Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
other: Published study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Although the test parameters documented do not totally comply with any specific testing guideline, they are well documented and scientifically acceptable. Thus, they are evaluated to be reliable to use.

Data source

Reference
Reference Type:
publication
Title:
Embryotoxic and teratogenic effects of CTAB, a cationic surfactant, in the mouse
Author:
B. Isomaa and K. Ekman
Year:
1975
Bibliographic source:
Fd Cosmet. Toxicol. Vol 13 pp 331-334

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Embryotoxicity and teratogenicity studies:
Female mice were mated overnight with males of the same strain. The presence of vaginal plugs was taken as evidence of pregnancy and the day a vaginal plug was found was designated day 1 of gestation. Aqueous CTAB in a dose of 10-5 or 35.0 mg/kg bw was administered as a single ip dose onday 8, 10, 12 or 14 of gestation. (These doses corresponded to 10 and 33% of the LD50).
The volume administered was 10ml/kg bw and control animals received an equal volume of water.
The animals were weighted at regular intervals and killed on day 19 of gestation. The foetuses and placentae were removed and the numbers of living and dead foetuses and the sex and weight of each foetus were recorded.
The foetuses were examined for external gross malformations under a stereomicroscope and then cleared in KOH and stained with Alizarin Red S for detection of skeletal anomalies and determination of the state of ossification.
The Student's t test was used for comparison of body weights and the binomal test for proportions was used to evaluate the frequency of anomalies and resorptions.

Distribution studies:
Pregnant mice were given an ip dose of CTAB (10% of the LD50) containing about 1µCi[14C] CTAB on day 14 of gestation. The animals were decapitated 1, 8 or 24 hr after the injection. Blood samples were collected and the placentae and foetuses were removed. Foetal livers and samples from maternal livers were taken for the determination of radioactivity. All samples were counted in a liquid scintillation spectrometer and the results were corrected for quenching by internal standardization.
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Cetyl trimethylammonium bromide (CTAB)
IUPAC Name:
Cetyl trimethylammonium bromide (CTAB)
Test material form:
not specified

Test animals

Species:
mouse
Strain:
NMRI
Details on test animals or test system and environmental conditions:
The animals were given commercial pellets and water ad. lib. and maintained in a 12 hour light-dark sequence under ambient conditions of 20± 1°C and 60 ± 5% humidity.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
other: aqueous solution
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Female mice were mated overnight with males of the same strain.
Duration of treatment / exposure:
single dose
Frequency of treatment:
Aqueous CTAB was administered as a single intraperitoneal dose on day 8, 10, 12 or 14 of gestation.
Duration of test:
The animals were killed on day 19 of gestation.
Doses / concentrations
Remarks:
Doses / Concentrations:
10.5 or 35.0
Basis:
other: mg/kg bw
No. of animals per sex per dose:
8 mice per group (4 groups: administation on day 8,10, 12 or 14 of gestation) per dose.
20 controls.
Control animals:
other: water
Details on study design:
The study includes also a distribution study:
Pregnant mice were given an ip dose of CTAB (10.5 mg/kg bw) containing about 1µCi [14C] CTAB on day 14 of gestation. The animals were decapited 1, 8 or 24 hours after the injection.

Examinations

Maternal examinations:
not examined in details, only body weight.
Ovaries and uterine content:
not examined
Fetal examinations:
The foetuses and placenta were removed and the numbers of living and dead foetus were recorded. The foetuses were examined for external gross malformations under a stereomicroscope and then cleared in KOH and stained with Alizarin Red S for detection of skeletal anomalies and determination of the state of ossification.
Statistics:
The Student's t test was used for comparison of body weights and the binomal test for proportions was used to evaluate the frequency of anomalies and resorptions.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:not examined

Details on maternal toxic effects:
No significant differences between controls and treated groups were in respect of maternal weight gain.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
CTAB treatment increased the number of dead implantations at the higher dose (35.0 mg/kg bw).

At both dose levels (10.5 and 35.0 mg/kg bw), CTAB increased the number of malformed foetuses and it was dose-dependent. The principal anomalies were cleft palate and minor skeletal defects.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Effects of CTAB administered ip to mice on day 8, 19, 12 or 14 of gestation.

 

Effects of a dose (mg/kg) of

  0 on day

10.5 on day

35.0 on day

Parameter 8-14ƚ 8 10 12 14 8 10 12 14
Maternal weight gainǂ(g¥) 22.56± 0.76 23.14 ± 1.09 23.74 ± 1.18 21.49 ± 1.26 21.53 ± 1.47 24.23 ± 0.98 19.84 ± 2.86 21.15 ± 1.23 20.08 ± 2.22
Live foetuses:                
 total no 212 84 88 84 93 64 69 80 81
% of implantations 95.1 93.3 89.8 92.3 94.9 65.3 71.9 88.9 79.4
Foetal weight (g¥) 1.26 ± 0.009 1.30 ± 0.012** 1.25 ± 0.017 1.27 ± 0.013   1.32 ± 0.015*** 1.25 ± 0.017 1.16 ± 0.013*** 1.21 ± 0.014**
Dead implantations:                  
total no. 11 6 10 7 5 34 27 10 21
% of all implantations 4.9 6.7 10.2 7.7 5.1 34.7*** 28.1*** 11.1* 20.6***
Malformed foetuses:                  
total no. 11 21 20 25 32 26 35 45 33
% of live foetuses 5.2 25.0*** 22.7 29.8*** 34.4*** 40.6*** 50.7*** 56.3*** 40.7***
no. with cleft palate 1 0 3* 4** 7*** 1 2 21*** 10***
no. with skeletal defectsǁ 10 21 18 28 31 25 36 39 41
Foetal sex ratio (M:F) 119:93 "39:45" "41:47" "49:35" "56:37" "27:37" "37:32" "43:37" "41:40"

ƚ Controls injected on day 8, 10, 12 or 14 are all treated as a single group.

ǂ Between day 1 and day 19 of gestation.

¥ Values are means for groups of eight mice (or 20 controls) ± SEM.

ǁ Minor skeletal defects including incomplete ossification of dorsal bones (frontals and parietals) of the skull and irregularities of the sternum. This incidence was not compared statistically with controls.

Values marked with asterisks differ significantly from those of the control group:*P<0.05; **P<0.01; ***P<0.001.

Table 2: Distribution of tadioactivity in maternal and foetal tissues after ip administration of [14C]CTAB in a dose of 10.5 mg/kg bw to pregnant mice on day 14 of gestation.

Amount of radioactivity (% of administered dose)
Tissue Time after injection (hr) 1 8 24
Maternal liver 39.25± 2.55 12.76 ± 1.52 2.36 ± 0.09
Total placentae 3.01 ± 0.10 3.20 ± 0.08 1.47 ± 0.10
Total foetuses 0.15 ± 0.04 0.49 ± 0.08 0.43 ± 0.03
Total foetal livers 0.05 ± 0.01 0.25 ± 0.03 0.14 ± 0.01

Values are means ± SEM for groups of three or four animals.

Results:

Only small amounts of radioactivity were found in the foetuses after ip administration of [14C]CTAB to pregnant mice on day 14 of gestation.

About 3% of the administered dose was in the placentae 1 hour after injection.

The decline of radioactivity in the placentae was slower than that in maternal blood and liver.

Applicant's summary and conclusion

Conclusions:
CTAB shows embryotoxicity and teratogenicity when administered intra peritoneally to pregnant mice at single doses corresponding to 10 or 33% of the LD50 (10.5 or 35.0 mg/kg bw). It is possible that the embryotoxicity and teratogenicity of CTAB is due to a disturbance of the functional integrity of the placenta.
Due the the administration route (i.p. of a strong irritant) the local effects of the subtances may very well be the cause of the effects seen. Thus no conclusion can be taken with regard to reproductive toxicity in relation to other and more relevante routes of exposure.
Executive summary:

Administrations of CTAB, a quaternary ammonium surfactant to pregnant mice in an ip dose corresponding to 10 or 33% of the LD50(10·5 or 35·0 mg/kg) increased the incidence of malformations, principally cleft palate and minor skeletal defects in the skull and sternum. At the higher dose, CTAB increased foetal mortality. Foetal body weight was increased by administration of CTAB on day 8 of gestation but was reduced by similar treatment on day 12 or 14. Very low levels of radioactivity were found in the foetuses after ip administration of [14C]CTABto pregnant mice on day 14 of gestation, the amounts probably being too small to cause teratogenic or toxic effects. An appreciable amount (about 3%) of the administered radioactivity was found in the placentae 1–8 hr after the injection.Cationic surfactants are known to affect the permeability of cells and tissues and it is possible that the embryotoxic and teratogenic effects of CTAB are due to a disturbance of the functional integrity of the placenta.

Thus, due the the administration route (i.p. of a strong irritant) the local effects of the subtances may very well be the cause of the effects seen. Thus no conclusion can be taken with regard to reproductive toxicity in relation to other and more relevante routes of exposure


Route: .live1