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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed study prior to GLP and OECD-TG, but meeting the necessary scientific and current guideline requirements.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1977
Report date:
1977
Reference Type:
publication
Title:
Demonstration of A-Alizarin staining of Bone
Author:
Hurley, L.
Year:
1965
Bibliographic source:
Supplement to Teratology Workshop Manual, Berkeley California, January 25-30, 1965, pp. 121-122
Reference Type:
publication
Title:
Teratology Principles and Techniques
Author:
Wilson, J. and Warkany, J.
Year:
1965
Bibliographic source:
University of Chicago Press, Chicago, Illinois, 1965, pp. 271-277

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: A study was initiated to determine the potential teratogenicity of phenothiazine upon gravid rats.
- See section material and methods for further details
GLP compliance:
no
Remarks:
Prior to GLP. However, a QA-statement is included.
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:
- Sponsor: West Chemical Product's, Inc.
- Lot No.: NB 36-155

Test animals

Species:
rat
Strain:
other: Albino
Details on test animals or test system and environmental conditions:
- Origin: Charles River
- Breeding: Breeding took place at the Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts
- Animals were shipped to the testing laboratory on day 5 of gestation
- Sex: only female rats were obtained for the study
- Food: Purina Rat Chow, Ralston Purina Co., St. Louis, Missouri, ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: corn oil
Details on exposure:
- Solutions were prepared by adding corn oil to the material
- Final concentrations were either 0.3, 1.0 or 3.0 percent phenothiazine (w/v)
- Doses were administered at 5 mL per kg of body weight
- Oral administration via gavage, by means of a hypodermic syringe equipped with a ball-tipped intubation needle
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
- Mating was done at the Charles River Breeding Laboratories, Inc., Notrh Wilmington, Massachusetts
- Copulation was confirmed by sperm-positive results of vaginal examinations
- Day zero is defined as the day of insemination
- Animals were shipped to the testing laboratory on day 5 of gestation
- All dams received were assigned to a treatment group, but only those confirmed pregnant were included in the reported data
Duration of treatment / exposure:
- All test animals were given the material daily from the 6th day of gestation period through the 15th day inclusive
Frequency of treatment:
- A total of 10 doses was applied
Duration of test:
- During mating and shipment on gestation day 5 animals were at the Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts
- From gestation day 5 until gestation day 20 animals were at the facilities of Industrial Bio-Test Laboratories
Doses / concentrations
Remarks:
Doses / Concentrations:
15 mg/kg/day, 50 mg/kg/day, 150 mg/kg/day
Basis:
actual ingested
All dams were weighed daily for the purpose of dosage administration
No. of animals per sex per dose:
21 treated female rats per group, not all were pregnant. Details see table "Outline of Experiment" in section "Any other information on material and methods incl. tables"
Control animals:
yes, concurrent vehicle
Details on study design:
- All females were sacrificed by chloroform asphyxation on the 20th day of gestation

Examinations

Maternal examinations:
BODY WEIGHT DATA
- Daily weighing for the purpose of dosage administration
- Data recorded and reported includes those at day 6 of gestation (initial dosing), day 9, day 12, day 15 (final dosing) and at day 20 (sacrifice)

MORTALITIY AND REACTIONS
- All noted maternal deaths and untoward behavioural reactions were recorded
Ovaries and uterine content:
- An incision was made in the abdominal wall and the full extent of both uterine horns were exposed immediately
- Implantation sites were counted, special attention being paid to resorption sites or any uterine abnormalities
- Number of corpora lutea was determined and recorded at this time
Fetal examinations:
REPRODUCTIVE AND EXTERNAL TERATOGENIC EFFECTS
- Fetal swellings were counted
- Number of viable fetuses present in the uterus was determined (spontaneous movement and a more ruddy color distinguishing live from dead animals)
- Fetuses were removed from the uterus by cutting the umbillical cord
- Blotting paper was used to remove excess amniotic fluid and blood prior to the weighing of the animals
- External examination paid special attention to the following abnormaities: please see table "Examined Abnormalities" in section "Any other information on materials and methods incl. tables".

FETAL SKELETAL AND INTERNAL DEVELOPEMENT
- All fetuses obtained were examined
- Evaluation of skeletal development was conducted using Alizarin staining according to Hurley et al., employing 2/3 of the fetuses of each sex from each litter
- Internal development was evaluated using free-hand razor blade section technique of Wilson et al. upon the remaining fetuses
- Please see references for a complete description
Statistics:
- Maternal body weights (gestation days 6, 9, 12, 15 and 20), maternal body weight gains (gestation days 6 through 15 and 6 through 20) and fetal body weight data consisted of a One-Way Analysis of Variance with significant effects disclosed by that treatment further studied by Scheffe's Multiple Comparison
- Statistical evaluation of the numbers of corpra lutea, implantation sites, resorption sites and fetuses (total number as well as the numbers of male and female fetuses) were conducted employing Chi-Square Test of Independence
Indices:
No data
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
See section "Remarks on results including tables and figures".


Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
See section "Remarks on results including tables and figures".

Effect levels (fetuses)

Remarks on result:
other: see Any other information on results incl. tables for details.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

A)    MATERNAL DATA

BODY WEIGHT DATA

-         Mean body weights are presented in the table “Body weight” in this section

-         The 15 mg/kg/day and the 50 mg/kg/day were comparable to the control

-         No statistically significant intergroup differences

-         Body weights in the 150 mg/kg/day group were less than those of the concurrent controls at gestation day 12 and 15, with day 12 results not being statistically significant

-         Body weight on gestation day 15 and body weight gain from gestation day 6 through 15 was significantly reduced in the 150 mg/kg/day group

Table: Body weight

Group [Dose Level/Day]

Body Weight [g]

Day of Gestation

Body Weight Gain [g]

6

9

12

15

20

Days 6 – 15

Days 6 – 20

Control

214

238

260

287

362

73

148

T-I [15 mg/kg]

217

238

259

286

365

69

148

T-II [50 mg/kg]

209

229

250

278

347

69

138

T-III [150 mg/kg]

212

226

246

267**

352

55**

140

** Statistically significant reduced at the 99% confidence level


MORTALITY

-         There were no maternal deaths

REACTIONS

-         No unusual behavioral reactions were observed, neither among control or test animals

-         One 150 mg/kg/day animal developed a sore on the right side of her neck, which has begun to heal prior to study termination

REPRODUCTIVE EFFECTS

-         Mean results are presented in table “Reproductive Effects” in this section

-         One control dam was found to have excessive amounts of blood in both uterine horns, another control in one uterine horn only

-         One dam of the 50 mg/kg/day group had brown-green colored placentas and left uterine horn

-         All other dams examined were free of gross uterine abnormalities

-         Numbers of corpora lutea, implantation sites, resorption sites and fetuses were similar for test and control dams

Table: Reproductive Effects

Group [Dose Level/Day]

Pregnant Females Examined

Autopsy Findings

(Mean/Female)

Females With One or More Resorption Sites

Corpora Lutea

Implantation Sites

Resorption Sites

Fetuses

Total

Percent

Control

20

10.7

9.8

1.0

8.8

8

40.0

T-I

[15 mg/kg]

18

10.5

9.2

0.4

8.8

6

33.3

T-II

[50 mg/kg]

21

10.9

9.7

0.1**

9.6

3

14.3

T-III

[150 mg/kg]

20

11.5

10.6

0.7

10.0

10

50.0

** Statistically significant reduced at the 99% confidence level

B) FETAL DEVELOPMENT DATA

BODY WEIGHTS

-         Body weights are presented in table “Fetal Body Weight Data” in this section

-         In utero body weights of fetuses from treated animals were essentially the same as from controls

-         There were no significant differences in sex ratios among pups from test and control litters

Table: Fetal Body Weight Data

Group [Dose Level/Day]

Number of Males

Mean Body Weight

Number of Females

Mean Body Weight

Control

97

3.7

79

3.4

T-I [15 mg/kg]

78

3.8

80

3.6

T-II [50 mg/kg]

101

3.7

100

3.6

T-III [150 mg/kg]

106

3.9

93

3.6

 

EXTERNAL DEVELOPMENT

-         External abnormalities noted among fetuses are listed in table “External Abnormalities” in this section

-         All other fetuses (except for those listed) were free of apparent malformations

-         Dam 38 of T-I exhibited an edematous late resorption

Table: External Abnormalities

Group [Dose Level/Day]

Finding

Control

-         Dam no 2: 1 fetus with hematoma

-         Dam no 6: 1 fetus with hematoma

-         Dam no 21: 1 fetus with hematoma

T-I [15 mg/kg]

-         Dam no 27: 6 of 11 fetuses with a hematoma

-         Dam no 33: 1 runt fetus

T-II [50 mg/kg]

-         Dam no 45: 2 of 8 fetuses with a hematoma

-         Dam no 59: 1 runt fetus

T-III [150 mg/kg]

-         Dam no 70: 3 of 10 fetuses with a hematoma

-         Dam no 76: 1 discolored (yellow) fetus

-         Dam no 78: 4 of 11 fetuses with a hematoma

-         Dam no 81: 1 runt fetus

 

SKELETAL DEVELOPMENT

-         Results of skeletal development are summarized in table “Fetal Skeletal Development” in this section

-         Control and treated groups compared favorably

-         No major skeletal anomalies were noted       

Table: Fetal Skeletal Development

Group [Dose Level/Day]

Fetuses Examined

Findings

Incidence

% of Total Examined

Control

120 (19)

*Incompletely ossified sternum section(s)

*Non-ossified sternum section(s)

*Supernumerary ribs

Angulated ribs

113 (19)

49 (14)

5 (2)

1

94.2

40.8

4.2

0.8

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

120 (19)

1

119 (19)

100.0

0.8

99.2

T-I

[15 mg/kg]

104 (18)

*Incompletely ossified sternum section(s)

*Non-ossified sternum section(s)

103 (18)

36 (15)

99.0

34.6

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

104 (18)

 

104 (18)

100.0

 

100.0

T-II

[50 mg/kg]

130** (21)

*Incompletely ossified sternum section(s)

*Non-ossified sternum section(s)

*Supernumerary ribs

Angulated and deteriorated ribs

Mal-formed parietal skull bones

Mal-formed interparietal skull bones

Mal-formed occipital skull bones

129 (21)

53 (18)

2 (2)

1

1

1

1

99.2

40.8

1.5

0.8

0.8

0.8

0.8

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

130 (21)

1

129 (21)

100.0

0.8

99.2

T-III

[150 mg/kg]

130 (20)

*Incompletely ossified sternum section(s)

*Non-ossified sternum section(s)

* Incompletely ossified frontal+parietal skull bones

*Supernumerary ribs

Angulated ribs

127 (20)

49 (17)

1

7 (4)

3 (1)

97.7

37.7

0.8

5.4

2.3

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

130 (20)

3 (1)

127 (20)

100.0

2.3

97.7

Note: Numbers in parentheses indicate the number of litters.
* Considered to be an incidental finding
** One fetus inadvertently destroyed during process.

INTERNAL DEVELOPMENT

-         Results of internal development are summarized in table “Fetal Internal Development” in this section

-          Findings in control and treated animals were similar, the incidence of findings within the normally expected range

-         Changes in the size of the atrium reflect naturally occurring variations commonly observed in the strain of rat employed and are considered to be incidental findings and are not considered to be treatment-related

Table: Fetal Internal Development

Group [Dose Level/Day]

Fetuses Examined

Findings

Incidence

% of Total Examined

Control

56 (19)

*Small atria

Renal caudal ectopia

Unilateral increased renal pelvic caviation

27 (15)

1

1

48.2

1.8

1.8

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

29 (15)

2 (2)

54 (19)

51.8

3.6

96.4

T-I

[15 mg/kg]

54 (18)

*Small atria

Unilateral increased renal pelvic caviation

16 (8)

1

29.6

1.9

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

16 (8)

1

53 (17)

29.6

1.9

98.1

T-II

[50 mg/kg]

70 (21)

*Small atria

27 (14)

38.6

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

27 (14)

  

70 (21)

38.6

100.0

T-III

[150 mg/kg]

69 (20)

*Small atria

Unilateral increased renal pelvic caviation

Bilateral increased renal pelvic cavitation

Slight edema

21 (4)

1

1

1

30.4

1.4

1.4

1.4

Total number of fetuses with findings

Total numbers of abnormal fetuses

Total number of fetuses with no abnormal findings

24 (15)

3 (3)

66 (19)

34.8

4.3

95.7

Note: Numbers in parentheses indicate the number of litters.
* Considered to be an incidental finding

Applicant's summary and conclusion

Conclusions:
There were no deaths or unusual behavioral reactions noted among the test or control animals. The results of the gestation day 20 sacrifice revealed no reproductive effect which could be correlated with the exposure to the tested material. There were no deaths or unusual behavioral reactions noted among the test or control animals. The results of the gestation day 20 sacrifice revealed no reproductive effect which could be correlated with the exposure to the tested material.
Executive summary:

A teratogenic study was conducted employing albino rats orally treated with 0, 15, 50 or 150 mg phenothiazine per kg body weight per day during gestation days 6 through 15 inclusive. The following results were obtained during the investigation.
Body weight and body weight gains of the control, 15 and 50 mg/kg/day group dams were similar throughout the investigation. The gestation day 15 body weights and the calculated body weight gains during the exposure period (gestation day 6 through 15) obtained for dams given 150mg phenothiazine per kg per day were significantly less than those of the concurrent control dams. The gestation day 12 body weights of these females were also slightly less than the control females, though these data, as well as all other body weights obtained among these dams were free of statistical significance. There were no deaths or unusual behavioral reactions noted among the test or control animals. The results of the gestation day 20 sacrifice revealed no reproductive effect which could be correlated with the exposure to the tested material.

The body weights of fetuses obtained from dams given phenothiazine were essentially the same as those of the concurrent control fetuses. The examination of fetuses for external developmental anomalies revealed no major structural abnormalities. The evaluations of fetal skeletal and internal development were similar for test and control group fetuses, the incidence of occurrences of findings being within the normally expected range experienced at the respective testing laboratory.