Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

Endpoint summary

Description of key information

A DEREK prediction on the skin sensitizing potential of SHR 1396 was negative and predicted the substance to be a non sensitizer.

SHR 1396 did not react with cysteine and lysine containing peptides resulting in a negative outcome for the DPRA. However, since precipitation was observed after the incubation period for both, SPCC and SPCL, it is not clear how much test substance remained in the solution to react with the peptides, therefore this results is uncertain and should be interpreted with due care.

In the KeratinoSens^TMassay SHR 1396 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) and was therefore concluded to be positive.

In the LLNA study there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, SHR 1396 was considered not to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

In silico assessment

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2 July 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

An in silico assessment is mentioned in the ECHA guidance as one of the non-animal tests that may be performed as part of a weight of evidence approach for the endpoint skin sensitization.

Qualifier:

no guideline required

Principles of method if other than guideline:

An in silico assessment was performed with DEREK NEXUS version 6.0.1.

GLP compliance:

no

Justification for non-LLNA method:

Since 11 October 2016 it is legally required to consider all available information for the endpoint skin sensitisation and to use a non in vivo test strategy based on in chemico, in silico and in vitro skin tests combined in a weight-of-evidence approach. An in vivo test (LLNA) is only allowed as last resort. DEREK results are adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.

Key result

Run / experiment:

other: SHR 1396

Parameter:

other: prediction of skin sensitization

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

SHR 1396 does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer (see prediction in study report).

Interpretation of results:

other: study is part of a weight of evidence approach and is not used for classification on its own

Conclusions:

SHR 1396 is predicted to be not sensitizing to the skin.

Executive summary:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, SHR 1396 does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4 June 2019 - 6 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of a weight of evidence.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.

Details on the study design:

TEST ITEM PREPARATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), and dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol and ethanol. Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 30.21 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1587 μL ACN after vortex mixing and 5 minute of sonication to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Storage: The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.
Incubation: the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.4 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.
Analysis: All samples were analyzed according to the HPLC method presented in Table 1. (See 'other information on materials and methods').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2. (See 'other information on materials and methods').

POSITIVE CONTROL
Cinnamic aldehyde
- Purity: 99.1%
- Batch: MKCB9907
- Expiry of batch: 30 November 2021

DATA EVALUATION
The concentration of SPCC or SPCL was spectrophotometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula: Percent Peptide Depletion= [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]x100. In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. (Table 3 in 'other information on materials and methods')

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r^2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Key result

Run / experiment:

other: Cysteine reactivity assay

Parameter:

other: Mean SPCC depletion (%)

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 1.3%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 76.3%

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: Mean SPCL depletion (%)

Value:

2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 1.9%

Positive controls validity:

valid

Remarks:

Mean percentage SPCL: 65.4%

Other effects / acceptance of results:

In the cysteine reactivity assay the test item showed 0.4% SPCC depletion while in the lysine reactivity assay the test item showed 2.8% SPCL depletion. The mean of the SPCC and SPCL depletion was
1.6% and as a result the test item 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.
Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.999	>0.99	1.000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.511 ± 0.003	0.50 ± 0.05	0.512 ± 0.007
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.496 ± 0.001	0.50 ± 0.05	0.505 ± 0.005
CV (%) for RC samples B and C	<15.0	1.3	<15.0	1.9
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	76.3	40.2-69.0	65.4
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.0	<11.6	0.8
SD of peptide depletion for the test item (%)	<14.9	0.6	<11.6	0.4

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
SHR 1396	0.4%	±0.6%	2.8%	±0.4%	1.6%	Negative: No or minimal reactivity

Interpretation of results:

other: study is part of a weight of evidence approach and is not used for classification on its own

Conclusions:

SHR 1396 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.

Executive summary:

A DPRA study was performed according to the OECD guidelines and following GLP principles, in order to determine the reactivity of SHR 1396 towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). 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 non-sensitizers. The relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, 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, a precipitate was observed. In the cysteine reactivity assay the test item showed 0.4% SPCC depletion while in the lysine reactivity assay the test item showed 2.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.6% 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. Since all acceptability criteria were met this DPRA is considered to be valid. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 April 2019 - 10 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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 KeratinoSensTM assay, which is recommended in international guidelines.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

March 2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

TEST SYSTEM
Model used: The KeratinoSens™ cell line, generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).
Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).
Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
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 (P+25).

ENVIRONMENTAL CONDITIONS
- Temperature used during incubation: humid atmosphere of 80 - 100% (actual range 34 - 96 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.9 - 37.9°C)

EXPERIMENTAL DESIGN
Testing was done with cells 80-90% confluent.
Cells were plated at a density of 10.000 cells/well in basic medium.
For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay.
Cells were incubated overnight.
The passage number used was P+3 in experiment 1 and P+5 in experiment 2.
Three wells per plate were left empty (no cells and no treatment) to assess background values.

EXPOSURE
After 24 hours of incubation, cells were exposed to the test substance. The medium was removed and replaced with 150 μL culture medium containing serum (but without Geneticin) to which 50 μL of the 25-fold diluted test substance and control items were added.
experiment 1: Concentration range of the test substance (μg/mL): 0.20, 0.39, 0.78, 1.6, 3.1, 6.3, 13, 25, 50, 100, 200, and 400.
experiment 2: Concentration range of the test substance (μg/mL): 0.12, 0.17, 0.26, 0.39, 0.59, 0.88, 1.3, 2.0, 3.0, 4.4, 6.7, and 10.
Concentration range of positive control EDMG (μg/mL): 7.8, 16, 31, 63, 125, and 250.
The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0°C containing 5.0 ± 0.5% CO2.

LUCIFERASE ACTIVITY MEASUREMENT
Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) were mixed together.
The assay plates were removed from the incubator and the medium was removed. Then 200 μL of the Steady-Glo 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
After 48 h exposure, medium was replaced with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1).
cells were incubated for 3-4 h at 37±1.0°C containing 5.0 ± 0.5% CO2. After that time, MTT was removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5

Remarks:

μg/mL

Value:

0.3

Positive controls validity:

valid

Remarks:

EC1.5: 19 μM

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

2.5

Positive controls validity:

valid

Remarks:

Imax: 2.70

Run / experiment:

other: Experiment 1

Parameter:

other: IC30

Remarks:

μg/mL

Value:

1.3

Positive controls validity:

not applicable

Run / experiment:

other: Experiment 1

Parameter:

other: IC50

Remarks:

μg/mL

Value:

1.5

Positive controls validity:

not applicable

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5

Remarks:

μg/mL

Value:

3.5

Positive controls validity:

valid

Remarks:

EC1.5: 52 μM

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

2.05

Positive controls validity:

valid

Remarks:

Imax: 2.87

Run / experiment:

other: Experiment 2

Parameter:

other: IC30

Remarks:

μg/mL

Value:

7

Positive controls validity:

not applicable

Run / experiment:

other: Experiment 2

Parameter:

other: IC50

Remarks:

μg/mL

Value:

9

Positive controls validity:

not applicable

Other effects / acceptance of results:

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 (19 μM and 52 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.70-fold and 2.87-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.7% and 6.1% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

study cannot be used for classification

Remarks:

Study is part of a weight of evidence approach

Conclusions:

SHR 1396 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this study.

Executive summary:

A KeratinoSens^TM assay was performed with SHR 1396 according to OECD guidelines and GLP principles in order to evaluate the ability of SHR 1396 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. SHR 1396 was dissolved/suspended in dimethyl sulfoxide at 40 mg/mL. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.20–400 μg/mL (2-fold dilution series) and in test concentrations of 0.12–10 μg/mL (1.5-fold dilution series) in the first and second experiment, respectively. The highest test concentration was the highest dose required in the current guideline. In the second experiment, a narrower dose-response analysis was performed using a lower dilution factor of 1.5-fold to investigate the induction in experiment 1 in more detail. No precipitate was observed at any dose level tested. Both experiments passed the acceptance criteria, i.e. the luciferase activity induction obtained with the positive control 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 (19 μM and 52 μM in experiment 1 and 2, respectively), a dose response was observed and the induction at 250 μM was higher than 2-fold (2.70-fold and 2.87-fold in experiment 1 and 2, respectively), and the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.7% and 6.1% in experiment 1 and 2, respectively), indicating that the test conditions were adequate and that the test system functioned properly.

SHR 1396 showed toxicity (IC30values of 1.3 μg/mL and 7 μg/mL and IC50values of 1.5 μg/mL and 9 μg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5values of 0.3 μg/mL and 3.5 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.50-fold and 2.05-fold in experiment 1 and 2 respectively. Therefore, SHR 1396 is classified as positive in the KeratinoSens^TMassay since positive results (>1.5-fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control. In conclusion, SHR 1396 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this study.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul - 06 Aug 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)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

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:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Females nulliparous and non-pregnant: yes.
- Microbiological status of animals: SPF-quality.
- Age at study initiation: 10 weeks old.
- Weight at study initiation: 19.6 to 23.0 grams.
- Housing: Animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days.
- Indication of any skin lesions: no.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The daily mean temperature during the study period was 22 to 23°C.
- Humidity (%): The daily mean relative humidity of 53 to 79%.
- Air changes (per hr): at least 10.
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From 17 Jul to 05 Aug.

Vehicle:

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

No. of animals per dose:

5 animals

Details on study design:

PRE-SCREEN TESTS:
Two test item concentrations were tested; a 50% (w/w) and 100% (w/w) concentration.
Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY

Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
Group 1 was treated with the vehicle (vehicle control group). Group 2, 3 and 4 were treated with 25%, 50% and 100% concentration, respectively.

TREATMENT PREPARATION AND ADMINISTRATION:

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing.
The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item.
- Induction: Test item (25 μL) was applied in the dorsal surface of both ears of each animal, for 3 consecutive days.
- Excision of the Nodes. In day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue processing for radioactivity: On day 6 and following the excision of the nodes, a single cell suspension of limph node cells was prepared in PBS. LNC were washed twice and DNA was precipitated using 5% trichloroacetic acid (TCA).
- Radioactivity measurements: On day 7, precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

ANALYSIS
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

INTERPRETATION
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated 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 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3) (see table 1 in 'any other information on material and methods')

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

2.5

Test group / Remarks:

100%

Cellular proliferation data / Observations:

PRE-SCREENING TEST:
- At a 50% and 100% test item concentration, no signs of systemic toxicity were noted and up to very slight erythema was observed and therefore the 100% concentration was selected as highest concentration for the main study.

MAIN STUDY

SKIN REACTIONS / IRRITATION:
The very slight erythema of the ears as shown by all test item treated animals on at 1 to 6 Days during the observation period was considered not to have a toxicologically significant effect on the activity of the nodes.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

MACROSCOPIC EXAMINATION OF LyMPH NODES:
The majority of auricular lymph nodes were considered normal in size, except for the nodes in two animals treated at 100%, which were considered to be slightly enlarged.
No macroscopic abnormalities of the surrounding area were noted for the majority of animals. Pale discoloration of the surrounding area of the lymph nodes of the animals treated at 100% was noted, which was considered not to have affected the DPM values of these animals.

DETAILS ON STIMULATION INDEX CALCULATION : DPM (Disintegrations Per Minute) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

Interpretation of results:

other: Not skin sensitising

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

In conclusion, since there is no indication that the test item elicited a SI ≥ 3 when tested up to 100%, SHR 1396 was considered not to be a skin sensitizer.

Executive summary:

A LLNA study was performed following OECD guidelines and GLP principles. Three experimental groups of five females were treated with test item concentrations of 15%, 50% and 100%. One control group of five females was treated with the vehicle (Acetone/olive oil 1:4 v/v). All animals were treated for 3 consecutive days. Three days after the last exposure all animals were injected with 3H-methyl thymidine for measuring radioactivity. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was calculated for each group. 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. No macroscopic abnormalities of the surrounding area were noted for the majority of animals. Pale discoloration of the surrounding area of the lymph nodes of the animals treated at 100% was noted, which was considered not to have affected the DPM values of these animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 717, 938 and 972 DPM, respectively. The mean DPM/animal value for the vehicle control group was 384 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 1.9, 2.4 and 2.5, respectively. Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, SHR 1396 was considered not to be a skin sensitizer. Based on these results, SHR 1396 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 Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A DEREK assessment, DPRA and KeratinoSens^TMassay 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.

A DEREK prediction on the skin sensitizing potential of SHR 1396 was negative. The DPRA was negative as no significant depletion ofsynthetic peptides containing either cysteine or lysine in combination with SHR 1396 was observed. The KeratinoSens^TM assay showed a biologically relevant activation of keratinocytes when exposed to SHR 1396 and was therefore positive. Due to the cytotoxic properties of SHR 1396, continuing with the performance of aU-SENS^TMassay would be scientifically unjustified as the outcome of the study is not expected to aid in the overall conclusion on skin sensitization for SHR 1396.

Since the current data-set is insufficient to conclude on skin sensitizing potential of SHR 1396 and further in vitro testing is not expected to yield information that allows a final conclusion on this endpoint, it is scientifically justified to omit further in vitro testing and to proceed with in vivo testing.

LLNA:

A LLNA study was performed following OECD guidelines and GLP principles. Three experimental groups of five females were treated with test item concentrations of 15%, 50% and 100%. One control group of five females was treated with the vehicle (Acetone/olive oil 1:4 v/v). All animals were treated for 3 consecutive days. Three days after the last exposure all animals were injected with 3H-methyl thymidine for measuring radioactivity. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was calculated for each group. 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. No macroscopic abnormalities of the surrounding area were noted for the majority of animals. Pale discoloration of the surrounding area of the lymph nodes of the animals treated at 100% was noted, which was considered not to have affected the DPM values of these animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 717, 938 and 972 DPM, respectively. The mean DPM/animal value for the vehicle control group was 384 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 1.9, 2.4 and 2.5, respectively. Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, SHR 1396 was considered not to be a skin sensitizer. Based on these results, SHR 1396 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 Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the LLNA study, SHR 1396 does not have to be classified and has no obligatory labelling requirement for skin sensitisation according to Regulation (EC) No 1272/2008 and its amendments.