Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: 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

Reason / purpose for cross-reference:

reference to same study

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.

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Version / remarks:

1988

Deviations:

yes

Remarks:

urinalysis is missing

GLP compliance:

yes (incl. QA statement)

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 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:

Carcinogenicity study (males)
corresponding to 2 mg a.i./kg bw/day

Dose / conc.:

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

Remarks:

Carcinogencity study (males)
corresponding to 6 mg a.i./kg bw/day

Dose / conc.:

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

Remarks:

Carcinogenicity 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:

Carcinogenicity study (females)
corresponding to 4 mg a.i./kg bw/day

Dose / conc.:

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

Remarks:

Carcinogenicity study (females)
corresponding to 14 mg a.i./kg bw/day

Dose / conc.:

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

Remarks:

Carcinogenicity study (females): reduced in several steps to 65 g/kg bw/day due to high mortality); corresponding to 40 (13) mg a.i./kg bw/day

Dose / conc.:

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

Remarks:

Interim pathology group (males): reduced in several steps to 65 due to high mortality; corresponding to 20 (13) 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

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.

Dermal irritation (if dermal study):

not examined

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 (carcinogenicity 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 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 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 linked to age-related nonneoplastic or neoplastic lesions in CD-1 mice but not 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 deemed to be a strong indicator for adaptation. Results are also shown in the table under 'Attachments'.

Relevance of carcinogenic effects / potential:

No treatment-related neoplastic lesions were observed up to and including the highest dose level.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogencity

Effect level:

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

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: No carcinogenic potential

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

35 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: No carcinogenic potential

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

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

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: No carcinogenic potential

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

65 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: No carcinogenic potential

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

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 (10^9/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 × 10^9/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 × 10^9/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	389.4^a	476.4	451.8^a	+
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 A6.7-4:Carcinogenicity study: Incidence of tumours

Parameter	Group 1		Group 2		Group 3		Group 4		Dose-response +/–
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
No. of animals with neoplasms	8	14	9	11	6	7	3	7
No of animals with> 1 neoplasm	2	0	0	0	0	0	1	3
Benign neoplasms	8	7	6	1	1	4	4	4
Malignant neoplasms	2	7	3	10	5	3	0	6
Malignant with metastasis	2	2	1	6	2	3	0	3

 

Conclusions:

The NOAEL for carcinogenicity was 7 mg a.i./kg bw/d in males and 13 mg a.i./kg bw/d in females. No treatment-related neoplastic lesions were observed.
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®-1mice. 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 Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid 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.

In addition, significant toxicity was seen in females at all dose levels, therefore 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 an overall NOAEL for chronic toxicity of Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid (20% a.i.) can be established at 10 mg /kg bw/d for male mice while no NOAEL was established for females due to the occurrence of toxicologically relevant histopathological findings at this dose in premature decedents.

The chronic NOAEL derived from the combined chronic toxicity and carcinogenicity study in mice is thus 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).

The NOAEL for carcinogenicity was 7 mg a.i./kg bw/d in males and 13 mg a.i./kg bw/d in females. No treatment-related neoplastic lesions were observed.

 

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 rlinked to age-related non-neoplastic or neoplastic lesions in CD-1 mice but not 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 considered to be very likely in mice.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

other: Pathological Reassessment

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Version / remarks:

1988

Deviations:

yes

Remarks:

urinalysis is missing.

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-C10–C16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Cas Number:

139734-65-9

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Amines, N-C10–C16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Test material form:

other: 20% of the pure active substance in water

Details on test material:

- Name of test material: DOPA-Glycinate

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Chronic study (males)
corresponding to 2 mg a.i./kg bw/day

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Chronic study (males)
corresponding to 6 mg a.i./kg bw/day

Doses / concentrations 3

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Chronic study (females)
corresponding to 4 mg a.i./kg bw/day

Doses / concentrations 5

Dose / conc.:

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

Remarks:

Chronic study (females)
corresponding to 12 mg a.i./kg bw/day

Doses / concentrations 6

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

Doses / concentrations 7

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:

Doses / concentrations 8

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

Examinations

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.

Results and discussion

Results of examinations

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'.

Effect levels

1

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

2

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

3

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'

4

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'

Target system / organ toxicity

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

Any other information on results incl. tables

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 (10^9/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 × 10^9/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 × 10^9/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	389.4^a	476.4	451.8^a	+
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

 

Applicant's summary and conclusion

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.