Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Version / remarks:
1982
Deviations:
no
GLP compliance:
no
Remarks:
At the time of the study conduct, GLP was not compulsory. However, the study was conducted in accordance with the principles of GLP.
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
- Name of test material: DOPA-Glycinate

Test animals

Species:
rat
Strain:
other: Sprague Dawley, Crl: CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: Males: 120–245 g; Females: 155–195 g
- Housing: in groups of max. 3 (males) or 4 (females)
- Diet (e.g. ad libitum): powdered diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 18 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 50 and 100 mg/kg bw/day based on test substance as manufactured (20% aqueous solution)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Observations
Clinical signs: Yes (at least once daily)
Mortality: Yes (at least once daily)

Body weight: Yes (weekly throughout the study and on the day of necropsy)

Food consumption: Yes (recorded daily throughout the dosing period for the rats in one cage and evaluated on a weekly basis)

Water consumption: no

Ophthalmoscopic examination: Yes
Number of animals:
a) all animals of all groups
b) all control and high dosed animals
Time points:
a) before treatment
b) final week of treatment
Parameters: Ocular fundus with macula lutea, papilla, ocular vessels, cornea, lens and vitreous body.

Haematology: Yes
Number of animals: all animals.
Time point: final week of study
Parameters: mean cell haemoglobin, packed cell volume, mean cell haemoglobin concentration, mean cell volume, haemoglobin concentration, total and differential white blood cell counts, prothrombin time

Clinical chemistry: Yes
Number of animals: all animals.
Time point: final week of study
Parameters: glutamate oxalacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), alkaline phosphatase (AP), sodium ions (NA+), potassium ions (K+), calcium (Ca++), phosphorus (P), chloride (CL-), total protein (TP), blood urea (BU), albumin, albumin/globulin ratio (A/G ratio), glucose, cholesterol, triglycerides, creatinine, total bilirubin.

Urinalysis: No

Sacrifice and pathology:
Organ weights: Yes
Organs: adrenals, kidneys, liver, ovaries, testes

Gross and histopathology: Yes
Number of animals: all animals
Time points: end of study
Gross necropsy: external examination including all orifices and a full macroscopic examination of all tissues and organs in situ.
Number of animals: all control and high dosed animals
Time points: end of study
Histopathology: adrenals, aorta, bone marrow, brain, cecum, colon, duodenum, epididymides, esophagus, eyes, heart, ileum, jejunum, lymph nodes, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, skeletal muscle, skin and mammary gland, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids, tongue, trachea, urinary bladder, uterus, vagina.
Kidneys, liver, lungs were examined in rats from all groups.


Statistics:
Body weight, organ weight/body weight ratios, haematology data: analysis of variance followed by Student-Newman-Keuls test
Food consumption: evaluation of group mean values; only.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed.
There were occasional findings related to the dosing procedure, such as bleeding of snout following application in one high dose male. Furthermore, staggering gait and disequilibrium were observed repeatedly in one male rat of the mid dose group. Other infrequent findings were a subcutaneous nodule of 7 mm in diameter on the right side of the snout in one male of the low dose group and broken incisor in one control female.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The majority of mortalities observed was due to the experimental procedure such as intubation error (2 control animals, 2 mid dose animals, 3 high dose animals) and ether anesthesia for blood sampling (2 low dose animals). In one female animal, the cause of demise could not be determined due to cannibalism. However, this death was considered to be incidental since there was no unequivocal evidence of any signs of toxicity prior to death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The group mean body weight of the male rats of the mid dose and the high dose group was statistically significantly decreased from week 9 to week 13 of treatment.
The overall body weight gain (week 1 to 13) was statistically significantly reduced in mid dose males (–19.8%) and high dose males (–27.7%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no noteworthy effect on overall food intake throughout the treatment period for female animals when compared to the corresponding control.
For male animals, the group mean food intake was slightly increased in the low dose group (+7.0%) and slightly reduced the mid ( 8.0%) and the high dose group (–10.1%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Congestion of the optic disk was observed in five males and in one female high dose animal in week 13. Ocular changes are known to be associated with increased intracranial pressure and concomitant edematous swelling. Since there were no remarkable histopathological changes in the eyes of all animals examined, these findings have to be considered as compound-related clinical symptoms, possibly secondary to treatment.
Other incidental findings were retinal hemorrhage of the optic disk or the fundus or between optic disk and macula (one control male, one male and two females of the high dose group), corneal scar (one control female), and optic disk palish coloured as defined by atrophy of optic nerve (one female of the high dose group).
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Major statistically significant haematological changes observed at week 13 were distinctively increased leucocytes associated with increased neutrophile granulocytes and decreased lymphocytes seen in all treated groups compared with the control groups. Generally, leucocytosis, granulocytosis and lymphopenia are indicative of inflammatory processes. Further evidence to support this view were microscopic findings such as small microabscesses consisting of focal areas of necrosis and mild lymphoid hyperplasia in the mesenteric lymph nodes in high dose animals of either sex.
Minor statistically significant haematological findings were slightly reduced packed cell volume (PCV) and slightly increased mean cell haemoglobin concentration (MCHC) in all males of the treated groups.
Furthermore, prothrombin time (PT) values were slightly but statistically significantly reduced in males of the mid dose group (–21%) and in males (–21.5%) and females (–32.3%) of the high dose group.
Results are presented in Table A6.4.1- 1.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase of alkaline phosphatase levels (AP) was observed in high dose females only. This marked hyperphosphatasemia was considered to be difficult to interpret since alkaline phosphatase is widely distributed in the body and is present in considerable concentration in many tissues. In addition, in the absence of other unequivocal indications to account for this sex specific alteration increased alkaline phosphatase levels have to be considered as of unknown biological significance.
Further blood chemistry findings (reduction in glucose, total protein, albumin, albumin/globulin ratio and cholesterol) were generally slight and may be interpreted as slight effects of the test article on carbohydrate, lipid and protein metabolism.
Results are presented in Table A6.4.1- 1.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute liver weight was statistically significantly decreased (17.7%) and the relative kidney weight was statistically significantly increased (+17.5%) in high dose males. Both effects have to be considered primarily as a secondary effect following decrease of body weights.
Other changes such as reduction of absolute testes weights of mid dose males and increased relative adrenal weights of high dose males were due to single remarkable individual values which are considered to be more incidental than treatment-related.
There were no statistically significant findings in absolute and relative organ weights in females.
Results are presented in Table A6.4.1- 1.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings. Bloody stomach content in one high dose male was related to the dosing procedure when occasional bleeding of the snout was observed throughout the last week of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic compound related lesions were observed in the high dose rats only.
The following lesions of mesenteric lymph nodes were observed:
- Foci of necrosis in 5 out of 7 males and in 6 out of 8 females of the high dose group
- Acute inflammation in 5 out of 7 males and in 6 out of 8 females of the high dose group
- Lymphoid hyperplasia in 5 out of 7 males and in 3 out of 8 females of the high dose group
According to the study authors, these mesenteric lymph node lesions were secondary to treatment with the high dose of the test compound. However, the type of reaction noted is typical of a bacterial lymphadenitis, which is not a direct toxic effect. No bacteria or other causative agents were observed in the lesions.
Since histopathological examination of mesenteric lymph nodes did not include group 2 and 3 animals, the effect of the test substance at dose levels of 25 and 50 mg/kg bw/day on this tissue could not be clarified.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
(20% aqueous solution, "product by process")
Sex:
male/female
Basis for effect level:
other: significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes at all dose levels
Dose descriptor:
LOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Any other information on results incl. tables

Table A6.4.1-1:Results of the 90-day study in rats.

Parameter

Control

25 mg/kg bw/da

50 mg/kg bw/da

100 mg/kg bw/da

Dose-response
+/–

 

m

f

m

f

m

f

m

f

m

f

Number of animals examined

10

10

10

10

10

10

10

10

 

 

Mortality

1/10

1/10

0/10

2/10

1/10

1/10

2/10

2/10

 

 

Group mean body weight [g]

 

 

 

 

 

 

 

 

 

 

Week 1

189

173

193

170

192

168

192

171

 

 

Week 9

438

262

454

258

402*

262

380*

254

 

Week 13

468

263

470

260

419*

271

399*

161

 

Overall body weight gain (week 1 to 13) [%]

278 g

89 g

+0.4

+1.1

–19.8*

+15.7

–27.7*

–1.1

 

 

 

 

 

 

 

 

 

 

 

 

Group mean food consumption [g/rat/cage/week]

 

 

 

 

 

 

 

 

 

 

Week 1

185

134

198

142

181

142

186

143

 

 

Week 13

183

123

182

134

156

118

146

113

 

 

Overall food intake (week 1–13) [%]

2362 g

1721 g

+7.0

+6.0

–8.0

+0.9

–10.1

–3.9

 

 

 

 

 

 

 

 

 

 

 

 

Ophthalmoscopy (No. of animals examined week 13)

9

9

n.e.

n.e.

n.e.

n.e.

8

8

 

 

Congestion of optic disk

0

0

 

 

 

 

5

1

+

 

Haematology (No. of animals examined week 13)

9

9

10

10

9

9

8

8

 

 

White Blood Cell Counts [1000/mm³]

15.3

10.5

17.2

12.5

24.2*

17.2*

24.6*

20.2*

+

+

Neutrophils [%]

12

8

21*

19*

36*

27*

38*

34*

+

+

Lymphocytes [%]

87

90

77*

80*

63*

72*

60*

66*

Packed Cell Volume [%]

45.3

42.7

42.2*

42.0

42.8*

42.0

42.5*

41.6

 

 

Mean Cell Haemoglobin Concentration [g/dL]

36.7

37.2

38.4*

37.8

37.8*

37.4

38.4*

37.8

 

 

Prothrombin Time [s]

18.6

19.5

17.4

18.2

14.7*

17.7

14.6*

13.2*

 

 

 

Clinical chemistry (No. of animals examined week 13)

9

9

10

10

9

9

8

8

 

 

Alkaline Phosphatase (U/L)

236.1

30.6

236.6

138.0

250.1

195.8

246.1

212.7*

 

+

Glucose [mg/dL]

124.6

102.9

124.8

104.8

106.7*

96.1

97.5*

104.6

 

 

Total protein [mg/dL]

6.8

7.1

6.8

7.1

6.3*

6.7*

6.1*

6.3*

 

 

Albumin [g/dL]

3.5

3.9

3.4

3.9

2.9*

3.4*

3.0*

3.1*

 

 

A/G ratio

1.1

1.2

1.0

1.3

0.9*

1.0*

1.0*

1.0*

 

 

Cholesterol [mg/dL]

70.3

87.6

75.7

91.7

60.5

67.8*

58.8

67.8*

 

 

Group mean body weight at necropsy [g]

482

272

486

270

421

270

396

262

 

 

Absolute liver weight [g]

Relative liver weight

14.88

3.09

8.99

3.30

15.85

3.26

9.18

3.41

13.38

3.12

9.26

3.44

12.25*

3.11

8.89

3.41

 

 

Absolute testes weight [g]

3.43

3.31

2.77*

3.17

 

 

Relative adrenals weight [%]

0.010

0.022

0.009

0.021

0.011

0.023

0.016*

0.026

 

 

Absolute kidney weight

Relative kidneys weight [%]

2.76

0.57

1.84

0.68

3.01

0.62

1.96

0.73

2.56

0.60

1.90

0.70

2.58

0.67*

1.86

0.71

 

 

*)         significantly greater or less than control mean (p < 0.05)

n.e.     not examined

a)         in terms of test substance as manufactured (20% aqueous solution)

 

 

Applicant's summary and conclusion

Conclusions:
Sprague Dawley rats were treated with DOPA-Glycinate for 90 consecutive days by daily oral gavage at dose levels of 0, 25, 50 and 100 mg/kg bw/day (as 20% aqueous solution), corresponding to 0, 5, 10 and 20 mg a.i./kg bw/d.
The LOAEL based on significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes at all dose levels was 25 mg/kg bw/day (as 20% aqueous solution), corresponding to 5 mg a.i./kg bw/d. No NOAEL could be determined. Therefore, a supplementary 90-day oral toxicity test utilising a modified dose range was conducted (A6.4.1/02).
Executive summary:

The oral 90-day toxicity study was carried out according to EPA 82-1 (1982).Four groups of 10 male and 10 female Sprague Dawley rats, respectively, were administered orally by gavage 0, 25, 50 and 100 mg/kg bw/day of DOPA-Glycinate (20% aqueous solution, substance as manufactured), corresponding to 0, 5, 10 and 20 mg a.i./kg bw/d. for 90 days.

The conduct of the study was similar to OECD 408 (1998) with the exception that (i) no NOAEL could be determined, (ii) no sensory reactivity test, no assessment of grip strength, no motor activity assessment and no functional observations were performed, (iii) the weight variation at the commencement of the study exceeded ± 20 % for males, (iv) weights of epididymides, uterus, thymus, spleen, brain and heart were not determined, and (v) the mesenteric lymph nodes were not examined for the low and mid dose group despite observed effects at the high dose group.

Treatment with the test substance at a dose level of 100 mg/kg bw/day produced toxic effects manifested by foci of necrosis, acute inflammation and lymphoid hyperplasia of the mesenteric lymph nodes in animals of either sex. Since histopathological examination of mesenteric lymph nodes did not include the low dose and mid dose animals, the effect of the test substance at dose levels of 25 and 50 mg/kg bw/day on this tissue could not be clarified.

Further treatment-related findings were reduction in body weight gain and overall food consumption in male animals receiving 50 and 100 mg/kg bw/day, ophthalmological findings (congestion of the optic disk) in five high dose male animals and one female, haematological changes (significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes) in males and females receiving 25, 50 and 100 mg/kg bw/day, slight blood chemistry changes (reduction in glucose, total protein, albumin, albumin/globulin ratio and cholesterol) within the normal range of background data in males and females treated with the test substance at 50 and 100 mg/kg bw/day, and a markedly increased alkaline phosphatase level in high dosed females only.

No treatment-related mortality or clinical signs occurred during the study.

Apart from slight leucocytosis, granulocytosis and lymphenia treatment with the test substance at a dose level of 25 mg/kg bw/day was very well tolerated and did not elicit any further toxic changes attributable to oral administration of the compound tested.

Based on a significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes at all dose levels, the LOAEL was 25 mg/kg bw/d (as 20% aqueous solution), corresponding to 5 mg a.i./kg bw/d.

No NOAEL could be determined based on the results of this study. Therefore, a supplementary 90-day oral toxicity test utilising a modified dose range was conducted (A6.4.1/02).