Registration Dossier

Administrative data

Description of key information

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. a-Ketoglutarate, di-Na is predicted to be not sensitizing to the skin.

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in ultrapure water at 100 mM. There was no evidence of co-elution of a-ketoglutarate, di-Na with either Cysteine or Lysine peptide. Peptide depletion was calculated as 6.4% and 2.8% in Cysteine and Lysine Assays, respectively, resulting in a mean peptide depletion of 4.6%. This value places a-ketoglutarate, di-Na in the Minimal Reactivity Class resulting in a DPRA prediction of non-sensitiser.

A valid Keratinosens assay was performed according to OECD 442D and GLP principles. The test item was dissolved in dimethyl sulfoxide at 200 mM. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.977 – 2000 µM (2-fold dilution series). The test item precipitated at the highest dose level tested. Two independent experiments were performed.

The test item showed no toxicity and no induction of the luciferase activity was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.28-fold and 1.29-fold in experiment 1 and 2, respectively. a-Ketoglutarate, di-Na is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations of =1000 µM.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
19 December 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE: DEREX NEXUS
2. MODEL (incl. version number): DEREK NEXUS 5.0.2.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: C(CCC(C([O-])=O)=O)([O-])=O.[Na+].[Na+]
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached
5. APPLICABILITY DOMAIN (OECD principle 3)

- Descriptor domain:The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule in Derek, a result of ‘no alerts fired’ is presented to the user. This can be interpreted as a negative prediction or that the query compound is outside the domain of the model. Which of these is more appropriate may depend on the endpoint of interest. For the endpoint of skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ‘no alerts fired’ may be extrapolated to a negative prediction

- Structural fragment domain: DEREK NEXUS is applicable to organic structures including their salts, e.g. sodium, potassium. For skin sensitisation, which features 80 alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ’no alerts fired’ may be extrapolated to a negative prediction. The current structure has no extraordinary features and is considered to fall within the applicability domain.

- Mechanism domain: as the prediction is ‘no alerts fired’ none of the mechanisms predicted in the 80 skin sensitisation alerts is applicable to this structure.

-Metabolic domain: no evident metabolism that might lead to skin sensitization is predicted for this structure

6. ADEQUACY OF THE RESULT
-Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.
Guideline:
other: REACH Guidance on QSARs R.6
Version / remarks:
Prediction on the potential for skin sensitization with the in silico model DEREK NEXUS, version 5.0.2.
Principles of method if other than guideline:
- Software tool(s) used including version: in silico model DEREK NEXUS version 5.0.2
- Knowledge Base: Derek KB 2015 2.0
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'
GLP compliance:
no
Specific details on test material used for the study:
SMILES:C(CCC(C([O-])=O)=O)([O-])=O.[Na+].[Na+]
Key result
Parameter:
other: alerts for skin sensitization
Remarks on result:
other: DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. a-Ketoglutarate, Di-Na (CAS Nr. 305-72-6) is predicted to be not sensitizing to the skin.
Interpretation of results:
other: Non-Sensitizer
Conclusions:
DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. a-Ketoglutarate, Di-Na (CAS Nr. 305-72-6) is predicted to be not sensitizing to the skin.
Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of a-Ketoglutarate, Di-Na (CAS Nr. 305-72-6) with the in-silico model DEREK NEXUS. In this assessment version 5.0.2 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts:The DEREK NEXUS system has been designed for the qualitative prediction of the possible toxicity of chemicals. The predictions made by DEREK NEXUS are intended as an aid to toxicological assessment and, where appropriate, should be used in conjunction with other methods. “No alerts fired” may be extrapolated to a negative prediction.

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item a-Ketoglutarate, Di-Na (CAS Nr. 305-72-6).

Therefore, substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

The result is adequate to be used in a weight-of-evidence approach together within chemico/in vitro studies to complete the endpoint skin sensitization.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21 March - 20 April 2017
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))
Version / remarks:
2015
Deviations:
yes
Remarks:
The guideline states that the HPLC run sequence should be set up in order to keep the HPLC analysis time less than 30 hours. For the Cysteine samples reported, the time between sample preparation and the injection of the last sample was 35 h and 15 min.
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
SUMMARY: DPRA measures the reaction of the test item with synthetic peptides containing cysteine (Ac-RFAACAA-COOH) or lysine (Ac-RFAAKAA-COOH). The custom peptides contained cysteine or lysine as the nucleophilic reaction centres and phenylalanine to facilitate HPLC detection. Test item and peptide were combined and incubated together for 24 h at room temperature. Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC immediately prior to the lysine peptide assay.

EXPERIMENTAL PROCEDURES

PEPTIDES:
Source: RS Synthesis
Batch:
- Cysteine: No. P161108-LC180433
- Lysine: No. P161108-LC107617
Purity:
-Cys: 95.2%
-Lys: 98.14%

BUFFERS USED:
- Phosphate buffer: ca 100 mM, pH 7.53
- Ammonium acetate buffer: ca 100 mM, pH 10.2

SOLUBILITY ASSESSMENT:
- ultrapure water was selected as the most suitable solvent for the test material

PREPARATION PEPTIDE STOCK SOLUTIONS:
- CYSTEINE: stock solution of 0.501 mg/mL (0.667 mM) in phosphate buffer
- LYSINE: stock solution of 0.518 mg/mL (0.667 mM) in ammonium acetate buffer

CYSTEINE PEPTIDE ASSAY:
-PREPARATION: test item was dissolved in ultrapure water and mixed by inversion and vortex until fully in solution. The concentration of the test solution corrected for purity, was 19.1 mg/mL (99.8 mM, 99.8% from the target). Cinnamic aldehyde was dissolved in acetonitrile with a concentration of 13.22 mg/mL (100 mM). All test item and control solutions were prepared immediately prior to use.
-PREPARATION OF THE SANDARD CURVE: Dilution buffer was prepared by mixing phosphate buffer (pH 7.5, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1) was prepared by mixing peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (from 0.534 to 0.0167 mM). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analysed by HPLC immediately prior to the cysteine peptide assay.
-REFERENCE CONTROL: Acetonitrile (250 µL) was mixed with peptide stock solution (750 µL). Three replicates of this were produced for Reference Control A. Reference Control B was prepared as described for Reference Control A. Three replicates were analyzed at the beginning of the testing run, and three at the end of the testing run, to demonstrate peptide stability over the analysis time. Reference Control C samples were prepared containing the solvent that the test item was dissolved in: three replicates containing acetonitrile (250 µL) and peptide stock (750 µL) and three replicates containing ultrapure water (50 µL), acetonitrile (200 µL) and peptide stock (750 µL). These samples were included in every assay run together with the samples containing test item. They are used to verify that the solvent does not impact upon peptide stability during the assay, and to calculate percentage peptide depletion.
- PEPTIDE ASSAY METHOD: The assay contained a 1:10 molar ratio of peptide to test item. Positive control or test item (50 µL) was mixed with acetonitrile (200 µL) and the peptide solution (750 µL). The vials were mixed by vortex. Co-elution controls were prepared by mixing together acetonitrile (200 µL), phosphate buffer (750 µL) and test item (50 µL). All test items and positive control samples were prepared in triplicate. All vials were stored in the dark at ambient temperature for ca 24 h until analyzed by HPLC.

LYSINE PEPTIDE ASSAY:
-PREPARATION: test item was dissolved in ultrapure water and mixed by inversion and vortex until fully in solution. The concentration of the test solution corrected for purity, was 19.1 mg/mL (99.7 mM, 99.7% from the target). Cinnamic aldehyde was dissolved in acetonitrile with concentration of 13.22 mg/mL (100 mM). All tets item and control solutions were prepared immediately prior to use.
- PREPARATION OF THE STANDARD CURVE: Dilution buffer was prepared by mixing ammonium acetate buffer (pH 10.20, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1) was prepared by mixing peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (from 0.534 to 0.0167 mM). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analyzed by HPLC.
- REFERENCE CONTROL: like for cysteine
- PEPTIDE ASSAY METHOD: The assay contained a 1:50 molar ratio of peptide to test item. Cinnamic aldehyde or test item (250 µL) were mixed with peptide solution (750 µL). The vials were mixed by inversion and vortex. Co-elution controls were prepared by mixing together ammonium acetate buffer (750 µL) and test item (250 µL). All vials were stored in the dark at ambient temperature for ca 24 h until analysed by HPLC.

CHROMATOGRAPHIC AND DETECTOR PARAMETERS
- Column: Phenomenex Luna C18 (2) (2 x 100 mm, 3 µm)
- Run Time: 20 min
- Mobile Phase Conditions: Mobile Phase A: trifluoracetic acid (0.1%, v/v) in Milli-Q H2O
Mobile Phase B: trifluoracetic acid (0.085%, v/v) in acetonitrile
- Flow Rate: 0.35 mL/min
- Column Temperature: 30°C
- Auto Sampler Temperature: Room temperature
- Injection Volume: 7 µL
- UV Wavelength: 220 nm
- HPLC Gradient: see below

CALCULATIONS:
The concentration of peptide remaining in each sample following incubation was calculated from integrated peak area, with reference to the peptide standard curve. Percent peptide depletion was calculated from the following formula:
Peptide Depletion (%) = 1 – ( Peak Area (Sample) / Mean Peak Area (Reference Control C)) x 100
Positive control results:
The mean depletion value for the positive control was 70.1% showing a high reactivity (Sensitizer)
Key result
Run / experiment:
other: DPRA cysteine and lysine prediction model
Parameter:
other: %peptide depletion (mean value)
Value:
4.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Cinnamic aldehyde
Remarks on result:
other:
Remarks:
Minimal reactivity (Non-Sensitizer)
Other effects / acceptance of results:
No co-elution of the test item with either peptide was observed.

SYSTEM SUITABILITY FOR THE CYSTEINE ASSAY
The calibration linearity, r2, for the cysteine standard curve was 0.9955. This met the acceptance criteria for r2 which was >0.990.

The mean peptide concentration of Reference Control A was 0.608 ± 0.003 mM (mean ± SD). The calculated peptide concentration in the Reference Control C samples was 0.599 ± 0.004 mM (acetonitrile) and 0.576 ± 0.005 mM (ultrapure water). These controls did not meet the acceptance criteria (0.5 ± 0.05 mM).

For the six Reference Control B and three Reference Control C samples in acetonitrile, the coefficient of variation (CV) was 1.7% (acceptance criteria for CV was <15%).

The mean percentage peptide depletion value of the three replicates for cinnamic aldehyde fell within the lower bound and upper bound values of 60.8% and 100.0% for cysteine, with a peptide depletion value of 84.6 ± 0.1% (mean ± SD).

Finally, the standard deviation of replicate test item samples was <14.9% for a-Ketoglutarate, di-Na (actual SD was 0.9%).

The data have been accepted despite the Reference Control A and Reference Control C samples not meeting acceptance criteria. This was due to an error in the preparation of the standard curve, leading to the top two concentrations being erroneous (lower than expected). As the top sample in the standard curve was the only sample with a higher nominal concentration than the reference controls, this could not be excluded from the calculations. The standard curve is used to calculate the concentration of peptide present in the Reference Control samples whereas peptide depletion is calculated from the peak area of the appropriate Reference Control C samples. The function of Reference Control A is to demonstrate the accuracy (or otherwise) of the standard curve, which it did. The function of the Reference Control C samples is to demonstrate peptide stability in the solvents. The peptide was shown to be stable from the consistent high (and historically acceptable) peak area values measured for all reference control samples.

Therefore, the concentration of peptide present in the standards does not have an impact on the calculation of peptide depletion, and furthermore all other acceptance criteria have been met, including a correct prediction for the positive control. The results are accepted and there is no impact on the outcome of the cysteine assay.

SYSTEM SUITABILITY FOR THE LYSINE ASSAY
The calibration linearity, r2, for the lysine standard curve was 1.0000. This met the acceptance criteria for r2 which was >0.990.

The mean peptide concentration of Reference Control A was 0.504 ± 0.001 mM (mean ± SD). The mean peptide concentration of Reference Control B analysed prior to the testing run was 0.504 ± 0.002 mM. The mean peptide concentration of Reference Control B analysed after the testing run was 0.493 ± 0.003 mM. The calculated peptide concentration in the Reference Control C samples was 0.502 ± 0.002 mM (acetonitrile), 0.499 ± 0.006 mM (ultrapure water). These samples met the acceptance criteria of 0.5 ± 0.05 mM. In addition, for the six Reference Controls B and three Reference Control C in acetonitrile, the CV was 1.1% (acceptance criteria for CV was <15%).

The mean percentage peptide depletion value of the three replicates for cinnamic aldehyde fell within the lower bound and upper bound values of 40.2% and 69.0% for lysine, with the SD <11.6%. The actual percentage peptide depletion value reported for cinnamic aldehyde was 55.6% ± 4.3% (mean ± SD). Finally, the standard deviation of replicate test item samples was <11.6% for the test item (actual SD was 1.1%).

PROTOCOL DEVIATIONS
-Prior to HPLC analysis, the samples should be inspected visually. This was not done. Samples were to be inspected for the presence of precipitate. This will only impact upon the test item samples, as all other samples were prepared according to the prescribed method, and do not have solubility concerns. Solubility of the test item was assessed previously, and not found to be an issue. Therefore, precipitates are very unlikely, and there is no impact on study integrity.
-The time between sample preparation and the last injection of a sequence should not exceed 35 h. For the Cysteine samples reported, the time between sample preparation and the injection of the last sample was 35 h and 15 min. The ECVAM protocol actually allows a far greater time window than this. In addition, the final sample was one of the replicates of Reference Control B, the purpose of which is to confirm the stability of the peptide over the analysis time. The CV of these samples met the acceptance criteria, therefore the peptide was stable over the duration of testing. There is no impact on study integrity.
- Details the acceptance criteria for the tests. In the cysteine assay these criteria were not met for the peptide concentration in Reference Control C and Reference Control A however, the data was accepted. The peptide concentration in the reference control samples was calculated from the Standard Curve. There was a suspected preparation error in the standard curve, leading to the top two concentrations being erroneous (lower than expected). Standard 2 was excluded due to being an outlier from the linear relationship. As the top sample in the standard curve was the only sample with a higher nominal concentration than the reference controls, this could not be excluded from the calculations. The standard curve is used to calculate the concentration of peptide present in the Reference Control samples whereas peptide depletion is calculated from the peak area of the appropriate Reference Control C samples. The function of Reference Control A is to demonstrate the accuracy (or otherwise) of the standard curve, which it did. The function of the Reference Control C samples is to demonstrate peptide stability in the solvents. The peptide was shown to be stable from the consistent high (and historically acceptable) peak area values measured for all reference control samples. Therefore, the concentration of peptide present in the standards does not have an impact on the calculation of peptide depletion, and furthermore all other acceptance criteria have been met, including a correct prediction for the positive control. The results are accepted and there is no impact on the outcome of the cysteine assay.

DEMONSTRATION OF TECHNICAL PROFICIENCY
Prior to use, Charles River Laboratories demonstrated technical proficiency in the DPRA test, using the panel of proficiency chemicals listed in OECD 442C (Toner, F, 2015).

 Test Item % Peptide Depletion Cysteine (Mean  ± SD) % Peptide Depletion Lysine (Mean ± SD) Mean of Cysteine and Lysine DPRA Classification (Cysteine and Lysine Prediction Model)
 Test Material  6.4 ±0.9 2.8 ± 1.1   4.6  Minimal Reactivity (Non-Sensitizer)
 Positive control  84.6 ±0.1  55.6 ±4.3  70.1 High Reactivity (Sensitizer)

Using the cysteine and lysine prediction model (see Table below) the test material was categorised as minimally reactive and a non-sensitiser.

Mean depletion values (Cys Lys)  Mean Depletion values (cys only) Reactivity classification  DPRA Prediction
 <6.38 %  <13.89%  Minimal

 Non Sensitizer

 6.38 -22.62%  13.89 -23.09%  Low  Sensitizer
 22.62 -42.47%  23.09%-98.24%  Moderate  Sensitizer
 >42.47  >98.24%  High  Sensitizer
Interpretation of results:
other: minimally reactive: non-sensitizer
Remarks:
Study will be used for classificatin in combination with other studies (Weight of Evidence)
Conclusions:
In conclusion, according to the DPRA cysteine and lysine prediction model alpha-Ketoglutarate, di-Na (CAS 305-72-6) was classified as minimally reactive and was, therefore, a non-sensitiser.
Executive summary:

Skin sensitisation is a type IV (delayed) hypersensitivity reaction that results from the interaction of a sensitising agent with host proteins to form an immunogenic complex.

Small molecules that can interact with proteins in this way are referred to as haptens, and are generally not immunogenic in isolation. Hapten-modified proteins are recognised as foreign by antigen presenting cells, leading to T-cell activation and localised inflammation at the site of all subsequent exposures to the hapten.

Most skin sensitising agents are electrophiles, i.e. will accept an electron pair from a nucleophile to form a covalent bond.  The amino acids cysteine and lysine are thought to be the nucleophiles most frequently modified in proteins during sensitisation, and the ability of small molecules to react with these amino acids forms the basis of the Direct Peptide Reactivity Assay (DPRA).

The objective of this study was to assess the peptide binding capability of test material using synthetic cysteine and lysine peptides and to classify the test material to one of the four reactivity classes leading to a DPRA prediction according to the following prediction model.

 Mean depletion values (Cys Lys)  Mean Depletion values (cys only) Reactivity classification  DPRA Prediction
 <6.38 %  <13.89%  Minimal

 Non Sensitizer

 6.38 -22.62%  13.89 -23.09%  Low  Sensitizer
 22.62 -42.47%  23.09%-98.24%  Moderate  Sensitizer
 >42.47  >98.24%  High  Sensitizer

The reaction of the test item with synthetic peptides containing cysteine (Ac-RFAACAA-COOH) or lysine (Ac-RFAAKAA-COOH) was performed.  The custom peptides contained cysteine or lysine as the nucelophilic reaction centres and phenylalanine to facilitate detection by HPLC analysis.

For each peptide assay, the test item was prepared at a concentration of 100 mM in ultrapure water as determined from the solubility experiment conducted under Charles River Study No. 799765 (Vinall, J, 2017).  The test item and peptides were combined and incubated together for ca 24 h at room temperature. Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC.  From the results obtained, a reactivity class was assigned and a DPRA prediction was made according to the above criteria.

The results obtained are presented in the following table:

 Test Item % Peptide Depletion Cysteine  % Peptide Depletion Lysine  DPRA Classification
 Test Material  6.4  ± 0.9  2.8  ± 1.1  Minimal Reactivity (Non-Sensitizer)
 Cinnamic Aldehyde (positive control) 84.6  ± 0.1   55.6 ± 4.3  Moderate Reactivity (Sensitizer)

The acceptance criteria for the test were fulfilled, with the exception that for the cysteine test, the peptide concentration calculated in the Reference Control A and Reference Control C samples was slightly high, due to an error in the preparation of the standard curve, leading to the top two concentrations being erroneous (lower than expected).  This does not have an impact on the calculation of peptide depletion, and furthermore all other acceptance criteria have been met, including a correct prediction for the positive control, therefore the results are accepted and there is no impact on the outcome of the cysteine assay.

In conclusion, according to the DPRA cysteine and lysine prediction model, alpha-Ketoglutarate, di-Na (CAS 305-72-6) was classified as minimally reactive and was, therefore, a non-sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20 February - 17 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
- Test concentrations: final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.977 µM (final concentration vehicle DMSO of 1%)
- All concentrations of the test item were tested in triplicate.
- Positive control: Ethylene dimethacrylate glycol, final concentration 7.81 to 250 µM (final concentration DMSO of 1%)
- Negative control: vehicle: 1% DMSO in exposure medium

- Test System A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland).

- Cell culture:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Manteinance Medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

- Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.3 °C).

EXPEIMENTAL DESIGN
- Plating of cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each test item, one plate was used for the luciferase activity measurements, and one parallel plate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 21 in experiment 1 and 25 in experiment 2.
- Treatment of cells
The medium was removed and replaced with fresh culture medium (150 µL culture medium containing serum but without Geneticin) to which 50 µL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. In total 2 experiments were performed.

- Luciferase activity measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).
- Cytotoxicity assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 µM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- Data analysis
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

- Data interpretation
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction


Positive control results:
• Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.64 and the EC1.5 42.9 µM.
• Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.47 and the EC1.5 36.3 µM.
Key result
Run / experiment:
other: 1
Parameter:
other: maximal average fold induction of luciferase activity (Imax)
Value:
1.28
Vehicle controls validity:
valid
Remarks:
No luminesce activity induction compared to the vehicle control was observed at any of the test concentrations
Positive controls validity:
valid
Remarks:
Imax: 2.64
Key result
Run / experiment:
other: 2
Parameter:
other: maximal average fold induction of luciferase activity (Imax)
Value:
1.29
Vehicle controls validity:
valid
Remarks:
No luminesce activity induction compared to the vehicle control was observed at any of the test concentrations
Positive controls validity:
valid
Remarks:
Imax: 4.47
Other effects / acceptance of results:
Acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
-The EC1.5 of the positive control was between 5 and 125 µM (42.9 µM and 36.3 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.64-fold and 4.47-fold in experiment 1 and 2, respectively).
-Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.2% and 9.8% in experiment 1 and 2, respectively).

alpha-Ketoglutarate, Di-Na showed no toxicity in both experimetns. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.

Table1          
Overview luminescence induction and cell viability of a-Ketoglutarate, Di-Na in Experiment 1 and 2

Concentration (µM)

0.977

1.95

3.91

7.81

15.6

31.3

62.5

125

250

500

1000

2000

Exp 1 luminescence

0.75

1.06

1.09

1.16

1.11

0.99

0.99

1.28

1.08

0.86

1.06

1.10

Exp 1 viability (%)

92.0

102.3

99.3

79.3

62.9

96.6

133.6

131.7

83.2

124.2

131.8

125.5

Exp 2 luminescence

0.83

0.92

0.96

1.02

1.03

1.08

1.02

1.12

1.13

1.21

1.16

1.29

Exp 2 viability (%)

88.2

75.7

93.8

97.3

92.7

91.0

96.6

99.1

100.6

104.3

109.1

108.6

 

Table2          
Overview luminescence induction and cell viability positive control Ethylene dimethacrylate glycol in Experiment 1 and 2

Concentration (µM)

7.81

15.6

31.3

62.5

125

250

Exp 1 luminescence

1.18

1.38

1.31

1.70***

2.06***

2.64***

Exp 1 viability (%)

76.7

92.4

112.1

111.4

106.4

99.1

Exp 2 luminescence

1.12

1.25

1.44

1.84***

2.30***

4.47***

Exp 2 viability (%)

110.8

113.6

120.6

122.5

117.8

118.1

***p<0.001 Students t test

 

Table3          
Overview EC1.5, Imax, IC30and IC50values

 

EC1.5 (µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

NA

1.28

NA

NA

Test item Experiment 2

NA

1.29

NA

NA

Pos Control Experiment 1

42.9

2.64

NA

NA

Pos Control Experiment 2

36.3

4.47

NA

NA

NA = Not applicable

Interpretation of results:
other: no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes
Remarks:
Study will be used for classificatin in combination with other studies (Weight of Evidence)
Conclusions:
In conclusion, the KeratinoSensTM assay is valid and a-Ketoglutarate, Di-Na (CAS No. 305-72-6) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of a-Ketoglutarate, Di-Na (CAS No. 305-72-6) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 14152300 of the test item was a white crystalline powder with a purity of 99.3%.  The test item was dissolved in dimethyl sulfoxide at 200 mM.  From this stock 11 spike solutions in DMSO were prepared.  The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.977 – 2000 µM (2-fold dilution series).  The test item precipitated at the highest dose level tested.  Two independent experiments were performed.

Both experiments passed the acceptance criteria:

-The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.

-The EC1.5 of the positive control was between 5 and 125 µM (42.9 µM and 36.3 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (2.64-fold and 4.47-fold in experiment 1 and 2, respectively).

-Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.2% and 9.8% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

The test item showed no toxicity (no IC30 and IC50 value) and no induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.  The maximum luciferase activity induction (Imax) was 1.28-fold and 1.29-fold in experiment 1 and 2 respectively.  a-Ketoglutarate, Di-Na is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of =1000 µM.

In conclusion, the KeratinoSensTM assay is valid and a-Ketoglutarate, Di-Na (CAS No. 30572-6) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

All three studies performed, DEREK, DPRA and KeratinoSens assay, conclude that alpha-ketoglutarate, di-Na is considered not to be a skin sensitizer.

Moreover, alpha-ketoglutarate (, di-Na) is an endogenous intermediate in the citric acid cycle, which takes place in the mitochondria of each eukaryotic cell, including those in the skin. It is a molecule involved in multiple metabolic and cellular pathways (Voet & Voet, 1995). When lesion of the skin occurs, necrosis of cells results in the skin being exposed to the cell content, and therefore to alpha-ketoglutarate (, di-Na). Consequently, alpha-ketoglutarate (, di-Na), an endogenous substance omnipresent in the body, is not considered to be a skin sensitizer.

In conclusion to alpha-ketoglutarate, di-Na (CAS No. 305-72-6) does not have to be tested further and does not have to be classified for skin sensitization according to Regulation 1272/2008 and amendments.

 

Ref: Voet, D & Voet, JG.Biochemistry, 2nd Edition, John Wiley & Sons, Inc.NY, 1995.