[No public or meaningful name is available]

Endpoint summary

Description of key information

An assessment of skin sensitisation potential of the registered substance has been performed based on the information available on the constituents, the substance was found to be non-sensitising 

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:

14 April - 06 May 2022

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 KeratinoSens™ test method)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Sponsor; Lot: 2021/3/17/1


- Purity, including information on contaminants, isomers, etc.:
UVCB


RADIOLABELLING INFORMATION (if applicable)
none

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature in a closed container

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
No correction was made for the composition/purity of the test material.
A solubility test was performed. The test material was suspended in DMSO to a final concentration of 40 mg/ml (black homogenous suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. The 100-fold dilution in DMEM glutamax of 40 mg/ml and 20 mg/ml showed moderate precipitate and was therefore not suitable to test. The 100-fold dilution of the 10 mg/ml DMSO stock showed slight precipitation. This concentration was selected as highest concentration for the main assay (limit of solubility).
In the main experiments the test material was suspended in dimethyl sulfoxide (DMSO) at 10 mg/ml (black homogenous suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098 and 0.049 µg/ml (final concentration DMSO of 1%). All concentrations of the test material were tested in triplicate. All formulations formed a clear solution.
In experiment 1 the test material precipitated at concentration 25 µg/ml and upwards at the start of the treatment and at 1.6 µg/ml and upwards at the end of the treatment period. In experiment 2 the test material precipitated at concentration 3.1 µg/ml and upwards at the start of the treatment and at 3.1 µg/ml and upwards at the end of the treatment period.
Test material concentrations were used within 2 hours after preparation.
Any residual volumes were discarded.

Preparation of the Positive Control:
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.4.1, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The formulation of the positive control was used in studies performed concurrently.


Preparation of the Vehicle Control
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
Treatment of Cells
The medium was removed and replaced with fresh exposure medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control materials were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2.

SEEDING AND INCUBATION
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh DMEM Glutamax containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C
± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

LUCIFERASE ACTIVITY MEASUREMENTS
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. The cells were washed with 100 µL PBS and then 100 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 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds)

Vehicle / solvent control:

DMSO

Negative control:

other: Vehicle control

Positive control:

other: Ethylene dimethacrylate glycol

Positive control results:

See any other information

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

7.1 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The test material showed toxicity (IC30 values of 7.1 µg/ml and 4.2 µg/ml and IC50 values of 37 µg/ml and 32 µg/ml in experiment 1 and 2, respectively). No biologically relevant 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 1.20-fold and 1.45-fold in experiment 1 and 2 respectively. The test material is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations <200 µg/ml with a cell viability of <70% compared to the vehicle control.

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

Both tests passed the acceptance criteria

Experiment 1

• The test material precipitated at concentration 25 µg/ml and upwards at the start of the treatment and at 1.6 µg/ml and upwards at at the end of the treatment period


• The test material showed toxicity. The calculated IC₃₀ was 7.1 µg/ml and the calculated IC₅₀ was 37 µg/ml.


• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The I_max was 1.20 and therefore no EC₅ could be calculated.


• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 3.86 and the EC₅ 25 µM.

Experiment 2

• The test material precipitated at concentration 3.1 µg/ml and upwards at the start of the treatment and at 3.1 µg/ml and upwards at the end of the treatment period


• The test material showed toxicity. The calculated IC₃₀ was 4.2 µg/ml and the calculated IC₅₀ was 32 µg/ml.


• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The I_max was 1.45 and therefore no EC₅ could be calculated.


• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 2.72 and the EC₅ 46 µM.

Both tests passed the acceptance criteria:

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


• The EC₅ of the positive control was within two standard deviations of the historical mean (25 µM and 46 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.86-fold and 2.72-fold in experiment 1 and 2, respectively).


• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.8% and 10% in experiment 1 and 2, respectively).

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

 

Table 1          
Overview Luminescence Induction and Cell Viability of Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres in Experiment 1 and 2

Concentration (µg/ml)	0.049	0.098	0.20	0.39	0.78	1.6	3.1	6.3	13	25	50	100
Exp 1 luminescence	1.07	1.17	1.15	1.10	1.11	1.20	1.15	1.08	0.98	0.90	0.67	0.63
Exp 1 viability (%)	105	99	95	102	93	83	75	71	61	53	46	43
Exp 2 luminescence	1.11	1.31	1.45	1.38	1.38	1.42	1.44	1.40	1.26	0.92	0.76	0.68
Exp 2 viability (%)	95	94	89	86	84	77	72	66	58	53	44	43

 

Table 2          
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.20	1.38	1.59^***	1.90^***	2.39^***	3.86^***
Exp 1 viability (%)	102	99	98	99	100	97
Exp 2 luminescence	1.15	1.16	1.37	1.65^***	2.07^***	2.72^***
Exp 2 viability (%)	94	87	83	83	79	74

^*** p<0.001 Student’s t test

 

Table 3          
Overview EC_1.5, I_max, IC₃₀ and IC₅₀ Values

	EC1.5 (µg/ml)	Imax	IC30 (µg/ml)	IC50 (µg/ml)
Test material Experiment 1	NA	1.20	7.1	37
Test material Experiment 2	NA	1.45	4.2	32
	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Pos Control Experiment 1	25	3.86	NA	NA
Pos Control Experiment 2	46	2.72	NA	NA

NA = Not applicable

 

Interpretation of results:

other: negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions

Conclusions:

In conclusion, Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres 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 Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensÔ assay.

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

Batch 2021/3/17/1 of the test material was a fine black powder. The test material was suspended in dimethyl sulfoxide (DMSO) at 10 mg/ml. 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 0.049 – 100 µg/ml (2-fold dilution series). The highest test concentration was considered to be the limit of solubility. In experiment 1 the test material precipitated at concentration 25 µg/ml and upwards at the start of the treatment and at
1.6 µg/ml and upwards after the treatment period. In experiment 2 the test material precipitated at concentration 3.1 µg/ml and upwards at the start of the treatment and at
3.1 µg/ml and upwards after the treatment period.

Both tests passed the acceptance criteria:

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


• The EC₅ of the positive control was within two standard deviations of the historical mean (25 µM and 46 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.86-fold and 2.72-fold in experiment 1 and 2, respectively).


• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.8% and 10% 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 material showed toxicity (IC₃₀ values of 7.1 µg/ml and 4.2 µg/ml and IC₅₀ values of 37 µg/ml and 32 µg/ml in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.20-fold and 1.45-fold in experiment 1 and 2 respectively. The test material is classified as negative in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations <200 µg/ml with a cell viability of <70% compared to the vehicle control.

In conclusion, Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres 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.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

Assessment based on available information

Type of information:

other: Assessment

Adequacy of study:

weight of evidence

Study period:

2022

Reliability:

other: Assessment

Rationale for reliability incl. deficiencies:

other: Assessment based on available information

Principles of method if other than guideline:

An assessment of skin sensitisation potential of the registered substance was performed based on the information available on the constituents (see attachment for full details).

Type of study:

other: All data available in literature for constituents

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Sponsor; Lot: 2021/3/17/1


- Purity, including information on contaminants, isomers, etc.:
UVCB


RADIOLABELLING INFORMATION (if applicable)
none

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature in a closed container

Key result

Remarks on result:

other: Not likely to be a sensitizer based on available information on the constituents.

Interpretation of results:

GHS criteria not met

Conclusions:

All known constituents of the registered substance can be concluded as non-sensitizers based on the available information in the literature or based on the general experience related to widespread and historical use. Due to the presence of unknown constituents in the substance, there is minor uncertainty in the skin sensitisation endpoint for the registered substance. However, based on the currently available information on the known constituents, the substance does not meet the sensitisation classification criteria of the CLP regulation. (See attachment for full assessment).

Executive summary:

An assessment of skin sensitisation potential of the registered substance has been performed based on the information available on the constituents.

 

Available data from animal studies suggest that carbon black, which is a major constituent of the registered substance, is not a skin sensitizer. Amorphous silica also does not appear to be skin sensitizer based on the available information. There is no animal data available for the crystalline form of silica, quartz; however, based on widespread and a long history of use of quartz, it is unlikely to be a skin sensitizer. Currently, no data on skin sensitisation potential of ZnS is available, however, ZnS can be presumed to be a non-sensitizer based on the information available for various highly as well as slightly soluble zinc salts. Similarly, there is currently no skin sensitization data available for CaO. However, CaO can be concluded to be a non-sensitizer based on supporting information provided in this assessment (see attachment).

 

It is re-emphasised here that the registered substance is a UVCB in which ~6% of the constituents are unknown. The ‘Unknowns’ accounts for inorganic and organic unknowns present at low individual concentrations (no one component present at >1% w/w) as determined by semi-quant XRF and CHN analysis (hydrogen and nitrogen). Due to the presence of unknowns, there is a minor uncertainty in the assessment. Nonetheless, CLP criteria sets the Generic Concentration Limit (GCL) of Category 1 skin sensitiser as ≥ 1%. Additionally, for category 1A skin sensitizers, the GCL is ≥ 0.1%. As stated above, none of the unknown constituents are present at >1% w/w therefore, it is highly unlikely that any of the unknown constituents will trigger a skin sensitization classification; however, there is still uncertainty because there could be an unknown constituent with category 1A classification present at level >0.1%.  

 

Overall, due to the presence of unknown constituents, the assessment of skin sensitisation endpoint for the registered substance demonstrates minor uncertainty. However, based on the currently available information on the known constituents, the substance does not meet the sensitisation classification criteria of the CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

OECD 442C study was not conducted because it is not suitable for UVCB substances. 

 

OECD 442E showed sedimentation of the test material after the incubation. The method does not appear to be suitable for the test material.

 

The substance was found to be negative in the OECD 442D test.

 

An assessment of skin sensitisation potential of the registered substance has been performed based on the information available on the constituents. Due to the presence of unknown constituents, the assessment of skin sensitisation endpoint for the registered substance demonstrates minor uncertainty. However, based on the currently available information on the known constituents, the substance does not meet the sensitisation classification criteria of the CLP regulation.

Justification for classification or non-classification

An assessment of skin sensitisation potential of the registered substance has been performed based on the information available on the constituents.

 

Available data from animal studies suggest that carbon black, which is a major constituent of the registered substance, is not a skin sensitizer. Amorphous silica also does not appear to be skin sensitizer based on the available information. There is no animal data available for the crystalline form of silica, quartz; however, based on widespread and a long history of use of quartz, it is unlikely to be a skin sensitizer. Currently, no data on skin sensitisation potential of ZnS is available, however, ZnS can be presumed to be a non-sensitizer based on the information available for various highly as well as slightly soluble zinc salts. Similarly, there is currently no skin sensitization data available for CaO. However, CaO can be concluded to be a non-sensitizer based on supporting information provided in this assessment (see attachment).

 

It is re-emphasised here that the registered substance is a UVCB in which ~6% of the constituents are unknown. The ‘Unknowns’ accounts for inorganic and organic unknowns present at low individual concentrations (no one component present at >1% w/w) as determined by semi-quant XRF and CHN analysis (hydrogen and nitrogen). Due to the presence of unknowns, there is a minor uncertainty in the assessment. Nonetheless, CLP criteria sets the Generic Concentration Limit (GCL) of Category 1 skin sensitiser as ≥ 1%. Additionally, for category 1A skin sensitizers, the GCL is ≥ 0.1%. As stated above, none of the unknown constituents are present at >1% w/w therefore, it is highly unlikely that any of the unknown constituents will trigger a skin sensitization classification; however, there is still uncertainty because there could be an unknown constituent with category 1A classification present at level >0.1%.  

 

Overall, due to the presence of unknown constituents, the assessment of skin sensitisation endpoint for the registered substance demonstrates minor uncertainty. However, based on the currently available information on the known constituents, the substance does not meet the sensitisation classification criteria of the CLP regulation.