Benzyl-C12-14-alkyldimethylammonium chlorides

Developmental toxicity / teratogenicity

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

batch number is not presented for the vehicle (purified water) in the study report; autolysed foetuses were not sexed at the time of hysterectomy and head sections were archived in the same way as brain sections.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

batch number is not presented for the vehicle (purified water) in the study report; autolysed foetuses were not sexed at the time of hysterectomy and head sections were archived in the same way as brain sections.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides

EC Number:

270-325-2

EC Name:

Quaternary ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides

Cas Number:

68424-85-1

Molecular formula:

C12-14H25-29-(CH3)2-C6H5-N.CL

IUPAC Name:

Quaternary ammonium compounds, benzyl C12-C16 (even numbered)-alkyldimethyl chlorides

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

KBL New Zealand White, Specific Pathogen Free (S.P.F.).

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories (Elevage Scientifique des Dombes, Châtillon-sur- Chalaronne, France).
- Sex: Female
- Age/weight at study initiation: 18-20 weeks; mean body weight of 3451 g (range: 2950 g to 4095 g).
- Number of animals per group:22
The animals were individually housed in stainless steel cages, in a barriered rabbit unit, under specific pathogen free (SPF) standard laboratory conditions.
. temperature : 18 ± 3°C
. relative humidity : 50 ± 20%
. light/dark cycle : 8h dark/16h light (5:00 - 21:00)
. ventilation : about 8 to 10 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Concentration in vehicle: 0, 0.3, 1 and 3 mg test substance/mL The test item dosage forms were prepared daily and stored at room temperature prior to use.
- Chemical analysis: The concentration of samples taken from each control and test substance dosage form prepared for use on the first day of treatment and on the last day of treatment was determined.
- Total volume applied: The quantity of dosage form administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 10 mL/kg/day was used.

Analytical verification of doses or concentrations:

not specified

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). The animals were supplied to CIT on day 1 post-coitum; consequently they were acclimated for a period of 5 days before the beginning of the treatment period.

Duration of treatment / exposure:

Day 6 to 28 post coitum

Frequency of treatment:

Daily

Duration of test:

23 d

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

vehicle alone

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

30 mg/kg bw/day

No. of animals per sex per dose:

22 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose-levels were selected following the results of a previously conducted range finding study (CIT/Study No. 26147RSL): no maternal toxic effects were recorded at 3, 10 or 30 mg a.s./kg/day, but marked toxicity was observed at 50 and 100 mg/kg/day, which resulted in the deaths of a high number of females in both high dosegroups. At these two latter dose-levels, clinical signs (orange coloured urine, soft faeces, soiled urogenital area, emaciated appearance…), body weight loss, lower food consumption and reddish colour of the stomach mucosa were observed in most females. Consequently, the dose-levels of 3, 10 and 30 mg/kg/day were selected for the main study.

Examinations

Maternal examinations:

- Body weight Yes: each female on days 2, 4, 5, 6, 9, 12, 15, 18, 22, 26 and 29 post-coitum.
- Food consumption: The quantity of food consumed by each female was recorded for 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.
- Clinical signs: Morbidity and mortality. From the start of the treatment period, each animal was observed at least once a day, at approximately the same daily time for the recording of clinical signs (including evidence of abortion/desorption).

Ovaries and uterine content:

- On Day 29 post-coitum, all the surviving dams were sacrificed and subjected to a macroscopic post-mortem examination. A gross examination of placentas was also performed. The fetuses were removed by hysterectomy and the uterus weighed. The net body weight gain was calculated. The litter parameters, namely, the number of corpora lutea, implantation sites, early and late resorptions, dead and live fetuses were recorded. The fetuses were weighed and submitted to external examination.

Fetal examinations:

- The live fetuses were killed and then subjected to a fresh dissection and detailed examination of soft tissue, including body, head and brain. The sex was determined. The carcasses were then fixed and the skeletons (including cartilage) stained and examined.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day:
- the relevant clinical signs concerned the deceased females and two prematurely sacrificed females. No other clinical signs were noted in females from this group.
At 10 mg/kg bw/day:
- there were no relevant clinical signs except for two females with blood in the bedding on Days 22 and 23 post-coitum or absence of feces from Day 26 post-coitum

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

At 30 mg/kg bw/day:
- three females died and two females were prematurely sacrificed for ethical reasons or abortion.
At 10 mg/kg bw/day:
- there were no deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day:
- body weight gain was transiently reduced (GD 9-12, -70% below the control, p<0.05) but returned to normal values thereafter.
At 10 mg/kg bw/day:
- reduction of maternal body weight gain did not reach statistical significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

At 10 mg/kg bw/day:
- reduction of food consumption did not reach statistical significance.

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:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

At 30 mg/kg bw/day:
- the necropsies revealed in 8/22 females: accentuated lobular pattern in the liver, pale liver, whitish areas and/or blackish deposits and/or edema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder.
At 10 mg/kg bw/day:
- the necropsies revealed in 5/22 females: dilated gall bladder, accentuated lobular pattern.
- pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

no effects observed

Details on results:

At 30 mg/kg/day:
- three females died and two females were prematurely sacrificed for ethical reasons or abortion,
- the relevant clinical signs concerned the deceased females and two prematurely sacrificed females. No other clinical signs were noted in females from this group.
- body weight gain was transiently reduced (GD 9-12, -70% below the control, p<0.05) but returned to normal values thereafter,
- the necropsies revealed in 8/22 females: accentuated lobular pattern in the liver, pale liver, whitish areas and/or blackish deposits and/or oedema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder,

At 10 mg/kg/day:
- there were no deaths. Reduction of maternal body weight gain and food consumption did not reach statistical significance,
- there were no relevant clinical signs except for two females with blood in the bedding on days 22 and 23 post-coitum or absence of faeces from day 26 post- coitum,
- the necropsies revealed in 5/22 females: dilated gall bladder, accentuated lobular pattern, - pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa,

At 3 mg/kg/day:
no signs of maternal toxicity.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The fluctuations noted in the mean number of corpora lutea and implantation sites were slight and not dose-related, they were consequently considered not to be treatment-related.

Details on maternal toxic effects:

At 3 mg/kg bw/day:
- no signs of maternal toxicity

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

3 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

gross pathology

other: based on dilated gall bladder, accentuated lobular pattern in liver, transient but severe body weight gain reduction on GD 9-12 and mortality at top dose

Remarks on result:

other: Blackish content in stomach and intestines is indicative of local corrosive effects of test substance related to the dosing of substance by gavage

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: general toxicity, no effects observed on reproductive organs

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The occurrence of some external malformations throughout all treated groups, including the controls, did not suggest any substance-related origin because of their low incidence, absence of dose-relationship and/or statistical significance.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant malformations was noted but the presence of a full supernumerary 13th pair of ribs (as a foetal variation), was markedly increased at 10 and 30 mg/kg bw/day. These differences in foetal or litter incidence, which were within background data, were most probably due to a low control value. The incidence of one other skeletal variation (unossified 5th sternebra) was significantly greater but in the low dose-group only.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The occurrence of some soft tissue malformations throughout all treated groups, including the controls, did not suggest any substance-related origin because of their low incidence, absence of dose-relationship and/or statistical significance.

Details on embryotoxic / teratogenic effects:

Conclusion: 
There were no treatment-related effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups. The test substance was not teratogenic in rabbits.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

general and developmental toxicity

Effect level:

30 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No treatment related developmental effect up to the highest dose

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Result: 

Range-finding study:

Groups of six mated female rabbits received daily oral administration at 0 (vehicle, purified water), 3, 10 or 30 mg C12 -16 ADBAC/kg bw/day from day 6 to day 28 post-coitum. In the absence of maternal toxicity, additional groups with dose levels 0, 50 and 100 mg C12 -16 ADBAC/kg bw/day were added.

C12 -16 ADBAC from Day 6-28 p.c. produced marked maternal toxicity at 50 and 100 mg/kg/day shown by high number of deceased females and severe clinical conditions in most animals.

At 30 mg/kg bw/day, slightly high mean number of post-implantation loss associated with slightly low mean number of live foetuses were recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg/day.

Based on these results, the 30 mg/kg bw/day dose-level was chosen as the high dose-level in the further developmental toxicity study (CIT Study No. 26148 RSL).

Main study:

The test substance was not teratogenic in rabbits.

- LO(A)EL maternal toxic effects:
10 mg/kg bw/d, based on necropsy findings in 5/22 animals. Incidence was increased to 8/22 at 30 mg/kg bw/day (dilated gallbladder 3/22, accentuated lobular pattern liver 3/22).
Foci (reddish/brownish) in the lung was also observed in 2 females, but also in view of findings in range finding study and parallel study with comparable compound, this can be caused by inadvertent presence of substance into the airways and not attributable to systemic toxicity. Incidence was not increased in the top-dose group. There is an indication of lower body weight gain, correlating to a lower food consumption, but that was not statistical significant and in the high-dose goup not different from the mid-dose group. Blackish content in stomack and intestines is indicative of local corrosive effects of test substance.

- NO(A)EL maternal toxic effects:
3 mg/kg bw/day. There seems to be a dose-response related increase in necropsy findings in stomach, intestine and liver. Dilated gallbladder incidence was not increased in highest dose group compared to mid-dose.

- LO(A)EL embryotoxic / teratogenic effects:
Based on the number of foetuses presenting malformations or variations and in the absence of a treatment-related increase of such observation, the embryo-fetal development was not considered to be affected by treatment.
- NO(A)EL embryotoxic / teratogenic effects:
30 mg/kg bw/day, being the highest tested dose.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the NOAEL for maternal toxicity and embryo-foetal development was established at 3 and 30 mg a.i./kg bw/day respectively in rabbits.

Executive summary:

A study was conducted to determine the developmental toxicity and teratogenicity of the test substance, C12-16 ADBAC (49.9% active in water), according to OECD Guideline 414 and US EPA OPPTS 870.3700, in compliance with GLP. The substance was administered to pregnant rabbits by gavage from Day 6 to 28 post-coitum at the dose-levels of 0, 3, 10 or 30 mg a.i./kg bw/day. Based on the results of a range-finding study, the test subsatnce was administered as a solution by daily oral administration at 3, 10 and 30 mg a.i./kg/day from day 6 to day 28 post-coitum. One group of 22 females received the vehicle alone (purified water) under the same experimental conditions and acted as a control group. A standard dosage-volume of 10 mL/kg body weight was used for each group. Food consumption and body weight were recorded daily throughout the study period. Clinical signs were checked each day. On day 29 post-coitum, all the surviving dams were sacrificed and subjected to a macroscopic post-mortem examination. The foetuses were removed by hysterectomy and the uterus weighed. The net body weight gain was calculated. The litter parameters, namely, the number of corpora lutea, implantation sites, early and late resorptions, dead and live foetuses were recorded. The foetuses were weighed and submitted to external examination. The live fetuses were killed and then subjected to a fresh dissection and detailed examination of soft tissue, including body, head and brain. The sex was determined. The carcasses were then fixed and the skeletons (including cartilage) stained and examined. The dose of 30 mg a.i./kg bw/day caused the death of three females, severe clinical condition or abortion in two other females and transient, lower maternal body weight gain. Necropsies revealed in 8/22 females accentuated lobular patterns in the liver, whitish areas and/or blackish deposits and/or oedema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder. At 10 mg/kg bw/day, also an indication of corresponding lower food consumption was observed. Relevant necropsy findings were noted in 5/22 females ((dilated gall bladder, accentuated lobular pattern, pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa). Blackish content in stomach and intestines was considered to be indicative of local corrosive effects of the test substance. No maternal toxicity or effects on litter data parameters or embryo-foetal development were noted at 3 mg/kg bw/day. Moreover, there were no effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups up to 30 mg a.i./kg bw/day. Under the study conditions, the NOAEL for maternal toxicity and embryo-foetal development was established at 3 and 30 mg a.i./kg bw/day respectively in rabbits (Gaoua, 2005).