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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2007-03-28 to 2007-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study. The study was performed comparable to OECD guideline 414, the findings are considered relevant and the present documentation is considered sufficient to evaluate the chronic toxicity of chlorhexidine base. The salt chlorhexidine digluconate was used instead of chlorhexidine base. Due to the high level of structural similarity it is considered that the derived data are relevant for the assessment of Chlorhexidine base.In addition, Chlorhexidine digluconate dissociates in aqueous solution to the chlorhexidine base and the gluconate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
D-gluconic acid, compound with N,N''-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediamidine (2:1)
EC Number:
242-354-0
EC Name:
D-gluconic acid, compound with N,N''-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediamidine (2:1)
Cas Number:
18472-51-0
IUPAC Name:
N',N'''''-hexane-1,6-diylbis[N-(4-chlorophenyl)(imidodicarbonimidic diamide)] - D-gluconic acid (1:2)

Test animals

Species:
rat
Strain:
other: CD/Crl:CD (SD)
Details on test animals or test system and environmental conditions:
Strain / Stock: CD® / Crl: CD (SD)
Adaptation period: 6 days
Age (on day 0 of pregnancy): 8 - 10 weeks
Body weight (on day 0 of pregnancy): 198 - 289 g
Diet: Commercial ssniff(R) R-Z V1324 ad lib.
Housing: Except during the mating period, the dams were kept singly in MAKROLON cages
Room temperature: 22°C ± 3°C
Relative humidity: 55% ± 15%
12 hours dark/12 hours light cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis of the test item-carrier mixtures gave 92.5 to 100.6 % of the nominal values.
Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. The male and female breeding partners were randomly chosen. Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm and the stage of oestrus cycle. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
day 6 to 19 of gestation
Frequency of treatment:
once daily
Duration of test:
necropsy at day 20 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30 or 100 mg/kg bw/d (Volume 5 mL/kg bw)
Basis:

No. of animals per sex per dose:
4 groups with 25 females each. Evaluated litters: 20 per group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Based on a dose-range-finding study

Examinations

Maternal examinations:
Clinical signs:
Individual animals were observed daily for mortality, behaviour, external appearance and nature of the faeces.

Body weight:
The weight of each rat was recorded on day 0 of gestation followed by daily weighings. The body weight gain was also calculated in intervals.

Food and drinking water consumption:
The quantity of food consumed per rat was recorded daily with the exception of gestation day 20 (necropsy). Also the relative food consumption (g/kg bw/d) was calculated.
Ovaries and uterine content:
Examinations at necropsy (day 20):
Removal of ovaries and uteri of the dams. Uteri (in toto) were weighed. Dissection with macroscopic examination of the internal organs and placentae of the dams at necropsy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.
The fetuses were removed and the following examinations performed:
-Macroscopic inspection (gross evaluation) of the placentae.
-Determination a.) of the number of fetuses and % per dam (alive and dead) and placentae, b.) of sex and viability of fetuses, c.) of number and size of resorptions, d.) of corpora lutea in the ovaries, implantations and location of fetuses in the uterus and e.) of gravid uterus weights, weights of fetuses and weights of placentae.
Fetal examinations:
Fetuses were inspected externally for damages, especially for malformations. 50 % of the number of fetuses in each litter were examinded for skeletal anomalies, while the remaining 50 % were examined for soft tissue anomalies.
Evaluation/parameters: sex ratio per litter, distribution in the uterine horns, absolute and mean number of fetuses alive per group, mean % of fetuses alive per group, mean % per sex and group, runts, fetuses with variations or retardations, retardation rate, and pre- and post-implantation loss.
Statistics:
BARTLETT chi-square test, DUNNETT test, STUDENT's t-test, FISHER's exact test and chi2-test with YATES' correction for continuity

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality:
None of the dams treated died prematurely.

Clinical signs:
Laboured breathing on several test days in one dam dosed with 30 mgkg bw/d. Pilo-erection and/or laboured breathing on one to several test days in 8 of 21 dams dosed with 100 mg/kg bw/d, pre-dominantly during the second half of gestation until end of treatment. Further findings in these dams were a haemorrhagic nose in two females and reduced motility in one rat on two or three treatment days.

Body weight:
No adverse effects at <= 30 mg/kg bw/d. Dosing with 100 mg/kg bw/d caused a reduction from gestation day 9 onwards until end of study. Significant reductions for mean maternal body weights of high-dosed dams on gestation days 9 to 20, for mean maternal body weight changes on gestation days 6 to 9 and 9 to 12 and for the net weight change from day 6 onwards.

Food consumption:
Reduced relative food intake in dams dosed with 100 mg/kg bw/d on gestation days 7 to 13.

Drinking water consumption:
No adverse effects in treated animals.

Necropsy findings:
Necropsy revealed no pathological findings in treated animals.
Significant reductions in uterus and carcass weights in dams dosed with 100 mg/kg bw/d.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Corpora lutea / implantation sites / resorptions / weight and number of live fetuses / placental weights:
No adverse effects on prenatal fetal development at doses of <= 30 mg/kg bw/d.
No dead fetuses or runts at termination of the study.
At the materno-toxic dose of 100 mg/kg bw/d, significant increases were noted for early, late and total resorptions and the number of fetuses was significantly reduced. There was also a slight decrease in fetal weights.

Malformations / Variations:
No adverse effects at any dose level.

Retardations:
At the materno-toxic dose of 100 mg/kg bw/d, the following effects were noted: retarded ossification for sternebra(e) (not ossified), caudal vertebral bodies (no body ossified), thoracic vertebral bodies (less than 13 bodies ossified), pelvic vertebral bodies (less than 5 bodies ossified), 5th metatarsalia (absence of ossification) and os ischii (not ossified).

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No teratogenic properties were noted during external/internal, skeletal and soft tissue examination up to the materno-toxic dose of 100 mg chlorhexidine digluconate/kg bw/d. There was no increase in the incidence of fetal malformations or variations. At the materno-toxic dose of 100 mg/kg bw/d, slight embryotoxic properties were noted in form of reduced fetal weights and increased incidences of skeletal retardations.
Executive summary:

In this prenatal developmental toxicity study, chlorhexidine digluconate was administered to female rats at dose levels of 0, 10, 30 or 100 mg/kg bw/d via gavage from the 6th to 19th day of gestation. First signs of maternal toxicity included laboured breathing at >= 30 mg/kg bw/d, while at 100 mg/kg bw/d pilo-erection, a haemorrhagic nose and/or reduced motility were noted. Furthermore, reductions were noted for the body weight, the net weight change and the food intake. The NOAEL for the fetuses was 30 mg/kg bw/d.

At the materno-toxic dose of 100 mg/kg bw/d, significant increases were noted for early, late and total resorptions and the number of fetuses was significantly reduced. In addition, slight reductions were noted for the fetal weights. The skeletal examination gave significantly increased fetal and/or litter incidences for skeletal retardations. The treatment caused no malformations or variations at any of the tested dose levels.