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Administrative data

immunotoxicity: short-term dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Immunotoxicity and allergic potential induced by topical application of dimethyl carbonate (DMC) in a murine model
Anderson SE, Franko J, Anderson KL, Munson AE, Lukomska E & Meade BJ
Bibliographic source:
J Immuntox 10, 59-66

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Concentration range-finding studies: Concentration range-finding studies were performed to select the concentrations of DMC to be used for dermal exposures. BALB/c mice were exposed topically to acetone vehicle or increasing concentrations of DMC in acetone on the dorsal surface of each ear (25 uL per ear) for three consecutive days. Animals were allowed to rest for 2 days following the last exposure and then weighed and examined for signs of toxicity, such as loss of body weight, fatigue/lack of activity, and ungroomed fur. DMC was tested at concentrations up to 100%. The maximum concentration selected for the subsequent studies was based on limits of toxicity.
For the hypersensitivity study, BALB/c mice (five mice/group) were topically treated with acetone vehicle, increasing concentrations of DMC or positive control [30% HCA (v/v; sensitization positive control) and 0.3% DNFB (v/v; irritancy positive control)] on the dorsal surface of each ear (25 uL per ear) once a day for three consecutive days. For the immune phenotyping and haematology studies, B6C3F1 mice (n = 5) were topically exposed to acetone or increasing concentrations of DMC (up to 100%) topically on the shaved backs (50 uL) once a day for 28 consecutive days. For analysis of the IgM response to SRBC, B6C3F1 mice (n = 6) were topically exposed to acetone or increasing concentrations of DMC (up to 100%) topically on the shaved backs (50 uL) once a day for 28 consecutive days. Cyclophosphamide (20 mg/kg in isotonic sterile saline) was included as the positive control for the analysis of the IgM response to SRBC and was injected intraperitoneally 4 days prior to sacrifice.
To determine the irritancy and sensitization potential of DMC, a combined local lymph node assay (LLNA) was conducted. DMC dosing concentrations (50–100%) and vehicle (acetone) were selected based on solubility and preliminary concentration range finding studies.
Phenotypic analysis of splenocytes: Spleen phenotypes were analysed using flow cytometry. Animals were euthanized by CO2 inhalation 24 h after the final exposure, weighed, and examined for gross pathology. Blood was collected in EDTA-coated vacutainer tubes following transection of the abdominal aorta and haematological analysis was conducted. The liver, spleen, kidneys, and thymus were removed, cleaned of connective tissue and weighed. For phenotypic analysis, the spleen was collected in phosphate-buffered saline and dissociated using the frosted ends of two microscope slides. Cell counts were performed using a Coulter Counter and 1 * 10E+6 cells per sample were added to the wells of a 96-well plate. Cells were washed using staining buffer and then incubated with Fc block. For analysis of T-cell sub-sets, cells were then treated with 100 μl of anti-mouse CD3e, anti-mouse CD4, and anti-mouse CD8a antibody solutions. For analysis of B-cells, cells received anti-mouse CD45R/B220 antibody. Parallel sets of cells received appropriate isotype controls diluted in staining buffer. The cells were then incubated on ice in the dark for 30 min. The cells were then washed and incubated with propidium iodide. After a final wash, cells were re-suspended in staining buffer and analysed with a Becton Dickinson LSR II flow cytometer using a propidium iodide. All assays were performed using the FACS DIVA software accompanying the flow system. For each sample, a minimum of 10,000 events was acquired.
Haematology: End-points analysed included peripheral erythrocyte and leukocyte counts, leukocyte differentials (lymphocytes, neutrophils, monocytes, basophils, and eosinophils), platelet counts, haematocrit and haemoglobin levels, mean corpuscular haemoglobin (MCH) and haemoglobin concentration (MCHC), mean corpuscular volume (MCP), mean platelet volume (MCV), and platelet distribution width (PDW).
Spleen in vivo response to the T-cell-dependent antigen SRBC: The primary IgM response to sheep red blood cells (SRBC) was enumerated using a modified haemolytic plaque assay. Four days prior to euthanasia on day 29, the mice were immunized with 7.5 * 10E+7 SRBC by intravenous injection. On the day of sacrifice, body and organ weights were recorded, and spleens were collected. Blood was also retrieved in serum collection tubes and stored for subsequent analysis of serum anti-SRBC IgM levels. Single cell suspensions of the spleens from individual animals were prepared in HBSS by disrupting the spleen between the frosted ends of microscopic slides. To identify the total number of spleen cells, 20 uL of cells were added to 10 ml of Isoton II diluent and two drops of Zap-o-globin were added to lyse red blood cells. Cells were then counted in the Coulter counter. Dilutions (1:30 and 1:120) of spleen cells were then prepared, and 100 uL of each dilution were added to test tubes containing a 0.5 mL warm agar/dextran mixture, 25 uL of 1:1 ratio of SRBC suspension, and 25 uL of 1:4 dilution guinea pig complement. Each sample was vortexed, poured into a petri dish, covered with a microscope coverslip, and incubated for 3 h at 37°C. The plaques (representing antibody-forming B-cells) were then counted. Results were expressed in terms of both specific activity (IgM PFC per 10E+6 spleen cells) and total activity (IgM PFC per spleen).
Serum IgM response to SRBC: Serum samples were analysed for anti-SRBC IgM using a commercially available ELISA kit. Test serum was diluted (1:200 to 1:1,600) and incubated in the micro-titer wells for 45 min at 25°C. The wells were subsequently washed, 100 uL horse-radish peroxidase-conjugated secondary antibody was added, and the plates incubated for a further 45 min at 25°C. Thereafter, the wells were washed to remove unbound antibodies, and 100 uL tetramethylbenzidine peroxidase (TMB) reagent was added to each well. The plates were then incubated for 20 min at room temperature before colour development was stopped by the addition of 50 uL of kit-provided Stop Solution. Optical density in each well was then measured spectrophotometrically at 450 nm. The concentration of the anti-SRBC IgM in the test samples was determined by comparison to a standard curve generated in parallel using SoftMax Pro software, and reported as units of anti-SRBC IgM (U/mL) plotted vs. absorbance values at 450 nm.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl carbonate
EC Number:
EC Name:
Dimethyl carbonate
Cas Number:
Molecular formula:
dimethyl carbonate
Test material form:
other: liquid
Details on test material:
Dimethyl carbonate was purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI)

Test animals

other: BALB/c and B6C3F1
Details on test animals or test system and environmental conditions:
Female BALB/c mice were used for the hypersensitivity studies, while female B6C3F1 mice were used to evaluate the IgM response to sheep red blood cells (SRBC).
All mice were purchased from Taconic (Germantown, NY) at 6–8 weeks of age. Upon arrival, the animals were allowed to acclimate for a minimum of 5 days. Each shipment of animals was randomly assigned to a treatment group, weighed, and individually identified via tail marking using a permanent marker. A preliminary analysis of variance on body weights was performed to insure an homogeneous distribution of animals across treatment groups. The animals were housed at a maximum of five mice/cage in ventilated plastic shoebox cages with hardwood chip bedding. NIH-31 modified 6% irradiated rodent diet and tap water was provided from water bottles, ad libitum. The temperature in the animal facility was maintained between 68–72°F and the relative humidity between 36–57%; a light/dark cycle was maintained at 12-h intervals.

Administration / exposure

Route of administration:
other: unchanged and acetone
Duration of treatment / exposure:
up to 28 days
Frequency of treatment:
once daily
Doses / concentrations
Doses / Concentrations: 0, 50, 75 or 100%
No. of animals per sex per dose:
Control animals:
Details on study design:
see above (principles of method)


Observations and clinical examinations performed and frequency:
see above (principles of method)
Sacrifice and pathology:
see above (principles of method)
Humoral immunity examinations:
see above (principles of method)
Specific cell-mediated immunity:
see above (principles of method)
Other functional activity assays:
see above (principles of method)
Positive control:
see above (principles of method)
Bartlett’s Chi Square test, one-way analysis of variance (ANOVA), Dunnett’s Multiple Range t-test, linear trend analysis

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
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 examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Gross pathological findings:
effects observed, treatment-related

Specific immunotoxic examinations

Specific cell-mediated immunity:
effects observed, treatment-related

Effect levels

Key result
Dose descriptor:
Effect level:
>= 75 other: %

Applicant's summary and conclusion

Using select immune function assays, the purpose of these studies was to evaluate the immunotoxicity of dimethyl carbonate following dermal exposure using a murine model. Following a 28 -day exposure, dimethyl carbonate produced a significant decrease in thymus weight at concentrations of >= 75%. No effects on body weight, haematological parameters (erythrocytes, leukocytes, and their differentials), or immune cell phenotyping (B-cells, T-cells, and T-cell sub-sets) were identified. The IgM antibody response to sheep red blood cell (SRBC) was significantly reduced in the spleen but not the serum. Dimethyl carbonate was not identified to be an irritant and evaluation of the sensitization potential, conducted using the local lymph node assay (LLNA) at concentrations ranging from 50–100%, did not identify increases in lymphocyte proliferation. These results demonstrate that dermal exposure to dimethyl carbonate induces immune suppression in a murine model.
Executive summary:

Dermal exposure to DMC was not found to be toxic at any concentration tested (details not reported), so that concentrations of up to 100% were tested in the subsequent studies. No ear swelling was observed in mice after dermal exposure and there was no increase in auricular draining lymph node proliferation identified after treatment. Therefore, it can be concluded that DMC has no irritancy or allergic sensitisation potential.

In contrast to body weight data, a statistically significant decrease in thymus weights was observed following exposure to 100% DMC (absolute weights at 0, 50, 75 or 100%: 55, 50, 44 and 40 mg; relative weights (% bw) at 0, 50, 75 or 100%: 0.26, 0.24, 0.20 and 0.18). No other significant changes in body or organ weight were observed. Although not statistically significant, there was a slight decrease in spleen weight. Dermal exposure to DMC did not alter any of the analysed haematological parameters, splenocyte numbers, or subpopulations.

For an evaluation for a possible immunosuppressive potential of DMC, the murine IgM response to SRBC was examined following a 28-day exposure. Statistically significant reductions in the PFC/spleen and specific (PFC/10E+6 cells) IgM antibody activity against SRBC were observed. Exposure of mice to 100% DMC resulted in a suppression of the values for PFC/spleen and PFC/10E+6 cells (33 and 46%, respectively, vs. values for vehicle-treated mice); 50% DIMETHYL CARBONATE resulted in suppressions of PFC/spleen (38%) and PFC/10E+6 cells (43%). While mice exposed to 75% DMC did not have a statistically significant reduction in antibody production, the levels appeared to be reduced compared to those associated with the vehicle controls. There was no change in serum anti-SRBC IgM antibody levels following exposure to DMC.