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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:
28th January 2019 - 6th February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
Recommended test system in the international OECD guideline for DPRA studies.

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene
EC Number:
830-582-9
Cas Number:
1951440-04-2
Molecular formula:
C32H50O2
IUPAC Name:
1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene
Test material form:
liquid: viscous
Specific details on test material used for the study:
Batch (Lot) Number: AS455433
Expiry date: 01 November 2020 (retest date) (taken from label)
Physical Description: Colourless to light yellow viscous liquid
Purity/Composition: 98.5%
Storage Conditions: At room temperature protected from light

Additional information
Test Facility test item number: 209996/A
Purity/Composition correction factor: No correction factor required

In chemico test system

Details on the study design:
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.

Experimental Design.

A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.4 mg of SPCC in 20.76 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. In addition, a RCcysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCIPA sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL IPA.

Sample Incubations:

After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and substance 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 23.6 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.
Prior to HPLC analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

HPLC Analysis.

SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:

System 1 (used for Cysteine Reactivity Assay):
Alliance separations module 2695 (Waters, Milford, MA, USA)
Dual λ absorbance detector 2487 (Waters)

System 2 (used for Lysine Reactivity Assay):
Alliance separations module 2695 (Waters, Milford, MA, USA)
Dual λ absorbance detector 2487 (Waters)

Results and discussion

Positive control results:
SPCL mean deplition for Cinnamic Aldehyde is 57.0% ± 0.7%. SPCC mean deplition for Cinnamic Aldehyde is 70.7% ± 0.3%.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: SPCC mean depletion (%)
Value:
1.8
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Precipitation observed following the incubation
Key result
Run / experiment:
other: 1
Parameter:
other: SPCL mean deplition (%)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Precipitation observed following the incubation

Any other information on results incl. tables

SPCC Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Substance Samples.

Sample code

Peak area

at 220 nm (µAU)

Concentration (mM)

SPCC Depletion

Peak area

at 258 nm (µAU)

Area ratio (A220/A258)

209996/A-cys-1

1866636

0.509

1.6%

49473

37.73

209996/A-cys-2

1870198

0.510

1.4%

49090

38.10

209996/A-cys-3

1852693

0.505

2.3%

49417

37.49

 

Mean

1.8%

NA

37.77

 

SD

0.5%

NA

0.31

SPCL Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Substance Samples.

Sample code

Peak area

at 220 nm (µAU)

Concentration (mM)

SPCL Depletion

Peak area

at 258 nm (µAU)

Area ratio (A220/A258)

209996/A-lys-1

1587711

0.483

0.0%

50991

31.14

209996/A-lys-2

1605124

0.488

0.0%

51014

31.46

209996/A-lys-3

1592852

0.484

0.0%

50692

31.42

 

Mean

0.0%

NA

31.34

 

SD

0.0%

NA

0.18

SD = Standard Deviation, NA = Not Applicable.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Precipitation of the substance was observed after the incubation period for both SPCC and SPCL, is not known how much of the substance remained in the solution to react with the peptides. Consequently, this negative result is uncertain.
Conclusions:
In the in chemico skin sensitisation assay the substance was negative and classified in the “no or minimal reactivity class”. However, since precipitation was observed before and after the incubation period for both SPCC and SPCL, this negative result is uncertain and should be interpreted with due care.
Executive summary:

The in chemico skin sensitisation study conducted in accordance with OECD TG 442C, 'In Chemico Skin Sensitization: Direct Peptide Reactivity Assay'. The substance was incubated with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) for 23.6 h at 25 °C. The SPCC and SPCL Percent Depletion Values were calculated. It was shown that after the incubation of substance with peptides there was a precipitate present for both SPCC and SPCL. The substance is classified in the “no or minimal reactivity class”. However, as the precipitation was observed it was not possible to conclude whether the sufficient levels of the substance had reacted with the peptides, therefore this negative result is uncertain and should be interpreted with due care.