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neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Clear description of method and results in a non-guideline and non-GLP study; no analytical information on the test substance.

Data source

Reference Type:
Differential expression of neuronal and inducible nitric oxide synthase in rat brain after subchronic administration of 3-monochloro-1,2-propanediol
Kim, Kisok
Bibliographic source:
Food and Chemical Toxicology 46 (2008) 955-960

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
The neurotoxic effects of 3-MCPD (10 or 30 mg/kg) administered for 13 weeks on the expression of two forms of nitric oxide synthase (NOS), neuronal NOS (nNOS), and inducible NOS (iNOS), in rat cerebral cortex and striatum were studied. Using immunocytochemistry, the number of nNOS-expressing neurons or the optical density of iNOS staining in sections from three coronal levels (bregma levels 1.0, -0.4, and -2.3 mm) were compared between 3-MCPD-treated and control rats.
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
Details on test material:
- Name of test material (as cited in study report): 3-monochloro-1,2-propanediol (3-MCPD)
- Obtained from Sigma Chemical Co., St. Louis, MO

Test animals

Details on test animals or test system and environmental conditions:
- Source: no data
- Age at study initiation: adult
- Weight at study initiation: 225 - 250 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data


Administration / exposure

Route of administration:
oral: unspecified
other: saline
Details on exposure:

VEHICLE: no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, 5 days a week
Doses / concentrations
Doses / Concentrations:
0, 10 or 30 mg/kg bw
nominal conc.
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle


Observations and clinical examinations performed and frequency:


Specific biochemical examinations:

For immunocytochemistry, the rats were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with saline solution, followed by a solution of 4% paraformaldehyde/1.5% sucrose in 0.1 M phosphate buffer (PB), pH 7.4. The brains were post-fixed overnight and cryoprotected in 20% sucrose. Using a freezing microtome, sections (40 um) were cut into 0.1 M PB or cryoprotectant solution. Immunocytochemistry was performed on free-floating sections in one batch. Prior to incubation with primary antisera, tissue sections were treated with 10% methanol and 3% hydrogen peroxide in 0.1 M PB, to inhibit endogenous peroxidase activity, and rinsed for 1–2 h in PB. Tissue sections were first incubated with either nNOS or iNOS antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 or 1:250, respectively, in phosphate-buffered saline (PBS) containing 0.5 mg/mL bovine serum albumin, 1.5% normal horse serum, and 0.3% Triton X-100, for 24 h at 4 deg C. The tissue sections were then thoroughly rinsed in PBS before incubation with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:200, at room temperature for 90 min. Following extensive rinsing, the tissue sections were incubated in an avidin–biotin complex solution diluted 1:100, at room temperature for 1 h. After extensive rinsing, immunoreactivity was visualized using a standard DAB procedure. Staining was intensified by incubating the tissue for 1 min in a solution of 0.02% 3,3'-diaminobenzidine tetra-hydrochloride and 0.01% H2O2, to yield a dark black reaction product. Sections were mounted onto gel-coated slides.

Quantification of immunocytochemistry:

The number of nNOS-positive neurons and the optical density of iNOS-positive staining were measured in one hemisphere of three representative coronal sections, at bregma levels 1.0, -0.4, and -2.3 mm, according to the Paxinos atlas, which included neocortical and striatal areas. For nNOS, only cells that were darkly and uniformly stained were counted as nNOS-positive neurons. Each entire neocortical subregion and randomly selected striatal areas were examined by light microscopy at 200x magnification and were charted using a microscope fitted with a drawing tube. For iNOS, all neocortical and striatal regions were included for analysis. Densitometric measurements using an NIH image program (ImageJ) were used to determine the staining density in the neocortex and striatum.
Neurobehavioural examinations performed and frequency:
Not applicable.
Sacrifice and (histo)pathology:
The rats were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with saline solution, followed by a solution of 4% paraformaldehyde/1.5% sucrose in 0.1 M phosphate buffer (PB), pH 7.4.
Positive control:
Not applicable.
Within-group and between-group comparisons were performed using either one-way ANOVA or repeated-measures ANOVA, as appropriate. When a statistical difference was detected by ANOVA, the Duncan multiple comparison test was used to define differences between the results. A value of P < 0.05 or P < 0.01 was deemed to be statistically significant. Mean values were calculated, together with the S.E. of the mean. All statistical analyses were conducted using the SAS 8.1 computer program (SAS Institute Inc., Cary, NC).

Results and discussion

Results of examinations

Clinical signs:
not specified
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
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Details on results:
nNOS immunoreactivity:

nNOS-immunoreactive neurons in controls and 3-MCPD-treated rats were well represented in all of the studied areas and were scattered throughout the cortex and striatum (Fig. 1B–G). Following treatment with 3-MCPD, the total numbers of nNOS-positive neurons in the neocortex and striatum were significantly decreased in the rostral part but were not significantly changed in the caudal part. The number of nNOS-positive neurons was significantly decreased after treatment with 3-MCPD (10 or 30 mg/kg), in the cortex and striatum at bregma level 1.0 mm (P < 0.01). At rostrocaudal bregma level -0.4 mm, treatment with 10 mg/kg of 3-MCPD slightly, but not statistically significantly, decreased the number of nNOS-positive neurons, whereas treatment with 3-MCPD at 30 mg/kg clearly showed a significant decrease in the numbers of nNOS-positive neurons in the cortex and striatum (P < 0.01). The total numbers of stained neurons in the cortex and striatum were not significantly changed by treatment with 3-MCPD at bregma level -2.3 mm.

iNOS immunoreactivity:

iNOS immunoreactivity was diffuse throughout the study area. In contrast to the differential decrease in the number of nNOS-positive neurons along the rostrocaudal extent observed with 3-MCPD, quantitative analysis of the mean density of iNOS staining revealed that the effects of 3-MCPD treatment produced a uniform increase in iNOS immunoreactivity throughout the rostrocaudal extent. In the cortex, 3-MCPD treatment significantly increased iNOS immunoreactivity at all three rostrocaudal levels studied (P < 0.01). Similarly, the optical density of iNOS staining in the striatum also showed a significant increase at all three levels following exposure to 3-MCPD (P < 0.01 or P < 0.05).

Effect levels

Dose descriptor:
Remarks on result:
not determinable
no NOAEL identified

Any other information on results incl. tables

In this study, the cell count data for nNOS and the optical density analysis for iNOS in selected coronal sections may reflect some degree of imprecision, because of the lack of a strict stereological evaluation. However, because the volume of the regions of interest was not changed after subchronic administration of 3-MCPD, the evaluation methods should not have affected the validity of the study results.

Applicant's summary and conclusion

In this study, 3-MCPD treatment changed the expression levels of nNOS and iNOS, key enzymes in NO production in neurons. NO plays an important role in linking synaptic and nonsynaptic transmission suggesting that impaired expression of NOS, induced by 3-MCPD treatment, may be neurotoxic because it disturbs the link between glutamatergic and monoaminergic neurons. Thus, the findings of this study suggest that alterations in NOS expression induced by subchronic 3-MCPD exposure may reflect changes in the molecular signal transduction of the gluatamate-NO-cGMP pathway in the neocortex and striatum, especially in the caudal brain area. In addition, these results suggest that the neurotoxic effects of 3-MCPD may be relevant to NO homeostatic disturbances, through differential expression of nNOS and iNOS throughout the rostrocaudal extent.

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