Protein hydrolyzates, milk

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 2014. EURL ECVAM Recommendation on the Keratinosens Assay

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: DB-ALM protocol 155: KeratinosensTM protocol

Principles of method if other than guideline:

Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 4 years experience with both the KeratinoSens and DPRA assays.

GLP compliance:

no

Remarks:

Conducted within the "spirit" of GLP (See Principles of method if other than guideline)

Type of study:

other: KeratinosensTM assay

Test material

Test material information

Constituent 1

Reference substance name:

Protein hydrolyzates, milk

EC Number:

296-575-2

EC Name:

Protein hydrolyzates, milk

Cas Number:

92797-39-2

IUPAC Name:

Protein hydrolyzates, milk

Test material form:

solid

In chemico test system

Details on the study design:

Test System(s):
The KeratinoSens™ cell line is derived from the human keratinocyte culture HaCaT. It contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene AKR1C2 [2].
The KeratinoSens™ cell line was developed by the testing lab and stored on liquid nitrogen. It was grown in 10 cm petri dishes as described in the SOP to 80% confluency prior to testing for 3 – 4
days. Cells were counted using a counting chamber and adjusted to the desired density. During seeding into 96-well plates, the cell suspension was gently stirred and cell sedimentation was avoided by repeatedly pipetting up and down to ensure homogeneous distribution of cells.

Basic Procedure:
Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a final level of 1% of the solvent DMSO containing the test substance. Each compound is tested at 12 concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent control, 1 well with no cells for background value and 5 wells with a dose response of the positive control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same set-up, and a forth parallel plate is prepared for cytotoxicity determination.

Positive control:
In each test Cinnamic aldehyde is included as positive control. It is tested in each test plate at five concentrations from 4 – 64 μM.

Endpoint & Endpoint Detection:
Two endpoints are measured: (i) Luciferase induction after a 48 h treatment with test substances and (ii) cytotoxicity as determined with the MTT assay recorded in a parallel plate with the same cell batch and made up with the same dilutions of the test substances.
Luminescence was read in a Promega Glomax Luminometer programmed to
i. add 50 μl of the luciferase substrate to each well,
ii. to then wait for 1 second and
iii. then to integrate the luciferase activity for 2 seconds.

Endpoint Value:
For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5-, 2- and 3- fold induction (EC1.5, EC2 and EC3) are calculated. For cytotoxicity the IC50 value is extrapolated.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP. The test plates are read by a plate reader, and the generated raw data are directly pasted into this template, and all data processing is performed automatically by this Excel sheet.
For both the PRESTO BLUE and the luciferase data, first the background value recorded in an empty well without added cells is subtracted.
For the PRESTO BLUE data the % viability is then calculated for each well in the test plate in relation to average of the six solvent control wells.
For the luciferase data the average value of the six solvent control wells is set to 1, and for each well in the test plate the fold induction is calculated in relation to this value.

The following parameters are then calculated from these processed raw data:
• Imax Maximal fold-gene induction of the luciferase gene over the full dose-response
• EC 1.5 Concentration in μM for 1.5-fold gene induction
• EC 2 Concentration in μM for 2-fold gene induction
• EC 3 Concentration in μM for 3-fold gene induction
• Pos / Neg Rating of substance according to prediction model
• reps. Positive number of independent repetitions positive / number of repetitions done
• IC50 Concentration in μM for 50% reduction of cell viability

Prediction Model
Substances are rated positive if the following conditions are met::
• The Imax indicates > 1.5-fold gene induction, and this induction is statistically significant above the solvent control in a particular repetition as determined by students T-test. The EC1.5 value is below 1000 μM in all three repetitions or in at least 2 repetitions. (If the Imax is exactly equal to 1.5, the substance is still rated negative and no EC1.5 value is calculated by the evaluation sheet.)
• At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC 1.5 determining value), the cellular viability is above 70%.
• There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

Testing of Proficiency chemicals and historical positive control data in the test facility:
The KeratinoSens assay was originally developed at the testing facility. Data for the Proficiency chemicals as defined by OECD TG 442d generated in the laboratory are summarized in the Givaudan report GCR 153’464 ’KeratinoSens assay: Proficiency testing at the testing facility.

Results and discussion

In vitro / in chemico

Results

Key result

Parameter:

other: Geometric Mean IC 50 (μM)

Value:

299.44

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Given is the IC50 value as the concentration in μM reducing the viability by 50%

Any other information on results incl. tables

         

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing
and assessment (IATA)[9]..
YOGURTENE 20 CQ had no toxicity for the KeratinoSens™ cells. In one of three repetitions, it did
induce the luciferase gene above a threshold of 1.5, while the other two repetitions are negative. It is
therefore considered a non-sensitizer according to the prediction model of the KeratinoSens™
assay

Executive summary:

YOGURTENE 20 CQ was not toxic for the KeratinoSens™ cells. In one of three repetitions, it did

induce the luciferase gene above a threshold of 1.5, while the other two repetitions are negative. It is

therefore considered a non-sensitizer according to the prediction model of the KeratinoSens™

assay.