(1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21st July 2021 to 13th August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™. (Last update: 23 July 2018).

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSens™ assay, which is recommended in international guidelines (e.g. OECD 442D).

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid

EC Number:

955-024-2

Cas Number:

2755812-45-2

Molecular formula:

C16 H23 F3 N2 O4

IUPAC Name:

(1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid

Test material form:

solid

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

A correction of 1.18% was made for the composition/purity of the test item.
A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (colourless solution). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax showed no precipitation. This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
In the main experiments the test item was dissolved in DMSO at 200 mM (colourless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 uM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.

ENVIRONMENTAL CONDITIONS
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 - 95%), containing 5.0 + 0.5% CO2 in air in the dark at 37.0 + 1.0°C (actual range 36.8 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO percentage was monitored once on each working day.

SUBCULTURING
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock.

PLATING OF CELLS
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+22 in experiment | and P+24 in experiment 2.

TREATMENT OF CELLS
The medium was removed and replaced with fresh exposure medium (150 wL culture medium containing serum but without Geneticin) to which 50 uL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours + 1 h at 37+1.0°C in the presence of 5% CO2.

LUCIFERASE ACTIVITY MEASUREMENT
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 wL of the SteadyGlo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

CYTOTOXICITY ASSESSMENT
For the KeratinoSens™ cell viability assay, medium was replaced after the 48 hour exposure time with fresh DMEM Glutamax containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide and cells were incubated for 3 - 4 hours at 37°C + 1.0°C in the presence of 5% CO. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Vehicle / solvent control:

DMSO

Negative control:

other: On each plate three blank wells were tested (no cells and no treatment).

Positive control:

other: Ethylene dimethacrylate glycol

Results and discussion

Positive control results:

Experiment 1
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.21 and the EC;.s 29 uM.





Experiment 2
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.03 and the EC;.s 37 uM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.42

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.46

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The KeratinoSens™ test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant equal or above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 uM).
b) The EC;,s of the positive control should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 uM should be higher than 2-fold. If the latter criterion is not fulfilled, the doseresponse of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.

In compliance with the OECD Guideline TG 442D. In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. (Adopted June, 2018), a KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
1. The Imax is equal or higher than (©) 1.5-fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity > 1.5-fold (1.¢. at the EC;.5 determining concentration)
3. The EC; 5 value is less than (<) 1000 uM (or < 200 ng/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 uM or 200 ug/mL and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, PF-07320267 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of PF-07320267 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay.
The study procedures described in this report were based on the most recent OECD guideline.
Batch JR-C200917012-D21001 of the test item was a white to off-white solid. A correction factor of 1.18 was used to correct for the purity. The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 — 2000 uM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.
Both tests passed the acceptance criteria:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was within two standard deviations of the historical mean (29 uM and 37 uM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 uM was higher than 2-fold (3.21-fold and 3.03-fold in experiment | and 2, respectively).
Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (11% in experiment 1 and 2).
Overall, it is concluded that the test conditions were adequate and that the test system functioned properly.
The test item showed no toxicity (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC).s value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.50-fold (1.496-fold without rounding off) and 1.42-fold in experiment 1 and 2 respectively.
The test item is classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 uM.
In conclusion, PF-07320267 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.