Escherichia coli dehalogenase catalyst

Endpoint summary

Description of key information

In vitro studies:

Three in vitro studies were conducted with Escherichia coli dehydrogenase catalyst.

- In the in chemico direct peptide reactivity assay (DPRA), under the given conditions, the test item showed high reactivity towards both peptides. Therefore, based on the results of this study, the test item was considered to be a skin sensitiser.

- In the in vitro KeratinoSens™ assay, under the given conditions, the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, based on these results of this study, the test item can be considered not to be a skin sensitiser.

- In the in vitro human cell line activation test (h-CLAT), the test item did upregulate the cell surface markers in at least two independent experiment runs. Therefore, based on the results of this study, the test item is considered to be a skin sensitiser.

In summary, Escherichia coli dehydrogenase catalyst was considered to be a skin sensitiser in two out of three of the in vitro studies; in the third in vitro study, it was considered not to be a skin sensitiser.

Review of the available evidence on enzymes and skin sensitisation:

The AIS/AMFEP Ad-hoc group concluded that enzymes should not be classified as skin sensitisers. This conclusion is based on the following considerations:

1 The results of predictive testing in man demonstrate that enzymes do not have a significant skin sensitisation potential for man.

2. In a clinical setting, enzymes have only very rarely been suggested as a possible cause of allergic contact dermatitis. Even in these few cases a causal relationship has never been proven. More commonly clinical studies have demonstrated that enzymes are not a cause of allergic contact dermatitis (ACD).

3. ACD has never been reported where there has been extensive occupational enzyme exposure in the detergent enzyme industries which, in the past, has led to respiratory sensitisation and/or irritant dermatitis.

4. Over a 25 year period, billions of consumers have had skin exposure to enzymes but there is no evidence that this exposure has given rise to skin sensitisation.

5. Whilst immunological contact urticaria (ICU) is not involved within the skin sensitisation classification, enzymes like all other foreign proteins may cause this response.

In addition, we have not received any reports of skin sensitisation from the use/handling of this substance.

Conclusion:

Although, Escherichia coli dehydrogenase catalyst was considered to be a skin sensitiser in two out of three of the vitro studies, a review of the available evidence on enzymes and skin sensitisation indicates that enzymes should not be classified as skin sensitisers. Therefore, based on a weight of evidence approach, Escherichia coli dehydrogenase catalyst is not considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

8th to 15th June 2018

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

Remarks:

No deviations that influenced the quality or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 11879017 (first solubility test), 4898318 (second solubility test and main experiments)
- Expiration date of the lot/batch: August 2018 (11879017), March 2019 (4898318)
- Purity test date: Not specified
- Storage condition of test material: ≤ - 20°C

Details on the study design:

Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials.< The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.
Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.

Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions.

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days, all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Data Analysis
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated according to the following formula: PPD = (1-(Peptide Peak Area in the Replicate Injection/Mean Peptide Peak Area in Reference Control C))*100
The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent, a co-elution was assumed and the evaluation would be adjusted accordingly.
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration different from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model), the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides.
The mean depletion of both peptides was 64.83%.
Depletion of the lysine peptide: 58.47%; SD of peptide depletion: 2.23%; CV of peptide depletion: 3.81%.
Depletion of the cysteine peptide: 71.18%; SD of peptide depletion: 0.06%; CV of peptide depletion: 0.08%.
Prediction Model 1 (cysteine peptide and lysine peptide / ratio 1:10 amd 1:50): mean peptide depletion: 64.83%, reactivity category: high, prediction: sensitiser.
Prediction Model 2 (cysteine peptide / test item ratio: 1:10): 71.18%, reactivity category: moderate reactivity, prediction: sensitiser.

Key result

Run / experiment:

other: Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)

Parameter:

other: mean peptide depletion

Value:

56

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Parameter:

other: mean peptide depletion

Value:

71.59

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: lysine peptide depletion (%)

Value:

87.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Co-elution control

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion (%)

Value:

56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Co-elution control

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria were met.

Please refer to document attached below (OECD 442C Study 179042 Tables of Results) .

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. The test item was soluble in water. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiments were prepared using water. All test item solutions were freshly prepared immediately prior to use.

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the maximum solubility of 12.5 mg/mL solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the maximum solubility of 12.5 mg/mL solution of the test item turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Co-elution with the Peptide Peaks

No co-elution of the test item with any of the peptide peaks was observed.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed high reactivity towards both peptides. The test item is considered as “sensitiser”.
The data generated with this test item should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study Escherichia coli dehalogenase catalyst was dissolved in water, based on the results of the pre-experiments. Since no molecular weight could be derived the test item was tested to its maximum solubility which was found to be 12.5 mg/mL. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC analysis.

All test item solutions were freshly prepared immediately prior to use.

For the maximum solubility solution of 12.5 mg/mL of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged and transferred into other vials prior to the HPLC analysis.

For the maximum solubility solution of 12.5 mg/mL of the test item turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged transferred into other vials prior to the HPLC analysis. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Precipitation of the test item with both peptide peaks was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (71.59%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.83%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13th to 29th June 2018

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)

Deviations:

no

Remarks:

No deviations that influenced the quality or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 119345-29-8
- Expiration date of the lot/batch: March 2019
- Purity test date: Not specified
- Storage condition of test material: ≤ - 20°C

Details on the study design:

Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use.
As the test item had no defined molecular weight, the test was performed using a pro forma molecular weight of 200 g/mol. A stock solution with a concentration of 40 mg/mL or 4% (w/v) was prepared by pre-weighing the test material into a glass vial and dissolving the test item in dist. water (Sigma; Lot No.: RNBF7110; RNBG3520). A 0.2 µm sterile filter was used to sterilise the test sample solution.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. Since the test item was dissolved in water, DMSO was added at a final concentration of 4% (v/v).
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Controls
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO (AppliChem; Lot No.: 0001179895) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001179895) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Cell line
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 02 in experiment 1; P 04 in experiment 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 +/- 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.

Composition of Media

Maintenance Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1943444) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0167F)
- 1% geneticin (final concentration: 500 µg/mL; Gibco Life Science, Cat. No. 10131-027, Lot No.: 1881779)

Assay Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1943444) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0167F)

Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1943444) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0167F)

Luciferase Assay System
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual.
Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501, Lot No.: 0000306966) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.
If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000246522; 0000254353) consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3520)

Further Reagents
MTT solution
- MTT (VWR, CAS No.: 298-93-1, Lot No.: 0977C002) stock solution: 5 mg/mL MTT in DPBS (Gibco Life Science; Lot No.: 1943446; 1971148)
SDS solution:
- 10% (w/v) sodium dodecyl sulfate (SDS; AppliChem, CAS No.: 151-21-3, Lot No.: 8X012218; 7J011684) in dist. water (Sigma; Lot No.: RNBG3520)
DPBS:
DPBS solution (without Ca2+/Mg2+) (Gibco Life Science; Lot No.: 1943446; 1971148)

Dose Groups
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.

Experimental Procedure
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 2) or over the weekend (experiment 1). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Positive control results:

Mean fold induction at each concentration:
Experiment 1:
4 uM: 1.26 (SD: 0)
8 uM: 1.39 (SD: 0.02)
16 uM: 1.62 (SD: 0.31)
32 uM: 2.24 (SD: 0.1)
64 uM: 5.41 (SD: 0.27)

Experiment 2:
4 uM: 1.28 (SD: 0.03)
8 uM: 1.40 (SD: 0.06)
16 uM: 1.83 (SD: 0.18)
32 uM: 2.32 (SD: 0.24)
64 uM: 5.60 (SD: 0.88)

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 (uM)

Value:

208.56

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 (uM)

Value:

440.38

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: EC1.5 (uM)

Value:

324.47

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: I max

Value:

1.84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: I max

Value:

2.55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: I max

Value:

2.19

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: IC30 (uM)

Value:

364.26

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: IC30 (uM)

Value:

102.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Run 2 and Mean

Parameter:

other: IC50 (uM)

Value:

243

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The acceptance criteria were met. See Table below under 'Any other information on results incl. tables).

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	107.0	99.8	103.4	5.1
	8.00	111.9	102.8	107.3	6.5
	16.00	114.0	107.5	110.7	4.6
	32.00	116.6	106.9	111.8	6.8
	64.00	112.8	96.9	104.9	11.2
Test Item	0.98	113.3	105.5	109.4	5.5
	1.95	113.1	100.1	106.6	9.2
	3.91	117.6	107.1	112.4	7.4
	7.81	120.8	106.8	113.8	9.9
	15.63	116.9	105.8	111.4	7.9
	31.25	114.6	98.5	106.6	11.4
	62.50	112.2	86.4	99.3	18.3
	125.00	93.0	60.6	76.8	22.9
	250.00	73.9	49.4	61.7	17.4
	500.00	65.3	51.1	58.2	10.0
	1000.00	64.7	54.0	59.4	7.5
	2000.00	84.4	90.7	87.6	4.4

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.25	1.27	1.23	1.25	0.02
	8.00	1.28	1.25	1.58	1.37	0.18
	16.00	1.33	1.40	1.47	1.40	0.07
	32.00	2.02	2.03	2.47	2.17	0.25	*
	64.00	4.42	5.04	6.19	5.22	0.90	*
Test Item	0.98	0.91	1.02	1.43	1.12	0.27
	1.95	1.46	0.97	1.07	1.17	0.26
	3.91	1.25	1.19	1.11	1.18	0.07
	7.81	1.41	1.13	1.29	1.28	0.14
	15.63	1.39	1.27	1.23	1.29	0.08
	31.25	1.15	1.22	1.20	1.19	0.04
	62.50	1.39	1.28	1.32	1.33	0.06
	125.00	1.52	1.29	1.42	1.41	0.12
	250.00	1.47	1.75	1.41	1.54	0.18
	500.00	1.82	1.47	1.66	1.65	0.18	*
	1000.00	2.07	1.62	1.83	1.84	0.23	*
	2000.00	1.54	1.62	1.75	1.63	0.11	*

* = significant induction according to Student’s t-test, p<0.05

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.26	1.26	1.31	1.28	0.03
	8.00	1.43	1.34	1.45	1.40	0.06
	16.00	1.92	1.63	1.95	1.83	0.18	*
	32.00	2.37	2.06	2.52	2.32	0.24	*
	64.00	6.03	4.59	6.19	5.60	0.88	*
Test Item	0.98	0.68	0.88	0.96	0.84	0.14
	1.95	0.83	1.09	1.07	0.99	0.15
	3.91	0.87	1.06	1.10	1.01	0.13
	7.81	0.96	1.20	1.12	1.10	0.12
	15.63	0.98	1.18	1.19	1.12	0.12
	31.25	1.05	1.31	1.15	1.17	0.13
	62.50	1.09	1.19	1.21	1.16	0.06
	125.00	1.20	1.20	1.34	1.25	0.08
	250.00	1.33	1.45	1.43	1.40	0.06
	500.00	1.51	1.50	1.58	1.53	0.05	*
	1000.00	1.92	2.13	1.91	1.99	0.13	*
	2000.00	2.12	2.66	2.86	2.55	0.38	*

* = significant induction according to Student’s t-test, p<0.05

Table 4: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.25	1.28	1.26	0.02
	8.00	1.37	1.40	1.39	0.02
	16.00	1.40	1.83	1.62	0.31
	32.00	2.17	2.32	2.24	0.10	*
	64.00	5.22	5.60	5.41	0.27	*
Test Item	0.98	1.12	0.84	0.98	0.20
	1.95	1.17	0.99	1.08	0.12
	3.91	1.18	1.01	1.09	0.12
	7.81	1.28	1.10	1.19	0.13
	15.63	1.29	1.12	1.21	0.12
	31.25	1.19	1.17	1.18	0.01
	62.50	1.33	1.16	1.25	0.12
	125.00	1.41	1.25	1.33	0.12
	250.00	1.54	1.40	1.47	0.10
	500.00	1.65	1.53	1.59	0.09	*
	1000.00	1.84	1.99	1.91	0.10	*
	2000.00	1.63	2.55	2.09	0.65

* = significant induction according to Student’s t-test, p<0.05

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	208.56	440.38	324.47	163.93
Imax	1.84	2.55	2.19	0.50
IC30[µM]	364.26	102.24	233.25	185.28
IC50[µM]	n.a.	243.00	243.00	n.a.

n.a.: not applicable

 

Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	15.1	pass	8.9	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	3.0	pass
EC1.5 PC	7 < x < 34 µM	18.06	pass	9.77	pass
Induction PC at 64 µM	2.00 < x < 8.00	5.22	pass	5.60	pass

Table 7: Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.6	3.5	96
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.4	0.6	96
EC1.5 PC	7 < x < 34 µM	18.5	6.0	96
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.5	96

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.
The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study Escherichia coli dehalogenase catalyst was dissolved in dist. water. As the test item had no defined molecular weight, the test was performed using a pro forma molecular weight of 200 g/mol. Based on this, a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (I_max) induction of 1.84 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 64.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.54) was found to be 250 µM. The corresponding cell viability was >70% (73.9%). The calculated EC_1.5 was 208.56 µg/mL.

In the second experiment, a max luciferase activity (I_max) induction of 2.55 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 90.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.53) was found to be 500 µM. The corresponding cell viability was <70% (51.1%). The calculated EC_1.5 was 440.38 µg/mL.

In experiment 1 only a slight, but not significant induction was observed at one slightly cytotoxic concentration and in experiment 2 significant induction was only observed for cytotoxic concentrations. Under the condition of this study the test item is therefore considered as non-sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20th to 31st July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 09 October 2017

Deviations:

no

Remarks:

No deviations that influenced the quality or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 119345-29-8
- Expiration date of the lot/batch: March 2019
- Purity test date: Not specified
- Storage condition of test material: ≤ - 20°C

Details on the study design:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 40 mg/mL, which was the highest soluble concentration.
Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.
The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. A stable suspension was formed when diluted 1:250 in cell culture medium.
The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls
A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.
Medium Control
A medium control was included in the test.
Solvent Controls
Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent previously determined.
Since the test item and the positive control were solubilized in DMSO, a DMSO control served as solvent control for the test item and for the positive control.
The solvent controls were diluted resulting in a final concentration of 0.2% (v/v) for DMSO.
Positive Control
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted resulting in a final DMSO concentration of 0.2% (v/v).

Test System
FACS
FACS: BD Canto II
Software: BD FACS DIVA 6.0
Voltage Settings: FSC: 300 V SSC: 250 V FITC: 400 V PI: 420 V
Threshold value of FSC: 5000
Compensation: PI - 14 % FITC FITC – 0 % PI

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 106 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 μg/mL streptomycin at 37 ± 1°C and 5% CO2.

Dose Groups
1. Medium Control: cell culture medium
2. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
3. Positive Control: 4 μg/mL DNCB
4. Test Item: 8 concentrations of the test item
(dose finding assay/ main experiment)
dose finding assay 1:
80.0, 40.0, 20.0, 10.0, 5.00, 2.50, 1.25, 0.63 μg/mL
main experiment 1 and 2:
80.00, 66.67, 55.56, 46.30, 38.58, 32.15, 26.79, 22.33 μg/mL

Pre-Experiments
Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 μg/mL and nickel sulphate at a final concentration of 100 μg/mL served as positive control while lactic acid at a final concentration of 1000 μg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.
Solvent Finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL.

Experimental Procedure
Dose Finding Assay
Starting from 40 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer).After washing, cells were re-suspended in 600 μL FACS buffer. 200 μL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 μg/mL.
The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration.
The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression
The test item was dissolved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 μL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 μg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.

Data Analysis
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.

Positive control results:

Experiment 1:
Concentration: 4.00 ug/mL
Cell Viability (CD86): 87.9%
Cell Viability (CD54): 87.1%
Cell Viability (Isotype IgG1); 87.8%
Mean Fluorescence Intensity (CD86): 9888
Mean Fluorescence Intensity (CD54): 2112
Mean Fluorescence Intensity (Isotype IgG1): 602
corrected Mean Fluorescence Intensity (CD86): 9286
corrected Mean Fluorescence Intensity (CD54): 1510
Relative Fluorescence Intensity (CD86): 391
Relative Fluorescence Intensity (CD54): 307
Ratio Isotype IgG1to [%] (CD86): 1643
Ratio Isotype IgG1to [%] (CD54): 351

Experiment 2:
Concentration: 4.00 ug/mL
Cell Viability (CD86): 83.1%
Cell Viability (CD54): 84.5%
Cell Viability (Isotype IgG1); 83.8%
Mean Fluorescence Intensity (CD86): 10005
Mean Fluorescence Intensity (CD54): 2272
Mean Fluorescence Intensity (Isotype IgG1): 573
corrected Mean Fluorescence Intensity (CD86): 9432
corrected Mean Fluorescence Intensity (CD54): 1699
Relative Fluorescence Intensity (CD86): 293
Relative Fluorescence Intensity (CD54): 327
Ratio Isotype IgG1to [%] (CD86): 1746
Ratio Isotype IgG1to [%] (CD54): 397

Key result

Run / experiment:

other: 1 and 2

Parameter:

other: Relative Fluorescence Intensity (RFI) of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1 and 2

Parameter:

other: Relative Fluorescence Intensity (RFI) of CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Acceptance Criterion:
All acceptance criteria were met (see below for details):

cell viability solvent controls [%]:
Range: >90
Experiment 1: 95.8 - 96.9, pass
Experiment 2: 94.8 - 96.6, pass

number of test dosed with viability >50% CD86
Range: ≥4
Experiment 1: 8, pass
Experiment 2: 8, pass

number of test dosed with viability >50% CD54
Range: ≥4
Experiment 1: 8, pass
Experiment 2: 8, pass

number of test dosed with viability >50% IgG1
Range: ≥4
Experiment 1: 8, pass
Experiment 2: 8, pass

RFI of positive control of CD86
Range: ≥150
Experiment 1: 391, pass
Experiment 2: 293, pass

RFI of positive control of CD54
Range: ≥200
Experiment 1: 307, pass
Experiment 2: 327, pass

RFI of solvent control of CD86
Range: <150
Experiment 1: 108, pass
Experiment 2: 98, pass

RFI of solvent control of CD54
Range: <200
Experiment 1: 99, pass
Experiment 2: 76, pass

MFI ratio CD86/IgG1 for medium control [%]
Range: >105
Experiment 1: 435, pass
Experiment 2: 603, pass

MFI ratio CD86/IgG1 for DMSO control [%]
Range: >105
Experiment 1: 455, pass
Experiment 2: 661, pass

MFI ratio CD54/IgG1for medium control [%]
Range: >105
Experiment 1: 176, pass
Experiment 2: 205, pass

MFI ratio CD54/IgG1for DMSO control [%]
Range: >105
Experiment 1: 174, pass
Experimental 2: 191, pass

Please refer to document attached below (OECD 442E Study 179044 Tables of Results) .

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.
The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study Escherichia coli dehalogenase catalyst was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 40 mg/mL to 0.31 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

80.00, 66.67, 55.56, 46.30, 38.58, 32.15, 26.79, 22.33 μg/mL

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained, cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 93.3% (CD86), 91.3% (CD54) and 93.2% (isotype IgG1 control) in the first experiment and to 92.4% (CD86), 90.6% (CD54) and 92.3% (isotype IgG1 control) in the second experiment.

In both experiments, the expression of the cell surface marker CD86 was upregulated above the threshold of 150% at all tested concentrations. Furthermore, in both experiments the expression of the cell surface marker CD54 was upregulated above the threshold of 200% at all tested concentrations.

Since the expression of both cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

Expert report

Type of information:

other: Expert report

Adequacy of study:

weight of evidence

Study period:

8th May 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

An expert statement from the AIS/AMFEP Ad-hoc group.

Type of study:

other: Expert report on several study types

Specific details on test material used for the study:

Not applicable

Details on test animals and environmental conditions:

Not applicable

Remarks on result:

other: Expert statement; no results available

Remarks on result:

other: Expert statement; no results available

Remarks on result:

other: Expert statement; no results available

Interpretation of results:

other: Enzymes should not be classified as skin sensitisers

Conclusions:

After review of all the available evidence the AIS/AMFEP Ad-hoc group concluded that enzymes should not be classified as skin sensitisers. This conclusion is based on the following considerations:
1 The results of predictive testing in man demonstrate that enzymes do not have a significant skin sensitisation potential for man.
2. In a clinical setting, enzymes have only very rarely been suggested as a possible cause of allergic contact dermatitis. Even in these few cases a causal relationship has never been proven. More commonly clinical studies have demonstrated that enzymes are not a cause of ACD.
3. ACD has never been reported where there has been extensive occupational enzyme exposure in the detergent enzyme industries which, in the past, has led to respiratory sensitisation and/or irritant dermatitis.
4. Over a 25 year period, billions of consumers have had skin exposure to enzymes but there is no evidence that this exposure has given rise to skin sensitisation.
5. Whilst immunological contact urticaria (ICU) is not involved within the skin sensitisation classification, enzymes like all other foreign proteins may cause this response.

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

Escherichia coli dehydrogenase catalyst is not considered to be a skin sensitiser based on a weight of evidence approach.