Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: (guideline, GLP study)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): BIOBANTM CS-1246 Antimicrobial
- Physical state: Clear, colorless liquid
- Analytical purity: Assumed to be 100% pure
- Lot/batch No.: 14400, 14401, 14402, 14403

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, INC. (Portage, MI, USA)
- Age at study initiation:81 days of age
- Weight at study initiation: Approximately 217-273 grams on gestation day 0
- Housing: All animals were housed indiviually housed in clean, wire-mesh cages suspended above cage board.
- Diet (ad libitum): Animals were supplied by diet of Purina certified Rodent Chow# 5002 in block form.
- Water (ad libitum): Tap water was provided to animals.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 69°F-75°F
- Humidity (%): 54-82%
- Air changes (per hr): 10-15
- Photoperiod: (12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
other: Deionized water
Details on exposure:
Preparation: The approprate amount of test article, AMINE CS-1246, for each group was weighed into 500 ml volumetric flasks. The flask was brought to volume with deionized water. The solution was inverted and shaken untill the test material was dissolved. The solution was transferred to a storage container and sufficient amount of vehicle was added to attain the appropriate concentration. Solutions for all dose groups were prepared weekly.
Administration: The test mixtures were administered orally by gavage, via a 16-gauge stainless stell gavage canula, once daily for 10 consecutive days initiating on gestation day 6 and continuing up to and including day 15 gestation. A dosage volume of 10 ml/kg was used for all dose levels.The control animals received 10 ml/kg deionized water on the same regimen. Indiviual dosages were based on the most recent body weights to provide correct mg/kg/dose.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See attachment-1 for details of dosing solution analysis.
Concentration inj vehicle: 0, 5, 25, 65 mg/mL
Details on mating procedure:
Length of mating period not specified. Females were placed in the home cages of the males until positive indication of mating was observed (presence of copulatory plug or a vaginal smear positive for sperm).
Duration of treatment / exposure:
Days 6 through 15 of gestation
Frequency of treatment:
Once daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
650 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25 animals/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of preliminary range finding study (WIL-129006)
- Rationale for animal assignment: random)

Examinations

Maternal examinations:
Body weights were recorded on gestation days 0, 6, 9, 12, 16, and 20. Animals were observed twice daily from days 0-20 of gestation. They were observed for signs of toxicity at the time of dosing and approximately one hour following dosing.No indications that food consumption was recorded.
Ovaries and uterine content:
The uterus was opened and the total number and location of all fetuses, early and late resorptions, and total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented. Uteri with no macroscopic evidence of nidation were excised, opened, and placed in ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
- External examinations: Yes: Fetuses were weighed, sexed, and tagged for identification.
- Soft tissue examinations: Yes: on approximately half of the fetuses
- Skeletal examinations: Yes: on approximately half of the fetuses
A detailed external examination of each fetus was conducted on the eyes, palate, and external orifices. Crown-rump length was recorded for late resorptions. Approximately half of the fetuses underwent a soft tissue examination, and the other half a skeletal examination. External, visceral, and skeletal findings were recorded as developmental variations or malformations.
Statistics:
All analyses were conducted using two-tailed tests for minimum significance levels of 5% comparing the treatment group to the control group. All group means were presented with standard deviations. Fetal sex ratios were compared by the chi-square test with Yates' correction factor. The numbers of litters with malformations and variations were compared by Fisher's exact test. The numbers of early and late resorptions, dead fetuses, and postimplantation losses were compared by the Mann-Whitney U-test. Mean numbers of corpora lutea, total implantations, viable fetuses, mean fetal body weights, maternal body weights, maternal body weights at each interval, maternal body weight gains, and food consumption were analyzed by a one-way ANOVA and Dunnett's test.
Indices:
None
Historical control data:
None

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Survival of the dams was 100% in all dose groups. The incidence of clinical findings did not suggest a relationship to test material administration. Mean maternal body weights were depressed in animals dosed with 650 mg/kg/day. No adverse effect on maternal body weights was seen at dose levels of 50 and 250 mg/kg/day. There were no treatment-related necropsy findings noted at any dose level.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 other: mg/kg bw
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Eighteen fetuses (in four litters) in the 650 mg/kg/day group had malformations. The majority of the malformations were abdominal wall defects and/or cleft palate, and were clustered in 2 litters. Two of the three late resorptions in an additional litter had similar malformations. Mean fetal weight was slightly decreased in the 650 mg/kg/day group when compared to controls (although within the historical control range). An increase in the percentage of fetuses with delayed ossification of sternebrae 5 and 6 was observed in both the high and the low dose group as compared to controls. These finding were within historical control values and there was no dose-reponse relationship. No other signs of embryotoxicity or fetotoxicity were apparent at any dose level.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
250 other: mg/kg bw
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Survival of the dams was 100% in all dose groups.  The incidence of clinical findings did not suggest a relationship to test material administration.  Mean maternal body weights were severely depressed in animals dosed with 650 mg/kg/day.  No adverse effect on maternal body weights was seen at dose levels of 50 and 250 mg/kg/day.  There were no treatment-related necropsy findings noted at any dose level.

Eighteen fetuses (in four litters) in the 650 mg/kg/day group had malformations.
  The majority of the malformations were abdominal wall defects and/or cleft palate, and were clustered in 2 litters.  Two of the three late resorptions in an additional litter had similar malformations.  Mean fetal weight was slightly decreased in the 650 mg/kg/day group when compared to controls (although within the historical control range); the decrease was due mainly to the two litters with most of the malformed fetuses.  An increase in the percentage of fetuses with delayed ossification of sternebrae 5 and 6 was considered biologically-relevant at the high dose.  No fetal anomalies were noted in the 50 and 250 mg/kg/day groups.  The incidence of fetal and developmental variations in the 50 and 250 mg/kg/day groups was comparable to those in the concurrent control group and in the historical control data of the testing facility.  No other signs of embryotoxicity or fetotoxicity were apparent at any dose level.

The NOAEL was reported to be 250 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
In conclusion, a dose level of 650 mg/kg/day caused maternal toxicity and developmental toxicity. However, this developmental toxicity was characterized by marked interlitter differences in suseptibility.THE NOAEL in this study was 250 mg/kg/day.
Executive summary:

Virgin female Crl:CD BR rats were dosed by oral gavage an aqueous solution of the test material daily from gestation day 6-15. Animals were observed twice daily from days 0-20 of gestation. They were observed for signs of toxicity at the time of dosing and approximately one hour following dosing. Body weights were recorded on gestation days 0, 6, 9, 12, 16, and 20.

 

Each surviving female was sacrificed on gestation day 20 and examined for gross abnormalities. The number of corpora lutea on each ovary was counted. The uterus was opened and the total number and location of all fetuses, early and late resorptions, and total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented. Uteri with no macroscopic evidence of nidation were excised, opened, and placed in ammonium sulfide solution for detection of early implantation loss.

 

Each fetus was weighed individually, sexed, and tagged for identification. A detailed external examination of each fetus was conducted on the eyes, palate, and external orifices. Crown-rump length was recorded for late resorptions. Approximately half of the fetuses underwent a soft tissue examination, and the other half a skeletal examination. External, visceral, and skeletal findings were recorded as developmental variations or malformations. Survival of the dams was 100% in all dose groups. Mean maternal body weights were depressed in animals dosed with 650 mg/kg/day; no adverse effect on maternal body weights was seen at dose levels of 50 and 250 mg/kg/day. There were no treatment-related necropsy findings noted at any dose level.

 

Eighteen fetuses (in four litters) in the 650 mg/kg/day group had malformations. The majority of the malformations were abdominal wall defects and/or cleft palate, and were clustered in 2 litters. Two of the three late resorptions in an additional litter had similar malformations. Mean fetal weight was slightly decreased in the 650 mg/kg/day group when compared to controls (although within the historical control range); the decrease was due mainly to the two litters with most of the malformed fetuses. An increase in the percentage of fetuses with delayed ossification of sternebrae 5 and 6 was considered biologically-relevant at the high dose. No fetal anomalies were noted in the 50 and 250 mg/kg/day groups. The incidence of fetal and developmental variations in the 50 and 250 mg/kg/day groups was comparable to those in the concurrent control group and in the historical control data of the testing facility. No other signs of embryotoxicity or fetotoxicity were apparent at any dose level. The NOEL for maternal toxic effects was reported to be 250 mg/kg/day. Embryotoxicity, characterized bydelayed ossification of sternebrae 5 and 6was seen in the high dose (650 mg/kg/day) group. The NOEL for embryotoxic effects is therefore 250 mg/kg/day with a LOAEL of 650 mg/kg/day (the highest dose tested).The other effects seen in the high dose group, i.e. cleft palate and abdominal wall defects, are not considered to be indicative of selective developmental toxicity but rather a cluster effect.