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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2001 to 1 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICCVAM NIH No. 99-4494 (The Murine Local Lymph Node Assay: a Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds)

Deviations:

not specified

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

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 [1,5c] pyrimidine)
- Appearance: yellowish solid powder

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 weeks
- Weight at study initiation: 18.1 - 22.2 g
- Housing: 2 per cage for a two day period then individually thereafter. Cages have wire mesh floors and are suspended above catch pans.
- Diet: Certified Rodent Diet, ad libitum
- Water: municipal water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 - 22.0 °C
- Humidity (%): 47.6 - 52.8 %
- Air changes (per hr): 12 - 15 air exchanges per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark (lights on 0600 to 1800)

IN-LIFE DATES: From 27 April 2001 to 1 May 2001

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0.3, 1 and 3 % test material in N,N,-dimethylformamide (DMF)

No. of animals per dose:

6 mice per dose

Details on study design:

SCREEN STUDY
A screen study was conducted to determine the minimally irritating concentration (MIC) - the highest concentration that produces approximately a 10 % ear swelling response.
- Compound solubility: the test material was not soluble in acetone:olive oil; therefore, DMF was the vehicle of choice for this study.
- Dosing solutions: the maximum amount of test material that could be suspended in DMF was 3.5 %. Therefore, concentrations for the screen were set at 3.5, 1.2, 0.35, 0.12, 0.035 and 0.012 % test material in DMF.
- Method: for the screen, six animals per group received 12.5 µL of the appropriate dosing solution on the dorsal and ventral portions of the left ear and an equal amount of DMF on the dorsal and ventral portions of the right ear. Prior to the administration of the dosing solutions and 24 hours after the last application, the thickness of each ear was measured using a micrometer. The MIC for the subsequent LLNA was set at 3.0 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Groups of 6 mice were topically exposed to one of three suspensions of test material in DMF (0.3, 1 and 3 %) for three consecutive days. Additional groups of 6 animals were exposed to the vehicle alone or a positive control. All mice received 12.5 µL of the appropriate treatment on the dorsal and ventral portion of both ears.
Five days following the initiation of the LLNA, all mice received an intravenous injection, via the lateral tail vein, of 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Approximately 5 hours later, the mice were sacrificed and the draining auricular lymph nodes were excised and pooled for each mouse. A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregation in PBS. The lymph node cells were washed twice in PBS and were placed in 5 % trichloroacetic acid (TCA) for approximately 70 hours. The cells were spun down and reconstituted in 5 % TCA and the tritiated thymidine incorporation was measured on a beta-scintillation counter as disintegrations per minute (dpm) per mouse.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE
A test material with a Stimulation Index of 3 or more can be considered a skin sensitiser.

Positive control substance(s):

other: 1 % dinitrochlorobenzene in DMF

Statistics:

The positive controls and treatment groups were compared to the vehicle control group using a one-way analysis of variance.

Results and discussion

Positive control results:

Animals treated with the positive control produced a SI of 30.9 ± 4.6, which is consistent with the material being a known contact sensitiser.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Treatment with the vehicle, DMF, resulted in a SI of 1.0 ± 0.1. Animals treated with 0.3, 1 or 3 % test material in DMF all produced an SI of less than 3. See Tables 1 and 2.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 1.

Any other information on results incl. tables

Table 1: Individual dpm and SI

Treatment	Animal no.	dpm	SI
0.3 % test material in DMF	2213	848.15	3.53
	2214	272.88	1.14
	2215	235.68	0.98
	2216	460.91	1.92
	2217	79.66	0.33
	2218	390.47	1.63
	Average	381.29	1.59
	Standard error	107.8	0.4
1 % test material in DMF	2219	393.59	1.64
	2220	205.07	0.85
	2221	386.45	1.61
	2222	No sample*	No sample*
	2223	237.98	0.99
	2224	274.95	1.15
	Average	299.61	1.25
	Standard error	38.5	0.2
3 % test material in DMF	2225	463.50	1.93
	2226	1175.13	4.90
	2227	146.38	0.61
	2228	178.37	0.74
	2229	382.07	1.59
	2230	420.53	1.75
	Average	461.00	1.92
	Standard error	152.4	0.6
Vehicle control (DMF)	2201	162.86	0.68
	2202	331.29	1.38
	2203	112.98	0.47
	2204	231.19	0.96
	2205	321.86	1.34
	2206	279.76	1.17
	Average	239.99	1.00
	Standard error	36.0	0.1
1.0 % dinitrochlorobenzene in DMF	2207	6951.56	28.97
	2208	12011.33	50.05
	2209	5908.15	24.62
	2209	8697.52	36.24
	2210	3999.27	16.66
	2211	6868.63	28.62
	Average	7406.24	30.86#
	Standard error	1114.1	4.6

An average background dpm of 82.80 was subtracted from each dpm determination

*sample tube broken

# significantly different from the vehicle control

Body Weights

Only 7 of the 18 animals treated with test material gained weight over the course of the study, There was no explanation for the body weight loss observed over the course of this study (see Table 2).

Table 2: Summary of Stimulation Index and % Body Weight Gain

Treatment group	SI ± SE	% Body Weight Gain ± SE
Vehicle	1.0 ± 0.1	-0.2 ± 1.4
1.0 % Dinitrochlorobenzene*	30.9 ± 4.6#	-0.4 ± 1.5
3 % Test material*	1.6 ± 0.4	0.6 ± 1.1
1 % Test material*	1.3 ± 0.2	0.4 ± 1.2
0.3 % Test material*	1.9 ± 0.6	-1.5 ± 0.9

* in DMF

# significantly different from the vehicle control

SI = Stimulation index

SE = Standard error

Irritation

The ear swelling response was variable but no treatment produced ear swelling much above 10 %. Treatment with the test material also produced no erythema.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the study the test material was not considered to be a a contact sensitiser at concentrations ≤3 %.

Executive summary:

A study was conducted to assess the test material as a potential contact sensitiser to female BALB/c mice in a Local Lymph Node Assay (LLNA) in accordance with the standardised guideline ICCVAM NIH No. 99-4494.

Following a screening study to assess the minimum irritating concentration (MIC), the LLNA study was performed using 3, 1 and 0.3 % test material in DMF. The test material (12.5 µL) was applied to the dorsal and ventral portion of the ears of six mice once a day for three consecutive days. In addition, six mice per group were treated with the vehicle or a positive control, 1 % dinitrochlorobenzene in DMF.

Five days following the initiation of the LLNA, all mice received intravenous injections, via their lateral tail vein, of 20 µCi of tritiated thymidine in 250 µL of phosphate buffer saline. Five hours later, the mice were sacrificed and their auricular lymph nodes were excised. Single cell suspensions of the lymph nodes were prepared and the radioisotope incorporation was measured on a beta-scintillation counter as disintegrations per minute (dpm) per mouse.

The mice treated with DMF produced a SI of 1.0 ± 0.1. The proliferative response of the animals treated with 1 % dinitrochlorobenzene produced a SI of 30.9 ± 4.6 which is consistent with a positive response and was significantly different from the mice treated with DMF. The SIs of mice treated with 3, 1 and 0.3 % of the test material in DMF were all less than 3.0.

Therefore, under the conditions of this study, the test material is not considered to be a contact sensitiser at concentrations ≤3 %.