Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD testing guideline compliant study. It is acceptable to use the data of melamine for melamine phosphate because phosphate is not relevant for acute systemic toxicity.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Melamine
- Chemical name: 2,4,6-triamino-1,3,5-triazine
- CAS Reg. No.: 108-78-1
- Molecular formula: C3H6N6
- Purity: 99.9 %
- Molecular weight: 126.12
- Melting point: 350 °C
- Vapour pressure: 4*10-6 Pa at 20 °C
- Specific gravity: 1.574 kg/L at 20 °C
- Solubility in water: 3.2 g/L at 20 °C
- Particle size range: 0.2 - 10 µm
- Stability under test conditions: unlimited

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species, strain: Rats, Crl:[WI]WU BR, SPF-reared.
Supplier: Charles River Wiga, Sulzfeld, Germany.
Number of animals and sex: 5 males and 5 females.
Age: 6 - 7 weeks at the time of delivery.
After delivery the animals were housed in animal room 15.07, checked for overt signs of ill health and abnormalities and kept in quarantine for 9 days.After approval of the lot (negative titres to microorganisms tested), they were transferred to animal room 6.0.04. The rats were randomly allocated to the cages, separated to sex.
Room number: 6.0.04.
Room temperature: Between 20.0 and 22.5 °C.
Relative humidity: Between 40 and 66 %.
Air exchange: 10 per hour.
Light: 12-hour light and 12-hour dark cycle.
Cages: Suspended stainless steel cages with wire mesh floor and front.
Feed: Rat & Mouse No. 3 Breeding diet RM3 from SDS Special Diets Services, Witham, England. Each batch of this diet is analysed by SDS for nutrients and contaminants.
Water: Tap water suitable for human consumption, supplied by N.V. Waterleidingbedrijf Midden-Nederland (WMN). Water samples are periodically analysed by WMN.
Identification: Earmark (tattoo) for the individual animals, letter and colour code for the animal groups.
Acclimatisation: 9 days.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Exposure chamber

Animals were exposed to the test atmosphere in a nose-only inhalation chamber, a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom. The inhalation chamber consisted of a cylindrical aluminium column, surrounded by a transparent cylinder.
The column had a volume of ca. 50 L and consisted of a top assembly with two mixing chambers, underneath a rodent tube section and the exhaust section at the bottom. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling, particIe size analysis, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (BatteIIe), positioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmosphere at the nose of the animals is prevented.


Generation of the test atmosphere

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. The test atmosphere was generated by passing the test material using a dry material helix feeder (Hethon, Hengelo, the Netherlands) to a jet mill (Institute's design). In order to guarantee a non-fluctuating output a stirring-rod was used within the helix feeder whereas a vibration device was placed just before the entrance to the jet milI. The jet mill was operated with dry pressurized air (Iess than 1% humidity); the test material was delivered using a slip stream of air conditioned room air.
In first instance, water mist produced by an ultrasonic nebulizer was added to obtain relative humidity values slightly above 30% to satisfy the required range of 30-70%. Using this set-up, however, the concentration dropped from nearly 5 g/m3 to about 2 g/m3 which was far below the required concentration (viz. > 5 g/m3).
Because of this concentration drop it was decided not to add water mist and to accept a low relative humidity to obtain the limit concentration of 5 g/m3. In addition, a particle size distribution measurement showed that particles were relatively large, viz. an MMAD of 6.2 µm with a geometric standard deviation of 1.9 was obtained. Apparently, although a jet mill was used to break up agglomerated particles, it was not sufficient to generate a test atmosphere consisting of respirable particles only (viz. almost 100% smaller than 5 µm, or with an MMAD of between 1 and 4 µm). However, since it was not expected to obtain higher numbers of smaller particles using other dispersing devices without possibilities to break up agglomerates, a jet mill was used during the present study.

Before exposure, the rate of airflow through the exposure unit was established at the input pressures used (5.0 bar for both jet and mill). During exposure, the pressure settings were recorded at regular intervals (approximately each half hour).
In this way, the total airflow was monitored indirectly through the aerosol generation system. The average amount of air passed through the exposure unit was 99.2 L/min including about 28 L/min from the slip stream. The concentration of the aerosol was controlled by the output of the feeder which was measured twice at three different settings the day after exposure. During exposure, the setting of the feeder was recorded at regular intervals (approximately each half hour) and at changes.
The generated aerosol was passed to the inlet of the exposure unit and directed downward through the mixing sections towards the animaIs. At the bottom of the unit the test atmosphere was exhausted. The period between the start of the generation of the test atmosphere and the start of exposure of the animals was 111 min.


Analysis of the test atmosphere

The actual concentrations in the test atmosphere were determined eight times (about twice per hour) by gravimetric analysis. Representative samples were obtained by passing 5 L test atmosphere samples at 5 L/min through fibre glass filters (Sartorius). Filters were weighed before sampling, loaded with aerosol particles, and finally weighed again. The actual concentration was calculated by dividing the amount of test material captured by the volume of the air sample taken.

The nominal concentration was determined by dividing the total amount of test material used by the total volume of air passed through the exposure unit.

ParticIe size distribution measurements were carried out once during preliminary testing and once during exposure using a 10-stage cascade impactor (Anderssen, Atlanta, USA) with the largest cut-off size of 32 µm. The Mass Median Aerodynamic Diameter (MMAD) and the mean geometric standard deviation (gsd) were calculated.

The temperature of the test atmosphere was recorded eight times during exposure at regular intervals (about twice per hour) using a RH/T device (TESTO 610, GmbH & Co, Lenzkirch, Schwarzwald, Germany). The oxygen concentration was checked once during exposure (Beryl, Cosma, Igny, France).
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5.19 g/m³
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
The rats were visually inspected just before exposure, for reactions to treatment during the exposure, shortly after exposure, and at least once daily during the observation period.
Body weights were recorded just prior to exposure (day 0), and on days 7 and 14.
At the end of the 14-day observation period, all rats were killed by exsanguination from the abdominal aorta under ether anaesthesia. All rats were necropsied and examined for gross pathological changes.
Statistics:
none

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 190 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortalities during exposure to the test item or during the observation period.

Clinical signs:
other: Changes in breathing pattern: Slightly decreased breathing frequency in all animals during the first hour of exposure, followed by moderately decreased breathing rate during the remaining exposure period. 1 hour after exposure blepharospasm was observed i
Body weight:
Overall body weight gain was considered to be normal for rats of this strain and age.
Gross pathology:
In all animals lung changes were noted: One or more petechiae in all males and one female, grey (and/or pale) discolouration in one male and three females, and white spots in one male and two females.
Thymic changes were observed in one male: Unilateral red thymus
Other findings:
none

Any other information on results incl. tables

Analytical results

 

The actual concentration of Melamine during the exposure based on gravimetric analyses is given in Table 1. The mean measured concentration during exposure was 5.19 +/- 0.45 g/m³.

 

The nominal concentration during exposure was calculated to be 12.3 g/m³, indicating a generation efficiency of 42 % .

 

The particle size distribution is given in Table 2. Ca. 66% of the particles present at the animals breathing zone had an aerodynamic diameter equal to or smaller than 8.2 µm, whereas ca. 35 % equal to or smaller than 5.0 µm. The Mass Median Aerodynamic Diameters (MMAD) was 6.6 µm with a mean geometric standard deviation of 2.5. These data were essentially the same as obtained during preliminary testing: an MMAD of 6.2 µm with a mean geometric standard deviation of 1.9.

 

The mean temperature during exposure was 20.9 +/- 0.2 °C (range 20.7-21.2 °C), the mean relative humidity was 28 +/- 4% (range 22-32 %) and the oxygen concentration was 21.4 %.

Applicant's summary and conclusion

Executive summary:

The acute inhalation toxicity of Melamine was observed by nose-only exposure of one group of five male and five female rats to a test atmosphere containing the limit concentration of 5.19 +/- 0.45 g test item per m³ for a single 4 -hour period. The Mass Median Aerodynamic Diameter was 6.6 µm. After exposure the animals were observed for 2 weeks. Clinical examinations resulted in changes in breathing pattern in all animals during the exposure to the test item and in blepharospasm in two females

shortly after the administration. From day 1 onwards until the end of the experiment on day 14 no abnormalities were observed in the animals. Overall body weight gain was considered to be normal and no mortality occurred. On day 14 after the administration all animals were necropsied. Abnormalities at necropsy consisted of lung changes (petechiae, discolouration and white spots) in all animals and thymic changes (unilateral red thymus) in one male.

It was concluded that the 4 -hour LC50 value of Melamine was higher than 5.19 g/m³ for both sexes under the present test conditions.