[1,2,4]Triazolo[1,5-c]pyrimidine, 2,2'-dithiobis[5-ethoxy-7-fluoro-

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 January 1994 to 31 January 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

(see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

yes

Remarks:

(see below)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 81-3 (Acute inhalation toxicity)

Deviations:

yes

Remarks:

(see below)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The Acute Inhalation Toxicity Study Guidelines specified by the Japan Ministry of Agriculture, Forestry and Fisheries (MAFF, 1985)

Deviations:

yes

Remarks:

(see below)

Principles of method if other than guideline:

The animals were exposed to a limit test concentration of 6.07 mg/L. This concentration exceeded the limit test concentration of 5 mg/L however the mean mass median aerodynamic diameter for the aerosol was 3.02 µm, which was within the 1 to 4µm targeted range for a respirable atmosphere.

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

EC Number:

695-187-4

Cas Number:

166524-75-0

Molecular formula:

C14H12F2N8O2S2

IUPAC Name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 2,2'-Dithiobis(5-ethoxy-7-fluoro[1,2,4]triazolo [1,5-c]pyrimidine)
- Synonym: DEDS
- Appearance: light beige powder

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: The rats were approximately 12 weeks old when exposed to the test material.
- Weight at study initiation: Mean male bodyweight at the start of the test: 230.0 g (standard deviation 8.8); mean female bodyweight at the start of the test: 151.3 g (standard deviation 3.1).
- Fasting period before study: No.
- Housing: The animals were housed two per cage in stainless steel wire cages during acclimation. Animals were singly housed during the 2-week post-exposure period.
- Diet (e.g. ad libitum): Certified Rodent Chow was available ad libitum except during exposure.
- Water (e.g. ad libitum): Tap water (municipal water supply) was available ad libitum except during exposure.
- Acclimation period: 3 weeks prior to exposure.

ENVIRONMENTAL CONDITIONS
Animals were housed in a room designed to maintain adequate environmental conditions concerning temperature and relative humidity and regulated for the specific species under study
- Photoperiod (hrs dark / hrs light): A 12-hour photoperiod was employed throughout the study.

IN-LIFE DATES: From: 7 January 1994 To: 31 January 1994

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Chambers
A 42 litre ADG nose-only chamber modified by the testing laboratory (30 cm diameter x 60 cm high) was used for this study. Compressed air supplied to the chamber was controlled by a system designed to maintain temperature at approximately 22 °C. The airflow was maintained at approximately 30 litres per minute, which was sufficient to provide the normal concentration of oxygen to the animals. The chamber was operated at a slight negative pressure relative to the surrounding area. Rats were exposed to the test material by a nose-only technique.

- Generation System
Aerosols were generated from powdered test material using a Jet Mill (Model 00 Jet-O-Mizer, Fluid Energy Aljet, Plumsteadville, PA). The nature of the test material was such that it easily and quickly bridged (would not flow to the auger screw) upon standing. Even with the feeder's built-in agitation, the test material would not feed once hand stirring ceased. Therefore, to maintain consistent chamber concentrations and feeding of the test material through the screw feeder to the jet mill, the contents of the feeder were continuously hand stirred. Also, to prevent any possible plugging of the jet mill, the test material was sieved prior to use using a U.S.A. standard testing sieve (opening 500 micrometers, 32 mesh).

- Chamber Monitoring
The air flow through the chamber was determined with a manometer. The manometer was calibrated with a gas meter (Model DTM-115, Singer Aluminum Diaphragm Meter, American Meter Division, Philadelphia, PA, USA) prior to the start of the study. Ambient room temperature, chamber humidity and chamber air flow values were recorded every 30 minutes during the 4 hour exposure period.
Chamber temperature, relative humidity and airflow were within expected values. Chamber relative humidity values in the range reported resulted from the use of compressed air as the total air supply through the dust generator to the chamber.

TEST ATMOSPHERE
- Mass Concentration
The mass concentration of aerosol present in the chamber was determined gravimetrically four times during the 4 hour exposure period. Each gravimetric sample was taken by drawing the chamber atmosphere, at a rate of 3 litres per minute for two minutes, through a vertical stainless steel tube which projected into the animal breathing zone and collecting the aerosol particles on a pre-weighed 0.45 micron Teflon filter (Schleicher and Schuell, Keene, NH). After each atmosphere sampling, the filter was reweighed to obtain the total weight of the particles. The time-weighted average (TWA) exposure concentration was calculated from these gravimetric measurements.

- Particle Size Determination
The aerodynamic particle size was determined twice during the exposure period. Each sample was taken by drawing samples from the animal breathing zone, at a rate of 3 litres per minute for two minutes, through a six-stage Cascade Impactor (Model 266, Sierra Instruments, Carmel Valley, CA, USA). The mass median aerodynamic diameter (MMAD) and geometric standard deviation (σg) were determined for each sample as well as the average of the two samples.

- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD of 3.02 µm, GSD 2.48.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

6.07 mg/L

No. of animals per sex per dose:

5 animals per sex

Control animals:

no

Details on study design:

The animals were acclimated to the nose cones for a single four hour period on on the day preceding exposure to the test material.

- Duration of observation period following administration: 14 days.
- Frequency of observations and weighing: Animals were weighed and examined prior to exposure to the test material. All animals were observed during the exposure period and daily during the two-week post-exposure period. The observations included an evaluation of the fur, eyes, mucous membranes and respiration. Behaviour pattern and nervous system activity were also assessed by specific observation for tremors, convulsions, salivation, lacrimation and diarrhoea, as well as lethargy and other signs of altered central nervous system function. An additional daily observation and routine monitoring on weekends were limited to husbandry procedures required to ensure the availability of food and water. All rats were weighed on test days 2, 4, 8, 11, and 15 during the two-week post-exposure period.
- Necropsy of survivors performed: yes. All rats were submitted for a complete necropsy examination on test day 15. The rats were anaesthetised with methoxyflurane and euthanised. The necropsy included examination of the eyes with a microscope slide using fluorescent illumination. No tissues were saved.

Statistics:

Means and standard deviations of animal bodyweights and chamber temperatures, relative humidities and airflows and chamber concentration (mean only) were calculated for descriptive purposes.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 6.07 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: MMAD 3.02 µm

Mortality:

No mortality was observed.

Clinical signs:

other: No clinical effects were observed during the exposure period or throughout the 14 day post-exposure period.

Body weight:

Mean bodyweights for male and female rats on test day 2 were decreased approximately 6 % from pre-exposure values. This initial weight loss is typical of the weight losses historically observed in this age and strain of rats exposed to non-lethal test atmospheres by the nose-only techniques used in this study. The animals subsequently gained weight throughout the remainder of the 2-week post-exposure period.

Gross pathology:

One female rat had a herniated left liver and a distended ovarian bursa both of which were spontaneous and not considered treatment-related. There were no visible lesions attributable to exposure noted in any of the animals necropsied on test day 15.

Any other information on results incl. tables

Table 1 Chamber Atmosphere Concentrations During Exposure

Exposure Time (minutes)	Gravimetric Sample Concentration (mg/L)*	TWA Analytical Concentration (mg/L)	Temperature (°C)**	Relative Humidity (%)**	Chamber Air Flow (LPM)***
57	6.23	6.07	22.0 ± 0.0	10.1 ± 1.0	30 ± 0
141	5.46
213	6.64

TWA: Time-weighted average

*Flow rates for gravimetric sampling were 3 litres per minute for 2 minutes

**Mean ± standard deviation, N = 8

***Mean ± standard deviation, N = 9

 

Table 2 Particle Size Data Determined Gravimetrically

Mass Obtained on Different Stages of the Cascade Impactor (mg)
Effective Cut-off Diameter (µm)	Mean*	Dataset A**	Dataset B**
17 11 6.1 3 1.3 0.68 0.45	0.294 0.399 1.409 4.406 2.552 0.507 0.930	0.281 0.428 1.494 4.897 2.781 0.502 1.036	0.306 0.369 1.324 3.915 2.323 0.511 0.824
Sum	10.497	11.419	9.572
Cumulative Percent of Aerosol Particles Less Than Stated Size (%)
Effective Cut-off Diameter (µm)	Mean*	Dataset A**	Dataset B**
0.68 1.3 3 6.1 11 17	8.86 13.69 38.00 79.97 93.39 97.19	9.07 13.47 37.82 80.70 93.78 97.53	8.61 13.95 38.22 79.12 92.95 96.80
MMAD (µm)	3.02	2.95	3.02
Sigma G	2.48	2.37	2.48

* Mean of dataset A and dataset B values

**Flow rates were 3 litres/minute for 2 minutes (Datasets A and B obtained approximately 110 and 172 minutes into exposure).

MMAD assumes particles were from a log normal distribution. Particle size for probit values of 4, 5 and 6 is determined from a linear regression of the data. The MMAD is equivalent to a probit of 5 and the geometric standard deviation is equal to square root (Probit 6/Probit 4).

 

Table 3 Bodyweight Data (g)

Sex	Animal Number	Days on Test
		1	2	4	8	11	15
Male	94A0001 94A0002 94A0003 94A0004 94A0005	217.3 225.2 231.6 238.7 237.0	207.3 212.0 219.0 225.6 218.9	219.4 223.6 228.0 236.6 229.0	234.6 233.5 240.9 247.6 246.1	242.5 243.2 248.7 252.2 252.6	252.1 251.8 258.6 259.2 260.1
	Mean SD N	230.0 8.8 5	216.6 7.1 5	227.3 6.4 5	240.5 6.4 5	247.8 4.8 5	256.4 4.1 5
Female	94A0006 94A0007 94A0008 94A0009 94A0010	148.2 148.4 152.3 151.8 155.6	135.6 144.6 140.0 141.5 148.9	145.3 148.9 149.0 152.0 158.0	149.2 151.1 151.7 154.3 162.3	152.6 152.0 155.1 156.3 161.5	157.3 152.0 158.7 161.2 166.0
	Mean SD N	151.3 3.1 5	142.1 5.0 5	150.6 4.8 5	153.7 5.1 5	155.5 3.8 5	159.0 5.1 5

 

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the 4 hour LC50 of the test material to male and female rats was greater than the concentration of 6.07 mg/L with a particle size distribution MMAD of 3.02 microns. As such, the test material requires no classification in accordance with EU criteria.

Executive summary:

The acute inhalation toxicity of the test material was determined in accordance with the standardised guidelines OECD 403, EU Method B.2, FIFRA Guideline No. 81-3 and the Acute Inhalation Toxicity Study Guidelines specified by the Japan Ministry of Agriculture, Forestry and Fisheries (MAFF, 1985).

A group of 5 male and 5 female Fischer 344 rats was exposed to a limit test concentration of 6.07 mg/L of the test material for four hours in a nose-only inhalation chamber and observed for two weeks post-exposure.

The mean mass median aerodynamic diameter for the aerosol was 3.02 µm, with a geometric standard deviation of 2.48. Approximately 11 % of the total mass of particles was less than 1 micron in size and 80 % of the total mass was less than 6 microns in size.

All animals survived the exposure as well as the two-week post-exposure period. A mean body weight loss of approximately 6 % was noted in both male and female animals on the day following the exposure to the test material. These animals subsequently gained weight without any other exposure-related effects noted.

Under the conditions of this study, the 4 hour LC50 of the test material to male and female rats was greater than the concentration of 6.07 mg/L with a particle size distribution MMAD of 3.02 microns. As such, the test material requires no classification in accordance with EU criteria.