Melamine

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Remarks:

Investigating whether neonatal ingestion of melamine leads to renal and vascular dysfunction in adulthood; and whether ingestion of melamine in pregnant rats leads to renal dysfunction in their offspring.

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Scientific investigation not meeting standards as in guideline studies.

Remarks:

No analyses of the experimental animal feed and of the drinking water was performed, especially no analyses for cyanuric acid are reported, which is essential for this type of studies with melamine, as synergistic effects are known on the one hand, and contaminations with cyanuric acid in studies were reported repeatedly on the other hand.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study investigates whether neonatal ingestion of melamine leads to renal and vascular dysfunction in adulthood; and whether ingestion of melamine in pregnant rats leads to renal dysfunction in their offspring. A combination of approaches employed includes functional studies in rat renal arteries, renal blood flow measurement by functional magnetic resonance imaging, assay for pro-inflammatory and fibrotic biomarkers, immunohistochemistry, and detection of plasma and renal melamine.

GLP compliance:

not specified

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Melamine

EC Number:

203-615-4

EC Name:

Melamine

Cas Number:

108-78-1

Molecular formula:

C3H6N6

IUPAC Name:

1,3,5-triazine-2,4,6-triamine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Provider: Sigma-Aldrich, StLouis, MO, USA

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sprague Dawley (SD) rats were used in the present study. Rats were housed in a temperature-controlled holding room (22-23 °C) with a 12-h light/dark cycle, and fed a standard chow and water.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

In order to study the dose-dependent effects of melamine, male 4-week-old rats were given melamine dissolved in water freely for 3 months at three dosages ( 60, 300 and 600 mg·kg-1 body weight·day-1) while in the control group the rats were given normal access to water.
To study the effects of melamine on offspring from melamine-treated pregnant rats, 2-month old female rats were given melamine at a high dose (600mg-kg-1 body weight-day- 1) for 2 weeks before they were mated with male rats and continued during gestation. The offspring given rise by these melamine-treated mothers were further divided into melamine-treated group (HDM + HDO) that the male pups kept on receiving high-dose melamine at 600 mg·kg-1 body weight-day-1 for another three months and vehicle-treated group (HDO) that the pups stopped receiving melamine.
The volume of water consumption was monitored daily and body weight was measured weekly to adjust the amount of melamine dissolved in drinking water in order to obtain the approximate melamine intake at three dosages ( 60, 300 and 600 mg-kg-1 body weight-day-1).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Male 4-week-old rats were given melamine dissolved in water freely for 3 months.
Female rats were given melamine at a high dose (600mg-kg-1 body weight-day- 1) for 2 weeks before they were mated with male rats and continued during gestation. The offspring given rise by these melamine-treated mothers were further divided into melamine-treated group (HDM + HDO) that the male pups kept on receiving high-dose melamine at 600 mg·kg-1 body weight-day-1 for another three months and vehicle-treated group (HDO) that the pups stopped receiving melamine.

Frequency of treatment:

Continously.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

for male rats

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

for male rats

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

for male rats

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

For the female rats before mating and during gestation.

Doses / concentrations 5

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

For male offspring of melamine treated mothers.

No. of animals per sex per dose:

No data.

Control animals:

yes, concurrent no treatment

Details on study design:

In order to study the dose-dependent effects of melamine, male 4-week-old rats were given melamine dissolved in water freely for 3 months at three dosages ( 60, 300 and 600 mg·kg-1 body weight·day-1) while in the control group the rats were given normal access to water.
To study the effects of melamine on offspring from melamine-treated pregnant rats, 2-month old female rats were given melamine at a high dose (600mg-kg-1 body weight-day- 1) for 2 weeks before they were mated with male rats and continued during gestation. The offspring given rise by these melamine-treated mothers were further divided into melamine-treated group (HDM + HDO) that the male pups kept on receiving high-dose melamine at 600mg·kg-1 body weight-day-1 for another three months and vehicle-treated group (HDO) that the pups stopped receiving melamine.
The volume of water consumption was monitored daily and body weight was measured weekly to adjust the amount of melamine dissolved in drinking water in order to obtain the approximate melamine intake at three dosages ( 60, 300 and 600 mg-kg-1 body weight-day-1).

Urine was collected using a metabolic cage.
After sacrifice of rats, weights of the heart and the kidneys were measured.
Systolic blood pressure was measured by the tail-cuff method and blood pressure was calculated from the average of 5 successive recordings.
Serum creatinine was measured by creatinine assay kit (Abcam) according to manufacturer's protocol.

Melamine was quantified in kidneys by high performance liquid chromatography (HPLC)
method.

Statistics:

Results were mean± SEM from different rats. Concentration-response curves were analyzed by non-linear regression curve fitting using two-way AN OVA followed by Bonferroni post-hoc tests (GraphPad Prism software, Version 4.0) to approximate Emax as the maximal response and pD2 as the negative logarithm of the drug concentration that produced 50% of Emar The protein expression was quantitated by densitometer and normalized to GAPDH. Images of immunohistochemistry staining were analyzed by Image} (NIH). Statistical significance were determined by two-tailed Student's t-test or one-way AN OVA followed by Bonferroni post-hoc tests when more than two treatments were compared. A P value of less than 0.05 was regarded as statistically significant.

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Various effects.

Observed effects

Basic parameters in melamine-treated rats:
Three-month oral consumption of melamine in drinking water at three dosages ( 60, 300 and 600 mg·kg-1 body weight·day-1) in SD adult male rats did not change the body weights. Melamine consumption (at medium and high dose) significantly elevated the concentration of melamine in both plasma and kidney samples. Melamine ingestion did not affect the systolic blood pressures, heart weight and weight of kidney on both sides did not change significantly.

Any other information on results incl. tables

Impaired endothelial function in renal arteries of melamine-treated rats in a dose-dependent manner:

In isolated SD rat renal arteries constricted with phenylephrine, the cumulative addition of acetylcholine (ACh) caused endothelium-dependent relaxations. Chronic consumption of melamine impaired the ACh-induced relaxations with reduction in maximum relaxation and higher concentrations of ACh turned relaxation into contractile responses. By contrast, the endothelium-independent relaxations in response to sodium nitroprusside (SNP) were identical in renal arteries isolated from vehicle control and melamine-treated rats. To dissect the possible mechanisms responsible for the impaired relaxations, the roles of COX-2, thromboxane prostanoid (TP) receptor and reactive oxygen species (ROS) were examined. In renal arteries from rats treated with melamine at three different doses, NS398 (COX-2 inhibitor), Sl8886 (TP receptor antagonist) and tiron plus DETCA (ROS inhibitor and scavenger) markedly improved ACh-induced relaxations in medium and high dose treatment groups and reduced the contractile phase in all the three-dosage groups.

It was also tested: the impact of melamine consumption on the appearance of endothelium-dependent contractions (EDCs) in response to ACh in renal arteries following acute treatment with NOS inhibitor, L-NAME. In non-treated vehicle control rats, ACh-induced contractions were negligible and low-dose melamine ingestion caused a small contraction. On the other hand, medium and high dose of melamine triggered large EDCs. Treatment with NS398, S18886, or tiron plus DETCA abolished the EDCs in renal arteries from medium- and high-dose melamine-treated rats.

 

Impaired renal cortical blood flow in melamine-treated rats:

Functional magnetic resonance imaging (fMRI) was used to detect the changes in morphology of kidneys and measure the renal blood flow. Regions of interest (ROI) were drawn in the renal cortex of both kidneys. The cortical enhancement was reduced in melamine-treated rats in a dose-dependent manner.

 

Increased pro-inflammatory and pro-fibrotic markers in renal arteries and kidney of melamine-treated rats:

Immunohistochemistry staining of kidney showed that melamine treatment led to markedly increased expression of fibronectin in glomeruli in a dose-dependent manner, suggesting the remodeling of kidney and arteries were taking place. This effect was further confirmed by Western blotting showing elevated fibronectin levels in both kidneys and renal arteries. The expression ofnTGF-β1 in kidney and renal arteries in melamine-treated rats were also upregulated, further indicating the remodeling response to melamine treatment. Furthermore, increased expression of bone morphogenic protein 4 (BMP4) and cydooxygenase-2 (COX-2) in both kidney and renal arteries following chronic melamine consumption suggesting both tissues may undergo an inflammatory process.

 

Consumption of melamine in pregnant rats led to renovascular dysfunction in their offspring:

The male offspring given rise by high-dose melamine-treated mothers kept on receiving high-dose melamine at 600 mg·kg-1 body weight-day-1 for another three months (HDM + HDO). HDM + HDO rats had markedly increased concentration of melamine in both plasma and kidney samples as compared with the offspring from the same high-dose melamine-treated mothers but stopped receiving melamine (HDO). Compared to rats receiving high dose melamine (HD), HDM + HDO rats have similar melamine concentrations in kidney, plasma, and urine, but slightly higher serum creatinine. Interestingly, although HDO rats did not receive melamine treatment, due to their exposure to melamine before birth, the melamine contents in kidney, plasma, and urine remained significantly higher than rats from vehicle group even after 3 months. The ACh-induced EDRs were greatly impaired in renal arteries from HDO rats as compared with vehicle control. The ACh-induced EDRs were further aggravated in renal arteries from HDM + HDO rats than those from HD rats. SNP-induced endothelium-independent relaxations were comparable in the three groups. The ACh-induced EDCs were enhanced in renal arteries from HDO rats as compared with vehicle control. And the EDCs further increased in HDM + HDO rats, indicating that embryonic exposure to melamine may have long term harmful effect on renovascular function. Accordingly, several pro-inflammatory markers including CCL2, TNF, and IL1β, although not increased in kidneys from HD rats, were all upregulated in HDM + HDO rats, showing that chronic inflammation in the kidney due to melamine ingestion was potentiated by embryonic exposure to melamine.

Applicant's summary and conclusion

Executive summary:

Melamine was dosed in the drinking water to male rats (0 - 60 - 300 - 600 mg/kg bw/d) and also to pregnant rats (0 - 600 mg/kg bw/d). This study investigates whether neonatal ingestion of melamine leads to renal and vascular dysfunction in adulthood; and whether ingestion of melamine in pregnant rats leads to renal dysfunction in their offspring.

A combination of approaches employed includes functional studies in rat renal arteries, renal blood flow measurement by functional magnetic resonance imaging, assay for pro-inflammatory and fibrotic biomarkers, immunohistochemistry, and detection of plasma and renal melamine.

The study provides mechanistic evidence showing that melamine reduces renal blood flow and impairs renal and vascular function associated with overexpression of inflammatory markers, transforming growth factor-β1, bone morphogenic protein 4 and cyclooxygenase-2 in kidney and renal vasculature. Melamine also induces renal inflammation and fibrosis.

Melamine causes nephropathies in offsprings from high dosed pregnant rat, as well as in neonatal rat high exposed to melamine afterbirth, thus supporting the clinical observations of kidney stone and acute renal failure in infants consuming melamine-contaminated milk products.