D-Gluconic acid, δ-lactone, reaction products with propanolamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Substance is exclusively used as an ingredient in cosmetic products and therefore no animal teating is allowed.

Test material

Test material information

Constituent 1

Reference substance name:

D-Gluconamide, N-(3-hydroxypropyl)-

Cas Number:

190445-18-2

Molecular formula:

C9H19NO7

IUPAC Name:

D-Gluconamide, N-(3-hydroxypropyl)-

Constituent 2

Reference substance name:

gluconic acid, aminopropanol salt

Molecular formula:

C9 H21 N1 O8

IUPAC Name:

gluconic acid, aminopropanol salt

Test material form:

liquid

Remarks:

50% solution in water

Details on test material:

% solids in water: 48.0 - 52.0

Specific details on test material used for the study:

ID: Samson 18
Appearance: liquid
CAS; na
Lot: Z333-94-475
STORAGE: ROOM TEMPERATURE
Purity: 50% in water

In vitro test system

Details on the study design:

Justification of test method:
In this assay, the test article is incubated concurrently in two separate buffers, one with cysteine (Ac-RF AACAA-COOH) and one with lysine (Ac-RF AAKAA-COOH). Reactive chemicals bind one or both of the peptides thereby reducing their free concentration levels. The disappearance of each peptide is measured by HPLC/UV. This method does not require any biological material such as enzymes in order for this reaction to take place. It is important to note that the cysteine peptide captures soft electrophiles, while the lysine peptide captures hard electrophiles. This makes the DPRA assay a good choice to screen for reactive chemicals which are associated with allergic contact dermatitis.
Preparation of samples:
- Test Article Stock: Client test article Samson 18 was received by Cyprotex lab staff and stored at room temperature. Samson 18 was provided at 50% (-2M) in water. Stock Samson 18 was diluted in water to 100 mM.
- Reference materials: Reference article stocks were prepared in acetonitrile (Optima LC/MS, 99.9%, Fisher, Waltham, MA, Lot No. 150041, CAS 75-05-8) as directed by the OECD guideline solvent.
- Peptides: A 0.667 mM stock solution of the lysine peptide was prepared by diluting the peptide with Lysine Reaction buffer, and a 0.667 mM stock solution of cysteine peptide was prepared by diluting the peptide in Cysteine Peptide Reaction buffer. Unprepared peptides were stored at approximately -80°C desiccated. Peptides were from LifeTein LLC (South Plainfield, NJ).
Reference substances: p-Benzoquinone (positive), Lactic Acid (negative) and 2,3-Butanedione (positive).
Method for evaluation of peptide depletion:
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. 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). All reactions were run in triplicate.
Reference control reactions were prepared by mixing 2.5 mL of acetonitrile with 7.5 mL of the respective buffer. 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. No precipitation was observed in either the test or reference article reactions. After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. 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.
Test Validity and Acceptance Criteria:
For data generated at Cyprotex, basic statistical analysis was performed on the data, which included means of replicates, standard deviations, and CV%. Values within a dose group that varied from the mean by more than _i.2 standard deviations were omitted from the final mean calculation. For the calculation of the DPRA score, negative depletion values are changed to zero prior to averaging the individual peptide depletions.
The results of each assay were evaluated and compared to reference controls. DPRA results were expressed as percent of control. All data were placed in a final datasheet and used to determine reactivity classification. Reference controls were within Cyprotex criteria.

Results and discussion

Positive control results:

% cysteine depletion: % Lysine Depletion: Response Class:
p-Benzoquinone = 99.4% p-Benzoquinone = 94.6% high
Lactic acid = 2.9% Lactic acid = 1.1% minimal
2,3-Butanedione = 83.8% 2,3-Butanedione = 23.8% high

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Main

Parameter:

other: % Cisteine depletion

Value:

0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Main

Parameter:

other: % Lysine depletion

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

System suitability was shown by examining the r2 value for the standard curves and the average, standard deviation and % coefficient of variation of the reference control samples. The calibration curves for both peptides were shown to have r2 values of greater than 0.99.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion Samson 18 was found to be minimally reactive with both the Cysteine and Lysine peptides, and was ranked as negative for sensitization potential.