3-chloropropane-1,2-diol

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to EPA OPPTS 870.7800 (Immunotoxicity) with the exception that animals were dosed over a 14 day period rather than the 28 days recommended by the guideline; no analytical information on the test substance was provided.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Evaluation of the potential immunotoxicity of 3-monochloro-1,2-propanediol in Balb/c mice I. Effect on antibody forming cell, mitogen-stimulated lymphocyte proliferation, splenic subset, and natural killer cell activity

Author:

Lee JK, Byun JA, Park SH, Kim HS, Park JH, Eom JH, and Oh HY

Year:

2004

Bibliographic source:

Toxicology 204 (2004) 1-11

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7800

Deviations:

yes

Remarks:

Animals were dosed over a 14 day period rather than the 28 days recommended by the guideline.

Principles of method if other than guideline:

MCPD was administered by gavage for 14 days at 0, 25, 50, and 100 mg/kg per day to female Balb/c mice. The antibody-mediated immune response to sheep red blood cells (SRBC) was assessed using the antibody-forming cell (AFC) assay, and splenic cell phenotypes were quantified by flow cytometry. Hematological and histopathological changes were assessed. Mitogen-stimulated spleen lymphocyte proliferation and natural killer (NK) cell activity were evaluated.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-chloropropane-1,2-diol

EC Number:

202-492-4

EC Name:

3-chloropropane-1,2-diol

Cas Number:

96-24-2

Molecular formula:

C3H7ClO2

IUPAC Name:

3-chloropropane-1,2-diol

Constituent 2

Reference substance name:

3-monochloro-1,2-propanediol

IUPAC Name:

3-monochloro-1,2-propanediol

Details on test material:

- Name of test material (as cited in study report): 3-Monochloro-1,2-propanediol (MCPD)
- Obtained from Sigma (USA)

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Toxicological Research (part of the Korea Food and Drug Administration in Seoul, Korea)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 17.6 - 21 g
- Fasting period before study: no data
- Housing: polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): housed in a room with controlled temperature
- Humidity (%): housed in a room with controlled humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
MCPD was dissolved in distilled water.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 50, or 100 mg/kg per day
Basis:
actual ingested

No. of animals per sex per dose:

7 - 8 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose of 100 mg/kg per day was chosen based on a preliminary range-finding experiment to identify the dose which did not cause mortality or inhibit 10% of body weight gain.

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and Day 14

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters examined: Erythrocytes, leukocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), hemoglobin distribution width (HDW), platelet, mean platelet volume (MPV), and differential leukocyte counts were determined with a hematological analyzer (ADVIA120, Bayer, Germany). Spleen and thymus cellularities were determined by counting with a hemocytometer.

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

Sacrifice and pathology:

Organ weights of the animals used in the antibody response were determined on the day of necropsy.

Thymus, spleen, mesenteric lymph nodes, bone marrow (femur), peyer’s patch (ileum), brain, kidney, liver, and adrenal gland were fixed in neutral, aqueous, phosphate-buffered 4% solution of formaldehyde, wax-embedded according to a routine processing protocol, and 5-um sections were cut and stained with hematoxylin and eosin (H&E). The tissue slides were examined microscopically.

Cell viabilities:

SPLEEN: Yes
- Method: Spleen cellularities were determined by counting with a hemocytometer.
- Dose groups: 0, 25, 50, and 100 mg/kg/day
- No. of animals: 8 per dose

THYMUS: Yes
- Method: Thymus cellularities were determined by counting with a hemocytometer.
- Dose groups: 0, 25, 50, and 100 mg/kg/day
- No. of animals: 8 per dose

BONE MARROW: No data

Humoral immunity examinations:

Antibody-forming cell (AFC) assay:
- Method: Mice were sensitized intraperitoneally 4 days prior to the assay with 5×10EE8 sheep red blood cells (SRBC) per mouse 0.2 mL of EBSS. Single cell suspensions of splenocytes were prepared in 3 mL EBSS. The number of antibody-forming cells was determined as follows: 0.05% DEAE–dextran was added into melted 0.5% agar in EBSS and maintained at 47 deg C throughout the assay. Then, 350 uL of melted agar was dispensed into 12 mm × 75 mm heated glass tubes, followed by the addition of 25 uL of an indicator SRBC, 100 uL of the spleen cell suspension, and 25 uL of guinea pig complement. The SRBCs were washed at least three times with EBSS before use. A 150 uL aliquot from the tube was then immediately pipetted onto a 100 mm × 20 mm Petri dish and the agar solution was covered with a 22 mm × 40 mm microscopic cover glass. The Petri dishes were placed at room temperature for several minutes to allow the agar to solidify, and were then incubated at 37 deg C for 3 h to form hemolytic plaques in a humidified 37 deg C incubator. Following the incubation, the number of AFCs was counted. The results were expressed as AFCs/10EE6 spleen cells or AFCs × 10EE3 per spleen. The body and organ weights of the animals used in the antibody response were determined on the day of necropsy.
- Doses: 0, 25, 50, and 100 mg/kg/day
- No. of animals: 7 per dose

Non-specific cell-mediated immunity:

NATURAL KILLER (NK) CELL ACTIVITY: Yes
- Method: YAC-1 target (2 × 10EE7 cells/mL) was stained with CFSE for 1 h at 37 deg C in 5% CO2. CFSE in the form of a 5 mM stock solution in DMSO was added to give a final concentration of 10 uM. The cells were washed three times in RPMI 1640 medium containing 10% FBS. One milliliter of 5 × 10EE5 cells/mL of the target cells were plated into 24-well microplate, then the effector cells (splenocytes) in 1 mL were added to the wells in triplicate at effector-to-target (E:T) cell ratios of 50:1. Following an 18-h-incubation at 37 deg C in 5% CO2, propidium iodide was added to give a final concentration of 0.5 ug/mL. The samples are measured by flow cytometry within 60 min. Five thousand target cells are collected and the results are given as percentage of dead targets gated in a dot-plot.
- Flow cytometric analysis was performed with a FACScan flow cytometer (Beckton Dickinson, USA). The FAC- Scan was equipped with a 488-nm-argon laser and detectors for forward scatter (FSC), side scatter (SSC), FL1 (band bass filter wavelength 530 nm), FL2 (585 nm), and FL3 (650 nm) fluoresceine emission in the green, red/orange, and long red parts of the spectra, respectively. Splenocytes stained with FITC-, PE-, and Cy chrome-labeled antibodies were detected on FL1, FL2, and FL3, respectively, while dead cells stained with PI were detected on FL2. Fluorescence overlap was compensated for electronically using splenocytes stained with a single color (FITC, PE, and PI), and 10,000 events were acquired and stored for each analysis. Splenocytes were identified by their characteristic appearance on a dot-plot of FSC versus SSC and electronically gated to exclude platelets, red cells, or dead cell debris. The gate was the same for both exposed and control mice. The results are shown as the percentage of positive cells within a gate.
- Dose groups: 0, 25, 50, and 100 mg/kg/day
- No. of animals: 7 per dose

Other functional activity assays:

SPLEEN CELL PROLIFERATION ASSAY (ANTI-CD3 MEDIATED T CELL PROLIFERATION)
- Method: Spleen cell suspensions were prepared in EBSS, washed, and resuspended in RPMI 1640 media containing 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL of penicillin, 100 ug/mL of streptomycin, 15 mM HEPES, and 50 uM 2-mercaptoethanol. The cell number was adjusted to 2 × 10EE6 cells/0.9 mL of culture media. Then, a 90 uL aliquot of the cell suspension was transferred to each well of a 96-well tissue culture plate. Each chemical in culture medium (10 uL) was directly added into the spleen cell culture. The cultures were incubated in the presence of given concentrations of either LPS or Con A. After the cells were cultured for 72 h at 37 deg C in humidified 5% CO2, the number of proliferating cells was determined with the MTS non-radioactive cell proliferation assay where 20 uL of freshly prepared MTS/PMS solution was added to each well, and after incubating it for 1–4 h, the absorbance at 490 nm was recorded.
- Dose groups: 0, 25, 50, and 100 mg/kg/day
- No. of animals: 7 per dose

CELL STAINING FOR PHENOTYPIC ANALYSIS
- Method: Single cell suspensions were prepared from the spleen of each treatment by gently rubbing the spleen through a nylon mesh filter. Cellular debris was removed and cells were washed in PBS and resuspended (10EE7 cells/mL) in FACS buffer (PBS, 10 mM HEPES buffer, 0.01% sodium azide, and 1% heat-inactivated fetal bovine serum). Cells were labeled in a 96-well plate (10EE6 cells per well) with 10 uL of each selected fluorochrome-conjugated monoclonal antibody (Ab). Double staining was performed for FITC-CD3e/PE-CD45R and FITC-CD3e/PE-DX5. Triple staining was performed for Cy-cychrome-CD3e/FITC-CD4/PE-CD8a. The stain for natural killer cell was performed with a DX5 mAb. NK cells express DX5 + CD3 -1.
- Dose groups: 0, 25, 50, and 100 mg/kg/day
- No. of animals: 7 per dose

Statistics:

Data are expressed as the mean ± S.D. The data were analyzed by a one-way analysis of the variance followed by Dunnet’s method as a post hoc test. Statistical analysis was performed using Sigma Stat ver. 2.0.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Gross pathological findings:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived the experimental period of 14 days - no MCPD-related deaths were observed.

BODY WEIGHT AND WEIGHT GAIN
There were no significant changes of body weight gain between the MCPD-exposed mice and the vehicle-control mice.

HAEMATOLOGY
There were no changes in the percentage of leukocytes. No significant changes were observed in the number and mean cell volume of erythrocytes, hemoglobin level, and hematocrit in treated mice. There were no significant changes of MCV, MCH, MCHC, RDW, and platelet in mice-treated with MCPD, compared to the vehicle control. No significant changes were observed in mice treated with MCPD on differential count of leukocytes.

GROSS PATHOLOGY
A 2-week period of administrating MCPD resulted in changes of thymus weight. Mice dosed with the high dose of MCPD (100 mg/kg/day) had reduced absolute and relative thymus weights. Similar changes were recorded in spleen weights (see table below). There were no significant changes of absolute and relative weights in liver, kidney, adrenal, and brain between the MCPD-exposed group and the control group.

CELL VIABILITIES
A suppressive effect of MCPD was observed in the thymus cellularity in animals treated with medium and high-MCPD doses. The spleen cellularity decreased in mice given high dose of MCPD.

HUMORAL IMMUNITY EXAMINATIONS
In the primary antibody response to sheep erythrocytes assay, a slight increase in AFCs per million spleen cells and AFCs × 10EE3 per spleen in mice treated with the low dose of MCPD (25 mg/kg/day) were observed. However, there were 397 +/- 41 in the vehicle control group, which was decreased to 387 +/- 31 in the medium dose group (50 mg/kg/day) and 256 +/- 42 in the high dose group (100 mg/kg/day), when the data were expressed as AFCs per million spleen cells. The reduction in the high dose group was statistically significant (P < 0.05). The percentage decrease was 3 and 36%, respectively. The results were the same as that of AFCs × 10EE3 per spleen.

NON-SPECIFIC CELL-MEDIATED IMMUNITY
Natural killer cell assay: At the effector:target ratio of 50:1, the percentage of cytotoxicity was 11.46 +/- 0.79% in the vehicle control group. This was statistically significantly (P < 0.05) decreased to 8.96 +/- 0.09 and 9.50 +/- 1.52% in animals exposed to MCPD at the dose levels of 50 and 100 mg/kg, respectively, which was a 22 and 17% reduction when compared to the vehicle control.

OTHER FUNCTIONAL ACTIVITY ASSAYS
Spleen cell proliferation assay (Anti-CD3 mediated T cell proliferation): There were no significant changes in T-cell proliferative activity of spleen lymphocytes in mice treated with MCPD to Con A and anti-CD3. There were no significant changes in the B-cell proliferative response to LPS in mice treated with MCPD.

Phenotypic analysis of splenic subset: There were no significant differences in the percentage of CD3+ and B220+ cells between the exposed mice and the control mice. No significant changes in the percentage of CD4+ and CD8+ cells between MCPD- exposed mice and control mice were observed. There were no significant differences in the percentage of DX5+ CD3-cells between the MCPD-exposed mice and the control mice.

Specific immunotoxic examinations

Cell viabilities:

effects observed, treatment-related

Humoral immunity examinations:

effects observed, treatment-related

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

effects observed, treatment-related

Other functional activity assays:

no effects observed

Other findings:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 50 - <= 100 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The major immune parameters affected in mice treated with 100 mg/kg/day were the antibody-forming cell responses, spleen and thymus cellularities, and NK cell activity.

Any other information on results incl. tables

Absolute and relative organ weight in female Balb/c mice orally exposed to 3-MCPD for 14 days (mean +/- S.D. [n = 8])

Parameter	Control	25 mg/kg 3-MCPD	50 mg/kg 3-MCPD	100 mg/kg 3-MCPD
Spleen (mg)	103.5 +/- 5.2	109.9 +/- 15.1	113.3 +/- 8.3	91.7 +/- 34.8
Spleen % of body wt.	0.48 +/- 0.02	0.50 +/- 0.07	0.49 +/- 0.03	0.41 +/- 0.15
Thymus (mg)	81.1 +/- 7.9	70.0 +/- 13.1	68.4 +/- 11.7	58.9 +/- 11.8*
Thymus % of body wt.	0.38 +/- 0.03	0.32 +/- 0.06	0.30 +/- 0.05	0.26 +/- 0.05*

 * Indicates significant difference from corresponding control (P < 0.05)

Distribution of splenocytes subset in female Balb/c mice orally exposed to 3-MCPD for 14 days (mean +/- S.D. [n=7])

Parameter (%)	Control	25 mg/kg 3-MCPD	50 mg/kg 3-MCPD	100 mg/kg 3-MCPD
CD3+ CD45R-	64.2 +/- 2.4	54.5 +/- 3.6	44.8 +/- 4.8*	60.2 +/- 4.8
CD45R= CD3-	13.6 +/- 2.2	13.5 +/- 1.0	12.0 +/- 0.4	15.9 +/- 1.9
CD3+ CD4+ CD8-	26.9 +/- 0.2	22.0 +/- 1.2*	24.6 +/- 0.8	23.0 +/- 2.3
CD3+ CD4- CD8+	12.1 +/- 0.1	12.1 +/- 0.5	12.0 +/- 0.7	12.0 +/- 0.7
DX5+ CD3-	4.16 +/- 0.11	3.61 +/- 0.27	4.38 +/- 0.3	3.97 +/- 0.5
CD3/CD45R ratio	2.51	1.83	1.74	2.9
CD4/CD8 ratio	1.74	2.17	1.88	2.21

 * Indicates significant difference from corresponding control (P < 0.05)

 

 

Applicant's summary and conclusion

Conclusions:

The immunotoxic potential of MCPD in mice was evaluated by examining the changes in the number of spleen cell subsets; the spleen IgM antibody response to the T-dependent antigen, SRBC; the spleen cell proliferative response to Con A, anti-CD3, and LPS; the spleen NK cell activity and histopathology. The results from the studies demonstrate that MCPD can modulate the immune response in the female Balb/c mice at a dose of 100 mg/kg when administered by oral gavage for 14 days. The major immune parameters affected were the antibody-forming cell responses, spleen and thymus cellularities, and NK cell activity.