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Toxicological information

Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: in compliance with GLP and method is relevant to well established guidelines as well as complete report is available.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
Limit test:

Test material

Details on test material:
Test substance is micronized from this substance.
The commertial product (>355 um) has practically no micronized particle size (<10 um .
-purity test date:1989-11-03
- Lot/batch No.: 3099997
- Stability under test conditions:
- Storage condition of test material:room temperature

Test animals

New Zealand White
Details on test animals or test system and environmental conditions:
Source of animal: Hazleton Research Products,Inc. (PA)
Housing: allhousing and care will confirm to the standards recommended by the Guide for the Care and Use of laboratory Animals.
1.temp: 68+-5 degF
2.humidity: 55%;-15%
3.fresh air: 10-12 changes per hour
4.light: 12h light/12h dark
5.record: once daily

Administration / exposure

Route of administration:
oral: gavage
CMC (carboxymethyl cellulose)
Details on exposure:

- Justification for use and choice of vehicle (if other than water): Biologically inert edible polymer to give viscousity to water for suspension.
- Concentration in vehicle: 0.9%
- Amount of vehicle (if gavage):2ml/kg bw /day
- Lot/batch no.: Fisher Scientific, Cincinnati, Ohio, Lot # 874544
- Purity:Not applicble
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Experimental System: HPLC
Solutions preparation: ca 0.025 g PMI dissoled in methanol in a 50 mL flask
The 0.0000, 0.1500, 0.7500 and 1.5000 mg/mL suspensions of N-phenylmaleimide used for dosing in this study in rabbits were analized on the first day of dosing, near the middle of the dosing period and near the end of the dosing period.
recovery range for PMI in o.5 % methylcellulose were 92.4-102.2%.
To be found in Appendix B sited on p-61 of the test report.
Details on mating procedure:
- Impregnation procedure: [artificial insemination ]
- Verification of same strain and source of both sexes: [ no (males were obtained from the same source and supplier as females)]
- Proof of pregnancy: [the day of insemination ]was referred to as [day 0] of pregnancy
- Any other deviations from standard protocol:none
-details : Semen was collected from the males and was diluted with 0.9% physiological saline and maintained in a water bath at 35-36deg F during insemination procedure . App. 0.5 ml of the diluted semen was introduced into females vagina. Semen fromone male was used to inseminate an equal number of females in each study group. Immediately following insemination, the females were administered human chorionic gonadotropin, via the marginal ear vein.
Duration of treatment / exposure:
Gestation day 6 through gestation day 18.
Frequency of treatment:
Single daily dose
Duration of test:
Animals were sacrificed on gestation day 29. Test were conducted from 1/10/90 ( physical examination ) to 7/05/90 (Fetal Skeltal Examinaton ). reporting followed thereafter to 1/24/91.
Doses / concentrations
Doses / Concentrations:
0.3, 1.5, 3.0
nominal conc.
mg/kg bw/d
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Prospected overt maternal effect at the highest dose selected.


Maternal examinations:
- Time schedule: daily
- Cage side observations checked in table [No.2] were included.

- Time schedule: daily

- Time schedule for examinations: on day 0, 6, 9, 12, 15, 19, 24 and 29

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- Sacrifice on gestation day #29
- Organs examined:Cranical, thoracitic, abdominal and pelvic aogans were observed as well as each overy was examined for corpora luttea,

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:Uteri with no macroscopic evidence of implants were placed in 10% aqueous ammonium sulfide for detection of early embryolethality.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ] /
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
All analyses were two-tailed with a minimum significunce level of 5%. One way analysisof variance folloed by Dunnett's test was used to analyse maternal and fetal data including body weights, food consumption, number of viable fetuses, implantation site and corpora lutea. Mann-Whitney U test was used to compare post-implantation loss, dead fetuses, and resorption. Fetal sex retios were analysed using chi-square test. Fisher'sExact test was used to analyse the incidence and number of fetal malformations (if exist) and variations utilizing the dam(litter) as the experimenta unit.
Indeces were not shown spesifically (except sex ratio) but incidences were individually compared among groups.
Historical control data:
There were no incidencefound that need to be judged by coppareing with historical data. However, historical control data are provided (Appendix O).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs of Toxicity were observed at the 1.5 and 3.0 mg/kgBW/day levels and included labored breathing, rales few feces, whitish colored mocoid material in the cage/tray and clear nasal discharge. No treatment related clinical signs were observed in the 0.3 mg/kg BW/day group. Statistically significant dose-dependent body weight losses occured at 1.5 and 3.0 mg/kgBW/day levels during the first three days of treatment (gestation day 6-9). These losses resulted in statistically reduced body weight gain at the 1.5 /kgBW/day level and statistically significant body weight loss at 3.0 mg/kgBW/day levelfor the entire treatment period (gestation days 6-19). Folloeing the completion of treatment(gestation days 19-29), body weught gain at these levels was significantly increaseed compared with the control group. Food consumption at the 1.5 and 3.0 mg/kgBW/day levels was statistically reduced for entire treatment period (gestation days 6-19),. Body weight gain and food consumption were similar between control and 0.3mg/kgBW/day level.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
>= 3 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
Effect level:
0.3 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

A dosage level of 0.3 mg/kg/day was considered a no observed-effect level (NOEL) for maternal toxicity.
A dosage level of 3.0 mg/kg/day, the highest dode tested in the study was considered a NOEL for developmental toxicity.
Executive summary:

Oral administration of N-Phenylmaleimide to pregnant rabbits during the period of major organogenesis produced dose-dependent maternal toxicity at dosage levels of 1.5 and 3.0 mg/kg/day.

Therefore, a dosage level of 0.3 mg/kg/day was considered a no observed-effect level (NOEL) for maternal toxicity.

A dosage level of 3.0 mg/kg/day was considered a NOEL for developmental toxicity.