Methyl 2,3-dibromopropionate

Endpoint summary

Description of key information

Skin sensitisation:

A DEREK assessment, DPRA assay and a KeratinoSensTM assay were available.

 

A DEREK prediction on the skin sensitizing potential of test item was positive based on the presence of an alkyl halide. The DPRA was positive as test item depleted all SPCC and SPCL. The substance was classified in the "high reactivity class". The KeratinoSensTM assay outcome was positive as a biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway was observed at low concentrations at non-cytotoxic concentrations. Based on the results, it is concluded that test item is a skin sensitizer. The EC3 of 4.4% was predicted based on structural analogues of test item was not sufficiently reliable to differentiate between skin sensitization category 1A and 1B. As no reliable potency prediction can be made on the available data and as the substance is classified as corrosive (category 1) no in vivo testing to determine potency on skin sensitization needs to be conducted.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018-11-20

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:

See attached justification

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version: DEREK NEXUS version 6.0.1
- Model(s) used: DEREK NEXUS
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Key result

Parameter:

other: EC3 / %

Value:

4.4

Remarks on result:

other: moderate sensitizer

Other effects / acceptance of results:

DEREK NEXUS version 6.0.1 yielded an alert for skin sensitization for the test item. The test item is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicted an EC3 for the test item of 4.4% (moderate sensitizer), based on local lymph node assay (LLNA) and the guinea pig maximisation test (GPMT) data of 15 closest structurally related substances that had a structural similarity between 15 and 44%.

Interpretation of results:

other: Sensitizing

Conclusions:

The test item is predicted to be sensitizing to the skin (plausible).

Executive summary:

The potential for skin sensitization of test item was predicted with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS version 6.0.1 yielded an alert for skin sensitization for the test item. The test item is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicted an EC3 for the test item of 4.4% (moderate sensitizer), based on local lymph node assay (LLNA) and the guinea pig maximisation test (GPMT) data of 15 closest structurally related substances that had a structural similarity between 15 and 44%.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-02-05 to 2019-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Specific details on test material used for the study:

Purity: 98%
Batch No.: 800327680

Details on the study design:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 46.54 mg for both the cysteine and lysine reactivity assay
- Purity: 98%

SOLVENT USED: Acetonitrile (ACN)

POSITIVE CONTROL USED
- Solutions for Cysteine Reactivity Assay: 750 µL Stock solution of 0.667 mM SPCC (Synthetic Peptide Containing Cysteine) + 200 µL ACN + 50 µL Cinnamic aldehyde solution (100 mM in ACN)
- Solutions for Lysine Reactivity Assay: 750 µL Stock solution of 0.667 mM SPCL (Synthetic Peptide Containing Lysine) + 250 µL Cinnamic aldehyde solution (100 mM in ACN)

CO-ELUTION CONTROL (CC):
- Solutions for Cysteine Reactivity Assay: 750 µL Phosphate buffer pH 7.5 + 200 µL ACN + 50 µL test solution (100 mM)
- Solutions for Lysine Reactivity Assay: 750 µL Ammonium acetate buffer pH 10.2 + 250 µL test solution (100 mM)

REFERENCE CONTROL
- Solutions for Cysteine Reactivity Assay: 750 µL of the 0.667 mM SPCC stock solution + 250 µL ACN.
- Solutions for Lysine Reactivity Assay: 750 µL of the 0.667 mM SPCL stock solution + 250 µL ACN.

NUMBER OF REPLICATES
Co-elution control (CC) samples: 1 for both the cysteine and lysine reactivity assay
Positive control samples: 3 each for both the cysteine and lysine reactivity assay
Test item: 3 each for both the cysteine and lysine reactivity assay
Reference Control samples: 3 each for both the cysteine and lysine reactivity assay

SAMPLE INCUBATIONS
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 23 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.

Positive control results:

Cysteine Reactivity Assay
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.1% ± 0.1%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
Lysine Reactivity Assay
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 64.8% ± 1.7%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC depletion%

Value:

100

Positive controls validity:

valid

Remarks on result:

other: the mean SPCC depletion of the test item

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL depletion%

Value:

93.8

Positive controls validity:

valid

Remarks on result:

other: the mean SPCL depletion of the test item

Other effects / acceptance of results:

- Precipitation: No precipitate or phase separation was observed in any of the samples while after incubation, a phase separation was only observed in the SPCC test item samples.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.

In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 93.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 96.9% and as a result the test item was considered to be positive in the DPRA and classified in the “highreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Interpretation of results:

other: positive

Conclusions:

The test item was considered to be positive in the DPRA and classified in the "high reactivity class" when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this study was to determine the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OCED Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

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 220 nm and 258 nm. 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.

 

Upon preparation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples while after incubation, a phase separation was only observed in the SPCC test item samples.

In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 93.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 96.9%.

 

As a result the test item was considered to be positive in the DPRA and classified in the "high reactivity class" when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-01-16 to 2019-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways.
The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is proposed to address this second key event. Skin sensitizers have been reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes. In the KeratinoSensTM cell line activation of the Nrf2 pathway results in luciferase expression which can be quantified easily.

Specific details on test material used for the study:

Purity: 98%
Batch No.: 800327680

Details on the study design:

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. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+13 in experiment 1 and P+6 in experiment 2.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL 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. Initially, experiment 1and 2 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 2 valid experiments were performed.

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 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
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37 °C ± 1.0 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Experiment 1:
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.71 and the EC(1.5) 20 µM.

Experiment 2:
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.12 and the EC(1.5) 56 µM.

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Remarks:

The maximal average fold induction of luciferase activity

Value:

3.27

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Remarks:

The maximal average fold induction of luciferase activity value

Value:

4.86

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
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 (20 μM and 56 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.71-fold and 2.12-fold in experiment 1 and 2, respectively).
Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (11% and 8.1% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Experiment 1

No precipitation was observed at the start and end of the incubation period in the 96-well plates.

The test item showed toxicity. The calculated IC30 was 30 μM and the calculated IC50 was 39 μM.

A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 3.27 and the EC1.5 9.6 μM.

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.71 and the EC1.5 20 μM.

Experiment 2

No precipitation was observed at the start and end of the incubation period in the 96-well plates.

The test item showed toxicity. The calculated IC30 was 12 μM and the calculated IC50 was 14 μM.

A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 4.86 and the EC1.5 5.7 μM.

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.12 and the EC1.5 56 μM.

Interpretation of results:

other: positive

Conclusions:

The test item showed toxicity (IC30 values of 30 μM and 12 μM and IC50 values of 39 μM and 14 μM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 9.6 μM and 5.7 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 3.27-fold and 4.86-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control.

Executive summary:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay based on the most recent OECD Guideline 442D.

The test item was dissolved 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). The highest test concentration was the highest dose required in the current guideline. In the second experiment, a more narrow 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. Two independent experiments were performed.

Both 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 (20 μM and 56 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.71-fold and 2.12-fold in experiment 1 and 2, respectively).

Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (11% and 8.1% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

The test item showed toxicity (IC30 values of 30 μM and 12 μM and IC50 values of 39 μM and 14 μM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 9.6 μM and 5.7 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 3.27-fold and 4.86-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control.

In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation:

Positive results in DEREK assessment, DPRA assay and KeratinoSensTM assay.

According to Regulation (EC) No 1272/2008, table 3.4.2, this substance should be classified as category 1 for this endpoint.