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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
According to OECD TG 442C (2015) in chemico and in vitro test methods have been considered scientifically valid for the evaluation of the skin sensistization hazard of chemicals. The DPRA is proposed as a first step to address the molecular intitiating event of the skin sensitization AOP, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine.

Test material

Constituent 1
Reference substance name:
Rosin, reaction products with acrylic acid
EC Number:
280-192-2
EC Name:
Rosin, reaction products with acrylic acid
Cas Number:
83137-13-7
Molecular formula:
N.A. - UVCB substance
IUPAC Name:
Rosin, reaction products with acrylic acid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot # 646729
- Expiration date of the lot/batch: 19 November 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Final preparation of a solid: Test material was prepared at 100 mM in acetone.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test material was solubilized in acetone and applied as a solution.

In chemico test system

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

The test article, MTDID 28640, was solubilized in acetone (48.0 mg in 1.589 mL acetone) to yield a final concentration of 100 mM and added to the peptide reactions.

DPRA was run according to Cyprotex SOP-2078. The cysteine peptide was prepared at 0.667 mM in cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer as outlined in OECD 442c. The reaction mix for cysteine peptide had a 1:10 test peptide to reference article ratio (0.5 mM cysteine to 5 mM reference article). The reaction mix for lysine peptide had a 1:50 peptide to reference article ratio (0.5 mM lysine to 25 mM reference article). Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile, acetone or water containing no reference or test articles.

Reference control reactions were prepared by mixing 2.5 mL of acetonitrile with 7.5 mL of the respective buffer. Aliquots of 1 mL were added to 9 glass vials for each peptide and placed at the beginning middle and end of the run sequence. These reactions were used to ensure the consistency of peptide detection during the run. A standard curve was prepared for both peptides by adding 400 μL of acetonitrile to 1600 μL of 0.667 mM peptide to make a 0.534 mM standard. This 0.534 mM standard was serial diluted in 20% acetonitrile/buffer to make a 6-point standard curve. A zero-peptide standard was also included in the standard curve.

After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. The samples were transferred to Western Michigan University Homer Stryker M.D. School of Medicine Innovation Center for peptide assessment via HPLC with gradient elution and UV detections at 220 nm using an Agilent 1100 HPLC equipped with a Photodiode Array. Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Column temperature was 30°C, injection volume 5 μL, and flow rate was 0.35mL/min. Sample analysis was initiated within 24 ±2 hours of the reaction start. Samples were injected once. The first sample was injected at 13:35 on 6/2/17. The final sample was injected at 17:41 on 6/3/17. After determination of peptide remaining in the analysis, percent depletion relative to vehicle controls was calculated relative to no test article (vehicle control) samples. Peptide reactivity was reported as percent depletion and was calculated using the following formula: % Depletion = (1-(test compound area/ vehicle control area)) x 100

Results and discussion

Positive control results:
2,3-Butanedione, the positive control, performed as expected in the assay and was ranked in the correct reactivity class according to the OECD guideline.

In vitro / in chemico

Results
Key result
Parameter:
other: % Depletion DPRA
Value:
7.9
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
MTDID 28640 showed a low level of reactivity with both peptides and was also positive for sensitization potential. It should be noted that a moderate level of precipitation in both the lysine and cysteine reactions was observed. Because reaction with the peptide was observed (despite precipitation) the OECD guideline does allow for prediction of sensitization potential.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the results of the study, MTDID 28640 is predicted to have sensitizing potential within this stage of the skin sensitization AOP.
Executive summary:

The sensitization potential of MTDID 28640 was examined using the Direct Peptide Reactivity Assay (DPRA). The test was performed under GLP conditions and followed the guidance in OECD 442C (2015). The test article, MTDID 28640, was solubilized in acetone to yield a final concentration of 100 mM. The cysteine peptide was prepared at 0.667 mM in cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer as outlined in OECD 442C. The reaction mix for cysteine peptide had a 1:10 test peptide to reference article ratio (0.5 mM cysteine to 5 mM reference article). The reaction mix for lysine peptide had a 1:50 peptide to reference article ratio (0.5 mM lysine to 25 mM reference article). Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile, acetone or water containing no reference or test articles. Aliquots of 1 mL solubilized test article were added to 9 glass vials for each peptide and placed at the beginning middle and end of the run sequence. These reactions were used to ensure the consistency of peptide detection during the run. A standard curve was prepared for both peptides by adding 400 μL of acetonitrile to 1600 μL of 0.667 mM peptide to make a 0.534 mM standard. This 0.534 mM standard was serial diluted in 20% acetonitrile/buffer to make a 6-point standard curve. A zero-peptide standard was also included in the standard curve. After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Column temperature was 30°C, injection volume 5 μL, and flow rate was 0.35mL/min. Sample analysis was initiated within 24 ±2 hours of the reaction start. Samples were injected once. After determination of peptide remaining in the analysis, percent depletion relative to vehicle controls was calculated relative to no test article (vehicle control) samples. Peptide reactivity was reported as percent depletion and was calculated using the following formula: % Depletion = (1-(test compound area/ vehicle control area)) x 100

MTDID 28640 showed a moderate level of precipitation in both the lysine and cysteine reactions. Reactions containing precipitate were spun at 400xg for 5 minutes prior to running HPLC analysis. Because reaction with the peptide was observed (despite precipitation) the OECD guideline allows for prediction of sensitization potential. A percent depletion DPRA of 7.9% was observed for MTDID 28640 indicating a low reactivity class (6.38% < Mean % Depletion ≤ 22.62%). Based on the results of the study, MTDID 28640 is predicted to have sensitization potential within this stage of the skin sensitization AOP.