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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1973-11-29 to 1974-07-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed study prior to GLP and OECD-TG meeting the necessary scientific and current guideline requirements.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1974
Report date:
1974

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Prior to establishment of OECD technical guidelines
- Objective of the study: Evaluation and characterisation of subchronic oral toxicity of pharmaceutical grade phenothiazine following oral administration via food to beagle dogs for 13 weeks; several doses were tested;
- See section material and methods for further details
GLP compliance:
no
Remarks:
Prior to GLP
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenothiazine
EC Number:
202-196-5
EC Name:
Phenothiazine
Cas Number:
92-84-2
Molecular formula:
C12H9NS
IUPAC Name:
10H-phenothiazine
Details on test material:
- Lot no.: 31106RB
- Description: fine, pale green powder with an unpleasant odour
- Receipt date: 1973-11-16
- Purity: Pharmaceutical grade, assumed 100% for purposes of dosage calculations

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Age: Young adults
- Health status: healthy
- Animals: 40 purebred beagles (20 males and 20 females)
- Selection: Dogs were selected on the basis of preliminary clinical laboratory screens
- Assignment to groups: Randomly assigned
- Food: Ground Wayne Dog Meal, ad libitum

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test material was incorporated into the basal laboratory died (Ground Wayne Dog Meal) on a weight by weight basis and mixed in a twin shell blender to provide the appropriate dietary levels for each group

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared each week
- Mixing appropriate amounts with (Type of food): Ground Wayne Dog Meal
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- Treatment: 13 weeks
- Exposure: via feed seven days a week
Frequency of treatment:
- Daily, feed was available seven days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
50ppm, 200ppm, 500ppm and 2000ppm
Basis:
nominal in diet
No. of animals per sex per dose:
4 male and 4 female dogs per dose
Control animals:
yes
Details on study design:
- Group assignment, please see table "Animal Groups and Dietary Levels" in section "Any other information on materials and methods"
Positive control:
No positive controls included

Examinations

Observations and examinations performed and frequency:
OBSERVATION AND RECORDS
- DAILY: Appearance, behaviour, appetit, elimination and signs of pharmacologic effect
- WEEKLY: Individual body weights and food and compound consumption

CLINICAL LABORATORY STUDIES (HEMATOLOGICAL, BIOCHEMISTRY, URINE ANALYSIS, SEE BELOW)
- Performed on all dogs initially and at four and 13 weeks

HEMATOLOGICAL STUDIES
- Hematocrit
- Hemoglobin
- Erythrocyte count
- Total and differential leukocyte count

BIOCHEMISTRY STUDIES
- Determinations of fasting blood sugar
- Blood urea nitrogen
- Bromsulphalein liver function test
- Serum glutamic-pyruvic transaminase

URINE ANALYSIS
- Specific gravity
- pH
- Glucose
- Ketones
- Total protein
- Bilirubin
- Microscopic examination of the sediment
Sacrifice and pathology:
TERMINATION
- After 13 weeks of treatment, all animals were sacrificed by exsanguination under surital anesthesia
- Complete necropsies were performed

ORGAN WEIGHTS (FROM EACH DOG)
- Thyroids
- Heart
- Liver
- Spleen
- Kidneys
- Adrenals
- Testes

TISSUE PRESERVATION (OBTAINED FROM EACH DOG)
- See Table: "Tissue Preservation" in section "Any other information on materials and methods"

HISTOPATHOLOGICAL EVALUATION
- Control and high dose group dogs (i.e. groups 1 and 4): All tissues listed in Table: "Tissue Preservation" in section "Any other information on materials and methods" were stained with hematoxylin and eosin and were examined microscopically
- 500ppm-group: spleen, bone marrow and rib junctiom were stained with hematoxylin and eosin and were examined microscopically
- 50ppm, 200ppm, 500ppm: liver and kidneys were stained with hematoxylin and eosin and were examined microscopically

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
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:
APPEARANCE, BEHAVIOUR, BODY WEIGHT CHANGES AND SIGNS OF COMPOUND EFFECT
- Body weight and food consumption are given in tables "Group mean body weights (kg.) at selected intervals" and "Average daily drug intake (mg/kg/day)" in section "Remarks on results including tables and figures".
- All of the test animals were comparable to the controls with respect to behaviour, appetite, elimination
- Body weight was either maintained (+/-5%) or gained while on study
- Rate of growth between groups was comparable
- Both males and females of the 500ppm group showed lower relative weight gains over the 13-week treatment period; however, these animals were slightly heavier than their counterparts at the initiation of the treatment
- Occasional instances of soft feces were observed; however, the occurence was among the control as well as the test animals and were considered to be of little significance
- Haircoat of the forelimbs, chest and dorsal thoraic areas of all high level test animals was stained orange in color beginning during the first week of study and contiunuing until termination
- Latter finding is probably a result of the oxidation of small amounts of phenothiazine adhering to the hair following feeding
- Food consumption was not adversely affected as a result of treatment
- Evaluation of food consumption data resulted in actual daily drug intake as indicated in table "Average daily drug intake (mg/kg/day)" in section "Remarks on results including tables and figures".

CLINICAL LABORATORY STUDIES (HEMATOLOGICAL DATA, BLOOD CHEMISTRY DATA, URINE ANALYSIS)

A) HEMATOLOGICAL DATA:
- Lowering of hematocrit, hemoglobin and erythrocyte count values in two male and one female 2000ppm dog at the four week interval
- Erythrocyte count in two further female 2000ppm dogs reduced
- At 13 weeks hematocrit, hemoglobin and erythrocyte count were again lowered in three male 2000ppm level dogs
- All remaining values were within acceptable normal limis and comparable with control values

B) BLOOD CHEMISTRY DATA:
- Slight lowering of blood sugar values at one female 200ppm and 500ppm dose dog, as well as in one male and in all 2000ppm female dogs
- All other parameters assessed at four and thirteen weeks were generally within acceptable parameters and comparable to control values

C) URINE ANALYSIS
- Generally unremarkable results
- Exception: Traces of sugar in one 2000ppm male at for weeks and four dogs (one 50ppm, two 200ppm and one 500ppm) at week 13

GROSS PATHOLOGY
- No consistent gross tissue changes which could be attributed to the administration of the test item
- Exception: dark colored spleen in all 2000ppm animals
- Please also see table "Summary of Necropsy Findings at 13 Weeks" in section "Remarks on results including tables and figures".

ORGAN WEIGHTS
- Examination of the data showed all values for the test animals to be within acceptable normal limits and comparable with control values
- Exception: spleen weights and spleen/body weight ratios of two 2000ppm female dogs were increased

MICROSCOPIC PATHOLOGY
- Tissue alterations included: marked splenic congestion with areas of increased extramedullary hematopoiesis
- Deposition of hemosiderin in the spleen, liver, kidney and bone marrow
- Increased cellularity of the bone marrow with a marked increase in erythroid elements
- These changes were related to the toxic effects of phenothiazine on the red blood cells and were present in all 2000ppm animals
- Hemosiderin deposition was present, but to a lower degree in the liver and kidney sections of three males and one female of the 500ppm group
- One 500ppm male dog also had slightly increased extramedullary hematopoiesis and hemosiderin pigment deposition in the spleen with a slight increase in erythroid elements in the bone marrow
- Changes in the spleen and bone marrow sections from the remaining 500ppm dogs were equivocal and within the range of normal variability

MICROSCOPIC PATHOLOGY - INCIDENTAL LESIONS
- Subendocardial hemorrhages in the hearts of one female control dog and one female 2000ppm dog
- A heart valve containing dilated vessels and small thrombi were found at one female 2000ppm dog
- Congenital defect of the auricle and heart valve of one female 500ppm
- Small cysts were present in the pituitary of one female 2000ppm dog
- Cystic area was present in th parathyroid of another female 2000ppm dog
- One female 500ppm dog had a congenital defect in one lobe of the lung resembling a severe bullous ephysema
- One male 50ppm dog had complete aplasia of the left kidney with compensatory hypertrophy of the right kidney
- Scattered incidence of splenic sucapsular hemorrhage and inflammatory cells in the livers and kidneys was seen in both control and treated animals




Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs; haematology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table: Group mean body weights (kg.) at selected intervals

Interval [weeks]

MALES

Dosage Level [ppm]

0

50

200

500

2000

0

8.9

9.1

9.3

10.8

8.3

4

9.8

10.7

10.7

11.0

9.3

8

10.2

11.3

11.1

11.2

10.0

13

10.6

11.9

11.7

11.6

10.1

% Increase

19

30

26

7

22

Interval [weeks]

FEMALES

Dosage Level [ppm]

0

50

200

500

2000

0

6.5

7.7

8.0

8.6

7.8

4

9.2

9.1

9.3

9.2

8.8

8

9.7

9.8

9.7

9.5

9.2

13

9.7

10.2

10.1

9.5

9.7

% Increase

49

32

26

10

24

 

Table: Average daily drug intake (mg/kg/day)

Feeding Level [ppm]

Drug intake (mg/kg/day)

MALES

FEMALES

0

0

0

50

1.54

1.59

200

6.06

6.82

500

16.93

17.68

2000

69.30

67.05

Table: Summary of Necropsy Findings at 13 weeks

Observation

Group no

1

2

3

4

5

Sex

M

F

M

F

M

F

M

F

M

F

 

Thyroids– Connected

 

 

 

 

 

1

 

 

 

 

Lung– Right cardiac lobe enlarged and distended with air. Portions are only thin membraneous remnants of the lung. Completely filled with air. Other portions resemble an alveolar Emphysema. Right diaphragmatic lobe smaller than normal

 

 

 

 

 

 

 

 

 

1

Heart– Subendocardial brush stroke reddening in left ventricle

 

 

 

 

 

 

 

1

 

 

Heart– Two small firm nodes in right atrioventricular valve

 

 

 

 

 

 

 

1

 

 

Heart– Heart worm in right ventricle

 

 

 

 

 

 

 

 

 

1

Heart– Left auricle thickened with lumen partially closed

 

 

 

 

 

 

 

 

 

1

Heart– Right atrioventricular valve thickened

 

 

 

 

 

 

 

 

 

1

Kidney– Left missing

 

 

1

 

 

 

 

 

 

 

Spleen– Black nodes at margin

 

 

 

2

3

 

 

 

 

 

Spleen– Grey discoloration on surface

 

 

 

 

1

 

 

 

 

 

Spleen– Dark in color

 

 

 

 

 

 

4

4

 

 

Spleen– Enlarged

 

 

 

 

 

 

 

1

 

 

Urinary Bladder– Red area on neck

 

 

 

 

 

 

1

 

 

 

Small intestine– Round worms

 

 

 

1

1

1

2

1

1

 

Applicant's summary and conclusion

Conclusions:
The animals responded well physiologically and the response of the animals at the 500ppm level is of minor biological significance.
Executive summary:

Phenothiazine (pharmaceutical grade) was evaluated for sub-chronic toxicity following dietary administration to four groups of three male and three female beagle dogs for 13 weeks at dietary levels of 50, 200, 500 and 2000ppm. A fifth group of dogs served as control and received only the basal laboratory diet. Parameters evaluated for compound induced effect included survival, appearance, appetite, elimination, body weight changes, clinical laboratory data, gross necropsy findings, organ weights and organ/body weight ratios, as well as microscopic pathology.
All control and test animals survived the study and there was no indication of a compound related effect among the test groups with regard to appearance with regard to appearance, behavior, appetite, elimination and body weight changes. The observation of orange-stained fur on the forelimbs, chest and dorsal thoracic area of the high level dogs was ascribed to the oxidation product resulting from small amounts of phenothiazine adhering to the fur following feeding. Hematological data suggest a decrease in values among the principal elements of the profile (hemoglobin, hematocrit and total erythrocyte counts). Three of the six high level exhibited slight decreases in values for these parameters at both four and 13 weeks. The effect of phenothiazine in producing a hemolytic anemia at high dosage levels is well documented in the literature and this observed effect is probably related to that response. Blood chemistry data revealed decreased values for fasting glucose in four of six high level dogs at week 4 which was not observed at week 13.
Necropsy findings were generally unremarkable except for notation of dark colored spleens in all of the high level dogs. Spleen weights and spleen/body weight ratios were elevated in two of three high dose females.
Microscopic examination of tissues revealed marked splenic congestion and extramedullary hematopoiesis of the spleen; deposition of hemosiderin in the spleen, liver, kidney and bone marrow and increased cellularity of the bone marrow, particularly in erythroid elements. These effects were observed in all high level dogs and are reflective of the hemolytic effect of phenothiazine on red blood cells. There was a marginal effect in the dogs at 500ppm, where four of six dogs demonstrated hemosiderin deposition in the liver and kidney. One male exhibited increased extramedullary hematopoiesis in the spleen. However, the other elements of the phenothiazine-induced effects (increase in cellularity of the bone marrow and a reflection of the effect in the hematological studies) suggest that the animals responded well physiologically and the response of the animals at the 500ppm level is of minor biological significance.