophages in the mesenteric lymph nodes from both sexes at 60 and 200 mg/kg bw/day. In addition, these findings were associated with an accumulation of foamy macrophages in the mucosal lactea from jejunum and ileum in both sexes at 200 mg/kg bw/day, an unreported finding in the original study report of the 90-day oral toxicity study in mice. This accumulation was considered to be directly related to accumulation of foamy macrophages in the mesenteric lymph nodes. There were no significant differences noted for the spleen and Peyer’s patches comparing the mean sums from controls with animals at 200 mg/kg bw/day. Therefore, a general effect of the test substance on the immune system was excluded. All findings were local in mesenteric lymph nodes. They were considered to be due to increased storage of the test item or its metabolite(s) as a consequence of uptake by macrophages via lactea and lymphatic vessels. Inflammatory lesions in the mesenteric lymph nodes were of minimal incidence and severity and were noted at 60 and 200 mg/kg bw/day only. In addition, no such findings were noted in the available 78-week study in mice. Even foamy macrophage agglomeration was not seen in mesenteric lymph nodes. Especially, there was no indication for inflammation in the lymph nodes after 78 weeks. Also in the available 90 -Day study in dogs, no inflammation was noted. It was therefore concluded, that the few cases of minimal inflammatory lesions in mesenteric lymph nodes from mice of the 13-week study were not related to treatment with the test item. Instead, an adaptation was deemed to be very likely in mice. As the histopathological renal changes were limited to the 200 mg/kg bw/day group and no enhanced lesions were reported in the kidneys from animals of the other available repeated-dose toxicity studies, especially the 78-Week mouse study, the kidneys were not part of the enhanced immune system evaluation. Taken together, the systemic NOAEL was newly determined to be 60 mg /kg bw/day for male and female mice, which corresponds to 12 mg active ingredient/kg bw/day based on the described effects on decreased body weight and food consumption and changes in haematological and biochemical parameters.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Version / remarks:
- 2001
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Stefano Morini® s.a.s., Polo d’Enza, Italy
- Age at study initiation: 6 months
- Weight at study initiation: Males: 8.7–12.4 kg, Females: 7.7–12.3 kg
- Housing: singly or by twos in kennels, inside yard and outside yard with a total floor space of 9 m²
- Diet: Commercial ssniff® Hd-H V3234; 40 g/kg b.w. of food were offered to each dog daily for two hours; in case of animals with poor appetite, the food was served for a longer period (up to 8 hours); the residue was removed and weighed
- Water: ad libitum
- Acclimation period: 4 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C - Route of administration:
- oral: gavage
- Vehicle:
- other: tap water
- Details on oral exposure:
- VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg b.w.
- Concentration in vehicle: 0.5, 1.5 and 4.5 mg a.i./mL - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- daily
- Dose / conc.:
- 25 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 5 mg a.i./kg bw/day
- Dose / conc.:
- 75 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 15 mg a.i./kg bw/day
- Dose / conc.:
- 225 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 45 mg a.i./kg bw/day
- No. of animals per sex per dose:
- 4
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose levels are based on the results of a 4-week dose-range-finding study in dogs.
- Fasting period before blood sampling for clinical biochemistry: overnight - Positive control:
- none
- Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout the study
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- recorded daily
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: visual appraisal daily
OPHTHALMOSCOPIC EXAMINATION: Yes
- All animals were examined before treatment and during test week 13.
- Parameters: Adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus.
HAEMATOLOGY: Yes
- Number of animals: all animals.
- Time points: before first dosing / in week 6 / at study termination
- Parameters: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative), differential blood count (absolute), reticulocytes, platelets, haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration
CLINICAL CHEMISTRY: Yes
- Number of animals: all animals.
- Time points: before first dosing / in week 6 / at study termination
- Parameters: albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, phosphate, protein (total), urea, calcium, chloride potassium sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, γ glutamyl transferase
URINALYSIS: Yes
- Number of animals: all animals
- Time points: before first dosing / in week 6 / at study termination
- Parameters: volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite, epithelial cells, leucocytes, erythrocytes, organisms, further constituents, crystalluria, colour, turbidity - Sacrifice and pathology:
- Organ weights: Yes
Organs: adrenal glands, brain, epididymis, gall bladder, heart, kidney, liver, ovary, spleen, testicle, thymus, thyroid (incl. parathyroids), uterus
Gross and histopathology
Gross pathology:
Number of animals: all animals
Time point: end of study
Gross necropsy: a full macroscopic examination of all tissues and organs in situ.
Histopathology:
Number of animals: all animals
Time point: end of study
Histopathology: epididymis, kidney, liver, ovary, prostate, testicle, thymus, uterus inc. cervix, vagina, parathyroids (if grossly enlarged)
Number of animals: all control and high dosed animals
Time point: end of study
Additional histopathology: adrenal gland, aorta abdominalis, bone, bone marrow, brain, caecum, eye with optic nerve, gall bladder, gross lesions observed, heart, intestine large, intestine small, lacrimal gland, lungs, lymph node, mammary gland, muscle, nerve, oesophagus, pancreas, pituitary, salivary glands, seminal duct, skin, spinal cord, spleen, stomach, thyroid, tissue masses or tumours, trachea, urinary bladder
An additional pathological assessment was conducted to address possible systemic effects on the immune system. For the enhanced immune system evaluation, the following organs were evaluated based on the Enhanced Histopathology of the Immune System by Elmore (2012) and the details given by Elmore (2006): spleen, thymus, mesenteric lymph nodes, cervical lymph nodes, and Peyer’s patches - jejunum (GALT – gut associated lymphoid tissue) (high dose and control animals only). - Other examinations:
- Auditory examination
All animals were examined before treatment and during test week 13.
Examination: Noise test - Statistics:
- Body weight, food consumption, haematology, clinical biochemistry, relative and absolute organ weights: Dunnett’s multiple t-test
Urinalysis: Student’s t-test
Histology: Fisher’s exact test - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 5.0 mg a.s./kg bw/d: None
At 15 mg a.i./kg bw/day, two females revealed emesis on one and five test days, respectively, 5 to 60 min after administration, starting on test day 8. Males were not affected.
At 45 mg a.i./kg bw/day repeated emesis up to 4 times daily was noted in male and female animals on 51 to 79 test days from test day 5 onwards until the end of the study. Emesis started immediately post administration to 6 hours post administration. Additionally, slight to moderate salivation was noted in all high dosed animals on 70 to 84 test days from test day 6 onwards starting before or 0 to 5 minutes after administration and lasting up to 6 hours. - Mortality:
- no mortality observed
- Description (incidence):
- None of the dogs died prematurely.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A treatment related decrease of body weight was noted in the animals administered 15 or 45 mg a.i./kg bw/day up to 33% in the high dose males and 40% i nthe high dosed females from test week 2 onwards. This change was more pronounced in the female animals. The group mean body weight of the high dosed females was statistically significantly decreased in test weeks 6 and 11 to 13. The body weight at autopsy was decreased in the male and female animals treated with 15 or 45 mg a.i./kg bw/day by 34% in the high dose males and 41% in the high dose females)
Body weight gain followed a similar pattern. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- The overall average of the weekly mean food consumption was slightly decreased in animals treated with 15 or 45 mg a.i./kg bw/day. Statistically significant reduction was noted in high dosed females in test weeks 2 to 4 and in intermediate dosed females in test weeks 2 and 9.
Drinking water consumption revealed no difference compared with the control. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No test item-related changes of the eyes and the optic region were observed.
One low and one intermediate dosed male animal showed reddened conjunctivae and/or chemosis. Due to the lack of dose dependency, these findings were considered to be incidental. - Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 5 mg a.i./kg bw/day: No test-item related changes in haematological parameters
In animals treated with 15 or 45 mg a.i./kg bw/day the following changes in haematological parameters were noted:
Increase of leucocytes:
- Week 6: in intermediate dosed females and in high dosed animals of both sexes (statistically significant)
- Week 13: in intermediate dosed females and in high dosed animals of both sexes
Decrease of reticulocytes:
- Week 6: in high dosed males (statistically significant) and in high dosed females
Increase of neutrophils (absolute):
- Week 6: in intermediate dosed males and females (statistically significant) and in high dosed animals of both sexes (statistically significant)
- Week 13: in intermediate dosed females and in high dosed animals of both sexes
Increase of monocytes (absolute):
- Week 6: in intermediate dosed females (statistically significant) and in high dosed males and females (statistically significant)
- Week 13: in intermediate dosed females and in high dosed animals of both sexes
Increase of large unstained cells (absolute):
- Week 6: in intermediate dosed females and in high dosed males and females (statistically significant)
- Week 13: in intermediate dosed females and in high dosed animals of both sexes
Increase of activated partial thromboplastin time:
- Week 6: in high dosed females
- Week 13: in high dosed animals of both sexes (statistically significant)
Statistically significant increase of lymphocytes (absolute) in intermediate dosed females and high dosed males is considered not to be test item related. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 5 mg a.i./kg bw/day: No test-item related changes in biochemical parameters
In animals treated with 15 or 45 mg a.i./kg bw/day the following changes in biochemical parameters were noted:
Decrease of albumin:
- Week 6: in high dosed male (statistically significant) and female animals
- Week 13: in intermediate dosed females (statistically significant) and in high dosed animals of both sexes (statistically significant)
Increase of cholesterol:
- Week 6: in high dosed animals of both sexes (statistically significant)
Decrease of protein (total):
- Week 13: in high dosed animals of both sexes (statistically significant)
Decrease of ALAT:
- Week 6: in intermediate dosed males and females (statistically significant) and in high dosed animals of both sexes (statistically significant)
- Week 13: in high dosed males (statistically significant) and females
Decrease of ASAT:
- Week 6: in high dosed animals of both sexes (statistically significant)
- Week 13: in high dosed males (statistically significant) and females
Statistically significant decrease of A/G ratio, creatinine, calcium, chloride and sodium was considered not to be test item related. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 5 or 15 mg a.i./kg bw/day: No test-item related changes
Animals treated with 45 mg a.i./kg bw/day showed a slight increase of the urinary pH value in test week 6 (statistically significant increase in females) and week 12/13, and increased haemoglobin content in the urine. The specific gravity and the urine volume were not changed. - 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):
- 5 mg a.i./kg bw/day: No test-item related changes in relative or absolute organ weights
In animals treated with 15 or 45 mg a.i./kg bw/day the following changes were observed:
- Decrease of absolute heart weight in the intermediate dosed animals of both sex and in the high dosed animals of both sexes (statistically significant)
- Decrease of absolute and relative thymus weight in the intermediate dosed females (statistically significant) and in the high dosed males and females (statistically significant)
- Further, relative organ weights of adrenals, brain, kidneys and thyroids were increased (partly statistically significant) caused by the reduced body weight - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 5 mg a.i./kg bw/day: No test-item related changes
15 mg a.i./kg bw/day:
-Males: pale or yellowish discoloured kidneys (2/4), yellow-stained discoloured liver (1/4)
-Females: No changes observed
45 mg a.i./kg bw/day:
-Males: pale or yellowish discoloured kidneys (1/4), yellow-stained discoloured liver (2/4), reddened pancreas (1/4), downsized prostate (2/4)
-Females: pale kidneys (2/4), yellow-stained discoloured liver (2/4), red discoloured pancreas (1/4), white-stained discoloured spleen (1/4), grey-yellow discoloured foci (1/4) in the right lung (and a consolidated central lobe) and/or downsized uterus (3/4), thymus (2/4) and/or spleen (1/4)
Other changes in a few male or female animals of the control or test item treated groups are considered to be incidental. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Animals of both sexes treated with 15 or 45 mg a.i./kg bw/day showed morphological lesions in the genital system which are considered to be test item related:
-Males: Increase of immature spermatozoa with reduction of mature spermatozoa in the epididymides, dose-related atrophy/inactivation of the prostate, minimal to moderate atrophy of the germinative epithelium in the testes (only in high dosed animals))
-Females: Dose-related minimal to moderate atrophy of uterus, ovary, vagina and/or cervix, atrophy of the uterine horns and the mucosa (only in high dosed animals)
Additionally, a minimal to moderate reduction of lymphoid tissue (involution) was noted in the cortex and the medulla of the thymus of male and female animals treated with 15 or 45 mg a.i./kg bw/day which are considered to be test item-related.
The effects related to the reproductive organs described above are considered to be secondary due to exceedingly strong systemic toxicity. A histopathological re-assessment by an expert reasons, that the histomorphological findings in the reproductive organs are not primary test-item related effects, but are of secondary nature to the exceedingly strong systemic effects of the registration substance in beagle dogs that are not fully mature at study termination.
The enhanced immune system evaluation (see also Tables 2 - 4 under 'Any other information on results incl. tables') revealed lower mean sums for increased mesenteric and cervical lymph node values. In females the mean sum was higher for increased mesenteric lymph node values at 45 mg/kg bw/day. These values were linked to stress-related atrophic changes as also described in the histopathological re-assessment report related to the reproductive findings. The differences of mean sums in thymus and spleen, namely in females, also reflected the described atrophy. There were no significant differences noted for the spleen and Peyer’s patches comparing the mean sums from controls with animals at 200 mg/kg bw.
In the mesenteric lymph nodes from two females at 45 mg/kg bw/day, there was a minimal accumulation of foamy macrophages that could not be excluded from being induced by treatment with the test item and it might has been a consequence of accumulation of foamy macrophages in the mucosal lactea from jejunum and ileum in both sexes at 45 mg/kg bw/day. These findings were consisted with alterations noted in the available 13-Week study in mice. However, no inflammation was noted in the intestinal mucosa nor in the mesenteric lymph nodes. Therefore it was concluded, that these effects were not related to treatment with the test item. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- The auditory examination revealed no indication of any impairment to the auditory acuity.
- Dose descriptor:
- NOAEL
- Effect level:
- 25 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- test mat. (20% aqueous solution, "product by process")
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 5 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- Key result
- Critical effects observed:
- no
- Conclusions:
- Beagle dogs were treated with the registration substance for 90 consecutive days by daily oral gavage at dose levels of 0, 5, 15 and 45 mg a.i./kg bw/day. Test item-related changes were observed starting at a dose of 15 mg a.i./kg bw/day, including decreased body weight and food consumption, changes in haematological and biochemical parameters, increase of urinary pH-value and haemoglobin content in urine (in the high dosed groups only). The NOAEL was 5 mg a.i./kg bw/day.
- Executive summary:
The oral 90-day toxicity study with the registration substance (as 20% aqueous solution) in a non-rodent species (dog) was carried out according to OECD guideline 409 and EC method B.27. Four groups of 4 male and 4 female Beagle dogs, respectively, were administered orally by gavage 0, 5, 15 and 45 mg/kg bw/day in terms of active ingredient diluted in vehicle (tap water) for 90 days.
None of the animals died prematurely. Two of four female animals treated with 15 mg a.i./kg bw/day of test item revealed emesis and all animals administered 45 mg a.i./kg bw/day showed emesis and salivation on most treatment days.
Test item-related changes were observed starting at a dose of 15 mg a.i./kg bw/day, including decreased body weight and food consumption, changes in haematological and biochemical parameters, increase of urinary pH-value and haemoglobin content in urine (in the high dosed groups only).
No changes compared to the vehicle control were noted for drinking water consumption and the ophthalmological and auditory examination.
Absolute organ weights of the heart and the thymus were significantly decreased in animals treated with the intermediate and high dose of the test item. Relative thymus weight was significantly decreased in high and intermediate dosed females. In almost all males treated with 15 and 45 mg a.i./kg bw/day the prostates were downsized and the kidneys, livers and pancreas showed discolouration. In high dosed females also discolouration of the kidneys, livers, pancreas, spleen and/or lungs and a reduction in size of uterus, thymus and/or spleen were observed. The histomorphological examination revealed dose-related atrophy in the male and female genital system in intermediate and high dosed animals. Slightly more pronounced involution of the thymus was observed in both sexes treated with the intermediate and high dose. The histomorphological findings were not considered by the pathologist to be a direct effect of treatment but were rather attributed to the severe systemic toxicity of the test substance in combination with stress associated with decreased food intake as well as reduced body weight and body weight gain.
Based on these results, the NOAEL was 25 mg/kg bw/day, corresponding to 5 mg/kg bw/day active ingredient.
An additional pathological assessment was conducted in 2022 to address possible systemic effects on the immune system. The following organs were evaluated: spleen, thymus, mesenteric lymph nodes, cervical lymph nodes, and Peyer’s patches - jejunum (GALT – gut associated lymphoid tissue) (high dose and control animals only). The enhanced immune system evaluation revealed lower mean sums for increased mesenteric and cervical lymph node values. In females, the mean sum was higher for increased mesenteric lymph node values at 45 mg/kg bw/day. These values were linked to stress-related atrophic changes as also described in the histopathological re-assessment report related to the reproductive findings. The differences of mean sums in thymus and spleen, namely in females, also reflected the described atrophy. There were no significant differences noted for the spleen and Peyer’s patches comparing the mean sums from controls with animals at 200 mg/kg bw. In the mesenteric lymph nodes from two females at 45 mg/kg bw/day, there was a minimal accumulation of foamy macrophages that could not be excluded from being induced by treatment with the test item and it might has been a consequence of accumulation of foamy macrophages in the mucosal lactea from jejunum and ileum in both sexes at 45 mg/kg bw/day. These findings were consisted with alterations noted in the available 13-Week study in mice. However, no inflammation was noted in the intestinal mucosa nor in the mesenteric lymph nodes. Therefore, it was concluded, that these effects were not related to treatment with the test item. Thus, from the enhanced immune system evaluation it was finally concluded that the changes observed in the different available repeated dose toxicity studies were limited to local reactions in the mesenteric lymph nodes as a consequence of an uptake and transfer of the test item by intestinal macrophage via lacteal lymph fluid. General efects on the immune system are excluded. Inflammatory lesions in the mesenteric lymph nodes were of minimal incidence and severity and were noted in the available 13-week study in mice at 60 and 200 mg/kg bw/day only. No such findings were observed in the available 78-Week study in mice. Even foamy macrophage agglomeration was not seen in mesenteric lymph nodes. Especially, there was no indication for inflammation in the lymph nodes after 78 weeks. In addition, in the current 90-Day study in dogs, no inflammation was noted. Therefore, it was concluded, that the few cases of minimal inflammatory lesions in mesenteric lymph nodes from mice of the 13-week study were not related to treatment with the test item. It was noted that macrophages agglomerated in the intestinal lactea, which is deemed to be the first step in the pathway of development of lesions. It is noteworthy, that no intestinal inflammation was encountered in mice after 78 weeks. Instead, an adaptation was deemed to be very likely for mice.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-1 (90-Day Oral Toxicity)
- Version / remarks:
- 1982
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Sprague Dawley, Crl: CD(SD)BR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 7 to 8 weeks (6 to 7 weeks for follow up study)
- Weight at study initiation: Males: 120–245 g; Females: 155–195 g (Males: 200–235 g; Females: 150–190 gfor follow up study)
- Housing: in groups of max. 3 (males) or 4 (females)
- Diet: powdered diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 18 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- other: distilled water
- Details on oral exposure:
- VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw/day - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- once daily
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 20 mg a.i./kg bw/day
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 10 mg a.i./kg bw/day
- Dose / conc.:
- 25 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 5 mg a.i./kg bw/day
- Dose / conc.:
- 12.5 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 2.5 mg a.i./kg bw/day (follow up study)
- Dose / conc.:
- 6.25 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 1.25 mg a.i./kg bw/day (follow up study)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Observations and examinations performed and frequency:
- Observations
- Clinical signs: Yes (at least once daily)
- Mortality: Yes (twice daily)
Body weight: Yes (weekly throughout the study and on the day of necropsy)
Food consumption: Yes (recorded daily throughout the dosing period for the rats in one cage and evaluated on a weekly basis)
Water consumption: no
Ophthalmoscopic examination: Yes (not performed in the follow up study)
Number of animals:
a) all animals of all groups
b) all control and high dosed animals
Time points:
a) before treatment
b) final week of treatment
Parameters: Ocular fundus with macula lutea, papilla, ocular vessels, cornea, lens and vitreous body.
Haematology: Yes
Number of animals: all animals
Time point: final week of study
Parameters: mean cell haemoglobin, packed cell volume, mean cell haemoglobin concentration, mean cell volume, haemoglobin concentration, total and differential white blood cell counts, prothrombin time
Clinical chemistry: Yes (not performed in follow up study)
Number of animals: all animals.
Time point: final week of study
Parameters: glutamate oxalacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), alkaline phosphatase (AP), sodium ions (NA+), potassium ions (K+), calcium (Ca++), phosphorus (P), chloride (CL-), total protein (TP), blood urea (BU), albumin, albumin/globulin ratio (A/G ratio), glucose, cholesterol, triglycerides, creatinine, total bilirubin.
Urinalysis: no - Sacrifice and pathology:
- Organ weights: Yes (not performed in follow up study)
Organs: adrenals, kidneys, liver, ovaries, testes
Gross and histopathology: Yes
Grosspathology:
Number of animals: all animals
Time points: end of study
Gross necropsy: detailed examination of external body surface, all orifices and a full macroscopic examination of all tissues and organs in situ
Histopathology:
Number of animals: all control and high dosed animals
Time points: end of study
Histopathology: adrenals, aorta, bone marrow, brain, cecum, colon, duodenum, epididymides, esophagus, eyes, heart, ileum, jejunum, lymph nodes, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, skeletal muscle, skin and mammary gland, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids, tongue, trachea, urinary bladder, uterus, vagina.
Kidneys, liver, lungs were examined in rats from all groups.
In the follow up group only samples of mesenteric lymph nodes were analysed. - Statistics:
- Body weight, haematology data: analysis of variance, Student-Newman-Keuls test
Food consumption: evaluation of group mean values; only indicates trends in and between the three groups. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- Main study: No treatment-related clinical signs were observed.
There were occasional findings related to the dosing procedure, such as bleeding of snout following application in one high dose male. Furthermore, staggering gait and disequilibrium were observed repeatedly in one male rat of the mid dose group. Other infrequent findings were a subcutaneous nodule of 7 mm in diameter on the right side of the snout in one male of the low dose group and broken incisor in one control female.
Follow up study: No treatment-related clinical signs of intoxication were reported throughout the study period in any group. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Main study: The majority of mortalities observed was due to the experimental procedure such as intubation error (2 control animals, 2 mid dose animals, 3 high dose animals) and ether anesthesia for blood sampling (2 low dose animals). In one female animal, the cause of demise could not be determined due to cannibalism. However, this death was considered to be incidental since there was no unequivocal evidence of any signs of toxicity prior to death.
Follow up study: No treatment-related mortalities were observed during the study.
The mortalities observed (one female treated with 6.25 mg/kg bw/day and 12.50 mg/kg bw/day, respectively) were due to the experimental procedure such as intubation error and ether anesthesia for blood sampling. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Main study: No treatment-related effects on body weights of female animals were observed.
The group mean body weight of the male rats of the mid dose and the high dose group was statistically significantly decreased from week 9 to week 13 of treatment.
The overall body weight gain (week 1 to 13) was statistically significantly reduced in mid dose males (–19.8%) and high dose males (–27.7%).
Follow up study: There was no effect on mean body weight throughout the study in any treated group and sex.
Details are presented in Table A6.4.1- 2. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Main study: There was no noteworthy effect on overall food intake throughout the treatment period for female animals when compared to the corresponding control.
For male animals, the group mean food intake was slightly increased in the low dose group (+7.0%) and slightly reduced the mid ( 8.0%) and the high dose group (–10.1%).
Follow up study: There was no noteworthy effect on overall food consumption throughout the treatment period for animals of either sex when compared to corresponding controls. Results are presented in Table A6.4.1- 2. - 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):
- Main study: Congestion of the optic disk was observed in five males and in one female high dose animal in week 13. Ocular changes are known to be associated with increased intracranial pressure and concomitant edematous swelling. Since there were no remarkable histopathological changes of all animals examined, these findings have to be considered as compound-related clinical symptoms, possibly secondary to treatment.
A pathological re-assessment confirmed that the congestion of the optic disk was either by chance or consequences of retro-orbital blood sampling. This conclusion is based on the fact that increased intracranial pressure would cause lesions like status spongiosus in the brain, neuronal necrosis or degeneration, and in more chronic lesions also gliosis. In addition, intracranial pressure may cause death. None of these lesions have been reported. Similar findings were also not noted in the other available repeated dose toxicity studies in mice and dogs. The retro-orbital blood sampling was the method of choice at the time the study was performed. Hemorrhage and inflammation in the retro-orbital region are common lesions and congestion is a related alteration.
Other incidental findings were retinal hemorrhage of the optic disk or the fundus or between optic disk and macula (one control male, one male and two females of the high dose group), corneal scar (one control female), and optic disk palish coloured as defined by atrophy of optic nerve (one female of the high dose group).
Follow up study: not examined - Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Main study: Major statistically significant haematological changes observed at week 13 were distinctively increased leucocytes associated with increased neutrophile granulocytes and decreased lymphocytes seen in all treated groups compared with the control groups. Generally, leucocytosis, granulocytosis and lymphopenia are indicative of inflammatory processes. Further evidence to support this view were microscopic findings such as small microabscesses consisting of focal areas of necrosis and mild lymphoid hyperplasia in the mesenteric lymph nodes in high dose animals of either sex.
Minor statistically significant haematological findings were slightly reduced packed cell volume (PCV) and slightly increased mean cell haemoglobin concentration (MCHC) in all males of the treated groups.
Furthermore, prothrombin time (PT) values were slightly but statistically significantly reduced in males of the mid dose group (–21%) and in males (–21.5%) and females (–32.3%) of the high dose group.
Results are presented in Table A6.4.1- 1.
Follow up study: No treatment-related haematological changes were observed.
No biological relevance was attributed to significantly increased values for red blood cell concentration in males of both test concentrations and the increased value for packed cell volume in high dose females.
Details are presented in Table A6.4.1- 2. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Main study: A statistically significant increase of alkaline phosphatase levels (AP) was observed in high dose females only. This marked hyperphosphatasemia was considered to be difficult to interpret since alkaline phosphatase is widely distributed in the body and is present in considerable concentration in many tissues. In addition, in the absence of other unequivocal indications to account for this sex specific alteration increased alkaline phosphatase levels have to be considered as of unknown biological significance.
Further blood chemistry findings (reduction in glucose, total protein, albumin, albumin/globulin ratio and cholesterol) were generally slight and may be interpreted as slight effects of the test article on carbohydrate, lipid and protein metabolism.
Results are presented in Table A6.4.1- 1.
Follow up study: 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):
- Main study: The absolute liver weight was statistically significantly decreased (17.7%) and the relative kidney weight was statistically significantly increased (+17.5%) in high dose males. Both effects have to be considered primarily as a secondary effect following decrease of body weights.
Other changes such as reduction of absolute testes weights of mid dose males and increased relative adrenal weights of high dose males were due to single remarkable individual values which are considered to be more incidental than treatment-related.
There were no statistically significant findings in absolute and relative organ weights in females.
Results are presented in Table A6.4.1- 1.
Follow up study: not examined - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Main study: There were no treatment-related macroscopic findings. Bloody stomach content in one high dose male was related to the dosing procedure when occasional bleeding of the snout was observed throughout the last week of treatment.
Follow up study: There were no treatment-related macroscopic findings and no abnormalities of mesenteric lymph nodes. All of the findings encountered were considered to have emerged spontaneously. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Main study: Microscopic compound related lesions were observed in the high dose rats only.
The following lesions of mesenteric lymph nodes were observed:
- Foci of necrosis in 5 out of 7 males and in 6 out of 8 females of the high dose group
- Acute inflammation in 5 out of 7 males and in 6 out of 8 females of the high dose group
- Lymphoid hyperplasia in 5 out of 7 males and in 3 out of 8 females of the high dose group
These mesenteric lymph node lesions were secondary to treatment with the high dose of the test compound. However, the type of reaction noted is typical of a bacterial lymphadenitis, which is not a direct toxic effect. No bacteria or other causative agents were observed in the lesions.
Since histopathological examination of mesenteric lymph nodes did not include group 2 and 3 animals, the effect of the test substance at dose levels of 25 and 50 mg/kg bw/day on this tissue could not be clarified. However, the observed effects on the mesenteric lymph nodes in the available repeated dose toxicity studies in mice and dogs were re-evaluated in 2022 in the context of possible systemic effects of the test substance on the immune system (see also discussions in the endpoint summary of the section Repeated dose toxicity). It was finally concluded that the changes observed in the different repeated dose toxicity studies of mice and dogs were limited to local reactions in the mesenteric lymph nodes as a consequence of an uptake and transfer of the test item by intestinal macrophage via lacteal lymph fluid. A general effect of the test substance on the immune system was excluded. Instead, an adaptation was deemed to be very likely in mice, which is also assumed to be the case for the rat.
Follow up study: There were no treatment-related macroscopic findings and no abnormalities of mesenteric lymph nodes. All of the findings encountered were considered to have emerged spontaneously. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Dose descriptor:
- NOAEL
- Effect level:
- 12.5 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- (20% aqueous solution, "product by process")
- Sex:
- male/female
- Basis for effect level:
- haematology
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 2.5 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- haematology
- Key result
- Critical effects observed:
- no
- Conclusions:
- Sprague Dawley rats were treated with the registration substance for 90 consecutive days by daily oral gavage at dose levels of 0, 25, 50 and 100 mg/kg bw/day (as 20% aqueous solution), corresponding to 0, 5, 10 and 20 mg a.i./kg bw/day.
The LOAEL based on significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes at all dose levels was 25 mg/kg bw/day (as 20% aqueous solution), corresponding to 5 mg a.i./kg bw/d. No NOAEL could be determined. Therefore, a supplementary 90-day oral toxicity test utilising a modified dose range was conducted. In this follow up study, Sprague Dawley rats were treated with the registration substance for 90 consecutive days by daily oral gavage at dose levels of 0, 6.25 and 12.5 mg/kg bw/day (as 20% aqueous solution), corresponding to 0, 1.25 and 2.5 mg a.i./kg bw/d. No treatment related adverse effects were observed in this study. The oral 90 day NOAEL (rat, male/female) of the registration substance was 2.5 mg a.i./kg bw/day. - Executive summary:
The oral 90-day toxicity study was carried out according to EPA 82-1 (1982). Four groups of 10 male and 10 female Sprague Dawley rats, respectively, were administered orally by gavage 0, 25, 50 and 100 mg/kg bw/day of the registration substance (20% aqueous solution, substance as manufactured), corresponding to 0, 5, 10 and 20 mg a.i./kg bw/day for 90 days.
The conduct of the study was similar to OECD 408 (1998) with the exception that (i) no NOAEL could be determined, (ii) no sensory reactivity test, no assessment of grip strength, no motor activity assessment and no functional observations were performed, (iii) the weight variation at the commencement of the study exceeded ± 20 % for males, (iv) weights of epididymides, uterus, thymus, spleen, brain and heart were not determined, and (v) the mesenteric lymph nodes were not examined for the low and mid dose group despite observed effects at the high dose group.
Treatment with the test substance at a dose level of 100 mg/kg bw/day produced toxic effects manifested by foci of necrosis, acute inflammation and lymphoid hyperplasia of the mesenteric lymph nodes in animals of either sex. Since histopathological examination of mesenteric lymph nodes did not include the low dose and mid dose animals, the effect of the test substance at dose levels of 25 and 50 mg/kg bw/day on this tissue could not be clarified. However, the observed effects on the mesenteric lymph nodes in the available repeated dose toxicity studies in mice and dogs were re-evaluated in 2022 in the context of possible systemic effects of the test substance on the immune system (see also the discussions in the endpoint summary of the section Repeated dose toxicity). It was finally concluded that the changes observed in the different repeated dose toxicity studies of mice and dogs were limited to local reactions in the mesenteric lymph nodes as a consequence of an uptake and transfer of the test item by intestinal macrophage via lacteal lymph fluid. A general effect of the test substance on the immune system was excluded. Instead, an adaptation was deemed to be very likely in mice, which is also assumed to be the case for the rat.
Further treatment-related findings were reduction in body weight gain and overall food consumption in male animals receiving 50 and 100 mg/kg bw/day, ophthalmological findings (congestion of the optic disk) in five high dose male animals and one female (regarded as either chance effects or secondary treatment-related effects as a consequence of retro-orbital blood sampling), haematological changes (significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes) in males and females receiving 25, 50 and 100 mg/kg bw/day, slight blood chemistry changes (reduction in glucose, total protein, albumin, albumin/globulin ratio and cholesterol) within the normal range of background data in males and females treated with the test substance at 50 and 100 mg/kg bw/day, and a markedly increased alkaline phosphatase level in high dosed females only.
No treatment-related mortality or clinical signs occurred during the study.
Apart from slight leucocytosis, granulocytosis and lymphenia treatment with the test substance at a dose level of 25 mg/kg bw/day was very well tolerated and did not elicit any further toxic changes attributable to oral administration of the compound tested.
Based on a significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes at all dose levels, the LOAEL was 25 mg/kg bw/d (as 20% aqueous solution), corresponding to 5 mg a.i./kg bw/d.
No NOAEL could be determined based on the results of this study. Therefore, a supplementary 90-day oral toxicity test utilising a modified dose range was conducted (A6.4.1/02). In this study, three groups of 10 male and 10 female Sprague Dawley rats, respectively, were administered orally by gavage 0, 6.25 and 12.5 mg/kg bw/day of the registration substance (20% aqueous solution, substance as manufactured) for 90 days.
The conduct of the study was consistent in all important aspects to OECD 408 (1998) with the exception that only 2 dose levels (6.25 and 12.50 mg/kg bw/day) instead of 3 were examined and that survival, general health, haematological parameters, gross necropsy and histopathology of mesenteric lymph nodes were investigated only. However, this study is a follow-up study to reference A6.4.1/01. Thus, the investigation of further parameters is not considered to be required.
No treatment-related clinical signs, mortalities, effects on food consumption and body weight, haematological changes, macroscopic lesions in any of the organs or tissues examined were observed during the study. Microscopic findings did not reveal any abnormalities of mesenteric lymph nodes.
Based on the results, the NOAEL in males and females was 12.50 mg/kg bw/d (as 20% aqueous solution), corresponding to 2.5 mg a.i./kg bw/d. Based on the results, the NOAEL in males and females was 12.50 mg/kg bw/day (as 20% aqueous solution), corresponding to 2.5 mg a.i./kg bw/day. In addition, these results confirm the NOAEL in males and females of 12.50 mg/kg bw/day (as 20% aqueous solution), corresponding to 2.5 mg a.i./kg bw/day.
Referenceopen allclose all
Mortality rates and survival rates at study termination
Group |
Mortality rates at study termination |
|
males |
Females |
|
1 Control |
34 |
8 |
2 Males: 10 mg/kg bw/d Females: 20 mg/kg bw/d |
16 |
40 ** |
3 Males: 30 mg/kg bw/d Females: 60 mg/kg bw/d |
40 |
74 ** |
4 Males: 100 / 35 mg/kg bw/d Females: 200 / 65 mg/kg bw/d |
74 ** |
74 ** |
** significantly different from control at p </= 0.01 (Fisher test)
Table A6.7-3:Results of the carcinogenicity study
Dose [mg/kg bw/d] |
0 |
0 |
10 |
20 |
30 |
60 |
100/35 |
200/65 |
|
|
|
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
Number of animals |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
|
|
Mortality (at study termination) |
|
|
|
|
|
|
|
|
|
|
Mortality rate [%] |
34 |
8 |
16 |
40** |
40 |
74** |
74** |
74** |
+ |
+ |
Mean survival time [test week] |
72.1 |
77.1 |
76.0 |
73.5 |
70.4 |
67.3** |
58.8 |
52.3** |
|
– |
Clinical signs |
|
|
|
|
|
|
|
|
|
|
Swollen/reddened eyes |
3 |
1 |
1 |
1 |
15 |
34 |
21 |
18 |
+ |
+ |
Ptosis |
4 |
2 |
2 |
0 |
12 |
19 |
15 |
7 |
+ |
|
Haematology |
||||||||||
Haemoglobin content (nmol/L) |
||||||||||
Week 13 |
9.47 |
9.69 |
9.52 |
9.60 |
8.92** |
9.26 |
8.70** |
8.65** |
– |
– |
Week 26 |
9.14 |
9.62 |
9.09 |
9.16 |
8.33** |
8.66** |
7.99** |
8.53** |
– |
– |
Week 52 |
8.38 |
8.42 |
8.53 |
8.18 |
7.91 |
8.22 |
8.19 |
8.18 |
– |
– |
Week 78/79 |
8.04 |
8.22 |
7.99 |
7.81 |
7.91 |
8.36 |
8.43 |
7.59 |
|
|
Haematocrit (%) |
||||||||||
Week 13 |
41.8 |
42.5 |
41.9 |
41.6 |
39.6** |
40.3** |
38.8** |
38.3** |
– |
– |
Week 26 |
44.5 |
46.1 |
44.4 |
43.8** |
41.0** |
42.1** |
39.5** |
41.9** |
– |
– |
Week 52 |
40.3 |
42.0 |
41.1 |
39.9 |
38.2 |
40.7 |
40.3 |
40.5 |
|
|
Week 78/79 |
39.3 |
40.2 |
39.4 |
38.0 |
39.8 |
41.1 |
42.0 |
37.5 |
|
|
Leucocytes (109/L) |
||||||||||
Week 13 |
5.20 |
3.51 |
5.66 |
3.76 |
4.53 |
4.26 |
5.41 |
4.10 |
|
|
Week 26 |
3.85 |
3.97 |
4.23 |
3.66 |
3.02 |
3.38 |
2.94 |
4.59 |
|
|
Week 52 |
2.19 |
3.72 |
2.28 |
4.68 |
2.56 |
5.01 |
2.36 |
5.80** |
|
+ |
Week 78/79 |
5.98 |
5.56 |
7.96 |
4.67 |
6.99 |
4.70 |
5.18 |
5.21 |
|
|
Differential blood count – neutrophilic granulocytes (no. of cells × 109/L) |
||||||||||
Week 13 |
0.978 |
0.557 |
1.032 |
0.893 |
0.955 |
0.862 |
1.442 |
1.418** |
|
+ |
Week 26 |
1.102 |
0.710 |
0.961 |
0.914 |
0.697 |
1.001 |
0.921 |
1.464** |
|
+ |
Week 52 |
0.671 |
0.781 |
0.535 |
0.957 |
0.624 |
1.322 |
0.805 |
1.366** |
|
+ |
Week 78/79 |
1.349 |
1.363 |
1.633 |
1.631 |
2.454 |
1.985 |
1.461 |
1.541 |
|
|
Differential blood count – monocytes (no. of cells × 109/L) |
||||||||||
Week 52 |
0.041 |
0.094 |
0.039 |
0.150 |
0.053 |
0.181** |
0.056 |
0.176** |
|
+ |
Clinical biochemistry |
||||||||||
ASAT (U/L) |
||||||||||
Week 26 |
84.1 |
93.4 |
85.1 |
108.7 |
88.1 |
134.0 |
224.1** |
198.9** |
+ |
+ |
Week 52 |
83.0 |
92.1 |
81.8 |
89.8 |
89.3 |
137.7** |
165.9** |
156.2** |
+ |
+ |
Week 78/79 |
93.6 |
133.7 |
92.9 |
101.2 |
173.3 |
182.2 |
221.6** |
165.8 |
+ |
|
LDH (U/L) |
||||||||||
Week 26 |
222.7 |
209.8 |
195.7 |
226.6 |
209.7 |
282.3 |
409.2** |
366.8** |
+ |
+ |
Week 52 |
261.3 |
177.8 |
212.0 |
176.3 |
216.1 |
276.8** |
312.5 |
241.9 |
+ |
+ |
Week 78/79 |
262.0 |
819.9 |
236.6 |
298.5 |
342.1 |
389.4a |
476.4 |
451.8a |
+ |
|
Macroscopic findings |
||||||||||
Spleen reduction |
1/50 |
0/50 |
3/50 |
0/50 |
2/50 |
1/50 |
11/50 |
3/50 |
+ |
|
Dilated urinary bladder |
0/50 |
0/50 |
0/50 |
0/50 |
0/50 |
0/50 |
1/50 |
1/50 |
|
|
Enlarged urinary bladder |
1/50 |
0/50 |
1/50 |
0/50 |
3/50 |
1/50 |
16/50 |
5/50 |
+ |
+ |
Tightly filled urinary bladder |
3/50 |
0/50 |
2/50 |
0/50 |
10/50 |
11/50 |
35/50 |
10/50 |
+ |
+ |
**) significantly different from control (p<0.01) a) no evaluation possible due to unusually high vales in the female control |
||||||||||
Table 1: Body weight
Changes in body weight compared to the control[%] |
||||
Test week |
Group 5 |
Group 6 |
||
males |
females |
males |
females |
|
2 |
-7.9 |
-9.6 |
-19.4* * |
-17.1* * |
3 |
-8.5 |
-12.6* * |
-23.2* * |
-15.5* * |
4 |
-9.5 |
-7.6 |
-23.5* * |
-15.5* * |
5 |
-8.3 |
-6.7 |
-19.6* * |
-15.5* * |
6 |
-8.1 |
-7.5 |
-19.4* * |
-16.3* * |
7 |
-12.5* * |
-7.9 |
-22.3* * |
-18.4* * |
8 |
-13.8* * |
-10.8 |
-30.6* * |
-19.1* * |
9 |
-15.7* * |
-12.5* * |
-27.7* * |
-23.1* * |
10 |
-18.2* * |
-12.5* * |
-30.0* * |
-26.3* * |
11 |
-21.4* * |
-15.3* * |
-32.0* * |
-27.1* * |
12 |
-24.2* * |
-12.2* * |
-34.5* * |
-27.3* * |
13 |
-25.9* * |
-16.1* * |
-33.8* * |
-29.6* * |
**=statistically significantatp<=0.01
Table 2: Changes of food consumption
Changes of food consumption compared to the control [%] |
||||
Test week |
Group 5 |
Group 6 |
||
males |
females |
males |
females |
|
1 |
none |
none |
+ 22* * |
+13 |
2 |
none |
none |
+37* * |
+16 |
3 |
+46* * |
+39* * |
+80* * |
+56* * |
4 |
+48* * |
+43* * |
+81* * |
+ 51* * |
5 |
+39* * |
+26 |
+57* * |
+32* * |
6 |
+43* * |
+18 |
+51* * |
+27 |
7 |
+30* * |
none |
+51* * |
+17 |
8 |
+42* * |
none |
+87* * |
+19 |
9 |
+40* * |
+15 |
+53* * |
+18 |
10 |
+33* * |
+18 |
+58* * |
+ 24 |
11 |
+32* * |
none |
+ 55* * |
+21 |
12 |
+23 |
none |
+ 49* * |
+ 22 |
13 |
+36* * |
none |
+ 52* * |
+19 |
**=statistically significantatp<=0.01
Table 3: Changes in haematology
Changes in haematological parameters compared to the control[%] |
||||
Parameter |
Group 5 |
Group 6 |
||
males |
females |
males |
females |
|
WBC |
none |
+167* * |
none |
+75 |
PCT |
none |
+59* * |
+27* * |
+65* * |
Neut(abs.) |
none |
+343* * |
+203* * |
+ 281* * |
Lym(abs.) |
none |
+133* * |
none |
none |
Baso(abs.) |
none |
+467* * |
none |
+200 |
**statistically significantat p<= 0.01
Table 4: Organ weight changes
Changes in absolute organ weights compared to the control[%]
|
||||
Organ |
Group 5 |
Group 6 |
||
males |
females |
males |
females |
|
Kidney, left |
-20 |
-13 |
-34** |
-17** |
Kidney, right |
-22** |
-13 |
-32** |
-16** |
**statistically significantat p<= 0.01
Table 5: Enhanced immune system evaluation: accumulation of foamy macrophages in lactea from jejunum/ileum in animals at 200 mg/kg bw/day
Sex/Sacrifice/Group |
Animal |
Grading |
Sex/Sacrifice/Group |
Animal |
Grading |
Males, |
81 |
0 |
Females, |
91 |
1 |
82 |
1 |
92 |
0 |
||
83 |
2 |
93 |
1 |
||
84 |
1 |
94 |
2 |
||
85 |
3 |
95 |
1 |
||
86 |
2 |
96 |
0 |
||
87 |
0 |
97 |
1 |
||
88 |
2 |
98 |
1 |
||
89 |
1 |
99 |
1 |
||
90 |
1 |
100 |
1 |
Table 6: Enhanced immune system evaluation: mean sums for increased/decreased values in lymphatic system tissues (13-week study in mice)
Organ/Sex/Sacrifice/Group |
Increased |
Decreased |
Mesenteric lymph node, Males, Terminal Control |
0.8 |
0 |
Mesenteric lymph node, Males, Terminal, 6 mg/kg bw/day |
0.1 |
0 |
Mesenteric lymph node, Males, Terminal, 20 mg/kg bw/day |
0.2 |
0.7 |
Mesenteric lymph node, Males, Terminal, 60 mg/kg bw/day |
0.9 |
1.2 |
Mesenteric lymph node, Males, Terminal, 200 mg/kg bw/day |
2.4 |
1.7 |
|
|
|
Mesenteric lymph node, Females, Terminal Control |
0.8 |
0 |
Mesenteric lymph node, Females, Terminal, 6 mg/kg bw/day |
0.5 |
0 |
Mesenteric lymph node, Females, Terminal, 20 mg/kg bw/day |
0.3 |
0 |
Mesenteric lymph node, Females, Terminal, 60 mg/kg bw/day |
0.5 |
0.5 |
Mesenteric lymph node, Females, Terminal, 200 mg/kg bw/day |
2.1 |
0 |
|
|
|
Spleen, Males, Terminal, Control |
0.9 |
1.6 |
Spleen, Males, Terminal, 200 mg/kg bw/day |
1.8 |
1.6 |
Spleen, Females, Terminal, Control |
2.3 |
0 |
Spleen, Females, Terminal, 200 mg/kg bw/day |
2.1 |
1.2 |
|
|
|
Peyer’s Patches (Jejunum), Males, Terminal, Control |
0 |
0 |
Peyer’s Patches (Jejunum), Males, Terminal, 200 mg/kg bw/day |
0 |
0 |
Peyer’s Patches (Jejunum), Females, Terminal, Control |
0 |
0 |
Peyer’s Patches (Jejunum), Females, Terminal, 200 mg/kg bw/day |
0.2 |
0.2 |
Table 7: Enhanced immune system evaluation: presence of foamy macrophages in mesenteric lymph nodes at 60 and 200 mg/kg bw/day
Sex/Sacrifice/Group |
Animal |
Grading |
Sex/Sacrifice/Group |
Animal |
Grading |
Males, |
All |
0 |
Females, |
All |
0 |
Males, |
All |
0 |
Females, |
All |
0 |
Males, |
All |
0 |
Females, |
All |
0 |
Males, |
61 |
2 |
Females, |
71 |
0 |
62 |
0 |
72 |
0 |
||
63 |
2 |
73 |
1 |
||
64 |
1 |
74 |
0 |
||
65 |
1 |
75 |
0 |
||
66 |
0 |
76 |
1 |
||
67 |
2 |
77 |
n.p. |
||
68 |
2 |
78 |
0 |
||
69 |
2 |
79 |
0 |
||
70 |
2 |
80 |
n.p. |
||
Males, |
81 |
2 |
Females, |
91 |
1 |
82 |
2 |
92 |
1 |
||
83 |
3 |
93 |
1 |
||
84 |
1 |
94 |
1 |
||
85 |
0 |
95 |
0 |
||
86 |
3 |
96 |
2 |
||
87 |
0 |
97 |
0 |
||
88 |
2 |
98 |
2 |
||
89 |
2 |
99 |
1 |
||
90 |
1 |
100 |
1 |
n.p.: not present
Table 1 Results of the 90-day oral toxicity study with registration substance in dogs.
Parameter |
Control |
5 mg a.i./kg bw/da |
15 mg a.i./kg bw/da |
45 mg a.i./kg bw/da |
Dose-response (+/–) |
|||||
|
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
Number of animals examined |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
|
|
Mortality |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
Group mean body weight [kg] |
|
|
|
|
|
|
|
|
|
|
Week 0 |
10.93 |
10.30 |
10.78 |
9.98 |
10.70 |
9.83 |
10.73 |
9.90 |
|
|
Week 6 |
13.83 |
12.63 |
14.10 |
11.55 |
12.93 |
10.68 |
10.48 |
8.50** |
− |
− |
Week 13 |
16.28 |
14.13 |
16.10 |
12.80 |
14.48 |
11.53 |
10.90 |
8.50** |
− |
− |
Overall body weight gain (week 0 to 13) [%] |
49.20 |
37.48 |
48.50 |
27.70 |
35.48 |
18.60 |
1.95 |
–13.28 |
– |
− |
Group mean food consumption, weekly [g/kg bw/day] |
|
|
|
|
|
|
|
|
|
|
Week 1 |
37.5 |
38.3 |
39.3 |
38.3 |
34.0 |
34.0 |
37.3 |
31.5 |
|
|
Week 13 |
29.0 |
28.5 |
27.5 |
28.5 |
24.3 |
27.0 |
16.5 |
18.0 |
– |
− |
Haematology (No. of animals examined) |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
|
|
White Blood Cell [109/L] week 6 |
12.20 |
9.13 |
15.98 |
10.68 |
16.00 |
18.93** |
22.75** |
21.08** |
+ |
+ |
White Blood Cell [109/L] week 13 |
12.53 |
9.93 |
16.73 |
14.35 |
14.88 |
19.65 |
20.73 |
17.03 |
+ |
+ |
Reticulocytes [% of the erythrocytes] week 6 |
12.25 |
10.25 |
14.75 |
8.50 |
10.25 |
9.50 |
6.00** |
5.25 |
– |
− |
Neutrophils (abs.) week 6 [109/L] |
6.29 |
4.44 |
8.89 |
5.83 |
10.12 |
11.35** |
15.72** |
14.89** |
+ |
+ |
Neutrophils (abs.) week 13 [109/L] |
7.48 |
5.24 |
10.33 |
9.20 |
9.42 |
11.96 |
12.62 |
11.44 |
+ |
+ |
Monocytes (abs.) week 6 [109/L] |
0.77 |
0.53 |
1.23 |
0.57 |
1.17 |
1.24** |
1.44 |
1.29** |
+ |
+ |
Monocytes (abs.) week 13 [109/L] |
0.70 |
0.54 |
1.10 |
0.90 |
1.06 |
1.20 |
1.60 |
1.05 |
+ |
+ |
Large unstained cells (abs.) week 6 [109/L] |
0.05 |
0.04 |
0.06 |
0.03 |
0.06 |
0.12 |
0.12 |
0.14** |
+ |
+ |
Large unstained cells (abs.) week 13 [109/L] |
0.05 |
0.06 |
0.08 |
0.07 |
0.09 |
0.18 |
0.12 |
0.15 |
+ |
+ |
Activated partial thromboplastin time week 6 [sec] |
11.75 |
11.35 |
11.78 |
11.68 |
11.50 |
11.58 |
12.35 |
12.63 |
+ |
+ |
Activated partial thromboplastin time week 6 [sec] |
10.48 |
10.80 |
11.05 |
11.10 |
11.05 |
11.40 |
12.95** |
12.95** |
+ |
+ |
Clinical Biochemistry (No. of animals examined) |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
|
|
Albumin, week 6 [g/L] |
29.58 |
30.23 |
30.15 |
30.43 |
28.15 |
28.88 |
26.15** |
27.28 |
– |
− |
Albumin, week 13 [g/L] |
31.33 |
31.53 |
30.83 |
31.88 |
28.75 |
28.45** |
24.75** |
27.00** |
– |
− |
Cholesterol, week 6 [mmol/L] |
3.15 |
3.37 |
3.80 |
3.34 |
3.74 |
3.74 |
4.90** |
4.97** |
+ |
+ |
Protein, week 13 [g/L] |
54.5 |
54.3 |
54.3 |
53.5 |
55.0 |
50.5 |
46.3** |
46.8** |
– |
− |
ALAT, week 6 [U/L] |
41.5 |
46.8 |
45.5 |
40.8 |
28.0 |
35.0** |
17.3** |
19.3** |
– |
− |
ALAT, week 13 [U/L] |
41.0 |
45.8 |
45.5 |
39.8 |
24.0 |
30.0 |
16.8** |
26.3 |
– |
− |
ASAT, week 6 [U/L] |
34.0 |
32.3 |
30.0 |
28.0 |
27.0 |
24.0 |
19.3** |
14.5** |
– |
− |
ASAT, week 13 [U/L] |
29.5 |
26.8 |
28.8 |
27.0 |
25.5 |
21.8 |
17.3** |
18.5 |
– |
− |
Urinalysis (no. of animals examined) |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
|
|
pH, week 6 |
6.15 |
6.00 |
6.30 |
6.33 |
6.25 |
6.10 |
7.08 |
7.55 |
+ |
+ |
pH, week 12/13 |
6.35 |
6.13 |
6.63 |
6.45 |
6.25 |
6.38 |
7.08 |
6.78 |
+ |
+ |
Macroscopic findings (no. of animals examined) |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
|
|
Pale-yellowish discoloured kidneys [no. of animals] |
0 |
0 |
0 |
0 |
2 |
0 |
1 |
2 |
+ |
+ |
Yellow-stained discoloured liver [no. of animals] |
0 |
0 |
0 |
0 |
1 |
0 |
2 |
2 |
+ |
+ |
Downsized - Prostate (pr) - Uterus (ut) - Thymus (th) - Spleen (sp) |
0 |
0 |
0 |
0 |
0 |
0 |
2 (pr) |
3 (ut) 2 (th) 1 (sp) |
+ |
+ |
Organ weights (No. of animals examined) |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
|
|
Heart (abs.) [g] |
113.08 |
108.30 |
112.15 |
105.30 |
98.85 |
90.75 |
76.45** |
68.10** |
– |
− |
Thymus (rel.) [g/kg bw] |
1.40 |
1.95 |
1.53 |
1.33 |
1.33 |
1.08** |
1.03 |
0.68** |
– |
− |
Thymus (abs.) [g] |
21.55 |
26.58 |
24.20 |
16.03 |
18.68 |
11.90** |
10.40 |
5.60** |
– |
− |
a) in terms of active substance (test substance as manufactured constituting a 20% (w/w) aqueous solution of the active matter. **) significantly greater or less than control mean (p < 0.01) abs. absolute rel. relative |
||||||||||
Table 2 Enhanced immune system evaluation: mean sums for increased/decreased values in lymphatic system tissues (13-week study in dogs).
Organ/Sex/Sacrifice/Group |
Increased |
Decreased |
Mesenteric lymph node, Males, Terminal Control |
3.3 |
0 |
Mesenteric lymph node, Males, Terminal, 45 mg/kg bw/day |
0.8 |
0 |
Mesenteric lymph node, Females, Terminal Control |
2 |
0 |
Mesenteric lymph node, Females, Terminal, 45 mg/kg bw/day |
0.5 |
0.5 |
|
||
Cervical lymph node, Males, Terminal Control |
2.3 |
0 |
Cervical lymph node, Males, Terminal, 45 mg/kg bw/day |
1.5 |
0 |
Cervical lymph node, Females, Terminal Control |
4 |
0 |
Cervical lymph node, Females, Terminal, 45 mg/kg bw/day |
0.5 |
2.3 |
|
||
Thymus, Males, Terminal, Control |
0 |
4.5 |
Thymus, Males, Terminal, 5 mg/kg bw/day |
0 |
2.8 |
Thymus, Males, Terminal, 15 mg/kg bw/day |
0 |
3.5 |
Thymus, Males, Terminal, 45 mg/kg bw/day |
0.3 |
4 |
|
||
Thymus, Females, Terminal, Control |
0.5 |
0.3 |
Thymus, Females, Terminal, 5 mg/kg bw/day |
0.3 |
2 |
Thymus, Females, Terminal, 15 mg/kg bw/day |
0 |
3.5 |
Thymus, Females, Terminal, 45 mg/kg bw/day |
0 |
8.8 |
|
||
Spleen, Males, Terminal, Control |
0.5 |
1.5 |
Spleen, Males, Terminal, 45 mg/kg bw/day |
0 |
4.5 |
Spleen, Females, Terminal, Control |
0 |
1 |
Spleen, Females, Terminal, 45 mg/kg bw/day |
0 |
1.5 |
|
||
Peyer’s Patches (Jejunum), Males, Terminal, Control |
0 |
0 |
Peyer’s Patches (Jejunum), Males, Terminal, 45 mg/kg bw/day |
0 |
0.8 |
Peyer’s Patches (Jejunum), Females, Terminal, Control |
0.5 |
0 |
Peyer’s Patches (Jejunum), Females, Terminal, 45 mg/kg bw/day |
0.3 |
0 |
Table 3 Enhanced immune system evaluation: presence of foamy macrophages in mesenteric lymph nodes at 45 mg/kg bw/day.
Sex/Sacrifice/Group |
Animal |
Grading |
Sex/Sacrifice/Group |
Animal |
Grading |
Males, |
25 |
0 |
Females, |
29 |
0 |
26 |
0 |
30 |
0 |
||
27 |
0 |
31 |
1 |
||
28 |
0 |
32 |
1 |
Table 4 Enhanced immune system evaluation: accumulation of foamy macrophages in lactea from jejunum/ileum lactea in animals at 45 mg/kg bw/day.
Sex/Sacrifice/Group |
Animal |
Grading |
Sex/Sacrifice/Group |
Animal |
Grading |
Males, |
25 |
1 |
Females, |
29 |
1 |
26 |
1 |
30 |
1 |
||
27 |
1 |
31 |
1 |
||
28 |
0 |
32 |
1 |
Table A6.4.1-1: Results of the 90-day study in rats.
Parameter |
Control |
25 mg/kg bw/da |
50 mg/kg bw/da |
100 mg/kg bw/da |
Dose-response |
|||||
|
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
Number of animals examined |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
|
Mortality |
1/10 |
1/10 |
0/10 |
2/10 |
1/10 |
1/10 |
2/10 |
2/10 |
|
|
Group mean body weight [g] |
|
|
|
|
|
|
|
|
|
|
Week 1 |
189 |
173 |
193 |
170 |
192 |
168 |
192 |
171 |
|
|
Week 9 |
438 |
262 |
454 |
258 |
402* |
262 |
380* |
254 |
– |
|
Week 13 |
468 |
263 |
470 |
260 |
419* |
271 |
399* |
161 |
– |
|
Overall body weight gain (week 1 to 13) [%] |
278 g |
89 g |
+0.4 |
+1.1 |
–19.8* |
+15.7 |
–27.7* |
–1.1 |
– |
|
|
|
|
|
|
|
|
|
|
|
|
Group mean food consumption [g/rat/cage/week] |
|
|
|
|
|
|
|
|
|
|
Week 1 |
185 |
134 |
198 |
142 |
181 |
142 |
186 |
143 |
|
|
Week 13 |
183 |
123 |
182 |
134 |
156 |
118 |
146 |
113 |
|
|
Overall food intake (week 1–13) [%] |
2362 g |
1721 g |
+7.0 |
+6.0 |
–8.0 |
+0.9 |
–10.1 |
–3.9 |
– |
|
|
|
|
|
|
|
|
|
|
|
|
Ophthalmoscopy (No. of animals examined week 13) |
9 |
9 |
n.e. |
n.e. |
n.e. |
n.e. |
8 |
8 |
|
|
Congestion of optic disk |
0 |
0 |
|
|
|
|
5 |
1 |
+ |
|
Haematology (No. of animals examined week 13) |
9 |
9 |
10 |
10 |
9 |
9 |
8 |
8 |
|
|
White Blood Cell Counts [1000/mm³] |
15.3 |
10.5 |
17.2 |
12.5 |
24.2* |
17.2* |
24.6* |
20.2* |
+ |
+ |
Neutrophils [%] |
12 |
8 |
21* |
19* |
36* |
27* |
38* |
34* |
+ |
+ |
Lymphocytes [%] |
87 |
90 |
77* |
80* |
63* |
72* |
60* |
66* |
– |
– |
Packed Cell Volume [%] |
45.3 |
42.7 |
42.2* |
42.0 |
42.8* |
42.0 |
42.5* |
41.6 |
|
|
Mean Cell Haemoglobin Concentration [g/dL] |
36.7 |
37.2 |
38.4* |
37.8 |
37.8* |
37.4 |
38.4* |
37.8 |
|
|
Prothrombin Time [s] |
18.6 |
19.5 |
17.4 |
18.2 |
14.7* |
17.7 |
14.6* |
13.2* |
|
|
|
||||||||||
Clinical chemistry (No. of animals examined week 13) |
9 |
9 |
10 |
10 |
9 |
9 |
8 |
8 |
|
|
Alkaline Phosphatase (U/L) |
236.1 |
30.6 |
236.6 |
138.0 |
250.1 |
195.8 |
246.1 |
212.7* |
|
+ |
Glucose [mg/dL] |
124.6 |
102.9 |
124.8 |
104.8 |
106.7* |
96.1 |
97.5* |
104.6 |
|
|
Total protein [mg/dL] |
6.8 |
7.1 |
6.8 |
7.1 |
6.3* |
6.7* |
6.1* |
6.3* |
|
|
Albumin [g/dL] |
3.5 |
3.9 |
3.4 |
3.9 |
2.9* |
3.4* |
3.0* |
3.1* |
|
|
A/G ratio |
1.1 |
1.2 |
1.0 |
1.3 |
0.9* |
1.0* |
1.0* |
1.0* |
|
|
Cholesterol [mg/dL] |
70.3 |
87.6 |
75.7 |
91.7 |
60.5 |
67.8* |
58.8 |
67.8* |
|
|
Group mean body weight at necropsy [g] |
482 |
272 |
486 |
270 |
421 |
270 |
396 |
262 |
|
|
Absolute liver weight [g] Relative liver weight |
14.88 3.09 |
8.99 3.30 |
15.85 3.26 |
9.18 3.41 |
13.38 3.12 |
9.26 3.44 |
12.25* 3.11 |
8.89 3.41 |
|
|
Absolute testes weight [g] |
3.43 |
– |
3.31 |
– |
2.77* |
– |
3.17 |
– |
|
|
Relative adrenals weight [%] |
0.010 |
0.022 |
0.009 |
0.021 |
0.011 |
0.023 |
0.016* |
0.026 |
|
|
Absolute kidney weight Relative kidneys weight [%] |
2.76 0.57 |
1.84 0.68 |
3.01 0.62 |
1.96 0.73 |
2.56 0.60 |
1.90 0.70 |
2.58 0.67* |
1.86 0.71 |
|
|
*) significantly greater or less than control mean (p < 0.05) n.e. not examined a) in terms of test substance as manufactured (20% aqueous solution) |
||||||||||
Table A6.4.1-2: Results of the 90-day study in rats, dose levels based on test substance as manufactured (20% aqueous solution).
Parameter |
Control |
6.25 mg/kg bw/day |
12.50 mg/kg bw/day |
Dose-response |
||||
|
m |
f |
m |
f |
m |
f |
m |
f |
Number of animals examined |
10 |
10 |
10 |
10 |
10 |
10 |
|
|
Mortality |
0/10 |
0/10 |
0/10 |
1/10 |
0/10 |
1/10 |
|
|
Group mean body weight [g] |
|
|
|
|
|
|
|
|
Week 1 |
213 |
167 |
216 |
173 |
216 |
168 |
|
|
Week 13 |
492 |
245 |
503 |
256 |
503 |
259 |
|
|
Group mean food consumption [g/rat/cage/week] |
|
|
|
|
|
|
|
|
Week 1 |
200 |
130 |
197 |
131 |
206 |
129 |
|
|
Week 13 |
171 |
115 |
179 |
108 |
162 |
108 |
|
|
Haematology |
|
|
|
|
|
|
|
|
Red Blood Concentration [mill/mm³] |
7.6 |
7.1 |
7.9* |
7.1 |
7.9* |
7.0 |
|
|
Packed Cell Volume [%] |
42.5 |
41.8 |
43.2 |
41.1 |
43.2 |
40.3* |
|
|
*) significantly greater or less than control mean (p < 0.05) |
||||||||
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 2 mg/kg bw/day
- Study duration:
- chronic
- Species:
- mouse
- Quality of whole database:
- The available information comprises adequate and reliable studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
Repeated dose toxicity: inhalation - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
For the assessment of the repeated dose toxicity of the registration
substance, sub-chronic studies in mice, rats and beagle dogs, as well as
a combined chronic toxicity and carcinogenicity study in mice are
available.
Rodent studies - sub-chronic
An oral 90-day toxicity study was carried out according to EPA
82-1 (1982). Four groups of 10 male and 10 female Sprague Dawley rats,
respectively, were administered orally by gavage 0, 25, 50 and 100 mg/kg
bw/day of the registration substance (20% aqueous solution, substance as
manufactured), corresponding to 0, 5, 10 and 20 mg a.i./kg bw/day for 90
days.
The conduct of the study was similar to OECD Guideline 408 (2018) with
the exception that (i) no NOAEL could be determined, (ii) no sensory
reactivity test, no assessment of grip strength, no motor activity
assessment and no functional observations were performed, (iii) the
weight variation at the commencement of the study exceeded ± 20 % for
males, (iv) weights of epididymides, uterus, thymus, spleen, brain and
heart were not determined, (iv) no hormon evaluation was performed and
(v) the mesenteric lymph nodes were not examined for the low and mid
dose group despite observed effects at the high dose group.
Treatment with the test substance at a dose level of 100 mg/kg bw/day
produced toxic effects manifested by foci of necrosis, acute
inflammation and lymphoid hyperplasia of the mesenteric lymph nodes in
animals of either sex. Since histopathological examination of mesenteric
lymph nodes did not include the low dose and mid dose animals, the
effect of the test substance at dose levels of 25 and 50 mg/kg bw/day on
this tissue could not be clarified. However, the observed effects on the
mesenteric lymph nodes in the available repeated dose toxicity studies
in mice and dogs were re-evaluated in 2022 in the context of possible
systemic effects of the test substance on the immune system (see also
discussions below). It was finally concluded that the changes observed
in the different repeated dose toxicity studies of mice and dogs were
limited to local reactions in the mesenteric lymph nodes as a
consequence of an uptake and transfer of the test item by intestinal
macrophage via lacteal lymph fluid. A general effect of the test
substance on the immune system was excluded. Instead, an adaptation was
deemed to be very likely in mice, which is also assumed to be the case
for the rat.
Further treatment-related findings were reduction in body weight gain
and overall food consumption in male animals receiving 50 and 100 mg/kg
bw/day, ophthalmological findings (congestion of the optic disk) in five
high dose male animals and one female, haematological changes
(significant increase of leucocytes and neutrophile granulocytes and
decrease of lymphocytes) in males and females receiving 25, 50 and 100
mg/kg bw/day, slight blood chemistry changes (reduction in glucose,
total protein, albumin, albumin/globulin ratio and cholesterol) within
the normal range of background data in males and females treated with
the test substance at 50 and 100 mg/kg bw/day, and a markedly increased
alkaline phosphatase level in high dosed females only.
Considering the observed ophthalmological findings, ocular changes are
known to be associated with increased intracranial pressure and
concomitant oedematous swelling. Since there were no remarkable
histopathological changes of all animals examined, these findings have
to be considered as compound-related clinical symptoms, possibly
secondary to treatment. A pathological re-assessment confirmed that the
congestion of the optic disk was either by chance or consequences of
retro-orbital blood sampling. This conclusion is based on the fact, that
increased intracranial pressure would cause lesions like status
spongiosus in the brain, neuronal necrosis or degeneration, and in more
chronic lesions also gliosis. In addition, intracranial pressure may
cause death. None of these lesions have been reported. Similar findings
were also not noted in the other available repeated dose toxicity
studies. The retro-orbital blood sampling was the method of choice at
the time the study was performed. Haemorrhage and inflammation in the
retro-orbital region are common lesions and congestion is a related
alteration. Thus, the described ophthalmological findings are not
regarded as primary effects related to test substance treatment.
No treatment-related mortality or clinical signs occurred during the
study.
Apart from slight leucocytosis, granulocytosis and lymphenia treatment
with the test substance at a dose level of 25 mg/kg bw/day was very well
tolerated and did not elicit any further toxic changes attributable to
oral administration of the compound tested.
Based on a significant increase of leucocytes and neutrophile
granulocytes and decrease of lymphocytes at all dose levels, the LOAEL
was 25 mg/kg bw/day (as 20% aqueous solution), corresponding to 5 mg
a.i./kg bw/day.
No NOAEL could be determined based on the results of this study.
Therefore, a supplementary 90-day oral toxicity test utilising a
modified dose range was conducted:
This study was carried out according to EPA 82-1 (1982).Three groups of
10 male and 10 female Sprague Dawley rats, respectively, were
administered orally by gavage 0, 6.25 and 12.5 mg/kg bw/day of the
registration substance (20% aqueous solution, substance as manufactured;
corresponding to 1.25 and 2.5 mg a.i./kg bw/day) for 90 days.
The conduct of the study was consistent in all important aspects to OECD
Guideline 408 (1998) with the exception that only 2 dose levels (6.25
and 12.50 mg/kg bw/day) instead of 3 were examined and that survival,
general health, haematological parameters, gross necropsy and
histopathology of mesenteric lymph nodes were investigated only.
However, this study is a follow-up study to reference A6.4.1/01. Thus,
the investigation of further parameters is not considered to be required.
No treatment-related clinical signs, mortalities, effects on food
consumption and body weight, haematological changes, macroscopic lesions
in any of the organs or tissues examined were observed during the study.
Microscopic findings did not reveal any abnormalities of mesenteric
lymph nodes.
Based on the results, the NO(A)EL in males and females was 12.50 mg/kg
bw/day (as 20% aqueous solution), corresponding to 2.5 mg a.i./kg
bw/day. In addition, these results confirm the LOAEL in males and
females of 25 mg/kg bw/day (as 20% aqueous solution), corresponding to 5
mg a.i./kg bw/day.
Furthermore, a 13-week dose-range-finding study for the combined chronic
toxicity/carcinogenicity study carried out similar to OECD 408 in mice
is available. Six groups of 10 male and 10 female CD-1 mice,
respectively, were administered by dietary administration at doses of 0,
6, 20, 60, 200 and 400 mg/kg bw/day (20% aqueous solution, substance as
manufactured), corresponding to 0, 1.2, 4, 12, 40 and 80 mg a.i./kg
bw/day for 90 days.
The conduct of the study was similar to OECD Guideline 408 (2018) with
the exception that (i) no sensory reactivity test, no assessment of grip
strength, no motor activity assessment and no functional observations
were performed and (ii) no hormone evaluation was performed.
One of 20 mice treated with 400 mg/kg bw/day died prematurely. In
addition, marginal changes in behaviour were noted at 400 mg/kg bw/day.
The body weight of the male and female animals treated with 200 or 400
mg/kg bw/day via the diet was reduced from test week 2 onwards. The body
weight gain and body weight at autopsy were reduced accordingly.
Treatment with 200 or 400 mg/kg bw/day caused changes in haematological
and biochemical parameters (increase of leucocytes, platelets,
neutrophilic granulocytes, lymphocytes and basophilic granulocytes,
urea, aspartate aminotransferase and decrease in creatinine). No test
item-related influences were noted during ophthalmological and auditory
examination. Macroscopic inspection at necropsy revealed test
item-related changes in form of haemorrhagic foci and haemorrhagic
deposits in the stomachs of male and of female animals treated with 20,
60, 200 or 400 mg/kg bw/day, which are suspected to result from the
corrosive properties of the registration substance. Additionally, the
absolute weights of the kidneys were decreased at 200 and 400 mg/kg
bw/day. The histopathological examination of male and female mice
treated with 20, 60 or 200 mg/kg bw/day showed a dose dependent increase
of macrophages in the mesenteric lymph nodes with vacuolization of the
cytoplasm. In some mice purulent inflammatory reactions were noted. The
animals treated with 200 mg/kg bw/day via the diet revealed a minimal to
moderate increase of basophilic tubular cells with interstitial
lymphocytic inflammatory reaction in the kidneys, which is considered to
be related to the administration of the test item.
The mean actual intake of the test item via the diet was 5.9, 19.4,
59.7, 214.0 and 448.4 mg test substance (20% solution)/kg bw/day for the
males and 6.2, 20.7, 63.5, 216.9 and 444.6 mg/kg bw/day for the females
at the nominal dose levels of 6, 20, 60, 200 and 400 mg/kg bw/day.
An additional pathological assessment was conducted in 2022 to address
possible systemic effects of the test substance on the immune system.
For the enhanced immune system evaluation, the following organs were
evaluated: spleen, mesenteric lymph nodes, Peyer’s patches - jejunum
(GALT – gut associated lymphoid tissue) (high dose and control animals
only). The enhanced immune system evaluation revealed higher mean sums
for increased and decreased mesenteric lymph node values in males at 60
and 200 mg/kg bw/day. In females, the mean sum was higher for increased
mesenteric lymph node values at 200 mg/kg bw/day. Increased values were
mainly due to few cases of granulocytic infiltrate and single cell
necrosis/apoptosis. These findings were related to the accumulation of
foamy macrophages in the mesenteric lymph nodes from both sexes at 60
and 200 mg/kg bw/day. In addition, these findings were associated with
an accumulation of foamy macrophages in the mucosal lactea from jejunum
and ileum in both sexes at 200 mg/kg bw/day, an unreported finding in
the original study report of the 90-day oral toxicity study in mice.
This accumulation was considered to be directly related to accumulation
of foamy macrophages in the mesenteric lymph nodes. There were no
significant differences noted for the spleen and Peyer’s patches
comparing the mean sums from controls with animals at 200 mg/kg bw/day.
Therefore, a general effect of the test substance on the immune system
was excluded. All findings were local in mesenteric lymph nodes. They
were considered to be due to increased storage of the test item or its
metabolite(s) as a consequence of uptake by macrophages via lactea and
lymphatic vessels. Inflammatory lesions in the mesenteric lymph nodes
were of minimal incidence and severity and were noted at 60 and 200
mg/kg bw/day only. In addition, no such findings were noted in the
available 78-week study in mice (described below). Even foamy macrophage
agglomeration was not seen in mesenteric lymph nodes. Especially, there
was no indication for inflammation in the lymph nodes after 78 weeks.
Also in the available 90-Day study in dogs (also described below), no
inflammation was noted. It was therefore concluded, that the few cases
of minimal inflammatory lesions in mesenteric lymph nodes from mice of
the 13-week study were not related to treatment with the test item.
Instead, an adaptation was deemed to be very likely in mice. As the
histopathological renal changes were limited to the 200 mg/kg bw/day
group and no enhanced lesions were reported in the kidneys from animals
of the other available repeated-dose toxicity studies, especially the
78-Week mouse study, the kidneys were not part of the enhanced immune
system evaluation.
In summary, the systemic NOAEL was determined to be 60 mg/kg bw/day for
male and female mice, which corresponds to 12 mg active ingredient/kg
bw/day based on the described effects on decreased body weight and food
consumption and changes in haematological and biochemical parameters.
Rodent studies - chronic
The chronic toxicity and carcinogenicity of the registration
substance (20% a.i.) was tested in CD®-1 mice. Fifty mice per sex per
group were fed the test item at low, intermediate and high dose levels
for 78 weeks. Due to increased mortality rate in the high dose group the
initial high dose level was successively reduced to 35 mg/kg bw/day in
males and 65 mg/kg bw/day in females. Towards the end of the study
dosing was discontinued in the intermediate dosed females and high dosed
males and females. A satellite group of 20 dosed animals per sex and 10
associated control animals per sex for chronic toxicity testing was
included for 52 weeks.
Treatment-related effects on survival were observed. The mortality rate
of the intermediate dosed female mice treated with 60 mg/kg bw/day was
statistically significantly increased from approximately test week 60
onwards. The male and female animals treated with the high dose of
100/35 (males) or 200/65 (females) mg/kg bw/day revealed an increased
mortality and a corresponding decreased survival rate from approximately
test weeks 38 (males) and 14 (females) onwards (statistically
significant at p ≤ 0.01 from test weeks 51 (males) and 26 (females)
onwards). The time-point of the occurrence of statistical significance
of the mortality rate was directly influenced by the low mortality rate
of the control group. The severely increased mortality rates noted for
these animals resulted in dose reductions in the high dose group during
the course of the study and, subsequently, in the discontinuation of
dosing of the high dosed animals from test weeks 68 (males) and 67
(females) onwards. Furthermore, the dosing of the intermediate dosed
females was discontinued from test week 76 onwards.
Body weights were unaffected in the low dose animals and in intermediate
dose males. The body weight of the intermediate dosed female mice (60
mg/kg bw/day) was below the body weight of the control group from
approximately test week 41 onwards (up to 15%, p ≤ 0.01 in test weeks 41
and 49 to 73). Treatment with the high dose led to a reduced body weight
of males and females from approximately test week 3 onwards in males (by
4% to 20%, on average 12%, p ≤ 0.01 in test weeks 3 to 67) and from test
week 7 onwards in females (by 4% to 17%, on average 11%, p ≤ 0.01 in
test weeks 7 to 17 and 21 to 77). The total body weight gain was
accordingly reduced in the female animals of the intermediate dose group
and in the male and female animals of the high dose group when compared
to the respective start values and to the control group.
Local changes to eyes were seen in the intermediate and high dose
animals, including swollen/reddened eyes and ptosis were seen. The
observed effects to the eyes are known properties of the test item as it
may cause serious damage to eyes (see chapter on IUCLID chapter 7.3
Irritation / corrosion). The local exposure of the eye to the test item
during the food intake or grooming periods is very likely as the test
item was administered via the diet.
Haematological examination revealed increased levels of leucocytes,
neutrophilic granulocytes and monocytes in high dosed female mice, which
are indicative of inflammatory processes. Clinical chemistry showed
increased levels of aspartate-aminotransferase and lactate-dehydrogenase
in treated animals. Increased absolute and relative liver weights were
noted for the female animals.
The macroscopic inspection did not reveal any test item-related changes
in the organs or tissues of the high dosed animals.
Histopathological examinations revealed the following treatment-related
findings:
Non-neoplastic lesions - carcinogenicity study
Test item-related systemic non-neoplastic lesions were noted in males
and females of all dose level groups (10, 30 or 100/35 mg /kg bw/day
(male animals) or 20, 60 or 200/65 mg/kg bw/day (female animals)) at
terminal sacrifice (high dose group) and in the prematurely deceased
animals (all dose groups):
- muscle degeneration with cellular reaction in the skeletal muscle
(leg) of the high dosed animals (100/35 (males) or 200/65 (females)
mg/kg bw/day) at terminal sacrifice, and of the low dosed prematurely
deceased females (20 mg /kg bw/day (11/20 – 55%)) and the intermediate
and high dosed prematurely deceased animals (30 or 100/35 mg (males) or
60 or 200/65 (females) mg/kg bw/day);
- muscle degeneration with cellular reaction in the bone (sternum) of
the high dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day)
at terminal sacrifice, and of the prematurely deceased males and females
of all dose groups;
- muscle degeneration with cellular reaction in the heart of the high
dosed animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at
terminal sacrifice, and of the prematurely deceased males and females of
all dose groups;
- large macrophages in the mesenteric lymph nodes of the high dosed
animals (100/35 (males) or 200/65 (females) mg/kg bw/day) at terminal
sacrifice, and of the prematurely deceased males and females of all dose
groups;
- atrophy with cellular reduction in the spleen (in some cases
correlating with macroscopic findings) of the high dosed females (200/65
mg/kg bw/day) at terminal sacrifice, and of the high dosed prematurely
deceased males (100/35 mg/kg bw/day) and the intermediate and high dosed
prematurely deceased females (60 or 200/65 mg/kg bw/day);
- dilatation of the urinary bladder (in some cases correlating with
macroscopic findings) of the intermediate dosed males (30 mg/kg bw/day)
and of the high dosed males and females (100/35 (males) or 200/65
(females) mg/kg bw/day) at terminal sacrifice, and of the intermediate
and high dosed prematurely deceased males (30 or 100/35 mg/kg bw/day).
- vacuolisation of epithelial cells in the epididymides of the high
dosed male (100/35 mg /kg bw/day) at terminal sacrifice, and of the
intermediate and high dosed prematurely deceased males (30 or 100/35
mg/kg bw/day). The highest incidence and severity of these lesions were
in general observed in the high dose group (animals treated with 100/35
(males) or 200/65 (females) mg/kg bw/day). This effect is not considered
as biologically relevant related to reproduction toxicity as the the
two-generation reproduction toxicity study did not show adverse effects
related to reproduction toxicity (see also section Toxicity to
reproduction for details). In addition, the dose-related vacuolisation
of the epithelial cells lining the epididymides were observed in the
presence of marked general systemic toxicity and reductions in body
weight and body weight gain, which were considered adverse. Moreover,
isolated epididymal vacuolisation is generally considered to be an age
related effect in mice.
Non-neoplastic lesions-carcinogenicity study - Interim Pathology
Study
Test item-related systemic non-neoplastic lesions were noted in males
and females treated the high dose of 100/65 (males) or 200/75 (females)
mg/kg bw/day at terminal sacrifice and in the prematurely deceased
animals:
- muscle degeneration with cellular reaction in the skeletal muscle
(leg) of males and females;
- muscle degeneration with cellular reaction in the heart of males and
females;
- large macrophages in the mesenteric lymph nodes of males and females;
- large macrophages in the small intestine (duodenum, jejunum, ileum) of
males and females;
- hyperplasia of the secretory cells of the bronchial epithelium in the
lungs of males and females;
- vacuolisation of epithelial cells in the epididymides of the male
animals. This effect is not considered as biologically relevant related
to reproduction toxicity as the two-generation reproduction toxicity
study did not show adverse effects related to reproduction toxicity (see
section Toxicity to reproduction for details). In addition, the
dose-related vacuolisation of the epithelial cells lining the
epididymides were observed in the presence of marked general systemic
toxicity and reductions in body weight and body weight gain, which were
considered adverse. Moreover, isolated epididymal vacuolisation is
generally considered to be an age related effect in mice.
There was no evidence of carcinogenicity.
Taken together, mice treated with 30 or 100/35 mg registration
substance (20% a.i.)/kg bw/day (males) and 60 or 200/65 mg /kg bw/day
(females) showed an increase of numbers of white blood cells and an
increased incidence of large macrophages in mesenteric lymph nodes.
Therefore, treatment of mice with high dose of the registration
substance is associated with indications of inflammatory processes. As
already pointed out, an additional pathological assessment was conducted
in 2022 to address possible systemic effects on the immune system. For
the enhanced immune system evaluation, the following organs were
evaluated: spleen, thymus, mesenteric lymph nodes, Peyer’s patches -
jejunum (GALT – gut associated lymphoid tissue), and bone marrow (high
dose and control animals only) (high dose and control animals only). The
enhanced immune system evaluation revealed that all changes noted in the
78-Week study (higher mean sums mainly for decreased mesenteric lymph
node values in interim and terminal sacrifices, minor differences in the
thymus, higher mean sums for decreased splenic values in the spleen at
both interim and terminal sacrifices, and in Peyer's patches at the
terminal sacrifice, minor decreased myeloid:erythroid ratio in both
sexes at 200/65 mg/kg bw in the bone marrow at the interim sacrifice)
are rather linked to age-related non-neoplastic or neoplastic lesions in
CD-1 mice but not related to treatment with the test item. From the
enhanced immune system evaluation, it was finally concluded that the
changes observed in the different available repeated dose toxicity
studies were limited to local reactions in the mesenteric lymph nodes as
a consequence of an uptake and transfer of the test item by intestinal
macrophage via lacteal lymph fluid. A general effect of the test
substance on the immune system is excluded. Inflammatory lesions in the
mesenteric lymph nodes were of minimal incidence and severity and were
noted in the available 13-week study in mice at 60 and 200 mg/kg bw/day
only (discussed above). No such findings were noted in the current
78-Week study in mice. Even foamy macrophage agglomeration was not seen
in mesenteric lymph nodes. Especially, there was no indication for
inflammation in the lymph nodes after 78 weeks. In addition, in the
available 90-Day study in dogs, no inflammation was noted (discussed
below). Therefore, it was concluded, that the few cases of minimal
inflammatory lesions in mesenteric lymph nodes from mice of the 13-week
study were not related to treatment with the test item. In addition, it
was noted that macrophages agglomerated in the intestinal lactea, which
is deemed to be the first step in the pathway of development of lesions.
It is noteworthy, that no intestinal inflammation was encountered in
mice after 78 weeks. Therefore, an adaptation was considered to be very
likely in mice.
In addition, mice of both sexes treated with high dose of the
registration substance and additional females treated with 60 mg/kg
bw/day registration substance (20% a.i.) showed a statistically
significant increase of levels of aspartate aminotransferase and lactate
dehydrogenase and a dose-dependent statistically significant increase of
findings of muscle degeneration with cellular reaction in the heart,
sternum and skeletal tissues. In low dose mice of both sexes no
statistically significant differences of biochemical parameters to the
controls were observed. Any histopathological findings in the low dose
mice that died prematurely (8/50 males and 20/50 females) were not
statistically significant. However, in the prematurely deceased females
treated with 20 mg/kg bw/day of the test item an increased incidence of
muscle degeneration in heart (7/20), sternum (3/20) and skeletal muscle
(11/20) were observed. In low dose prematurely deceased males treated
with 10 mg/kg bw/day test item only one single incidence of muscle
degeneration in sternum (2/8) and heart (1/8) were observed. No
treatment-related neoplastic lesions were observed and it is concluded
that the registration substance has no carcinogenic potential in mice.
The study reported above does not conform to the requirements of OECD
Guidance document 35 or OECD Guidance document 116 in that excessive
toxicity including mortality (>50%) occurred at both the intermediate
dose (females) and high dose levels (males and females). This study is
therefore not strictly acceptable as a negative carcinogenicity study.
As significant toxicity was seen in females at all dose levels, an
overall study NOAEL for a chronic study could not therefore be
established. Thus, based on the observed findings in the interim
pathology study (after one year of treatment) and in the carcinogenicity
study a NOAEL for chronic toxicity can be established at 10 mg
registration substance (20% a.i.)/kg bw/day corresponding to 2 mg
a.i./kg bw/day for male mice. The LOAEL in females was 20 mg
registration substance (20% a.i.)/kg bw/day corresponding to 4 mg
a.i./kg bw/day.
Non-rodent study – sub-chronic
An oral 90-day toxicity study with the registration substance (as
20% aqueous solution) in a non-rodent species (dog) was carried out
according to OECD guideline 409 and EC method B.27. Four groups of 4
male and 4 female Beagle dogs, respectively, were administered orally by
gavage 0, 5, 15 and 45 mg/kg bw/day in terms of active ingredient
diluted in vehicle (tap water) for 90 days.
None of the animals died prematurely. Two of four female animals treated
with 15 mg a.i./kg bw/day of test item revealed emesis and all animals
administered 45 mg a.i./kg bw/day showed emesis and salivation on most
treatment days.
Test item-related changes were observed starting at a dose of 15 mg
a.i./kg bw/day, including decreased body weight and food consumption,
changes in haematological and biochemical parameters, increase of
urinary pH-value and haemoglobin content in urine (in the high dosed
groups only).
No changes compared to the vehicle control were noted for drinking water
consumption and the ophthalmological and auditory examination.
Absolute organ weights of the heart and the thymus were decreased in
animals treated with the intermediate and high dose of the test item.
In almost all males treated with 15 and 45 mg a.i./kg bw/day the
prostates were downsized and the kidneys, livers and pancreas showed
discolouration. In high dosed females also discolouration of the
kidneys, livers, pancreas, spleen and/or lungs and a reduction in size
of uterus, thymus and/or spleen were observed. These changes are
considered to be test item-related.
The histomorphological examination revealed dose-related atrophy in the
male and female genital system in intermediate and high dosed animals.
Slightly more pronounced involution of the thymus was observed in both
sexes treated with the intermediate and high dose. Atrophy of the
germinative epithelium of the testes in high dosed males was considered
to be treatment-related. However, the histomorphological findings were
not considered to be a direct effect of treatment but were rather
attributed to the severe systemic toxicity of the test substance in
combination with stress associated with decreased food intake as well as
reduced body weight and body weight gain in dogs that were not fully
mature at study termination.
Based on these results, the NOAEL was 25 mg/kg bw/day (as 20% aqueous
solution), corresponding to 5 mg a.i./kg bw/day.
An additional pathological assessment was conducted in 2022 to address
possible systemic effects on the immune system. The following organs
were evaluated: spleen, thymus, mesenteric lymph nodes, cervical lymph
nodes, and Peyer’s patches - jejunum (GALT – gut associated lymphoid
tissue) (high dose and control animals only). The enhanced immune system
evaluation revealed lower mean sums for increased mesenteric and
cervical lymph node values. In females, the mean sum was higher for
increased mesenteric lymph node values at 45 mg/kg bw/day. These values
were linked to stress-related atrophic changes as also described in the
histopathological re-assessment report related to the reproductive
findings. The differences of mean sums in thymus and spleen, namely in
females, also reflected the described atrophy. There were no significant
differences noted for the spleen and Peyer’s patches comparing the mean
sums from controls with animals at 200 mg/kg bw. In the mesenteric lymph
nodes from two females at 45 mg/kg bw/day, there was a minimal
accumulation of foamy macrophages that could not be excluded from being
induced by treatment with the test item and it might has been a
consequence of accumulation of foamy macrophages in the mucosal lactea
from jejunum and ileum in both sexes at 45 mg/kg bw/day. These findings
were consisted with alterations noted in the available 13-Week study in
mice described above. However, no inflammation was noted in the
intestinal mucosa nor in the mesenteric lymph nodes. Therefore, it was
concluded, that these effects were not related to treatment with the
test item. Thus, from the enhanced immune system evaluation it was
finally concluded that the changes observed were limited to local
reactions in the mesenteric lymph nodes as a consequence of an uptake
and transfer of the test item by intestinal macrophage via lacteal lymph
fluid. Inflammatory lesions in the mesenteric lymph nodes were of
minimal incidence and severity and were noted in the available 13-week
study in mice at 60 and 200 mg/kg/day only. No such findings were
observed in the available 78-Week study in mice. Foamy macrophage
agglomeration was not seen in mesenteric lymph nodes. Especially, there
was no indication for inflammation in the lymph nodes after 78 weeks. In
addition, in the current 90-Day study in dogs, no inflammation was
noted. Therefore, it was considered, that the few cases of minimal
inflammatory lesions in mesenteric lymph nodes from mice of the 13-week
study were not related to treatment with the test item. It was noted
that macrophages agglomerated in the intestinal lactea, which is deemed
to be the first step in the pathway of development of lesions. It is
noteworthy, that no intestinal inflammation was encountered in mice
after 78 weeks. Instead, an adaptation was deemed to be very likely.
Discussion
Toxicologically significant systemic effects in a 90-day study in
rats occurred at oral doses (gavage) of 5 mg a.i./kg bw/day and above.
Toxic effects were unspecific, manifested by reduction in body weight
gain (50 and 100 mg/kg bw/day, males), and reduction in food consumption
(50 and 100 mg/kg bw/day, males). Marked leucocytosis, granulocytosis
and lymphopenia as well as minor blood chemistry changes were noted at
25, 50 and 100 mg/kg bw/day. No other toxic effects were observed. Since
administration of 2.5 mg a.i./kg bw/day was well tolerated by the rats,
the NO(A)EL in rats was determined as 2.5 mg a.i./kg bw/day.
Toxicologically significant systemic effects in a 90-day study in mice
occurred at dietary dose levels of 40 mg a.i./kg bw/d and above. One of
20 mice treated with 400 mg/kg bw/day died prematurely. In addition,
marginal changes in behaviour were noted at 400 mg/kg bw/day. The body
weight of the male and female animals (200 or 400 mg/kg bw/day) was
reduced from test week 2 onwards. Accordingly, the body weight gain and
body weight at autopsy were reduced. Treatment with 200 or 400 mg/kg
bw/day caused changes in haematological and biochemical parameters
(increase of leucocytes, platelets, neutrophilic granulocytes,
lymphocytes and basophilic granulocytes, urea, aspartate
aminotransferase and decrease in creatinine). Macroscopic inspection at
necropsy revealed test item-related changes in form of haemorrhagic foci
and haemorrhagic deposits in the stomachs of male and of female animals
treated with 20, 60, 200 or 400 mg/kg bw/day, which are suspected to
result from the corrosive properties of the registration substance.
Additionally, the absolute weights of the kidneys were decreased at 200
and 400 mg/kg bw/day and in the histopathological observation the
animals treated with 200 mg/kg bw/day via the diet revealed a minimal to
moderate increase of basophilic tubular cells with interstitial
lymphocytic inflammatory reaction in the kidneys, which is considered to
be related to the administration of the test item. The systemic NOAEL
was determined to be 60 mg/kg bw/day for male and female mice, which
corresponds to 12 mg active ingredient/kg bw/day based on the described
effects on decreased body weight and food consumption and changes in
haematological and biochemical parameters.
Toxicologically significant systemic effects in a 90-day study in beagle
dogs occurred at oral doses (gavage) of 15 mg a.i./kg bw/day and above.
Two of four female animals treated with 15 mg a.i./kg bw/day of test
item revealed emesis and all animals administered 45 mg a.i./kg bw/day
showed emesis and salivation on most treatment days. Test item-related
changes included decreased body weight and food consumption, changes in
haematological and biochemical parameters, increase of urinary pH-value
and haemoglobin content in urine (in the high dosed groups only).
Absolute organ weights of the heart and the thymus were significantly
decreased in animals treated with the intermediate and high dose of the
test item. Relative thymus weight was significantly decreased in high
and intermediate dosed females. In almost all males treated with 15 and
45 mg a.i./kg bw/day the prostates were downsized and the kidneys,
livers and pancreas showed discolouration. In high dosed females also
discolouration of the kidneys, livers, pancreas, spleen and/or lungs and
a reduction in size of uterus, thymus and/or spleen were observed. Based
on these results, the NOAEL was 25 mg/kg bw/day, corresponding to 5
mg/kg bw/day active ingredient.
In a combined chronic toxicity and carcinogenicity study in mice
administration of ≥ 60 mg/kg bw/day registration substance (20% a.i.)
(females) and 100/200 mg/kg bw/day (males/females) via diet caused
significantly increased mortality. Therefore, the initial high dose
levels were reduced during the study period as follows: 100 reduced to
35 mg /kg bw/day for males and 200 reduced to 65 mg/kg bw/day for
females and finally treatment was suspended at about week 60. The
intermediate dose was also suspended in week 76 for females. The low
doses were not affected by increased mortality and could therefore be
left constant over the study period. Treatment-related effects on
survival, body weight and local changes to eyes, including
swollen/reddened eyes and ptosis, were observed (intermediate and high
dose). Haematological examination revealed increased levels of
leucocytes, neutrophilic granulocytes and monocytes in high dosed female
mice, which are indicative of inflammatory processes. Evaluation of
clinical chemistry showed increased levels of aspartate-aminotransferase
and lactate-dehydrogenase in treated animals. Histopathology revealed
degeneration with cellular reaction in skeletal muscle, sternum and
heart, large macrophages in mesenteric lymph nodes, vacuolisation of
epithelial cells in the epididymides, atrophy with cellular reduction in
spleen and dilatation of the urinary bladder (intermediate and high dose
animals). In addition, in the prematurely deceased low dose females (20
mg/kg bw/day of the registration substance (20% a.i.), corresponding to
4 mg a.i./kg bw/day) an increased incidence of muscle degeneration in
heart (7/20), sternum (3/20) and skeletal muscle (11/20) were observed.
Based on the histopathological alterations the chronic NOAEL is
established at 2 mg a.i./kg bw/day in males. No NOAEL was established
for females as significant histopathological alterations were seen in
premature decedents at this dose level (LOAEL = 4 mg a.i./kg bw/day).
The overall dose descriptors for chronic repeated dose toxicity
are the NOAEL of 2 mg a.i./kg bw/day in males and the LOAEL of 4 mg
a.i./ kg bw/day in females.
Justification for classification or non-classification
Due to the re-assessment of the findings in organs, which seemed to be
affected by the registration substance (eyes, mesenteric lymph nodes as
well as male/female genital systems) and the outcome, that all these
findings are not regarded as adverse effects caused by the treatment
with the registration substance, the available data do not meet the
criteria for classification in accordance with CLP, EU GHS (Regulation
1272/2008/EC) and are therefore conclusive but not sufficient for
classification.
For the new assessment, an enhanced immune system evaluation of the
existing histological sections of the available sub-chronic and chronic
toxicity / carcinogenicity studies was performed. The examination
revealed that the observed changes were limited to local reactions in
the mesenteric lymph nodes. Inflammatory lesions were of minimal
incidence and severity in the dose-range finding study with mice (90-day
study) while they were not observed in the chronic studies. The
assessing pathologist concluded therefore, that this is a strong
indicator for adaptation, instead of being adverse.
In addition, the ophthalmological findings in the 90-day repeated dose
study with rats were re-assessed. From this evaluation it was concluded,
that the observed ocular changes consisting of congestion of the optic
disc in only one sub-chronic study with rats were either by chance or
consequence of retro-orbital blood sampling. The latter was the blood
sampling method of choice at the time the study was performed.
Therefore, congestion of the optic disc is not regarded as a systemic
effect by the test substance.
Atrophy findings in the male/female genital systems in dogs were also
re-assessed. They were also not considered to be a direct effect of
treatment but were rather attributed to be of secondary stress-related
nature in dogs that were not fully mature at study termination. In
addition, vacuolisation of epithelial cells in the epididymides of mice
was observed in the presence of marked general toxicity including
mortality and are therefore also regarded as no primary effects related
to test substance treatment.
Annex I, Part 3.8.2.1.8 of Regulation (EC) No 1272/2008 describes
effects that do not support classification for STOT RE category 1 and 2.
Absence of “significant” toxicity and adaptive responses that are not
considered toxicologically relevant justify no classification.
Considering the nature and severity of the described effects on eyes,
mesenteric lymph nodes and male/female genital systems it was concluded
that a classification for repeated dose toxicity is scientifically not
justified according to CLP criteria.
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