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

Endpoint summary

Description of key information

The skin sensitization potential for Reaction mass of 3-hydroxypropan-1-aminium D-gluconate and N-(3-hydroxypropyl)-D-gluconamide was evaluated with FiberHance BM Solution (50%) in a Direct Peptide Reactivity Assay (DPRA) and a KeratinoSens assay.

The DPRA was conducted in accordance with Organization for Economic Co-Operation and Development Testing Guideline (OECD TG) 442C. FiberHance was tested at 100mM concentration and no reactivity was found with either the cysteine of the lysine peptide. Therefore, FiberHance was not rated as a sensitizer in the DPRA.

The KeratinoSens assay was conducted in accordance with OECD Guideline for the testing of chemicals: In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method. FiberHance tested at concentration up to 2000 μM in triplicates and was determined not to have a sensitizing potential under the condition of this assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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

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.

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

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

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

Remarks:

No specification of incubation conditions.

GLP compliance:

not specified

Remarks:

The study was performed according to national and/or international guidelines. The performing laboratory is GLP certified and test procedures are generally following GLP guidance and rules.

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

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

Specific details on test material used for the study:

ID: Samson 18

Details on the study design:

Protocol:
KeratinoSens cells were plated on 96-well tissue culture treated black walled clear bottom polystyrene plates, 125μL per well. The cells were left for 24 h to adhere and the media was replaced with a low serum medium prior to dosing with the test compound at a range of 12 concentrations (see Assay Summary for details). Each compound is dosed on two separate plates, one to determine luminsence (Keap1-Nrf2-ARE pathway) and a second to determine cytotoxicity (MTT cell viability assay). Following an incubation period of 48 h, the cells were either lysed and assessed for the luciferase reporter gene expression using a luminescent assay (Luciferase Assay Kit, Promega) and the BioTek luminometer or loaded with MTT [yellow; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]. After 4 hour incubation the resulting formazan from
the MTT cell viability assay was solubilised in a 10% SDS solution. The plates were then scanned at 570nm using a SunriseTM microplate absorbance reader (TECAN).

Data Interpretation
The KeratinoSensTM measures the induction of a luciferase gene expression (Keap1-Nrf2-ARE pathway) alongside cell viability following 48h of treatment with the test compound. A compound is rated a skin sensitizer in the KeratinoSens assay if the reporter gene expression is induced above a fold of 1.5 (EC1:5) at concentrations below 1000μM (or 200 μg/mL) with no observed reduction in cell viability of greater than 30%. Increased reporter gene expression in cells with less than 70% cellular viability (IC30) could be due to cell stress than rather than a potential skin sensitizing effect.

Study details:
- Incubation time: 48h
- Concentrations (μM): 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000
- Replicates per concentration: 3
- Cell model: KeratinoSens
- Vehicle: Water
- MW: 253.0
- KeratinoSens vehicle SD: 14% (passed), 5% (passed), 12% (passed)
- Certified on: 2015-09-28

Positive control results:

Compound Exp. 1 Exp. 2 Exp.3 Mean ± SD Flag Sensitizing
cinnamic aldehyde 6.91 22.3 17.7 15.6±7.90 3/3 Yes (+)
sodium dodecyl sulfate >31.3 >31.3 >31.3 >31.3 0/3 No ()

Run / experiment:

other: 2

Parameter:

other: An increase of the reporter gene expression is related to the activation of the transcription factor Nrf2 indicating skin sensitizing potential.

Value:

2 000

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: An increase of the reporter gene expression is related to the activation of the transcription factor Nrf2 indicating skin sensitizing potential.

Value:

2 000

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 3

Parameter:

other: An increase of the reporter gene expression is related to the activation of the transcription factor Nrf2 indicating skin sensitizing potential.

Value:

2 000

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A confirmatory clinical Human Repeat Insult Patch Test was conducted in accordance with the Good Clinical Practices. FiberHance was prepared in a 20% dilution (10% active) in water and patched to 213 subjects 3 times per week for a total of 9 applications (induction phase). The patched sites were evaluated prior to re-application. Approximately 2 weeks after the final induction patch, a challenge patch was applied to a virgin site and evaluated 1 and 3 days after the challenge phase. Under the conditions of this study, FiberHance indicated no potential for dermal irritation or allergic contact sensitization at the concentration tested. Based on the results of the studies, FiberHance is unlikely to induce skin sensitization at concentration up to 20% as supplied corresponding with 10% Reaction mass of 3-hydroxypropan-1-aminium D-gluconate and N-(3-hydroxypropyl)-D-gluconamide.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of in-vitro testing and the Human Repeat Insult Patch Test, Reaction mass of 3-hydroxypropan-1-aminium D-gluconate and N-(3-hydroxypropyl)-D-gluconamide does not meet the criteria for classification as sensitizing.