2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

Endpoint summary

Description of key information

A study was performed to assess the skin sensitisation potential of the test substance according to the OECD Guideline 429. Under the conditions of the  assay, the test substance, tested in a suitable vehicle was shown not to have skin sensitisation potential in the local lymph node assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2016 to 21 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females (if applicable) nulliparous and non-pregnant: YES
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes (supplied Datesand Ltd, PO Box 45, Manchester).
- Diet (e.g. ad libitum): ad libtum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): Appproximately 15 changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light, 12 hours dark

Vehicle:

other: 1% Pluronic L92 in distilled water

Concentration:

0 (vehicle control) 10, 25 and 50 % w/w

No. of animals per dose:

Four per dose

Details on study design:

PRE-SCREEN TESTS:
- Ear thickness measurements: The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
Three groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in 1% pluronic L92 in distilled water. On day 6, all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.
- Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Terminal procedures
Five hours following the administration of 3HTdR, all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group, the draining auricular lymph nodes were excised and processed. 3HTdR incorporation was measured by β-scintillation counting.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the vehicle control nodes (Stimulation Index). The test item would be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to vehicle control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation would be classified as a ‘non-sensitiser’.

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the disposable pipette tip. A further group (vehicle control group) of four mice received the vehicle alone in the same manner.

Positive control substance(s):

other: No concurrent positive contol for the study. Test responsiveness is assured in the laboratory by periodicaly running positive control studies.

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships.
Data were first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data were both normally distributed and had homogeneity of variances were met, a parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Positive control results:

Results from a separate the positive control study run the month previous to this study demonstrated that the test system was responding as expected.

Key result

Parameter:

SI

Remarks:

concentration: 10% w/w

Value:

1.28

Test group / Remarks:

Negative

Key result

Parameter:

SI

Remarks:

concentration: 25% w/w

Value:

1.19

Test group / Remarks:

Negative

Key result

Parameter:

SI

Remarks:

concentration: 50% w/w

Value:

1.16

Test group / Remarks:

Negative

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or vehicle control animals during the test.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding vehicle control group animals over the same period.

Preliminary Screening Test

The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.

Interpretation of results:

GHS criteria not met

Conclusions:

A study was performed to assess the skin sensitisation potential of the test substance according to the OECD Guideline 429. Under the conditions of the assay, the test substance, tested in a suitable vehicle was shown not to have skin sensitisation potential in the lymph node assay.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test substance in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear, according to the OECD Guideline 429. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, which is the maximum OECD recommended concentration for solid test items, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay (LLNA). Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a suspension in 1% pluronic L92 in distilled water at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

No mortality or signs of systemic toxicity was observed in the test item treated animals during the study. No treatment related effects were observed on body weight. The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 1.28, 1.19 and 1.16 for the 10, 25 and 50%w/w test concentrations, respectively.

In conclusion, under the conditions of the assay, the test substance tested in a suitable vehicle was shown not to have skin sensitisation potential in the lymph node assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was found to be not sensitising in a reliable and valid local lymph node assay in accordance with OECD TG 429. It can be concluded that the substance does not need to be classified in accordance with Regulation (EC) No. 1272/2008.