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immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Scientific investigation not meeting standards as in guideline studies. The interpretation of the results seems to be a bit euphoric and not always based on facts.
Remarkable: The diet was confirmed negative for both melamine and cyanuric acid.

Data source

Reference Type:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The objective of this study was to investigate the potential effects of melamine on humoral immunity with or without cyanuric acid in mice. Only the melamine part is referred here.
GLP compliance:
not specified
Limit test:

Test material

Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity: >99%
Supplied by Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China)

Test animals

other: Kumming
Details on test animals and environmental conditions:
Healthy male mice of the Kunming strain (N = 56) weighing 18- 22 g were purchased from Liaoning Changsheng Biological Technology Co., Ltd. The mice were acclimated for 7 days.. The animals were housed in a controlled laboratory conditions maintained at 20-24 °C temperature, 35-65% relative humidity and a 12-h light and dark cycle. Standard laboratory food (experimental animal center of Liaoning University of Traditional Chinese Medicine, Shenyang, China) and water were provided ad libitum throughout the study. The diet was confirmed negative for both melamine and cyanuric acid as described method by Heller and Nochetto (2008).

Administration / exposure

Route of administration:
oral: gavage
physiological saline
Details on exposure:
Administrations by gastric gavages for 30 days.
In the control group, the mice were given 1 mL physiological saline.
Duration of treatment / exposure:
30 days
Frequency of treatment:
Dosing once every 2 days.
Doses / concentrationsopen allclose all
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Animals were assigned randomly to seven groups (each group, N = 8 ).
There are one control group, three melamine groups (low, middle, and high doses) and three mixture groups of melamine and cyanuric acid (low, middle, and high doses).
Control animals:
yes, concurrent vehicle
Details on study design:
After 30 days, all experimental animals received 0.1 mL SRBC (1%) by intraperitoneal injection to stimulate the body humoral immune response.
After 24 h of intraperitoneal injection with SRBC, the blood was collected from orbital sinus of each mouse, and then the mice were sacrificed via cervical dislocation.
The spleens were collected immediately from each mouse.


Humoral immunity examinations:
Analysis of blimp-1 protein and gata-3 gene expression and the contents of IgG, sIgA and complement C3:
To demonstrate potential expression changes of blimp-1 and gata-3 proteins, Western blotting was performed as the method described by Posadas et al. (2010). Anti-13-actin antibody (Sigma, MO, USA) was used as an internal control.
The real-time quantitative RT-PCR ( qPCR) was used to demonstrate potential mRNA expression change of gata-3 gene.
To determine the content changes of IgG, slgA and complement C3, sera were separated from blood samples by centrifugation at 3000 rpm for 20 min at room temperature, and were stored at -80 °C for further use. The concentrations of IgG, slgA and complement C3 in each sample were determined by commercially available ELISA kits.

Analysis of plasma cells expressing CD138 and Tfh cell expressing CD4+ CXCR5+:
Lymphocytes were isolated from the spleen of each mouse using the method described by Cox et al. (2002). Cells were incubated with PE anti-mouse CD138, FITC anti mouse CD4 and PE anti mouse CXCR5 at 4 °C for 20 m in in a dark environment, respectively. A protocol without any anti-body was used as control. Subsequently, the cells were washed for three times in PBS-buffer (pH 7.3) and were fixed with fresh 4% paraformaldehyde solution. Lastly, the cells were analyzed on a FACScan machine (BD Biosciences, CA, USA). Approximately 10 000 cells were examined for each sample.

Content analysis of IL-21, IL-4, IL-6 and IL-10 cell factors of Th2 cell subsets in mouse serum:
Commercially available ELISA kits were used to evaluate the content changes of IL-21, IL-4, IL-6 and IL-10 of Th2 cell subset in mouse serum.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related

Specific immunotoxic examinations

Humoral immunity examinations:
effects observed, treatment-related
Description (incidence and severity):
Effect of melamine on plasma cell expressing CD 138: Based on the analysis by flow cytometry and compared with control group, although no significant
difference for CD138 was recorded in the melamine-treated groups with different doses (2, 10 and 50 mg/kg/2 days, respectively, P > 0.05), there was a decreasing tendency in the CD138 expression with the increasing of melamine dosage.

Effect of melamine on blimp-1 protein expression and the contents of IgG, sIgA and C3 in mice: The animals treated with melamine with different doses (2, 10 and 50 mg/kg/2 days, respectively) exhibited a decreasing (not significant) tendency in the expression of blimp-1 protein. For the contents of lgG, an increasing tendency (significant only in the low dose) was recorded in the melamine alone treated groups with increasing doses. No significant difference was found in the content of C3 or sIgA.

Effect of melamine on the expression rate CD4+cxcRs+ of spleen in mice: No dose dependent significant effects on the expression rate ofCD4+cxcRs+ were observed.

Effect of melamine on the content of/L-21, /L-4, IL-6, and IL-10 cell factors of Th2 cell subsets in serum: A dose related and significant decreases of IL-6 and IL-10 content was obtained.

Effect of melamine on the expression of gata-3 gene in spleen of mice: In the qPCR reactions a satisfactory single dissolve curve was obtained for both gata-3 gene and ~-actin gene (internal control), respectively. However, no significant difference was observed in comparison with control. The same as for the Western blotting results of gata-3 protein.

Applicant's summary and conclusion

Executive summary:

Mice were dosed by gavage with 0 - 2 - 10 - 50 mg melamine per kg and day for 30 days, every second day. After 30 days, all experimental animals received 0.1 mL SRBC (1%) by intraperitoneal injection to stimulate the body humoral immune response.

In comparison to control group, a significantly lower content of plasma cells expressing CD138 were observed with both middle and high doses of melamine. Moreover, the data showed that melamine suppressed the production of IL-6 and lL-10 in a dose-dependent manner. The results from the present study suggested that the exposure to melamine alone had certain humoral immunotoxicity in mice, especially when ingested in high dosage.