Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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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:
other: Guideline study with acceptable restrictions, GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Version / remarks:
1988
Deviations:
yes
Remarks:
urinalysis is missing.
Qualifier:
according to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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 (e.g. ad libitum): commercial ssniff® R/M-H V1530 diet, ad libitum
- Water (e.g. ad libitum): 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Carcinogenicity study (males): 0, 10, 30, 100 (reduced in several steps to 35 due to high mortality) mg/kg bw/d; corresponding to 0, 2, 6 and 20 (7) mg a.i./kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
Carcinogenicity study (females): 0, 20, 60, 200 (reduced in several steps to 65 due to high mortality) mg/kg bw/d; corresponding to 0, 4, 12 and 40 (14) mg a.i./kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
Interim pathology group (males): 0, 100 (reduced in several steps to 65 due to high mortality) mg/kg bw/d, corresponding to 0 and 20 (13) mg a.i./kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
Interim pathology group (females): 0, 200 (reduced in several steps to 75 due to high mortality) mg/kg bw/d, corresponding to 0 and 40 (15) mg a.i./kg bw/d
Basis:

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) 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
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)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
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).

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.

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.

Effect levels

open allclose all
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
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
20% aqueous solution, "product by process"
Sex:
female
Basis for effect level:
other: see 'Remark'
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

Critical effects observed:
not specified

Any other information on results incl. tables

Dose [mg/kg bw/d]

0

0

10

20

30

60

100/35

200/65

 

 

 

m

f

m

f

m

f

m

f

m

f

Number of animals

50

50

50

50

50

50

50

50

 

 

Mortality (at study termination)

 

 

 

 

 

 

 

 

 

 

Mortality rate [%]

34

8

16

40**

40

74**

74**

74**

+

+

Mean survival time [test week]

72.1

77.1

76.0

73.5

70.4

67.3**

58.8

52.3**

 

Clinical signs

 

 

 

 

 

 

 

 

 

 

Swollen/reddened eyes

3

1

1

1

15

34

21

18

+

+

Ptosis

4

2

2

0

12

19

15

7

+

 

Haematology

Haemoglobin content (nmol/L)

Week 13

9.47

9.69

9.52

9.60

8.92**

9.26

8.70**

8.65**

Week 26

9.14

9.62

9.09

9.16

8.33**

8.66**

7.99**

8.53**

Week 52

8.38

8.42

8.53

8.18

7.91

8.22

8.19

8.18

Week 78/79

8.04

8.22

7.99

7.81

7.91

8.36

8.43

7.59

 

 

Haematocrit (%)

Week 13

41.8

42.5

41.9

41.6

39.6**

40.3**

38.8**

38.3**

Week 26

44.5

46.1

44.4

43.8**

41.0**

42.1**

39.5**

41.9**

Week 52

40.3

42.0

41.1

39.9

38.2

40.7

40.3

40.5

 

 

Week 78/79

39.3

40.2

39.4

38.0

39.8

41.1

42.0

37.5

 

 

Leucocytes (109/L)

Week 13

5.20

3.51

5.66

3.76

4.53

4.26

5.41

4.10

 

 

Week 26

3.85

3.97

4.23

3.66

3.02

3.38

2.94

4.59

 

 

Week 52

2.19

3.72

2.28

4.68

2.56

5.01

2.36

5.80**

 

+

Week 78/79

5.98

5.56

7.96

4.67

6.99

4.70

5.18

5.21

 

 

Differential blood count – neutrophilic granulocytes (no. of cells × 109/L)

Week 13

0.978

0.557

1.032

0.893

0.955

0.862

1.442

1.418**

 

+

Week 26

1.102

0.710

0.961

0.914

0.697

1.001

0.921

1.464**

 

+

Week 52

0.671

0.781

0.535

0.957

0.624

1.322

0.805

1.366**

 

+

Week 78/79

1.349

1.363

1.633

1.631

2.454

1.985

1.461

1.541

 

 

Differential blood count – monocytes (no. of cells × 109/L)

Week 52

0.041

0.094

0.039

0.150

0.053

0.181**

0.056

0.176**

 

+

Clinical biochemistry

ASAT (U/L)

Week 26

84.1

93.4

85.1

108.7

88.1

134.0

224.1**

198.9**

+

+

Week 52

83.0

92.1

81.8

89.8

89.3

137.7**

165.9**

156.2**

+

+

Week 78/79

93.6

133.7

92.9

101.2

173.3

182.2

221.6**

165.8

+

 

LDH (U/L)

Week 26

222.7

209.8

195.7

226.6

209.7

282.3

409.2**

366.8**

+

+

Week 52

261.3

177.8

212.0

176.3

216.1

276.8**

312.5

241.9

+

+

Week 78/79

262.0

819.9

236.6

298.5

342.1

389.4a

476.4

451.8a

+

 

Macroscopic findings

Spleen reduction

1/50

0/50

3/50

0/50

2/50

1/50

11/50

3/50

+

 

Dilated urinary bladder

0/50

0/50

0/50

0/50

0/50

0/50

1/50

1/50

 

 

Enlarged urinary bladder

1/50

0/50

1/50

0/50

3/50

1/50

16/50

5/50

+

+

Tightly filled urinary bladder

3/50

0/50

2/50

0/50

10/50

11/50

35/50

10/50

+

+

**) significantly different from control (p<0.01)

a) no evaluation possible due to unusually high vales in the female control

 

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)

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 DOPA-Glycinate (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 ou 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: R-phrase 41). 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 administeredviathe 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).

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.

There was no evidence of carcinogenicity.

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 DOPA-Glycinate has no carcinogenic potential in mice.

The study reported above does not conform to the requirements of OECD 45 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.

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 DOPA-Glycinate (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 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).