Melamine

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Discrepancies and contradictions are found within the publication, as well as severe deficiencies of the design. E.g. the use of only one dose of the test substane and a control. The signs reported and the signs not reported raise doubts on the reliability of the studies. E.g.: An et al. wrote "However, the symptoms of piloerection and haematuria as well as reduced spontaneous activity were observed in most of melamine rats". These signs of sickness and especially the reduced spontaneous activity, raises doubts on the validity of at least the water maze experiments, because it is obvious that sick animals and those with reduced spontaneous activity will perform insufficiently in a water maze experiment, compared to the control animals. Data supporting the reliability and sensitivity of the test method (i.e. positive and historical control data)" are missing in the paperl. 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:

2011

Materials and methods

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

The study examines the effect on hippocampus and the possible mechanism induced by melamine in vivo. To address the hypothesis that melamine would impair the hippocampal function in vivo and then induce cognitive deficits, male Wistar rats were used to establish an animal model and melamine administered at a dose of 300 mg/kg/day for 4 weeks. Morris water maze test was employed to evaluate the learning and memory. The long term potentiation from Schaffer collaterals to CA1 region in the hippocampus was recorded.

GLP compliance:

not specified

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:

not specified

Details on test material:

Purity: 99.5 %.Supplier: Yingda Sparseness & Nobel Reagent Chemical Factory, Tianjin, PR China.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Age: 3 weeks.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1 %

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once each day.

Doses / concentrations

Remarks:

Doses / Concentrations:
300 mg/kg/d
Basis:
nominal conc.

No. of animals per sex per dose:

8 rats in the control group; 10 rats in the melamine group.

Control animals:

yes, concurrent vehicle

Examinations

Observations and clinical examinations performed and frequency:

Body weight every third day.

Specific biochemical examinations:

Biochemical assay in hippocampus
After LTP performance, the brain of each rat was removed and weighted, and then the hippocampus in the right side of brain was dissected out and rinsed in 0.1 M phosphate buffer (pH 7.4). After being weighted, the hippocampus was homogenized with ice-cold saline to be 10 % (w/v) homogenates. The mixtures were homogenized using a glass homogenizer for 5 min and centrifuged at 3000 rpm at 4 °C for 15 min. The supernatant was collected and stored at -70 °C for the determination of melamine content.

Neurobehavioural examinations performed and frequency:

Examination for neurological deficits such as head tilt, motor deficits, hemiparalysis and other physical changes such as spontaneous activity and piloerection.

Other examinations:

Morris water maze (MWM) experiment 24 h after the last gavage, all rats were trained and tested in MWM to monitor their spatial learning and memory for six consecutive days. The water maze consisted of a 1.5-m diameter circular tank divided into 4 equal quadrants (I-IV), and there was a 10-cm diameter platform submerged 2 cm below the water surface in the center of quadrant III.

In vivo long term potentiation (LTP) test
Electrophysiological tests were performed after Morris water maze assessment. The animals were anesthetized with urethane and then were placed in a stereotaxic frame for surgery and recording. Briefly, at the rat left head side, two small holes were drilled in the skull for the recording and stimulating electrodes inputting respectively. The bipolar stimulating electrode was implanted into the hippocampus Schaffer collaterals region, and the recording electrode was implanted into hippocampus CA 1 region. Test stimuli were delivered to the Schaffer collaterals every 30 s at an intensity that evoked a response of 70% of its maximum. Once the response stabilized, sampling was made under low-frequency stimulations (0.05 Hz) for 20 min as the baseline. After recording baseline, high frequency stimulation (8 pulses at 100 Hz for 6s repeated 30 times) was delivered and then the field excitatory post- synaptic potentials (fEPSPs) were amplified (100 x), filtered at 5.5 kHz, digitized and collected at 20 kHz sample frequency every 60 s for 60 min. Initial data measurement was performed in Clampfit 9.0. The fEPSPs slope (20-80 % level of the falling phase) was used to measure synaptic efficacy. LTP test took two days in melamine group and other two days in control group, respectively.

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Gross pathological findings:

not examined

Neuropathological findings:

effects observed, treatment-related

Details on results:

There were no significant differences of both brain weight and hippocampus weight between melamine group and control group. Furthermore, no sign of neurological deficits was found in melamine group. However, the symptoms of piloerection and haematuria as well as reduced spontaneous activity were observed in most of melamine rats.
There was a higher level of melamine in hippocampus in melamine group compared with that in control group.
Morris water maze experiment results:
During place navigation, the performance of all rats improved with training. There was a significant effect of melamine treatment, the escape latencies were prolonged significantly in melamine group compared with those in control group. Similar results were shown on swimming speeds and there were no marked differences of day, day x group and group. In addition, the swimming speeds of each group remained constantly throughout testing, with no significant difference was found between two groups on each day. In spatial probe test, it could be seen that there was a marked effect of melamine treatment. Statistical results revealed that both the platform crossings and the quadrant dwell time were decreased in melamine group compared with those in control group.
LTP from Schaffer collaterals to CA1
In LTP test, stimulation of Schaffer collaterals evoked a basal fEPSPs in hippocampus CA1 and high frequency stimulation induced LTP of the stimulated synapses for at least 1 h. The fEPSPs slopes increased immediately after high frequency stimulation and stabilized to a level above the baseline period. Moreover, it was found that fEPSPs slopes were significantly smaller in melamine group than those in control group.

Effect levels

Dose descriptor:

LOEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other:

Applicant's summary and conclusion

Conclusions:

Melamine dosed at 300 mg/kg bw/d for 28 days had a toxic influence on hippocampus, which induced the learning and memory deficits.

Executive summary:

The study examines the effect on hippocampus and the possible mechanism induced by melamine in vivo. To address the hypothesis that melamine would impair the hippocampal function in vivo and then induce cognitive deficits, male Wistar rats were used to establish an animal model and melamine administered at a dose of 300 mg/kg/day for 4 weeks. Morris water maze test was employed to evaluate the learning and memory. The long term potentiation from Schaffer collaterals to CA1 region in the hippocampus was recorded.

The result of the Morris water maze test showed that there were significant deficits of learning and memory induced by melamine. The long term potentiation test presented that field excitatory postsynaptic potentials (fEPSPs) slopes were significantly lower in melamine group compared to that in control group. In conclusion, melamine had a toxic influence on hippocampus, which induced the learning and memory deficits. It suggested that the potential mechanism was associated with impairments of synaptic plasticity.