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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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Reason / purpose:
reference to same study
Principles of method if other than guideline:
The studywas designed to investigate the potential adverse effects of maternal exposure to chloroacetonitrile on fetal liver in mice.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
99%, SigmaAldrich (St. Louis, Missouri, USA)
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
Animals were maintained on a regular diet and water as well as a 12-h light-dark cycle without any stressful stimuli. Room temperature was kept at 23 ± 2°C. All animal manipulations were performed between 8-10 a.m.
Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
Chloroacetonitrile was dissolved in corn oil and was given at respective doses of 12.5, 25, or 50 mg/kg/day orally in a dosing volume of 8 ml/kg from GD 6 to GD 18.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
not specified
Duration of treatment / exposure:
13 days
Frequency of treatment:
daily
Duration of test:
18 days
Dose / conc.:
12.5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
At gestation day (GD) 6, 24 pregnant mice were divided into four groups (6 mice/group). The first group served as a control group and was given corn oil (8 ml/kg/day) from GD 6 to GD18. Chloroacetonitrile was dissolved in corn oil and was given to groups 2, 3, and 4 at respective doses of 12.5, 25, or 50 mg/kg/day orally in a dosing volume of 8 ml/kg from GD 6 to GD 18. At the end of the experiment (GD 18), the animals in all groups were sacrificed by carbon dioxide asphyxiation, and abdomen of each mouse was opened transversally.
Maternal examinations:
Abdominal cavity of each mouse was examined and pregnancy status was confirmed.
Ovaries and uterine content:
The number of total implants, resorptions, and live and dead fetuses were recorded.
Fetal examinations:
All live fetuses were dissected from the uterus and were evaluated for bodyweight. Fetal abdomens were opened and livers were rapidly dissected out. Livers from each group (control and 25 mg/(kg day) chloroacetonitrile-treated group) were divided into two halves. One half was used for the determination of the biochemical parameters. The remaining livers were fixed in neutral buffered 10% formalin and then embedded in paraffin. Sagittal sections from all groups (5 µm thick) were stained by hematoxylin–eosin (H & E) and periodic acid-Schiff stain (PAS) stains for use in subsequent histologic studies (Keirnan, 1999).
GSH and GSSG were analyzed by HPLC-electrochemical (EC) analysis (Krien et al., 1992).
Intercellular MDA was determined by a slight modification of previously described method (Richard et al., 1992).
For the assessment of intercellular 8-OHdG, DNA was isolated from fetal livers according to the method described by Laws and Adams (1996).
Fetal liver tissues from treated and control animals were subjected to quantitative methods to detect apoptosis.
Caspase-3 protease activity was measured spectrofluorometrically by a modification of previously described methods (Ramachandran et al., 2001).
Protein was determined using BioRad protein reagent (Bradford, 1976) using bovine serum albumin as standard.
Statistics:
Data are presented as the means±SD for each experimental endpoint. Fetal survival and body weight relative to control were analyzed using one-way ANOVA followed by Dunnett’s post hoc analysis. For further analyses the non-paired Student’s t-test was used. Statistical significance was accepted at p < 0.05.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Implantations/litter:
Control: 12.1±1.35
12.5 mg/kg: 11.6±1.41
25 mg/kg: 11.5±1.88
50 mg/kg: 11.0±2.33
Total litter losses by resorption:
not specified
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Resorptions/litter:
Control: 0.5 ± 0.04
12.5 mg/kg: 0.6 ± 0.4
25 mg/kg: 0.5 ± 0.09
50 mg/kg: 1.1 ± 0.04
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The dose of 25 mg/kg of chloroacetonitrile did not affect viability of fetuses and reduced fetal body weight to 80% of the control value.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Control: 1.14 ± 0.20
12.5 mg/kg: 0.88 ± 0.11 (P < 0.05)
25 mg/kg: 0.88 ± 0.10 (P < 0.05)
50 mg/kg: 0.85 ± 0.12 (P < 0.05)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Fetal body weight was significantly decreased with all doses of chloroacetonitrile (12.5, 25, and 50 mg/kg), compared with control group.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Live fetuses/litter:
Control: 10.8 ± 1.29
12.5 mg/kg: 10.9 ± 1.44
25 mg/kg: 10.7 ± 1.31
50 mg/kg: 7.55 ± 1.88 (P < 0.05)
Changes in sex ratio:
not examined
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
GSH (nmole/mg protein):
Control: 149.7 ± 1.34
Chloroacetonitrile: 90.1 ± 0.90 (p < 0.05)

GSSG (nmole/mg protein):
Control: 0.83 ± 0.08
Chloroacetonitrile: 2.2 ± 0.20 (p < 0.05)

MDA (nmole/mg protein):
Control: 142 ±6
Chloroacetonitrile: 219 ± 4 (p < 0.05)

DNA oxidation (8-OHdG/dG×10E5):
Control: 5.1 ± 0.11
Chloroacetonitrile: 10.4 ± 0.09 (p < 0.05)
Details on embryotoxic / teratogenic effects:
Chloroacetonitrile-induced oxidative stress and redox imbalance in fetal liver were determined by assessing GSH and GSSG levels following in utero exposure. The data indicate that exposure to chloroacetonitrile resulted in a significant reduction in GSH levels in liver tissues (≈40% of control). This was accompanied by a 2.6-fold increase in GSSG levels as compared to control value. Levels of MDA were assessed as markers to elucidate lipid peroxidation. In-utero exposure
to chloroacetonitrile significantly elevated MDA levels in fetal liver tissues by 51%. Further, 8-OHdG was determined as a marker of oxidative DNA damage. The data indicate a significant increase in the levels of 8-OHdG in genomic DNA of fetal liver tissues of mice in the chloroacetonitrile-treated group (≈2-fold over control). The result of the apoptosis assessment indicates a significant increase (160.1% of control) in DNA fragmentation in chloroacetonitrile-treated fetal mouse liver as compared to control. Apoptosis was further confirmed by assessing activity of caspase-3 in cytosolic fractions of fetal livers by quantitating 7-amino-4-methylcoumarin from Ac-DEVD-aminocoumarin, a colorimetric substrate specific for caspase-3. The results illustrated that caspase-3 activity was significantly
increased (132.2% of control) following chloroacetonitrile treatment.

No histopathological changeswere detected. Liver of chloroacetonitrile-treated fetal mouse showed hepatocytes with vacuolated cytoplasm (V), karyolysis,

i.e., absence of nucleoli (N) and karyorrhexis, i.e., fragmented marginally displaced chromatin (C). Hematopoietic cells and congested central vein (CV) were also detected. Liver section from control fetal mice shows PAS-positive staining of the hepatocytes for glycogen deposition. However, livers of chloroacetonitrile-treated fetal mice show depletion of glycogen content of the hepatocytes specially those of the centerlobar zones.

Conclusions:
Maternal exposure to chloroacetonitrile adversely affects mouse fetal livers as evidenced by induction of oxidative stress, apoptosis and histopathological changes.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Principles of method if other than guideline:
The study was designed to investigate the potential adverse effects of intrauterine exposure to chloroacetonitrile on fetal body weight and development of the musculoskeletal system in mice.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
99%, SigmaAldrich (St. Louis, Missouri, USA)

Test animals

Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
Animals were maintained on a regular diet and water as well as a 12-h light-dark cycle without any stressful stimuli. Room temperature was kept at 23 ± 2°C. All animal manipulations were performed between 8-10 a.m.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
Chloroacetonitrile was dissolved in corn oil and was given at respective doses of 12.5, 25, or 50 mg/kg/day orally in a dosing volume of 8 ml/kg from GD 6 to GD 18.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Not specified
Duration of treatment / exposure:
13 days
Frequency of treatment:
daily
Duration of test:
18 days
Doses / concentrationsopen allclose all
Dose / conc.:
12.5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
At gestation day (GD) 6, 24 pregnant mice were divided into four groups (6 mice/group). The first group served as a control group and was given corn oil (8 ml/kg/day) from GD 6 to GD18. Chloroacetonitrile was dissolved in corn oil and was given to groups 2, 3, and 4 at respective doses of 12.5, 25, or 50 mg/kg/day orally in a dosing volume of 8 ml/kg from GD 6 to GD 18. At the end of the experiment (GD 18), the animals in all groups were sacrificed by carbon dioxide asphyxiation, and abdomen of each mouse was opened transversally.

Examinations

Maternal examinations:
Abdominal cavity of each mouse was examined and pregnancy status was confirmed.
Ovaries and uterine content:
The number of total implants, resorptions, and live and dead fetuses were recorded.
Fetal examinations:
All live fetuses were dissected from the uterus and were evaluated for body weight and external malformations. To examine internal and skeletal variations and malformations, they were randomly distributed in two groups. Approximately one-half of the available fetuses, from each group, were fixed in 95% ethanol, cleared with 1.0% KOH stained with alizarin red, and examined for skeletal malformations and variations. Lorke's criteria were applied for evaluation of developmental skeletal anomalies.
The remaining fetuses (from control group and 25 mg/kg chloroacetonitrile-treated group) were fixed in neutral buffered 10% formalin and then embedded in paraffin. Sagittal sections of fetuses from all groups (5 μm thick) were stained by hematoxylin-eosin (H & E), Masson's trichrome, alizarin red, and alcian blue stains for use in subsequent morphometric and histologic studies. The diameter of longitudinally-cut skeletal muscles was measured by viewing H & E-stained sections. Various fields were chosen and 50 readings were obtained. Using the measuring field menu from alizarin redstained sections, one vertebra in each chosen field
was enclosed inside a small measuring frame. Then, the area of calcified cartilage was masked by a blue binary color to be measured. The same field was used to measure the non-calcified cartilage area using the color detect menu.
Statistics:
Statistical analysis was performed using one-way ANOV A followed by Tukey Kramer as a post hoe test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Implantation/litter:
Control: 11.9 ± 1.44
12.5 mg/kg: 11.8 ± 1.53
25 mg/kg: 11.9 ± 1.92
50 mg/kg: 9.1 ± 1.27
Total litter losses by resorption:
not specified
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Resorptions/litter:
Control: 0.5 ± 0.4
12.5 mg/kg: 0.6 ± 0.4
25 mg/kg: 0.5 ± 0.09
50 mg/kg: 1.1 ± 0.09
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The dose of 25 mg/kg of chloroacetonitrile did not affect viability of fetuses.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Control: 1.14 ± 0.20
12.5 mg/kg: 0.88 ± 0.11 (P < 0.05)
25 mg/kg: 0.88 ± 0.10 (P < 0.05)
50 mg/kg: 0.85 ± 0.12 (P < 0.05)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Fetal body weight was significantly decreased with all doses of chloroacetonitrile (12.5, 25, and 50 mg/kg), compared with control group.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Live fetuses/litter:
Control: 10.8 ± 1.29
12.5 mg/kg: 10.9 ± 1.44
25 mg/kg: 10.7 ± 1.31
50 mg/kg: 7.55 ± 1.88 (P < 0.05)
Changes in sex ratio:
not examined
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External anomalies/litter:
Control: 0.0
12.5 mg/kg: 0.0
25 mg/kg: 0.0
50 mg/kg: 0.5 ± 0.04
External anomalies include subcutaneous edema and/or hemorrhage in extremities.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeleton anomalies/litter:
Control: 0.4 ± 0.01
12.5 mg/kg: 0.4 ± 0.02
25 mg/kg: 0.7 ± 0.01
50 mg/kg: 0.9 ± 0.01 (P < 0.05)
Skeleton anomalies include absence or delayed ossification of caudal vertebrae.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral anomalies/litter:
Control: 0.0
12.5 mg/kg: 0.0
25 mg/kg: 0.0
50 mg/kg: 0.4 ± 0.02 (P < 0.05)
Visceral anomalies include hemorrhage in thorax and/or abdominal cavity.
Details on embryotoxic / teratogenic effects:
A daily dose of 50 mg/kg of chloroacetonitrile resulted in an obvious elevation of external and visceral anomalies. Skeleton anomalies were significantly enhanced in the groups receiving 25 or 50 mg/kg. Chloroacetonitrile in lower doses (12.5 or 25 mg/kg) did not exhibit any observable adverse effects on the number of live fetuses. Pregnant mice receiving chloroacetonitrile in a daily dose of 50 mg/kg showed reduced number of live fetuses.
Histological examination of fetal axial skeleton indicated that chloroacetonitrile resulted in delayed appearance of endochondral ossification centers, widening of the vertebrae, and destruction of the calcified zone. In addition, the skeletal muscle fibers were markedly distorted, were small in size, and were widely separated by connective tissue. Both connective tissue perimysium and endomysium were less cellular compared with control sections. The histological findings were further confirmed by assessing the morphometric changes.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
12.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
skeletal malformations
Dose descriptor:
LOAEL
Effect level:
12.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes

Applicant's summary and conclusion

Conclusions:
Intrauterine exposure to low levels of chloroacetonitrile decreases fetal body weight and induces malformations in the musculoskeletal system in mice.