N-(4-chlorobenzhydryl)piperazine

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Production of Fetal Rat Malformations by - Norchlorcyclizine and Chlorcyclizine after Intrauterine application

Author:

A. L. WILK

Year:

1969

Bibliographic source:

Teratology Vol 2, Is 1, PP 55-65, feb 1969

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

To evaluate the teratologic potential of Norchlorcyclizine, in female Sprague-Dawley pregnant rats after Intrauterine application.

GLP compliance:

not specified

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-chlorobenzhydryl)piperazine

EC Number:

206-137-4

EC Name:

N-(4-chlorobenzhydryl)piperazine

Cas Number:

303-26-4

Molecular formula:

C17H19ClN2

IUPAC Name:

N-(4-chlorobenzhydryl)piperazine

Test material form:

solid

Details on test material:

- Name of the test material: 1-[(4-chlorophenyl)(phenyl)methyl]piperazine
- Common Name: Norchlorcyclizine
- IUPAC name: 1-[(4-chlorophenyl)(phenyl)methyl]piperazine
- Molecular weight: 286.804 g/mol
- Molecular formula: C17H19ClN2
- Substance type: Organic
- SMILES Notation: c1([C@@H](c2ccccc2)N2CCNCC2)ccc(Cl)cc1
- InChI: 1S/C17H19ClN2/c18-16-8-6-15(7-9-16)17(14-4-2-1-3-5-14)20-12-10-19-11-13-20/h1-9,17,19H,10-13H2
- Physical State: Solid (white to pale yellow)

Specific details on test material used for the study:

- Name of the test material: 1-[(4-chlorophenyl)(phenyl)methyl]piperazine
- IUPAC name: 1-[(4-chlorophenyl)(phenyl)methyl]piperazine
- Molecular weight: 286.804 g/mol
- Molecular formula: C17H19ClN2
- Substance type: Organic
- Smiles: c1([C@@H](c2ccccc2)N2CCNCC2)ccc(Cl)cc1
- Inchi: 1S/C17H19ClN2/c18-16-8-6-15(7-9-16)17(14-4-2-1-3-5-14)20-12-10-19-11-13-20/h1-9,17,19H,10-13H2

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: No data available.
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: Females:200 grams
- Fasting period before study: - No data available.
- Housing: - No data available.
- Diet (e.g. ad libitum): No data available.

- Water (e.g. ad libitum): No data available.

- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
No data available.

Administration / exposure

Route of administration:

other: intrauterine application

Vehicle:

other: HCl

Details on exposure:

Details on exposure
Millipore filters (HA 0.45µ) were cut into strips 4 X 0.2 cm and then dipped into 0.01 ml of aqueous solutions containing 50 µg/ filter squares concentrations of norchlorcyclizine HCl. The filter strip completely absorbed this volume and was dried under a stream of air. The strip was placed on a mold of the same dimensions, which was marked into 20 squares each measuring 0.2 X 0.2 cm and the filter strip was cut into squares. and implanted over the fetal side or placenta on day 13 of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to test for variation in the concentration of the compounds in the individual filter squares each square was placed in 1 ml of IN HC1 and the acid extracted compound read at 230 m µ in a spectrophotometer. An expected concentration of 50 or 90 µ g had a maximum variation of ± 10 µg. Filter squares similarly prepared with stoichiometric amounts of HCl were used as controls

Duration of treatment / exposure:

on days 11-16 of gestation days

Frequency of treatment:

on days 11-16 of gestation days

Duration of test:

on days 11-20 of gestation days

Doses / concentrations

Remarks:

50µg(0.05mg/filter ),90µg(0.09mg/filter )

Control animals:

yes, concurrent vehicle

Results and discussion

Effect levels

Dose descriptor:

LOAEL

Effect level:

0.05 other: mg/filter

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

gross pathology

other: malformation like Cleft palate and limb abnormalities

Remarks on result:

other: Developmental effects observed

Observed effects

Gross malformations were noted by observation and the fetuses were weighed and then placed in 80% ethanol. After alcohol fixation the fetuses were cleared of most soft tissue with 1% KOH and stained with 0.5% alizarin red S. The stained specimens were examined for skeletal malformations. The effect of the surgical procedure on the maternal weight gain from days 13-20 and on the fetal weight on day 20. Postoperational survival of pregnant rats was excellent. Pregnant operated rats gained approximately one-third as much weight as did unoperated pregnant animals and operated fetuses had a mean weight of about 3.3g on day 20while that of unoperated fetuses was 3.9 g. Fetal survival after surgery and insertion of HCl-containing filters was over 60% on every day except day 14 when it was approximately 42%. Cleft palate was produced at rates of 29 and 35% after norchlorcyclizine (50ug/filter) was inserted centrally on the fetal side on day 13 or 14 respectively. When the concentration of norchlorcyclizine was increased to 90 or 100 ug/filter and implanted centrally or over the head area of fetuses on day 15, less than 3% cleft palate was observed. Another malformation frequently seen following intrauterine application of norchlorcyclizine, was a limb malformation, which affected either the left forelimb or left hindlimb depending on whether the filter was placed centrally or over the hindlimb area of the fetus. The severity of the malformation ranged from simple fusion of the digits to complete absence of a paw. Left forelimb anomalies occurred at a rate of 24 and 38% after norchlorcyclizine (50 ug/filter) was inserted centrally on day 13 or 14 respectively and at a rate of about 3% after norchlorcyclizine- filter insertion on day 11, 12, 15, or 16A positive relation between filter position over the fetus and cleft palate production was also found .When norchlorcyclizine (50 ug/filter) was inserted over the head area on the fetal side 42% of the fetuses had cleft palates and 13% had hindlimb malformations. Filters with similar concentration of norchlorcyclizine inserted over the left hind limb, however, resulted in 11% cleft palate and 55% left hind limb malformation. Gross observation of whole and cleared fetuses showed that the heads of some fetuses treated with test material filters were misshapen and the bodies more squat than normal. the control filters produced no malformation when implanted on any of the experimental days. Similarly no abnormalities resulted after norchlorcyclizine-filter insertion over the placenta on day 13.Hence

Any other information on results incl. tables

Effectofintrauterine surgical procedureonmaternalandfetal weights

Day and treatment	No. of pregnant rats	Mean maternal wt gain between day 13-20 (g ±S.E)	No of fetuses	Mean wt. Of day-20 fetuses (g ±S.E)
None	8	68 ±5.6	58	3.9 ±0.05
13 HCl control (F.S.)*	25	20± 3.0	95	3.2 ±0.10
13 Nor CC(F.S.)**	30	28 ±4.2	130	3.2 ± 0.10
13 Nor CC(P.S.)*	21	28 ± 5.4	102	3.4 ±0.05

*Filters placed over the fetal side (F.S.); over the placenta(P.S.).

**Nor CC=norchlorcyclizine HCI, approx. 50 µg/filter.

Malformations produced aftez norchlmcyclizineHCZor HCl filter insertion on the fetal side over the headorthe left hindlimb

Day and treatment	Position of filter	No. of site treated	Survival	Cleft palate	% limb malformed *
					Left Hind limb	Right hind limb	Left fore limb
14 HCl	Head	60	32	0	0	0	0
14 Nor CC (50µg/filter )	Head	85	36	42	13	3	3
14 Nor CC (50µg/filter )	Hind limb	128	46	11	55	2	4
15 HCl	Head	35	90	0	0	0	0
15 Nor CC (50µg/filter	Head	78	83	3	0	0	0

*Right forelimb never malformed

Malformations produced following central insertion of compound-impregnated millipore filters

Day and treatment	No. of site treated	Survival (%)	Cleft palate (%)	Left forelimb* malformed (%)
11 HCl	31 F.S.**	80	0	0
11 Nor CC	48 F.S.	81	0	0
12 HCl	45 F.S.	93	0	0
12 Nor CC	135 F.S.	71	0	0
13 HCl	128 F.S.	74	0	0
13 Nor CC	200 F.S.	65	29	24
13 Nor CC(90µg/filter )	63 F.S.	6	75	75
14HCl	85 F.S.	42	0	0
14 Nor CC	72 F.S.	46	35	38
15 HCl	65 F.S.	66	0	0
15 Nor CC(90µg/filter )	79 F.S.	84	0	2
16 HCl	58 F.S.	74	0
16 Nor CC(90µg/filter )	89 F.S.	74	0	3

*The left forelimb was the only limb malformed following central insertion.

**Filters placed over the fetal side (F.S.); over the placenta(P.S.).

NorCC, norchlorcyclizine HC1. approx. 50µg/filter, unless noted otherwise

 

Applicant's summary and conclusion

Conclusions:

LOAEL was considered to be 0.05mg/kg/day on the bases of teratognic potential . When female Sprague-Dawley rats were exposed with Norchlorcyclizine (303-26-4) through the 11-16 day of gestation after Intrauterine application

Executive summary:

The teratognicity of Norchlorcyclizine (303-26-4)was studied in female Sprague-Dawley rats when they were exposed through the 14-15 day of gestation after Intrauterine applicationof test material.In this technique of intrauterine application a compound, which by passes the metabolism of the adult rat and directly subjects the fetus to microgram concentrations of the compound for a known period of time. Pregnant Sprague-Dawley rats were used on days 11-16 of gestation. Vaginal smears were taken to determine pregnancy with the appearance of sperm designated as day 0 of gestation. Semisterile operational procedure was followed. Animals weighing approximately 200 g were anesthetized with an intraperitoneal injection of1ml of a 10% pentobarbital sodium solution supplemented, when necessary, with light ether inhalation. A midline abdominal incision was made and the double horned uterus was exteriorized.Asmall cut was then made through the uterine wall and a Millipore filter square impregnated with norchlorcyclizine HCl, or HCl was inserted either on the yolk sac over the intact amniotic sac (fetal side) or on the placenta .Once inserted, the filters remained in place until after day 16 when fetal movements seemed to dislodge them. In each pregnant rat, fetuses in one uterine horn were usually subjected to experimental filters and the HCl-containing, control filter was placed over fetuses in the other horn. Prior to day 14 of gestation the position of the embryo cannot be seen through the uterus and the filters were always inserted centrally over the fetus. On days 14 and 15 it was possible to insert the filters centrally, over the hindlimb, or over the head area .All fetuses observed on days 14, 15, and 16 appeared to lie with their left side uppermost and the right side toward the placenta. After these uterine manipulations the uterus was returned to the abdominal cavity, the muscle layer sutured, and the skin clamped with stainless steel clips. The animal was then placed on its back in a warm recovery area and when it recovered from the anesthesia it was returned to the animal room and gestation was allowed tocontinue to day 20.Pregnant rats were weighed and killed on day 20 and the young removed.

 

Gross malformations were noted by observation and the fetuses were weighed and then placed in 80% ethanol. After alcohol fixation the fetuses were cleared of most soft tissue with 1% KOH and stained with 0.5% alizarin redS.The stained specimens were examined for skeletal malformations. The effect of the surgical procedure on the maternal weight gain from days 13-20 and on the fetal weight on day 20. Postoperational survival of pregnant rats was excellent. Pregnant operated rats gained approximately one-third as much weightasdid unoperated pregnant animals and operated fetuses had a mean weight of about 3.3g on day 20while that of unoperated fetuses was 3.9 g. Fetal survival after surgery and insertion of HCl-containing filters was over 60% on every day except day 14 when it was approximately 42%. Cleft palate was produced at rates of 29 and 35% after norchlorcyclizine (50ug/filter) was inserted centrally on the fetal side on day 13 or 14 respectively. When the concentration of norchlorcyclizine was increased to 90 or 100 ug/filter and implanted centrally or over the head area of fetuses on day 15, less than 3% cleft palate was observed. Another malformation frequently seen following intrauterine application of norchlorcyclizine, was a limb malformation, which affected either the left forelimb or left hindlimb depending on whether the filter was placed centrally or over the hindlimb area of the fetus. The severity of the malformation ranged from simple fusion of the digits to complete absence of a paw. Left forelimb anomalies occurred at a rate of 24 and 38% after norchlorcyclizine (50 ug/filter) was inserted centrally on day 13 or 14 respectively and at a rate of about 3% after norchlorcyclizine- filter insertion on day 11, 12, 15, or 16Apositive relation between filter position over the fetus and cleft palate production was also found .When norchlorcyclizine (50 ug/filter) was inserted over the head area on the fetal side 42% of the fetuses had cleft palates and 13% had hindlimb malformations. Filters with similar concentration of norchlorcyclizine inserted over the left hind limb, however, resulted in 11% cleft palate and 55% left hind limb malformation. Gross observation of whole and cleared fetuses showed that the heads of some fetuses treated with test material filters were misshapen and the bodies more squat than normal.The control filters produced no malformation when implanted on any of the experimental days. Similarly no abnormalities resulted after norchlorcyclizine-filter insertion over the placenta on day 13.Hence LOAEL was considered to be 0.05mg/kg/day on the bases of teratognic potntional. WhenfemaleSprague-Dawley rats were exposed withNorchlorcyclizine (303-26-4)through the 13-14 day of gestationafter Intrauterine application