2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide

Endpoint summary

Description of key information

Determination of the skin sensitisation potential of CDA-1 was carried out with the in vitro tests and followed up with an in vivo LLNA test.

In the OECD 442D ARE-Nrf2 Luciferase Test (18ENV18, XCellR8, 2018) the human skin sensitisation potential of CDA-1 was assessed using the validated in vitro method: the KeratinoSensTM test to determine keratinocyte activation. CDA-1 was classified as positive according to the KeratinoSens prediction model.

A second in vitro test (GS40HP, Envigo CRS GmbH, 2018) was performed to assess the skin sensitisation potential of CDA-1. The Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of CDA-1 which formed a stable suspension/dispersion in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The test item was considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Due to the low solubility of the test item and pH sensitivity issues, the third in vitro test, OECD 442C Direct Peptide Reactivity Assay could not be performed.

Based on the above information the skin sensitisation hazard could not be determined from the available in vitro tests. Therefore, the in vivo local lymph node assay (LLNA) test ( MB52RQ, Envigo, 2018) has been conducted to conclude on the classification. The test was designed to assess the skin sensitisation potential (delayed type hypersensitivity) of the test item in the mouse following topical application to the dorsal surface of the ear.

In this study Stimulation Indices (S.I.) of 1.26, 1.11, and 1.25 were determined with the test item at concentrations of 5, 10, and 25% in PG, respectively. Therefore the test item CDA-1 was not a skin sensitiser under the test conditions of this study. The result led to a conclusion that the substance CDA-1 is not a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 July 2018 - 26 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT).

Version / remarks:

October 2017

Deviations:

yes

Remarks:

The cytotoxicity measurement and est of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry. The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor/ 102Z5
- Expiration date of the lot/batch: 31 July 2018
- Purity: 99.9%

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design: The purpose of the Human Cell Line Activation Test (h-CLAT) is to assess the skin sensitising potential of CDA-1 in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The test item concentrations for the main experiment (h-CLAT) of CDA-1 are determined by XTT tests.
The highest test item concentration for the main experiment (h CLAT) of CDA-1 was previously determined by two XTT tests
The maximum concentration of test item was 5000 µg/mL in culture medium, as tested by a solubility test.

For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 µg/mL.

- The following concentrations of the test item were tested in the main experiments (h-CLAT):
43, 51, 61, 74, 89, 106, 128 and 153 µg/mL
The test item with a log Pow of 1.41 was tested in 3 independent runs.

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first h-CLAT run exceeded the positive criteria.

Run / experiment:

other: 1

Parameter:

other: The RFI of CD86 is greater or equal than 150%

Value:

150

Negative controls validity:

not valid

Positive controls validity:

not valid

Run / experiment:

other: 1

Parameter:

other: The RFI of CD54 is greater or equal than 200%

Value:

200

Negative controls validity:

not valid

Positive controls validity:

not valid

Run / experiment:

other: 2

Parameter:

other: The RFI of CD86 is greater or equal than 150%

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 2

Parameter:

other: The RFI of CD54 is greater or equal than 200%

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: The RFI of CD86 is greater or equal than 150%

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: The RFI of CD54 is greater or equal than 200%

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of the first run. However, the RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in the second and third independent run. Therefore the h CLAT prediction is considered negative for the tested test item in this h-CLAT

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item CDA-1 with a log Pow of 1.41 did not activate THP-1 cells up to a concentration of 106 µg/mL (limited by observed precipitations in the two highest concentrations of all three independent runs) under the test conditions of this study. Therefore the test item is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 June 2018 to 22 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor/ 102Z5
- Expiration date of the lot/batch: 31 Jul 2018

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Positive control results:

All of the acceptance criteria were met, and there was a dose-dependent increase of induction with the Positive Control therefore, the results are considered as valid.

Run / experiment:

other: 1

Parameter:

other: luciferase induction

Value:

1.5

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 2

Parameter:

other: luciferase induction

Value:

1.5

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: luciferase induction

Value:

1.5

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Remarks:

The KeratinoSensTM test's method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by the UN GHS, for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical into UN GHS category 1.

Conclusions:

The test item was classified as positive using the KeratinoSens prediction model.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September 2018 - 18 September 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)

Version / remarks:

adopted 22 July 2010

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

updated 06 July 2012

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor/102Z5
- Purity: 99.9%
- Expiration date of the lot/batch: 31 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, in the dark
(only valid for storage, may be used/formulated in light)
- Stability in Solvent: Not indicated by the Sponsor

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age (at beginning of treatment): Pre-test: 8 - 9 weeks , Main study: 9 - 10 weeks

- Weight at study initiation: Pre-test: 17.6 - 18.3g, Main study: 17.5 - 22.8g
- Housing: In groups; All animals belonging to the same experimental group were kept in one cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): approx. 45-65% (except for deviation*)
* The relative humidity in the animal room was between approximately 33-65 % instead of 45–65% for several hours.
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

5, 10 and 25%

No. of animals per dose:

4 (main test)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 25 suspension in Propylene glycol (PG)
- Irritation: No signs
- Systemic toxicity: No signs
- Ear thickness measurements: Yes
- Erythema scores: No visible erythema

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: B42
- Criteria used to consider a positive response:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.
The test item was placed into a mortar on a tared balance and PG was added whilst grinding (weight per weight).
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment by stirring.
The preparations were made freshly before each dosing occasion.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Positive control substance(s):

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

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2018.

Key result

Parameter:

SI

Value:

1.26

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

1.11

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.25

Test group / Remarks:

25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA :
The precipitates were resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
DETAILS ON STIMULATION INDEX CALCULATION
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
EC3 CALCULATION :
The EC3 value could not be calculated, since all S.I.´s were below the threshold value of 3.
CLINICAL OBSERVATIONS:
No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.
BODY WEIGHTS:
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item CDA-1 was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test item CDA-1 was not a skin sensitiser under the in vivo LLNA test conditions (MB52RQ, Envigo, 2018) therefore it is concluded that the test item CDA-1 is not classified for the skin sensitisation hazard.