1,2-dihydro-3H-1,2,4-triazol-3-one

Endpoint summary

Description of key information

The molecular initiating event of skin sensitization pathway covering Direct Peptide Reactivity Assay (DPRA) (OECD 442C, 2015) reported triazolone to be negative (Fleet 2018, Envigo Ref. GD06BG).

The second key event, which takes place in the keratinocytes the KeratinoSensTM test method (OECD 442D, 2015) reported triazolone to be negative also (Belot 2018, Envigo Ref. LP04VC-17ENV18).

However, in vitro Human Cell Line Activation Test (h-CLAT) (OECD 442E, 2017) considered triazolone positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP) (Roth 2018, Envigo Ref. JK61FM).

The QSAR prediction suggested Triazolone to be a skin sensitizer

As weight of evidence of the in silico and in chemico/in vitro assessments it was concluded that triazolone could be a skin sensitizer. Therefore, to assess the skin sensitization potency of the assumed sensitizer triazolone, a LLNA study (OECD 429) was justified.

In a Local Lympn Node Assay carried out in accordance with OECD Guideline 429, groups of four mice were treated with the test item at concentrations of 2.5%, 5% or 10% w/w in dimethyl sulfoxide. 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).

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (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.

There were no deaths and no signs of systemic toxicity were noted in the test or control animals during the test. The body weight change of the test animals was comparable to that observed in the corresponding control group animals over the same period.

The Stimulation Index (S.I.))expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group was found to be 0.5, 0.82 and 0.57 for test concnetrations of 2.5%, 5% and 10%, respectively.  

As the S.I. was less than 3 at all concentrations tested, the test item was considered to be a non-sensitizer under the conditions of the test.

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 Aug 2018 - 22 Aug 2018

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)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch: 0727/16
Purity: 99%

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited
- Females: nulliparous and non-pregnant
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: randomly allocated to cages
- Diet (e.g. ad libitum): Free access to food
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

Vehicle:

dimethyl sulphoxide

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: freshly prepared as a solution in dimethyl sulfoxide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
- Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a maximum attainable concentration of 10% w/w in dimethyl sulfoxide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily.
Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
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. 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 sensitization.

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item at concentrations of 2.5%, 5% or 10% w/w in dimethyl sulfoxide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. 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 tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

- Criteria used to consider a positive response: Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

not specified

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in dimethyl sulphoxide = 25
Stimulation index = 8.47
Result = Positive

α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

Key result

Parameter:

SI

Remarks:

2.5% w/w

Value:

0.5

Test group / Remarks:

Negative

Key result

Parameter:

SI

Remarks:

5% w/w

Value:

0.82

Test group / Remarks:

Negative

Key result

Parameter:

SI

Remarks:

10% w/w

Value:

0.57

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 control animals during the test.

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

Table 1. Results from main test (stimulation index)

Concentration (%w/w) in dimethyl sulfoxide	Stimulation Index	Result
2.5	0.50	Negative
5	0.82	Negative
10	0.57	Negative

The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitizer under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)