Phenothiazine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- See short description on "Developmental toxicity / teratogenicity"

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

67 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

See section "Developmental toxicity / teratogenicity".

Short description of key information:
- Sub-chronic oral feed study with dogs
- Prior to implementation of GLP and OECD guidelines
- Dose-dependent effects on the haematological system
- Result: No effects on reproductive organs were found up to the highest dose tested, which was 2000ppm (representing an actual substance intake of 67 mg/kg body weight/day (for females) and 69 mg/kg body weight/day (for males))
- See section "Repeated dose toxicity: oral" for further details on study results

Effects on developmental toxicity

Description of key information

- Teratogenicity study with rats
- Prior to implementation of GLP and OECD guidelines
- Foetal skeletal and internal development were similar for test and control animals 
- No effects on development of foetuses were found at doses three times higher the doses resulting in maternal toxicity
- The results of gestation day 20 sacrifices revealed no reproductive effect which could be correlated with the exposure to the tested material

Link to relevant study records

Reference

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.

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

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

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

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

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

Details on maternal toxic effects:

Maternal toxic effects:yes

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

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

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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

Remarks on result:

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

150 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Phenothiazine was tested in two studies for its possible teratogenic potential. Both studies were conducted prior to GLP and OECD guideline implementation, but allow for a robust evaluation of teratogenicity due to administration of phenothiazine. As a result of both studies no effects on development of foetuses were found at doses three times higher the doses already resulting in maternal toxicity, hence the resulting NOAEL is greater than 150 mg/kg body weight/day (study with rats) or 300 mg/kg body weight/day (study with mice). In the latter study numbers of resorption sites were slightly increased after phenothiazine administration when compared to controls. No indications for effects on foetal skeletal and internal development due to phenothiazine were found. The results on gestation day 20 sacrifices revealed no reproductive effect which could be correlated with the exposure to the tested material. Effects on reproductive organs were additionally assessed in the sub-chronic oral study with dogs. Here, no effects on reproductive organs were found up to the highest dose tested, which was 2000 ppm (representing an actual substance intake of 67 mg/kg body weight/day (for females) and 69 mg/kg body weight/day (for males)).All these studies were conducted via oral route of exposure. This route, however, is of low relevance for the present registration according to Commission Regulation (EC) No 1907/2006 (REACH). The same is true for inhalation, since phenothiazine is solely sold or imported as prills. As for dermal route of exposure, only indirect information on adsorption through the skin is available. In an acute dermal toxicity study (newly conducted as a limit test for the present registration) neither mortality nor any other sign of systemic toxicity was found, hence making it unlikely that dermal adsorption is comparable to gastrointestinal adsorption. Therefore, it is reasonable to assume that there is an additional margin of safety between the NOAELs identified in both studies, which cover only oral route of exposure.

The present studies lack some information about the test item used, e.g. the purity of the test item and consequently the impurities are not given. However, starting material for a synthesis of phenothiazine is diphenylamine, which is the most relevant impurity. Following the summary and conclusion drawn from the European Union Risk Assessment Report on diphenylamine (final version approved on 2008-05-29), teratogenic potential of diphenylamine in tests with rat and rabbit is not indicated, even not at maternally toxic doses.

The present studies do not exhibit any teratogenic effect. Theoretically, this might be caused by dilution of the test item phenothiazine with its precursor substance diphenylamine as main impurity. However, this is regarded as unlikely, due to the nature of the test material as precursor of medicinal substances (at the time the study was conducted), where usually high purities are demanded. Potential dilution should therefore not be an issue. In conclusion the results of the present study are regarded as reliable, not indicating a teratogenic potential of the test substance phenothiazine.

As a result of the literature review two further publications were found on possible effects on developmental/reproductive toxicity of phenothiazine. One of these publications (Telford et al., 1967) reported about effects on resorption in rats. However, there is crucial information missing in the publication, making it not assign a reliability score. The same is true for the publication of Bournias-Vardiabasis et al. (1983) where phenothiazine was one of 100 chemicals to evaluate the Drosophila embryonic cell culture test as a screening tool for teratogenicity.

In a MultiCASE computational assessment of the potential teratogenicity of phenothiazine it was concluded that derived from the ICSAS rules phenothiazine is not expected to be teratogenic in man.

Toxicity to reproduction: other studies

Additional information

See section "Developmental toxicity / teratogenicity".

Justification for classification or non-classification

Based on the present studies available classification of phenothiazine as toxic to reproduction according to the provisions laid down in Commission Regulation (EC) No 1272/2008 is not justified.