Registration Dossier

Toxicological information

Acute Toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 24, 2011 - April 28, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
temporary deviations from the minimum level of temperature and relative humidity occurred
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
yes
Remarks:
temporary deviations from the minimum level of temperature and relative humidity occurred
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
yes
Remarks:
temporary deviations from the minimum level of temperature and relative humidity occurred
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.
Deviations:
yes
Remarks:
temporary deviations from the minimum level of temperature and relative humidity occurred
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no

Test material

Constituent 1
Reference substance name:
Melam
IUPAC Name:
Melam
Constituent 2
Chemical structure
Reference substance name:
2,2'-iminobis[4,6-diamino-1,3,5-triazine]
EC Number:
222-695-1
EC Name:
2,2'-iminobis[4,6-diamino-1,3,5-triazine]
Cas Number:
3576-88-3
Molecular formula:
C6H9N11
IUPAC Name:
2-N-(diamino-1,3,5-triazin-2-yl)-1,3,5-triazine-2,4,6-triamine
Details on test material:
- Name of test material (as cited in study report): Melam
- CAS: 3576-88-3
- Substance type: White powder
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI (Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 9 weeks
- Weight at study initiation (pre-administration):
Females: 167-202 g
Males: 247-272 g
- Fasting period before study: not applicable
- Housing:
Before exposure: Group housing of five animals per sex per cage in labelled Macrolon cages (type IV; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
After exposure: Group housing as described above, except that a paper sheet was introduced into the cage covering the bedding and cage enrichment to prevent suffocation in case of bad health condition. At the end of the Day of exposure the paper sheet was removed.
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except during exposure to the test substance
- Water (e.g. ad libitum): ad libitum, except during exposure to the test substance
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.322.0°C, except for approximately 3 hours during the acclimatization period of the highest exposure group during which the temperature dropped down to 15.3 °C. As laboratory historical data do not indicate an effect of the deviations, the study integrity was not adversely affected by the deviations.
- Humidity (%): 23 - 73%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 24 February 2011; To: 21 March 2011

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: no vehicle used
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dynamic (continuous flow) system, based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consisted of three animal sections with eight animal ports each. Each animal port had its own atmosphere inlet and exhaust outlet.
- Exposure chamber volume: not applicable
- Method of holding animals in test chamber: the animals were placed in restraining tubes and connected to the animal ports.
- Source and rate of air: the source was by a dried pressurized air inlet and the number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen supply to the animals.
- Method of conditioning air: not applicable
- System of generating particulates/aerosols: administering the test substance to a stream of dried pressurized air using a combination of a spiral feeder (Randcastle Extrusion Systems, Cedar Grove, NJ, USA) and air mover (AIR-VAC, Milford, CT, USA) generated an aerosol. The rotation speed of the feeder was varied to obtain the desired exposure concentration. The aerosol was passed through a cyclone which allowed larger particles to settle. For the highest concentration the mean air flow was 32 L/min. For the lower concentration, the primary aerosol was diluted with pressurized air before it entered the exposure chamber and the mean total air flow was 35 L/min.
- Method of particle size determination: the particle size distribution was characterized twice during each exposure period. The samples were drawn (2 L/min) from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (SKC 225-713, fiber glass, SKC Omega Specialty Division, Chelmsford, MA, USA) and a fiber glass back-up filter (SEC-290-F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test substance collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined 1
- Treatment of exhaust air: from the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.
- Temperature, humidity, pressure in air chamber: the temperature and relative humidity were measured with a humidity and temperature indicator (E+E Elektronik, Engerwitzdorf, Austria) and were recorded after the animals were placed in the experimental set-up and at 30 minute intervals after initiation of the exposure. The probe was inserted in a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The temperature of the atmosphere was between 20.3 and 20.6 degrees Celsius and relative humidity was between 24 and 38%. These conditions were considered appropriate for this relatively short 4 hours exposure duration.

TEST ATMOSPHERE
- Brief description of analytical method used: not applicable
- Samples taken from breathing zone: no

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: the particle size distribution was characterized twice during each exposure period. The samples were drawn (2 L/min) from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (SKC 225-713, fiber glass, SKC Omega Specialty Division, Chelmsford, MA, USA) and a fiber glass back-up filter (SEC-290-F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test substance collected were measured gravimetrically.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
For the exposure to 5 mg/L, the MMAD was 3.7 µm (gsd 1.6) and 3.5 µm (gsd 1.8) respectively.
For the exposure to 1 mg/L, the MMAD was 3.3 µm (gsd 1.9) and 3.7 µm (gsd 1.8) respectively.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: no details provided
Analytical verification of test atmosphere concentrations:
no
Duration of exposure:
4 h
Concentrations:
Target concentrations were based on the cut off concentration values specified in the UN and EC classification guidelines. One group of five animals of each sex were exposed for 4 hours to a target concentration of the test substance of 5.5 mg/L and one group to 1.1 mg/L (the limits of the guideline were increased with 10% in order to avoid the mean actual concentration to fall below the cut off value due to experimental variation).
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
other: not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of weighing: days 1 (pre-administration), 2, 4, 8 and 15.
- Necropsy of survivors performed: yes, all animals were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded. Particular attention was given to any changes in the respiratory tract
- Other examinations performed:
During exposure: three times during exposure for mortality, behavioural signs of distress and effects on respiration.
After exposure: on Day 1, one and three hours after exposure and once daily thereafter until Day 15. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).
Statistics:
No statistical analysis was performed

Results and discussion

Preliminary study:
Not applicable
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
- All animals exposed to 5 mg/L were found dead or sacrificed for ethical reasons on the day of exposure
- No mortality occurred in the animals exposed to 1 mg/L
Clinical signs:
other: - For the exposure to 5 mg/L, labored respiration was seen during exposure. After exposure, lethargy, hunched and/or flat posture, slow breathing, laboured respiration, piloerection, pale skin and/or ptosis were seen among the animals on Day 1 - For the e
Body weight:
Overall body weight gain in surviving males and females was within the range expected for rats of this strain and age used in this type of study.
Gross pathology:
- For the exposure to 5 mg/L, macroscopic post mortem examination of the animals that were found dead or sacrificed for ethical reasons during the study revealed that the upper part of the trachea was blocked with hard white contents. The lungs showed many dark red foci
- For the exposure to 1 mg/L, no abnormalities were found at macroscopic post mortem examination of the animals

Any other information on results incl. tables

- For the exposure to 5 mg/L, the mean actual concentration was 4.7 ± 0.8 mg/L. The nominal concentration was 25.9 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 18%

- For the exposure to 1 mg/L, the mean actual concentration was 1.1 ± 0.2 mg/L. The nominal concentration was 5.0 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 22%

- The concentration measurements equally distributed over time showed that the substance concentrations were sufficiently stable

Applicant's summary and conclusion

Interpretation of results:
practically nontoxic
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
In an acute inhalation study in rats, Melam, CAS No 3576-88-3 was administered as a dust by inhalation for 4 hours to two groups of five male and five female Wistar rats at 5 mg/L and 1 mg/L. None of the animals survived the exposure to 5 mg/L and therefore a 4h-LC50 of >1 mg/L was found. Deposits of the test substance in the upper trachea for the high dose group animals might have impaired the respiration in these animals and contributed to their mortality. Based on this result, the substance was classified by the authors as harmful (Category 4) according to the CLP Regulation.
But when applying the OECD Guidance Document on Acute Inhalation Toxicity of 2009, Section 51, it is concluded that the effects seen at a concentration of 5 mg/L, which according to OECD "greatly exceeds real-world human exposure", are probably artefacts, not caused by toxicity, but by aggregation of the hygroscopic melam particles in the airway. Therefore no classification as to acute inhalation is proposed.
Executive summary:

In an acute inhalation study conducted in accordance with OECD 403 and GLP, Melam was administered as an dust by inhalation for 4 hours to two groups of five male and five female Wistar rats at 5 mg/L and 1 mg/L.

Animals were subjected to daily observations and determination of body weights on Days 1, 2, 4, 8 and 15. Macroscopic examination was performed on the day of death or after terminal sacrifice (day 15).

Results:

For the exposure to 5 mg/L, the mean actual concentration was 4.7 ± 0.8 mg/L. The nominal concentration was 25.9 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 18%. For the exposure to 1 mg/L, the mean actual concentration was 1.1 ± 0.2 mg/L. The nominal concentration was 5.0 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 22%. The concentration measurements equally distributed over time showed that the substance concentrations were sufficiently stable

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice. For the exposure to 5 mg/L, the MMAD was 3.7 µm (gsd 1.6) and 3.5 µm (gsd 1.8) respectively. For the exposure to 1 mg/L, the MMAD was 3.3 µm (gsd 1.9) and 3.7 µm (gsd 1.8) respectively.

All animals exposed to 5 mg/L were found dead or sacrificed for ethical reasons on the day of exposure. No mortality occurred in the animals exposed to 1 mg/L. For the exposure to 5 mg/L, labored respiration was seen during exposure. After exposure, lethargy, hunched and/or flat posture, slow breathing, laboured respiration, piloerection, pale skin and/or ptosis were seen among the animals on Day 1. For the exposure to 1 mg/L, no clinical signs were seen during exposure. After exposure, lethargy, hunched, slow breathing and piloerection were seen in all animals between Days 1 and/or 4. Overall body weight gain in surviving males and females was within the range expected for rats of this strain and age used in this type of study.

For the exposure to 5 mg/L, macroscopic post mortem examination of the animals that were found dead or sacrificed for ethical reasons during the study revealed that the upper part of the trachea was blocked with hard white contents. The lungs showed many dark red foci. The toxicological relevance can be doubted since deposits of the test substance in the upper trachea were seen. This physical obstruction might have impaired the respiration in these animals and contributed to their mortality. However, the possibility that systemic toxicity contributed to the mortality cannot be excluded, according to the authors.

For the exposure to 1 mg/L, no abnormalities were found at macroscopic post mortem examination of the animals. In this acute inhalation study in rats, a 4h-LC50 of >1 mg/L was therefore found.

It was considered by the author that the inhalatory LC50, 4h value of MELAM in Wistar rats exceeds 1 mg/L.

For classification, the OECD Guidance Document on Acute Inhalation Toxicity Testing, Series on Testing and Assessment Number 39, 2009, Section 51 was taken into account additionally:

"Achieving the GHS limit concentration of 5 mg/L is technically challenging for most aerosols and greatly exceeds real-world human exposure. It can be difficult or impossible to generate a respirable (MMAD of 1-4 µm) liquid or solid aerosol at this concentration without encountering experimental shortcomings. As aerosol concentration increases, particle size also increases due to the aggregation of solid particles or coalescing of liquid particles. ... At very high concentrations, dry powder aerosols ... tend to form conglomerates in the proximal nose causing physical obstruction of the animals airways (e.g., dust loading) and impaired respiration which may be misdiagnosed as a toxic effect. ...". It is therefore concluded by the registrant that the obstructions observed are an artefact and not caused by toxicity. Therefore also, no classification as to acute inhalation is proposed for melam.