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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:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Didecyldimethylammonium chloride
EC Number:
230-525-2
EC Name:
Didecyldimethylammonium chloride
Cas Number:
7173-51-5
Molecular formula:
C22H48N Cl
IUPAC Name:
N,N-Didecyl-N,N-dimethylammonium chloride
Test material form:
liquid

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Elevage Scientifique des Dombes, Châtillon-sur-Chalaronne, France)
- Age at study initiation: 18-20 wk
- Weight at study initiation: mean body weight of 3494 g (range: 3105 g to 3940 g).
- Housing: individually housed in stainless steel cages, in a barriered rabbit unit, under specific pathogen free (SPF) standard laboratory

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3°C
- Humidity: 50 ± 20%
- Air changes: 8 to 10 cycles/h of filtered, non-recycled air
- Photoperiod: 8h dark/16h light (5:00 - 21:00)


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Concentration in vehicle: 0, 1.33, 4, 10.66 mg/mL. The test item dosage forms were prepared daily and stored at room temperature prior to use.
Total volume applied: 3 mL/kg/d

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of DDAC in dosage form: The concentration of samples taken from each control and test item dosage form prepared for use on the first day of treatment and on the last day of treatment was determined. Values were found to be in agreement with nominal concentration ± 10%.
Details on mating procedure:
Animals were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as day 0 post-coitum (p.c.) or day 0 of gestation (GD 0).
Duration of treatment / exposure:
Day 6 to 28 post coitum
Frequency of treatment:
Daily
Duration of test:
Upto 28 d of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 4, 12 and 32 mg DDAC/kg/d
Basis:
nominal conc.
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a preliminary study of effects on embryo-fetal development in rabbits (CIT/Study No. 26153 RSL), in which the test item, DDAC, was administered to pregnant female rabbits, from Day 6 to Day 28 of pregnancy at the dose-levels of 4, 16 or 32 mg DDAC/kg/d.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 2, 4, 5, 6, 9, 12, 15, 18, 22, 26 and 29 post-coitum

FOOD CONSUMPTION: Yes
- Recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-18, 18-22, 22-26 and 26-29 post-coitum.
Any obvious spillage of food was documented.

POST-MORTEM EXAMINATIONS: Yes

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
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
- Examination of fetuses: Carried out only in the first 16 litters (females with at least one live fetus). The other litters will be discarded without further investigations.
Statistics:
Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
Indices:
Assessment of data
Data are expressed as group mean values ± standard deviation (maternal body weight and food consumption, fetal body weight, number of corpora lutea, implantations, fetuses and resorptions) or as proportions (pre-implantation loss, post-implantation loss and fetal findings). Whenever necessary, the experimental unit of comparison was the litter. Data on non-pregnant females were not included in the group mean calculations.

The following calculations were made:
Net body weight (presented as carcass weight): body weight on day 29 minus gravid uterine weight.
Net body weight change: (body weight on day 29 minus body weight on day 6) minus gravid uterine weight.

Pre-implantation loss: Number of corpora lutea - Number of implantation sites x 100 / Number of corpora lutea
Post-implantation loss: Number of implantation sites - Number of live fetuses x 100 / Number of implantation sites
Fetal or litter incidence: Total number of fetuses or litters with a particular finding x 100 / Total number of fetuses or litters examined
Mean proportion of affected fetuses: Sum of proportion of fetuses affected in each litter x 100 / Total number of litters examined
Historical control data:
No data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
When given 12 mg DDAC/kg/day, colored urine and loud breathing was observed in one female from GD 22. These signs were attributed to treatment with the test item. At 32 mg DDAC/kg/day, the number of animals showing clinical signs was greater than in the control group, and most of these clinical signs (colored urine, soft feces, soiled urogenital area) were recorded at the end of the gestation period, along with an absence of feces observed for three animals. Presence of blood in the bedding was noted in two females on GD 16 or 22 and one female was emaciated (E30669, GD 29). No other clinical signs were considered to be treatment-related, absence of feces is commonly recorded at the end of pregnancy and was therefore not considered to be treatment-related at 4 mg DDAC/kg/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
32 mg DDAC/kg/day group
Female E30668 was found dead after treatment on GD 25. Abdominal breathing from GD 24 and coldness to the touch on GD 25 were noted. During GD 18-22, food consumption was reduced and the animal lost 210 g. Dilated stomach with liquid and findings in the lungs (brownish foci, reddish areas and firm consistency) were seen at necropsy. Female E30673 was prematurely killed on GD 22 following signs of poor health status (hypotonia and coldness to the touch on GD 22). Liquid feces were seen from GD 20, soiled urogenital area from GD 21 and blood in bedding on GD 22. This animal lost weight from GD 6 (GD 6-22: -485 g), but the body weight loss was more pronounced from GD 18 (GD 18-22:-235 g) and it did not eat from GD 15. At necropsy, gall bladder was dilated and cecum was distended with gas. Female E30687 was found dead on GD 22, one fetus was found in the bedding on that day and blood in the bedding was noted from GD 21. Liquid feces with soiled urogenital area were seen from GD 20. During GD 15-18, food consumption was reduced and the animal lost 170 g. Numerous findings were detected at necropsy in the stomach (brownish foci, deposit in the mucosa) and in the intestines (cecum distended with gas and liquid contents). The gall bladder was dilated.

12 mg DDAC/kg/day group
Female E30652 aborted on GD 28. Absence of feces were noted from GD 22. During GD 15-26 this animal lost 380 g and ate only 26 g/day. At autopsy, the intestines were dilated (brown liquid content in cecum) and the gall bladder was reduced in size.

4 mg DDAC/kg/day group
Animal E30629 was found dead on GD 10. Gavage of this animal was difficult on GD 9 (traces of blood were in evidence on the gavage tube). On GD 10, the animal presented loud and abdominal breathing and oral administration of the dosage form was not possible. During GD 6-9, this animal lost 135 g and ate only 48 g. The autopsy revealed brownish areas in the lung lobes. Taking into account that this death occurred after gavage difficulties, a direct relationship to DDAC was improbable, and this death was considered incidental. Female E30633 was found dead on GD 26 without ante-mortem clinical signs. Food consumption and body weight change were normal on the days preceeding death. Necropsy of the lungs showed many brownish foci, several reddish areas and a firm consistency of the organ. In view of these last findings, this death could be consecutive to accidental aspiration of the test item into the lungs. Therefore a direct relationship to repeated administration of DDAC could be ruled out.

Control group
Female E30618 aborted on GD 23 (8 placentas in the bedding). Abnormal breathing was recorded from GD 18. Absence of feces and blood in the bedding were observed from GD 21. From GD 15, a marked reduction in food consumption (GD 15-18: 70 g/day, GD 18-22: 8 g/day) and a body weight loss (GD 15-22: -660 g) were recorded. No findings were seen at the autopsy. This abortion was considered to be spontaneous.

In absence of a dose-related increase of death/abortion at 4 and 12 mg DDAC/kg/day and with the abortion of one female in the control group, the mortality at 4 and 12 mg DDAC/kg/day were considered to be most probably incidental and not related to treatment with the test item. At 32 mg DDAC mg/kg/day, death/abortion in 3 females were ascribed to the treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 32 mg/kg/day, a body weight loss was noted from GD 6 to 9 and concerned ten females in comparison with two females of the control group. Therefore, the body weight gains during all intervals were lower than in the control group, showing statistical significance from GD 6 to 12, GD 6 to 15 and GD 6 to 26. At 32 mg/kg/day, the body weight gain during the whole dosing period was statistically lower (-56%, p<0.01) than in the control group. At 12 mg/kg/day, the body weight gain was slightly reduced during many intervals of the dosing period, reaching statistical differences from GD 6 to 15 and GD 6 to 26 and corresponds to -29% from GD 6 to 29 (whole dosing period) when compared to controls. At 4 mg/kg/day, no marked effects were noted in the body weight gains. The net body weight change (reflecting the maternal body weight change independently of the uterus weight) was lower in the treated groups than in the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 32 mg/kg/day, the mean food consumption was lower than the treated group values during the whole dosing period, differences being more marked at the beginning and at the end of the dosing period (GD 6-9, -22%, p<0.001; GD 9-12, -18%, p<0.001; GD 26-29, -37%, not statistically significant when compared to control values). At 4 and 12 mg/kg/day, very slight reductions were noted at different intervals, none of them were statistically significant when compared to control values.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The uterus weight of treated females at the low dose-level of DDAC was similar to that of the control group, while the weight of the gravid uterus of treated females at 12 and 32 mg DDAC/kg/day was slightly lower. These differences were correlated with lower numbers of fetuses.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy findings in the intestines (all treated groups), stomach and gall bladder (high-dose group only) were considered to be related to the treatment with DDAC. No other treatment-related findings were observed. There were no findings at the gross examination of placentae in any females.

Maternal developmental toxicity

Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
When compared with the control value, the number of dead fetuses was higher (and statistically significant when expressed as a total number) at 32 mg DDAC/kg/day. This difference led to a lower number of live fetuses, and a high mean post-implantation loss. All these mean values for this group were outside CIT background data. It is worth noting that the number of autolyzed fetuses was higher than in the control group.
The high number of autolyzed fetuses recorded in group 4 (32 mg DDAC/kg/day) was due to three litters (E30667, E30669 and E30670) with 5, 13 and 6 autolyzed fetuses. Although the number of dead fetuses recorded in the control group was higher than the CIT background data (mean per female= 0.2), all the differences noted in the litter data described above for the group treated at 32 mg DDAC/kg/day was attributed to treatment with the test item. The modification of these parameters in the low dose group was not considered to be treatment-related in the absence of obvious dose-relationship. The number of live fetuses in the intermediate dose-group and the control group were similar. The percentage of male fetuses and the fetal body weights were unaffected by treatment.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
32 mg DDAC/kg/d:
Evident signs of maternal toxicity (3 mortality), abortion, clinical signs at the end of the gestation; reduction in food consumption and body weight loss) were noted. Necropsy findings concerned the intestines, the stomach and the gall bladder.
Clinical signs were observed in 11/19 animals and consisted of colored urine (5/19), soft feces (4/19), absence feces (3/19), blood in bedding (2/19), and one incidence of loud breathing. One animal showed an emaciated appearance at the end of the study. Autopsy of the surviving animals revealed effects on intestines (8/19: distended with gas, dilated, brownish content), stomach (2/19: dilated, thickened wall, brown/whitish area/deposit), gall bladder (1/19: dilated) and lungs (1/19: brownish foci and reddish areas).

12 mg DDAC/kg/d:
One animal aborted on GD28 after period with absence of feces, weight loss, and autopsy showed dilated intestines and gall bladder reduced in size. One animal was not pregnant. Clinical signs in the remaining surviving animals consisted of absence of feces at end of pregnancy (2/10), loud breathing (1/20) and reddish colored urine from GD22 (1/20). Observations at autopsy: brownish foci in lungs (1/20), accentuated lobular pattern liver (2/10), thickened wall uterus (2/10), and brownish content intestines with liquid in cecum (2/10).

4 mg DDAC/kg/d:
Two animals found dead (GD 10 and 26) both displaying brownish areas/foci in lungs indicating possible aspiration of test item. Clinical signs in the remaining pregnant animals comprised of absence of feces observed at GD29 or from GD28 (3/20). Autopsy showed brownish foci in lungs (1/20), effects on intestines (2/20), and a pale liver (1/20).

Control:
One animal aborted on GD23 after a period of reduced food consumption, abnormal breathing, absence of feces, and blood in bedding. No autopsy findings. Of the remaining pregnant animals at term observations included blood in bedding (2/21), cutaneous lesion (2/20) and immobilized hind limb (1/20). Autopsy showed brownish foci in lungs in 3/20. (Net) body weight changes are difficult to interpret due to the big inter-individual variation. There are also large intra-individual differences between periods of the gestation period. Overall, there seems to be a dose related effect, tending to lower body weights at higher dose levels which corresponding to lower food consumption.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 4 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: effects secondary to local effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal body weights were unaffected by treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of male fetuses were unaffected by treatment.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of fetuses showing external malformations was 1, 0, 2 and 0 distributed in 1, 0, 2 and 0 litters at 0, 4, 12 or 32 mg DDAC/kg/day. This distribution was not considered to be treatment-related. Variations were also distributed in different groups including control group and with no significant dose-related increase.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No dose- or treatment-related increases were noted for any skeletal malformations. At 32 mg DDAC/kg/day, the number of litters with fetuses with incomplete ossification of the sixth sternebrae was higher than control (p<0.05 when expressed as litter incidence). Taking into account that this variation is commonly observed in fetuses of this strain of rabbit, and that incidences were within the CIT background data range, it was not considered to be a consequence of treatment with DDAC. No treatment-related changes were observed at cartilage examination in any of the litters examined. Therefore, it could be considered that the general ossification of fetuses was not affected by treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The only malformations detected at visceral examination were considered to be spontaneous in origin as affecting one fetus in the 0 and 12 mg DDAC/kg/day. The presence of whitish colored fluid in the abdomen (variation) of 5 fetuses from the same litter (5/6 fetuses doe E30667) treated at 32 mg DDAC/kg/day was considered to be most probably related to the test-treatment (statistically significant), as this finding was not observed in any of the fetuses from control group, and both the fetal and litter incidence were outside CIT background data.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 12 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: based on decreased number of live foetuses, increased post-implantation loss and a decreased foetal body weight at 32 mg/kg bw/day
Remarks on result:
other: prenatal effects only seen as unspecific consequence of maternal distress

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
32 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
yes

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOAEL for maternal toxicity and embryotoxic/teratogenic effects of the test substance were determined to be 4 and 12 mg a.i./kg bw/day respectively.
Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the test substance, DDAC (40% active in water) in rabbits by oral gavage route, according to OECD guideline 414 and EPA OPPTS 870.3700, in compliance with GLP. The test substance at concentrations of 0, 4, 12 and 32 mg a.i./kg bw/day was administered by gavage, to groups of 20 female New Zealand White rabbits from Day 6 to 28 post coitum. All females were submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of placentas was also undertaken. Fetuses were examined for skeleton and soft tissue malformations. When given 12 mg a.i./kg bw/day, coloured urine and loud breathing was observed in one female from GD 22 each. Both animals however, showed no findings at autopsy. At 32 mg a.i./kg bw/day, the number of animals showing clinical signs was greater than in the control group, and most of these clinical signs (coloured urine, soft faeces, soiled urogenital area) were recorded at the end of the gestation period, along with an absence of faeces observed for three animals. Presence of blood in the bedding was noted in two females on GD 16 or 22 and one female showed an emaciated appearance on GD 29. There was a big variability in litter data. The low dose group showed higher number of dead fetuses, post implantation loss and lower fetal weight compared to control. On the other hand, the number of live fetuses was even higher than of control. At the mid-dose, observations were comparable to control again. The high-dose group of 32 mg a.i./kg bw/day resulted to an increased post-implantation loss, decrease in live fetuses with a decreased fetal body weight, and high number of dead fetuses. Upon examination of the soft tissue, presence of whitish fluid in the abdomens of 5/6 fetuses (one litter) was observed. No relevant malformations or variations were observed during the fetal evaluation. At lower concentrations, litter parameters were not affected by the test treatment and no evidence of treatment-related findings were noted at skeletal examinations of the fetuses. Overall, there seems to be a dose related effect, tending to lower body weights at higher dose levels which corresponding to lower food consumption related to palatability of the compound in the diet. Under the study conditions, the NOAEL for maternal toxicity and embryotoxic/teratogenic effects of the test substance were determined to be 4 and 12 mg a.i./kg bw/day respectively (Chevalier, 2005).