cis-N-methyl-N-(1H-pyrrolo[2,3-d]pyrimidin4-yl)cyclobutane-1,3-diamine phosphate (1:1)

Endpoint summary

Description of key information

Skin sensitisation was conducted as per a weight of evidence based approach using DPRA, KeratinoSens and LLNA.

A DEREK assessment, a DPRA and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation(EU)2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. The DEREK assessment yielded no alerts on skin sensitization for the substance and predicted PF-03817968-09 to be a non-sensitizer. The DPRA was negative, as the substance did not show significant covalent binding with lysine and cysteine containing peptides. However, the DPRA could not be tested up to the recommended concentration and this affects the reliability of the outcome. Therefore as a result of this a LLNA in-vivo was considered necessary to assess the skin sensitisation endpoint

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The experimental start date was 03 Dec 2018, and the experimental completion date was 11 Dec 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Run / experiment:

other: SPCC depletion

Parameter:

other: Cysteine 1:10 / Lysine 1:50

Value:

1.7

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Negative: No or minimal reactivity

Run / experiment:

other: SPCL depletion

Parameter:

other: Cysteine 1:10 / Lysine 1:50

Value:

0.6

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Negative: No or minimal reactivity

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.
PF-03817968-09 was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since the DPRA study was performed using a test item stock solution with a concentration of 25 mM instead of the required 100 mM, no firm conclusion on the lack of reactivity can be drawn from this negative result.

Executive summary:

The objective of this study was to determine the reactivity ofPF-03817968-09 towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220nmand 258nm.SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and nonsensitizers. The study procedures described in this report were based on the most recent OECD guideline. No appropriate solvent was found to dissolve the test item at a concentration of 100 mM. Milli-Q water(MQ)was found to be an appropriate solvent to dissolve the test item at a concentration of 25 mM and was, after consultation with the Sponsor, used in this Direct Peptide Reactivity Assay (DPRA) study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and CMQ, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 1.7% SPCC depletion while in the lysine reactivity assay the test item showed 0.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.1% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. PF-03817968-09 was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since the DPRA study was performed using a test item stock solution with a concentration of 25 mM instead of the required 100 mM, no firm conclusion on the lack of reactivity can be drawn from this negative result.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The experimental start date was 29 Mar 2019, and the experimental completion date was 24 Apr 2019

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22 to 23°C with an actual daily mean relative humidity of 40 to 46%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10% w/w, 25% w/w, 40% w/w

No. of animals per dose:

Three groups of five animals were treated with one test item concentration per group.

Positive control substance(s):

other: Propylene glycol

Key result

Parameter:

SI

Value:

ca. 0.9

Variability:

+/- 0.1

Test group / Remarks:

10% w/w

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Key result

Parameter:

SI

Value:

ca. 0.6

Variability:

+/- 0.1

Test group / Remarks:

25% w/w

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Key result

Parameter:

SI

Value:

ca. 0.4

Variability:

+/- 0.1

Test group / Remarks:

40% w/w

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits aSI ≥ 3 when tested up to 40%,PF03817968-09 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give aSI=3) (if any) exceeds 40%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for
testing for contact hypersensitivity (see Appendix 2). Based on these results,PF-03817968-09 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be
classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whetherPF-03817968-09 induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report. The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2010), EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay". EPA, OPPTS 870.2600 (2003) “Skin Sensitization”. Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25% and 40% test item concentration, no signs of systemic toxicity were noted and up to very slight irritation was observed. Therefore, a 40% concentration was selected as highest concentration for use in the main study. In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 40% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Propylene glycol (PG)). Three days after the last exposure, all animals were injected with 3H-methylthymidineand after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index(SI) was subsequently calculated for each group. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 10%, 25% and 40% were 875, 597 and 405 DPM, respectively. The mean DPM/animal value for the vehicle control group was 936 DPM. TheSIvalues calculated for the test item concentrations 10%, 25% and 40% were 0.9, 0.6 and 0.4, respectively. Since there was no indication that the test item elicits aSI ≥ 3 when tested up to 40%,PF03817968-09 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give aSI=3) (if any) exceeds 40%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay is an appropriate model for testing for contact hypersensitivity. Based on these results,PF-03817968-09 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The experimental start date was 23 Nov 2018, and the experimental completion date was 11 Jan 2019.

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)

Qualifier:

according to guideline

Guideline:

other: EURL ECVAMDB-ALMProtocol n° 155: KeratinoSensTM. (Adopted March, 2018)

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

Three independent experiments were performed. The cells were in these experiments
incubated withPF-03817968-09 in a concentration range of 0.98 – 2000 µM (2-fold dilution
steps) in experiment 1 and 1.4 – 125 µM (1.5-fold dilution steps) in experiment 2 and 3 for 48
hours ± 1 h.The activation of the ARE-dependent pathway was assessed by measuring the
luminescence induction compared to the vehicle control. In addition, the viability was
assessed with an MTT assay

Key result

Run / experiment:

other: 0.98 – 2000 µM

Negative controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

PF-03817968-09 showed toxicity (IC30 value of 66 µM and IC50 value of 85 µM). A dose-related induction of the luciferase activity (EC1.5 value of 30 µM) was measured and the maximum luciferase activity induction (Imax) was 7.71-fold.

Key result

Run / experiment:

other: 1.4 – 125 µM

Negative controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

PF-03817968-09 showed toxicity (IC30 value of 25 µM and IC50 value of 42 µM). A dose-related induction of the luciferase activity (EC1.5 value of 29 µM) was measured and the maximum luciferase activity induction (Imax) was 4.88-fold.

Key result

Run / experiment:

other: 1.4 – 125 µM

Negative controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

PF-03817968-09 showed toxicity (IC30 value of 28 µM and IC50 value of 51 µM). A dose-related induction of the luciferase activity (EC1.5 value of 26 µM) was measured and the maximum luciferase activity induction (Imax) was 24.50-fold.

Other effects / acceptance of results:

In the first experiment,PF-03817968-09 showed toxicity (IC30 value of 66 µM and IC50 value
of 85 µM). A dose-related induction of the luciferase activity (EC1.5 value of 30 µM) was
measured and the maximum luciferase activity induction (Imax) was 7.71-fold. Since the
induction ≥ 1.5-fold was observed in the range of toxic to non-toxic concentrations this led to
an individual run conclusion of inconclusive. In the second experiment the test item showed
toxicity (IC30 value of 25 µM and IC50 value of 42 µM). A dose-related induction of the
luciferase activity (EC1.5 value of 29 µM) was measured and the maximum luciferase activity
induction (Imax) was 4.88-fold. However, the induction was only observed at test
concentrations with cell viability < 70%, this led to an individual run conclusion of Negative.
The first and second experiment were not concordant and therefore an additional third
experiment was needed. In the third experiment the test item showed toxicity (IC30 value of
28 µM and IC50 value of 51 µM). A dose-related induction of the luciferase activity (EC1.5
value of 26 µM) was measured and the maximum luciferase activity induction (Imax) was
24.50-fold. Since the induction was only observed at test concentrations with cell viability <
70%, the individual run conclusion was negative. Overall the test item is classified as
negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were
observed at test concentrations with a cell viability of > 70% in 2 out of 3 experiments.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion,PF-03817968-09 is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.

Executive summary:

The objective of this study was to evaluate the ability ofPF-03817968-09 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 GR13472 ofPF-03817968-09 was a white to off-white solid.PF-03817968-09 was dissolved/suspended in dimethyl sulfoxide 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 µM (2-fold dilution series). In the follow up experiments, a more narrow dose-response analysis was performed using a lower dilution factor of 1.5-fold to investigate the induction at 63 µM in experiment 1 in more detail. No precipitate was observed at any dose level tested. Three independent experiments were performed. All experiments 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 (5.5 µM, 104 µM and 29 µM in experiments 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (43.61-fold, 2.23-fold and 2.48-fold in experiment 1, 2 and 3, respectively). • Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.4%, 8.2% and 7.7% in experiment 1, 2 and 3, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly. In the first experiment,PF-03817968-09 showed toxicity (IC30 value of 66 µM and IC50 value of 85 µM). A dose-related induction of the luciferase activity (EC1.5 value of 30 µM) was measured and the maximum luciferase activity induction (Imax) was 7.71-fold. Since the induction ≥ 1.5-fold was observed in the range of toxic to non-toxic concentrations this led to an individual run conclusion of inconclusive. In the second experiment the test item showed toxicity (IC30 value of 25 µM and IC50 value of 42 µM). A dose-related induction of the luciferase activity (EC1.5 value of 29 µM) was measured and the maximum luciferase activity induction (Imax) was 4.88-fold. However, the induction was only observed at test concentrations with cell viability

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

in silico, in chemico and in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: OECD 442E Myeloid U937 Skin Sensitization Test (U-SENSTM)

Qualifier:

no guideline required

Guideline:

other: DEREK NEXUS

GLP compliance:

yes

Type of study:

other: DEREK assessment, Direct Peptide Reactivity Assay, KeratinoSensTM assay.

Parameter:

other: DPRA and KeratinoSen

Remarks:

DPRA was negative but could not be tested up to the recommended concentration. Keratinocytes assay showed activation (ARE)-dependent pathway. Activation or cytotoxicity cannot be determined and outcome not clear. LLNA test required

Remarks on result:

not determinable

Key result

Parameter:

SI

Remarks:

10% concentration

Value:

ca. 0.9

Key result

Parameter:

SI

Remarks:

25% concentration

Value:

ca. 0.6

Key result

Parameter:

SI

Remarks:

40% concentration

Value:

ca. 0.4

Interpretation of results:

study cannot be used for classification

Conclusions:

The DEREK assessment did not yield any alerts on skin sensitization for the structure of the substance and thereforePF-03817968-09 is predicted to be a non-sensitizer. The DPRA was concluded negative because no reactivity ofPF-03817968-09 towards SPCL and SPCC was observed. However, the substance was tested at a four times lower concentration (25mM) than is recommended and therefore the results should be interpreted with care. In the KeratinoSensTM assay the first and second run were inconclusive and negative, respectively. These outcomes were discordant and therefore an additional run was
initiated. The outcome of the third run was negative and thus the KeratinoSensTM assay overall outcome was negative. A closer examination of the individual runs shows a dose dependent increase in activation of keratinocytes at non cytotoxic concentrations. It cannot be reliably determined whether the activation is caused by cells becoming activated or if it is a cellular response to cytotoxic stress. As the mechanism behind the test outcome remains unknown, the results from the KeratinoSensTM assay are not appropriate to assess the skin sensitizing properties for this substance. Further in vitro testing with a U-SENSTM assay is considered not to give additional information as the cytotoxic properties are expected to negatively affect the dendritic cells and preclude a reliable outcome of the assay. Although three negative test results were obtained, the data is found to be insufficient for a reliable and unambiguous conclusion on the skin sensitizing properties ofPF-03817968-09. As further in vitro testing will not lead to a conclusion it is scientifically justified to continue testing in vivo.

Executive summary:

The objective of this study was to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information, including in silico, in chemico and in vitro data. A DEREK assessment, a DPRA and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation(EU)2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. The DEREK assessment yielded no alerts on skin sensitization for the substance and predicted PF-03817968-09 to be a non-sensitizer. The DPRA was negative, as the substance did not show significant covalent binding with lysine and cysteine containing peptides. However, the DPRA could not be tested up to the recommended concentration and this affects the reliability of the outcome. Keratinocytes exposed to PF-03817968-09 showed activation of antioxidant/electrophile responsive element (ARE)-dependent pathway. Whether this is activation or cytotoxicity cannot be determined. The outcome of the KeratinoSensTM assay is therefore not appropriate to aid in assessing the skin sensitizing properties for this substance. Although three negative test results were obtained, the data set is concluded not sufficiently reliable to conclude on the skin sensitizing properties ofPF-03817968-09. Performance of a U-SENSTM assay is considered to give no additional information, as cytotoxicity is expected, which precludes a scientifically reliable outcome. As further in vitro testing will not lead to an overall conclusion, it is scientifically justified to address the skin sensitizing properties with an in vivo test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)