1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

Chamber humidity values lower than recommended due to use of dry air; no impact on study outcome

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

yes

Remarks:

Chamber humidity values lower than recommended due to use of dry air; no impact on study outcome

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japan MAFF, Notification No. 12 Nousan-8147 (2000)

Deviations:

yes

Remarks:

Chamber humidity values lower than recommended due to use of dry air; no impact on study outcome

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

EC Number:

619-749-5

Cas Number:

730979-19-8

Molecular formula:

C16H20FN5

IUPAC Name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: young adults, approx. 2-3 months
- Weight at study initiation: males 184-210 g, females 176-194 g
- Housing: individually in conventional Makrolon Type IIIH cages on low dust wood granulate from SPSS
- Diet (e.g. ad libitum): standard fixed-formula diet KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): municipal tap water from polycarbonate bottles, ad libitum
- Acclimation period: at least 5 days; during this period rats were also acclimated to the restraining tubes (at least 1x2 h and 2x4 h)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 40-60
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

3.35 µm

Geometric standard deviation (GSD):

1.97

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the aerosolized test item in Plexiglas exposure tubes applying a directed-flow nose-only exposure principle. Tubes were chosen that accommodated the animals' size. These tubes were designed so that the rat's tail remained outside the tube, thus restrained-induced hyperthermia can be avoided. The chambers used are commercially available (TSE, 61348 Bad Homburg).

- Exposure chamber volume: about 3.8 L (two-chamber system: inner diameter = 14 cm, outer diameter = 35 cm, height = 25 cm). Details of this modular chamber and its validation have been published previously (Pauluhn, 1994).

- Method of holding animals in test chamber: Restrained in individual exposure tubes chosen to accomodate the animals' size.

- Source and rate of air: Compressed air was supplied by Boge compressors at a pressure of 180 kPa, providing more than 200 air exchanges per hour. Under such test conditions steady state was attained within the first minute of exposure. The ratio between the air supplied and exhausted was chosen so that approximately 80-90% of the supplied air was removed via the exhaust system. The remainder provided adequate dead-space ventilation for the exposure tubes. At each exposure port a minimal air flow rate of 0.75 L/min was provided. During the exposure period air flows were monitored continuously and, if necessary, readjusted to the conditions required. To identify exposure-related effects, comparisons with approriate controls are conducted. Controls were exposed to an atmosphere using essentially similar exposure conditions as were used for the test item (15 L air/min inlet air flow; conditioned dry air; duration of exposure = 1x4 h; 5 males and 5 females per group).

- Method of conditioning air: The compressed air was conditioned (i.e. freed from water, dust and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.

- System of generating particulates/aerosols: Wright-Dust Feeder, used for dry powder dispersion with compressed air (28 L/min). Due to the limited dustiness the test item was micronised first to optimise respirability. Then the test item was first filled into a reservoir of the device and subsequently compressed to a pellet using approximately 1 metric ton by a Carver laboratory press (F.S. Carver INC. Wabash, IN 46992, USA). From this pellet defined amounts of the test item were scraped off with approximately 2.15 revolution/min and were then effectively dispersed using pressurized air (approx. 180 kPa). In order to generate an atmosphere with a MMAD below 4 µm a cyclone was utilized. As this generator uses dry air, the humidity in the inhalation chamber is influenced.

- Method of particle size determination: The samples for the analysis of the particle size distribution were also taken in the vicinity of the breathing zone. During the exposure two samples were taken, which was the maximum feasible due to the gravimetrical samples taken for concentration monitoring. The particle-size distribution was analyzed using an ANDERSEN cascade impactor. The different particle sizes were collected on individual impactor stages (aluminia plates with silicone layer). These plates were subjected to gravimetric analysis using a digital balance. Mass percentages of the different particle sizes were calculated from the mass of particles found on the respective impactor stages and the total mass found. For the determination of the MMAD the mass percentage of a certain size stage was added to the percentages of the stages below and plotted graphically against particle size in a probability scale against a log particle-size scale. The MMAD was determined graphically by drawing a straight line best fitting the plotted points (linear regression); the estimated MMAD is the particle size at which the line crosses the 50% probability mark.

The GSD was calculated as the ratio of the particle sizes at which the linear regression line crosses the 84.1% mark and the 50% mark.

- Treatment of exhaust air: The exhaust air was purified via cotton-wool/HEPA filters. These filters were disposed of by Bayer AG.

- Temperature, humidity, pressure in air chamber: Temperature and humidity control was performed using a computerised system (Hydra, Fluke-Philips). The test atmosphere temperature and humidity were measured at the exposure location using a FTF-sensor (Elka-Elektronik, Lüdenscheid). Data were recorded automatically at intervals of 5 min. Mean temperature was 21.7 °C (22.6 °C control), mean humidity was <5.5% (<5.1% mean control).

TEST ATMOSPHERE
- Brief description of analytical method used: Test substance concentration was determined by gravimetric analysis (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany). The weight of the filters was determined by the use of a digital balance. The number of samples taken was sufficient to characterise the test atmosphere and was adjusted so as to accomodate the sampling duration and/or the need to confirm specific concentration values. Optimally, samples were collected after the equilibrium concentration had been attained in hourly intervals. The integrity and stability of the aerosol generation and exposure system was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA). Samples were taken continuously from the vicinity of the breathing zone.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): clean, conditioned air

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Gravimetric evaluation

Duration of exposure:

4 h

Concentrations:

2300 mg/m³ (2000 mg/m³ target concentration)

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Appearance and behaviour of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were made once a day (morning). Body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter. Individual weights were also recorded at death, if applicable.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs: assessments from restraining tubes were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, and severe respiratory signs). Following exposure, observations were made and recorded systematically; individual records are maintained for each animal. Cage-side observations included, but were not limited to, changes in the skin and fur, eyes, mucus membranes, respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death was recorded as precisely as possible, if applicable. Since these signs can only be assessed adequately from freely moving animals, no specific assessment was performed during exposure while animals were restrained. Each rat was first observed in its home cage and then individually examined. The following reflexes were tested, based on recommendations made by Irwin (1968): visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back). Further examinations comprised body weight and rectal temperature (the latter measured shortly after cessation of exposure, approximately within 30 min after the end of exposure) and gross pathological changes.

Statistics:

Necropsy findings from the respiratory tract of surviving rats, if present, were evaluated using the pair-wise Fisher test after the RxC chi-squared test. The Fisher test was only performed if differences occurred between groups in the RxC chi-squared test or if a frequency value of < 5 was calculated. This procedure was performed in accordance with Gad and Weil (1982). For calculation of the unilateral p value a symmetrical distribution was assumed (p unilateral = (p bilateral)/2).
Means and single standard deviations of body weights were calculated. Mean body weights were also depicted graphically as a function of time. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) was used.
Particle size (MMAD) was determined by linear regression of cumulated mass percentages of the respective size stages plotted against log particle-size including least square analysis. The GSD was determined as the ratio of the particle sizes at which the linear regression line crossed the 84.1% and the 50% mark.
Data of rectal temperature measurements were statistically evaluated using the ANOVA procedure (vide infra).
If calculation of a median lethal concentration (1C50) was possible, it was performed by computer (PC) according to the method of Rosiello et al. (1977) as modified by Pauluhn (1983). This method is based on the maximum-likelihood method of Bliss (1938). If only 2 pairs of values with greater than 0% lethality and less than 100% were available then the first linear approximation was based on these values, and a chi²-homogeneity test was not performed. In this case the interpolated concentration at 50% lethality was designated the approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (1994) was used for calculation, if applicable.

Results and discussion

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2 300 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: maximum technically attainable concentration ensuring an MMAD<4µm

Effect levels 2

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.3 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: maximum technically attainable concentration ensuring an MMAD <4µm

Mortality:

No mortality occurred.

Clinical signs:

other: 2000 mg/m³: Piloerection, ungroomed hair-coat, mydriasis, red tears, bradypnea, laboured breathing patterns, irregular breathing, motility reduced, limp, tremor, high-legged staggering gait, saltatory spasm (clonic), chewing movements, non-specific behavi

Body weight:

Statistical evaluation of the body weight of the rats revealed a significant decrease of mean body weights 1 day after the inhalation exposure of both control and test group, which is more pronounced in the test group; thereafter mean body weights constantly increase in both groups, which is a common observation due to the stress of restraint during exposure. Comparison of body weight development between the control and the test groups revealed significant body weight changes at isolated timepoints. However, the observed body weights of the animals during the course of the study closely resembled those of untreated animals of a comparable age as can be seen in the age-reference data from Harlan-Winkelmann provided in the study. Therefore, the observed effects cannot unequivocally be attributed to the test substance treatment.

Gross pathology:

Individual gross-pathological examinations of the rats indicated a lung with one light colored area in two female control rats. This was of no relevance for the toxicological evaluation of the test item. No findings were made in the males of the control group and all animals of the test group.

Other findings:

- Other observations: Statistical comparison between the control and the exposure group revealed significant decrease of body temperature for the rats of the test group (mean temperature 38.0 °C vs. 34.3 °C in males and 38.0 °C vs. 31.7 °C in females). As restraint is known to affect body temperature in form of hyperthermia this finding has to be attributed to test substance treatment.

Any other information on results incl. tables

Generation conditions and characterisation of chamber atmosphere (Mean values):

	Group 1	Group 2
Target conc. (mg/m³)	0	2000
Gravimetric conc. (mg/m³)	Control air	2300
Dust Generator Type	-	Wright Dust Feeder II
Inlet air flow (L/min)	15	28
Exhaust Air flow (L/min)	13	24
Temperature (mean, °C)	22.6	21.7
Rel. Humidity (mean, %)	<5.1	<5.5
MMAD (µm)	-	3.35
GSD (µm)	-	1.97
Aerosol mass <3 µm (%)	-	43.5
Mass recovered (mg/m³)	-	1975

The real-time aerosol monitoring with the RAS II photometer and filter analyses of the test atmosphere from the breathing zone indicated that the exposure conditions were temporally stable over the 4-h exposure period.

Analysis of the aerosol particle-size distribution from the breathing zone samples demonstrates that the aerosol generated was respirable. Internationally recognized recommendations such as of SOT (1992) were fulfilled, in regard to the requirement MMAD<4 µm.

Conclusion:

The LC50 for acute inhalation toxicity of the test substance was determined to be in excess of 2.3 mg/L for an exposure period of 4 hours. Due to the limited dustiness of the test substance this concentration reflected the maximum technically attainable concentration assuring a MMAD<4µm. Therefore, the test substance does not have to be classified for acute inhalation toxicity according to the criteria of the DSD and the CLP regulation as it did not induce any lethality at this concentration. The test substance has a low inhalation toxicity in rats.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008