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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:
1992-10-09 to 1992-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethylbis[2-[(1-oxooctadecyl)oxy]ethyl]ammonium chloride
EC Number:
267-382-0
EC Name:
Dimethylbis[2-[(1-oxooctadecyl)oxy]ethyl]ammonium chloride
Cas Number:
67846-68-8
Molecular formula:
C42H84NO4.Cl
IUPAC Name:
N,N-dimethyl-2-(stearoyloxy)-N-[2-(stearoyloxy)ethyl]ethanaminium chloride
Specific details on test material used for the study:
- Name of test material (as cited in study report): E 4429.03
- Physical state: liquid
- Analytical purity: 10% aqueous dispersion Diethyl ester dimethyl ammonium chloride at pH2.5-2.6
- Purity test date: 1992-16-06
- Lot/batch No.: E-4429.03
- Expiration Date of the lot/batch: 1993-02-04
- Other: Stable at pH 2.5-2.6. Store at room temperature, in the dark.

Test animals

Species:
rat
Strain:
other: Rat WIST HanIbm: WIST (SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: No data. (Age at pairing: 12 weeks, minimum).
- Weight at study initiation: No data. (189-228 g at day 0 post coitum).
- Fasting period before study: N/A.
- Housing: individually in Makrolon cages (type-3) with wire mesh tops and standarized granulated softwood bedding (Lignocel, Schill AG, CH 4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum): pelleted standard Kilba 343 rat/mouse maintenance diet ("Kilba", Klingentalmuehle AG, CH 4303 Kaiseraugust/Switzerland) ad libitum. (Batch no. 65-92).
- Water (e.g. ad libitum): tap water in bottles ad libitum.
- Acclimation period: 10 days (minimum).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 (air-conditioned).
- Humidity (%): 40-70.
- Air changes (per hr): 10-15.
- Photoperiod (hrs dark / hrs light): 12-hrs artificial fluorescent light/12 hours dark.

IN-LIFE DATES: From: 1992-10-09 To: 1992-11-21

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: NOAEL emBe-distelled water, previously adjusted with hydrochloric acid to pH 2.5-2.6
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The mixtures of the test substance and vehicle were prepared daily before administration. The test substance was weighed into a glass beaker on a tared precision balance (Mettler PE 360) and the vehicle added (w/v). The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer. All animals received a dose volume of 10 mL/kg bw with a daily adjustment of the individual volume to the actual body weight.

VEHICLE: Bi-distilled water (previously adjusted with hydrochloric acid to pH 2.5-2.6).
- Amount of vehicle (if gavage): 10 mL/kg.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance/vehicle dilutions were taken on two occasions during the dosing period of the study and sent to the Sponsor for analysis (concentration, homogeneity and stability over 2-hrs).
Details on mating procedure:
- Impregnation procedure: cohabitation.
- If co-housed:
- M/F ratio per cage: 1:1.
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data.
- Verification of same strain and source of both sexes: no data. (The male rats used for mating were in the possession of the testing laboratory. The fertility of these males was proved and was continuously controlled).
- Proof of pregnancy: sperm in vaginal smear/vaginal plug referred to as day 0 post coitum.
- Any other deviations from standard protocol: After mating, female rats were removed and allocated to the test groups.
Duration of treatment / exposure:
Day 6 through 15 post coitum.
Frequency of treatment:
Once daily in the morning.
Duration of test:
Females were sacrificed on day 21 post coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control

Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25/group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of the dose range-finding sudy (RCC Project 326158--see cross reference section).
- Rationale for animal assignment (if not random): Mated rats were assigned to the different groups using a computer-generated random algorithm.
- Rationale for method of administration: International guidelines recognize the efficacy of oral administration.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: The animals were checked at least twice daily for any mortalities. The animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recordered daily, from day 0 until day 21 post coitum.
- Body weight gains from days 0-6 p.c., 6-9 p.c., 9-12 p.c., 12-16 p.c., 6-16 p.c., 16-21 p.c., and 6-21 p.c. were calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Food consumption was recorded for the following periods: days 0-6, 6-9, 9-12, 12-16, and 16-21 post coitum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food consumed per period/days per period: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes. Any female sacrificed or found dead during the study was subjected to gross macroscopic examination of all internal organs.
- Sacrifice on day # 21 post coitum by CO2 asphyxiation. Fetuses were removed by Caesarean section.
- Organs examined: Emphasis on the uterus and its contents, position of the fetuses in the uterus, and number of corpora lutea.
- Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes.
- Gravid uterus weight: Yes. (The uteri (and contents) of all females with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain).
- Number of corpora lutea: Yes.
- Number of implantations: Yes. If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible haemorrhagic areas of implantation sites.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.
- Other: position of the fetuses was recordered.
Fetal examinations:
The fetuses were removed from the uterus, sexed, weighed individually, examnined for gross external abnormalities and allocated to one of the following procedures: Wilson's slicing technique or skeletal examination.

- External examinations: Yes: all per litter.
- Soft tissue examinations: Yes: half per litter. Wilson's slicing technique for examination of the viscera and brain. One half of the live fetuses from each litter were fixed in a mixture of ethyl alcohol, formol and acetic acid. After examination, the sections were preserved in a solution of ethyl alcohol and glycerine (one fetus/container).
- Skeletal examinations: Yes: half per litter. Fetuses were placed in a solution of potassium hydroxide for clearing and stained with alizarin red S (modified technique). The skeletons were examined and all abnormalities were recordered. The specimens were preserved individually in plastic bags.
- Head examinations: No data.
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data: means and standard deviations, univariate one-way analysis of variance, Dunnett-test, Steel-test, Fisher's Exact test for 2x2 tables. Individual values, means, standard deviations and t-statistics were rounded off before printing.
Indices:
N/A.
Historical control data:
N/A.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Description (incidence and severity):
In group 4 (1000 mg/kg bw/day), one female had bleeding from the vagina on days 15-16 post coitum. At scheduled Caesarean section, this female had one empty implantation site, only. No abnormal findings were noted in the dams of groups 1 (0 mg/kg bw/day), 2 (50 mg/kg bw/day), or 3 (250 mg/kg bw/day) with the exception of the dam in group 2 (50 mg/kg bw/day) mentioned above.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test substance related deaths were noted. One female in group 2 (50 mg/kg bw/day), was found dead in the evening of day 10 post coitum. At first daily inspection in the morning, this female had ruffled fur, ataxia, tremor and bleeding from the vagina. This single finding in the low dose group was considered to be incidental.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The differences in mean body weights between the control group and any dose group did not reveal any test substance related effect. In all groups the body weight gain and the corrected body weight gain (corrected for uterus weight) were similar. The dams of group 4 (1000 mg/kg bw/day), had statistically significantly increased mean body weights on day 5, between days 7-13 and on day 15 post coitum. These findings were considered to be a consequence of the slightly increased initial mean body weight: 213 g compared to 207 g in the control group, on day 0 post coitum.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Up to and including the highest dose level of 1000 mg/kg bw/day, food consumption of the dams was similar in all groups. No test substance related effects were noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related abnormal findings were noted at terminal necropsy of the dams in any group. One female in group 2 (50 mg/kg bw/day) which was found dead on day 10 post coitum had reddish lungs with dark red foci, reduced right kidney and enlarged left kidney with nodules and calcus in pelvis (both kidneys with pelvic dilation) and in the urinary badder dark red discoloration of the mucosa and urine with hemorrhagic contents were noted. No abnormal findings were noted in the dams of groups 1 (0 mg/kg bw/day), 3 (250 mg/kg bw/day), or 4 (1000 mg/kg bw/day).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Values for post-implantation loss were: 4.8 and 8.7 in groups 1 (0 mg/kg bw/day) and 4 (1000 mg/kg bw/day), respectively. The latter value attained statistical significance. Although the post-implantation losses were within the normal range of the historical control data of this strain of animals, two additional females (which were not included in the statistical analysis) had total post-implantation losses (i.e. implantation sites, only). Therefore, the increased post-implantation loss in group 4 (1000 mg/kg bw/day) was considered to be a slight effect of treatment with the test substance.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
In groups 2 (50 mg/kg bw/day) and 3 (250 mg/kg bw/day), none of the reproduction parameters as assessed by the mean number per dam of corpora lutea and implantation sites, pre- or post-implantation losses, and mean number of fetuses per dam were adversely affected by treatment with the test substance.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal body weights were similar in all groups. No test substance related effects were evident.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
In all groups, the sex ratios of the fetuses were not affected by the treatment with the test substance.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related abnormal findings were noted. Neither the type nor the incidences of the abnormal findings listed below indicated test substance related effects: In each group 1 (0 mg/kg bw/day) and 3 (250 mg/kg bw/day), one fetus with reduced mouth and shortened and tapering lower jaw was noted. In group 2 (50 mg/kg bw/day), one runt (fetal weight < 2.5 g) was noted. In group 4 (1000 mg/kg bw/day), one fetus with several abnormal findings--generally oedematous, cranioschisis with excencephaly, protusion of the tongue, eventration and both eyes partially missing--was noted.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidences of abnormal findings at skeletal examination were as following: 2/145 (=1%) fetuses in 2/25 litters in group 1 (0 mg/kg bw/day); 10/141 (=7%) fetuses in 6/24 litters in group 2 (50 mg/kg bw/day); 4/136 (=3%) fetuses in 4/25 litters in group 3 (250 mg/kg bw/day) and 4/116 (=3%) fetuses in 4/21 litters in group 4 (1000 mg/kg bw/day).
Mainly: wavy or fused ribs, abnormally or incompletely ossified sternebrae, dumbbell shaped or hemicentric thoracic vertebral body and in two fetuses in group 2 (50 mg/kg bw/day) fused os interparietale and occipitale were noted. Neither the type nor the incidences of these abnormal findings indicated test substance related effects. In all groups, the stage of the skeletal development of the fetuses was similar. The few statistically significant differences (on individual basis, only) between the control group and the dose group 4 (1000 mg/kg bw/day) in the ossification grade of the phalangeal nuclei did not indicate any test substance related effect. All values evaluated in this study were in the normal range of the historical control data of this strain of animals.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related abnormal findings were noted at visceral examination of the fetuses. In group 3 (250 mg/kg bw/day), in the fetus which had reduced mouth, shortened and tapering lower jaw at external examination a small palatoschisis was additionally observed at visceral examination. No abnormal findings were noted in groups 1 (0 mg/kg bw/day), 2 (50 mg/kg bw/day), or 4 (1000 mg/kg bw/day).

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: embryotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
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

N/A

Applicant's summary and conclusion