Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Endpoint summary

Description of key information

Based on animal welfare considerations a combination of non-animal methods in form of an adverse outcome pathway was performed to assess skin sensitisation potential of the test item. These non-animal approaches contain the molecular initiating event of skin sensitisation (in-chemicoprotein reactivity-OECD TG 442c) and the intermediate events (in-vitroARE-Nrf2 Luciferase Test Method OECD TG 442d and dendritic cell activation OECD 442e).

Due to the equivocal results of these test methods and the limited applicability especially for the tested metallic compounds further in-vivo testing (LLNA-OECD TG 429) for assessing skin sensitisation potential of the test item was performed. Based on the LLNA the test item can be considered as a moderate skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-03 to 2018-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species/strain: healthy CBA/CaOlaHsd mice
Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
Sex: female (nulliparous and non-pregnant)
Age at the beginning of the study: 8-9 weeks
Number of animals: 5 mice / group; 5 mice / prescreen test
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Vehicle:

dimethyl sulphoxide

Remarks:

Sigma-Aldrich, lot no. SHBG5154V, expiry date: 03/2019

Concentration:

Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
6.25%, 12.5% and 25% (w/v)
The preparations were made immediately prior to each dosing.

No. of animals per dose:

5 animals/dose group, 5 animals/control group
5 mice / prescreen test

Details on study design:

Prescreen Test
Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item in the vehicle was found to be 25% in DMSO (Sigma-Aldrich, lot no. SHBF2742V, expiry date: 17/03/2019).
 
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema and scored. Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.

Four animals were treated by topical application with the test item on three consecutive days at the following concentrations to the entire dorsal surface of each ear:
Animals no. 51 and no. 52 were treated with a test item concentration of 25% (suspended in DMSO)
Animals no. 53 and no. 54 were treated with a test item concentration of 12.5% (suspended with DMSO)
One further animal was treated with 100% DMSO and served as negative control (animal no. 55).
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured.
During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal.
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test.
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
6.25%, 12.5% and 25% (w/v)
The preparations were made immediately prior to each dosing.
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
6.25%, 12.5% and 25% (w/v)
The preparations were made immediately prior to each dosing.

Controls
DMSO was used as vehicle and served as negative control.
For animal welfare reasons the negative control was shared.
Positive controls are performed periodically. The recent results are included in the report.

Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (latest version, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).


Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical application of
25 µL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals was carried out
approximately 48 hours after the first application.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1. 
Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of
3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. Shortly before sacrificing the
thickness of the ears of all animals was measured for a third time. The draining auricular lymph nodes were excised, individually pooled for each
animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was
prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension
was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of
macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution
was transferred into scintillation vials and stored at room temperature overnight.
Determination of Incorporated 3H-Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM).
Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for
each animal.
This measurement was performed twice. The initial measurement is considered invalid due to technical reasons. The second measurement was
performed approximately one month after first measurement. As the 3-H half-life time is 12.32 years an influence on the measured activity is
excluded.

Positive control substance(s):

other: Phenylenediamine

Statistics:

Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM).
If outliers were identified, these values were not included in the calculation of the stimulation indices. As at least four values per group are required
for the evaluation of the results, the outlier test was not repeated to detect further outliers.

Positive control results:

A shared positive control was performed concomitantly, using 5 animals.
1% phenylenediamine (Sigma-Aldrich, lot no.: WXBC3008V, expiry date 01/03/2019) in DMSO was used as positive-control substance. The Stimulation Index was 8.4.

Key result

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

One of the three tested concentrations exceeded the stimulation index of 3. The stimulation index at a concentration of 6.25% was 0.5 The stimulation index at a concentration of 12.5% was 1.3 The stimulation index at a concentration of 25% was 3.2

Key result

Parameter:

SI

Value:

0.5

Test group / Remarks:

Stimulation with a concentration of 6.25%

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Stimulation with a concentration of 12.5%

Key result

Parameter:

SI

Value:

3.2

Test group / Remarks:

Stimulation with a concentration of 25%

Key result

Parameter:

EC3

Value:

23.64

Remarks on result:

other:

Remarks:

calculated EC3 = 23.64%

All animals survived throughout the test period without showing any clinical signs.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item was tested in dose groups of 6.25%, 12.5% and 25%. The test item showed a stimulation index of >3 at a concentration of 25%. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 23.64%, which provides an estimation of sensitisation potency.
The results of the radioactivity determination are supported by the second endpoint, the means of the lymph node weights per group, which showed increased values for the 25% test group compared to the negative control values.
According to Commission Regulation (EU) No 286/2011 (CLP) as well as GHS (Globally Harmonized Classification System) the test item Sanodure Fast Gold L dried has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Executive summary:

On the basis of the test results given below and in conformity with the criteria given in Commission Regulation (EU) No 286/2011 the substance should be:

classified into sub-category 1A
classified into sub-category 1B	X
unclassified

On the basis of the test results given below and in conformity with the criteria given in GHS (Globally Harmonized Classification System) the substance should be:

classified into sub-category 1A
classified into sub-category 1B	X
unclassified

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 6.25%, 12.5% and 25% (w/v), each suspended in dimethyl sulphoxide (DMSO).

Species/strain:                     mice, CBA/CaOlaHsd

Number of animals:            20 / main test

Vehicle:                         DMSO

Summary Results

There was no mortality and there were no significant clinical observations or effects on body weights.

One of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of       6.25% was     0.5

The stimulation index at a concentration of       12.5% was     1.3

The stimulation index at a concentration of       25%    was     3.2

There was a relevant increase in lymph node weight in the 25% test group compared to the negative control group.

The mean weight of the lymph node

for the 6.25% test group was                 1.9 mg

for the 12.5% test group was                 2.5 mg

for the 25% test group was                    3.2 mg

for the negative-control group was        2.5 mg

Conclusion

The test item Sanodure Fast Gold L was tested in dose groups of 6.25%, 12.5% and 25%. The test item showed a stimulation index of >3 at a concentration of 25%. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 23.64%, which provides an estimation of sensitisation potency. Based on this value the test item can be considered as a moderate sensitiser.

The results of the radioactivity determination are supported by the second endpoint, the means of the lymph node weights per group, which showed increased values for the 25% test group compared to the negative control values.

According to Commission Regulation (EU) No 286/2011 (CLP) as well as GHS (Globally Harmonized Classification System) the test item Sanodure Fast Gold L dried has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-04 to 2017-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Version / remarks:

January 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Details on the study design:

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.

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 66.77%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

3.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4835.2368	0.5340	4453.7617	0.5340
STD2	2314.6648	0.2670	2214.0920	0.2670
STD3	782.4597	0.1335	1103.6776	0.1335
STD4	525.4504	0.0667	542.1525	0.0667
STD5	266.4071	0.0334	261.3623	0.0334
STD6	135.9798	0.0167	131.8409	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1381.2087	0.1629	71.03	71.19	0.17	0.24
	1364.7275	0.1611	71.37
	1374.1948	0.1621	71.18
Test Item	0.0000	0.0109	100.00	100.00	0.00	0.00
	0.0000	0.0109	100.00
	0.0000	0.0109	100.00

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1562.8215	0.1884	62.85	62.34	0.45	0.72
	1598.4821	0.1926	62.00
	1591.5383	0.1918	62.17
Test Item	3995.6775	0.4796	3.75	3.76	0.45	11.87
	3976.6111	0.4773	4.21
	4013.6008	0.4818	3.31

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

Categorization of the Test Item

Predicition Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	51.88	High Reactivity	sensitizer	100.00	High Reactivity	sensitizer
Positive Control	66.77	High Reactivity	sensitizer	71.19	Moderate Reactivity	sensitizer

Conclusions:

In this study under the given conditions the test item showed high reactivity towards the cysteine peptide and might be considered as sensitiser. It should be noted that according to the guideline 442C metal compounds might not fall within the applicability domain of the DPRA. However, the data generated with this test 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:

In the present study the test item was dissolved in dist. water based on the results of the pre-experiments and a 100 mM stock solution was prepared. 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.

For the 100 mM stock solution of the test item no turbidity or precipitation 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. A slight precipitate was observed for the samples of the test item .Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation 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. Phase separation was observed for the samples of the positive control including the co-elution control of the positive control. Samples were not centrifuged prior to the HPLC analysis.

No co-elution of test item with the peptide peaks was observed, however, a slight precipitation was observed in the cysteine peptide experiment. Whereas negative results in combination with a precipitation cannot be evaluated as absence of sensitising potential, positive results can still be used. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

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% (51.88%). Based on prediction model 1 the test item might 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 66.77%.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-04 to 2017-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

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.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.55 in experiment 1; 3.62 in experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Remarks:

I max

Value:

44.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration

Remarks:

250 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

115.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Concentration 250 µM

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

37.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration

Remarks:

250 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

78.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Concentration 250 µM

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

1.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

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	100.7	97.2	99.0	2.5
	8.00	104.3	115.1	109.7	7.6
	16.00	106.8	106.5	106.6	0.2
	32.00	118.1	98.9	108.5	13.6
	64.00	122.0	99.4	110.7	16.0
Test Item	0.98	128.3	108.3	118.3	14.1
	1.95	134.1	108.1	121.1	18.3
	3.91	139.9	123.2	131.5	11.8
	7.81	159.4	141.3	150.3	12.8
	15.63	189.8	167.6	178.7	15.7
	31.25	216.6	188.2	202.4	20.0
	62.50	214.6	179.1	196.9	25.2
	125.00	185.5	131.3	158.4	38.3
	250.00	115.5	78.4	97.0	26.3
	500.00	31.2	33.4	32.3	1.5
	1000.00	7.2	9.3	8.3	1.5
	2000.00	0.1	0.2	0.2	0.1

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	1.08	1.13	1.21	1.14	0.07
	8.00	1.18	1.14	1.25	1.19	0.06
	16.00	1.34	1.30	1.40	1.35	0.05
	32.00	1.58	1.61	1.73	1.64	0.08	*
	64.00	2.39	2.60	2.66	2.55	0.14	*
Test Item	0.98	1.72	1.77	1.73	1.74	0.02	*
	1.95	1.47	1.94	1.81	1.74	0.24	*
	3.91	1.27	1.95	2.05	1.76	0.42	*
	7.81	2.11	2.72	2.22	2.35	0.33	*
	15.63	1.69	2.78	2.97	2.48	0.69	*
	31.25	3.35	4.10	4.17	3.87	0.46	*
	62.50	4.94	6.67	6.03	5.88	0.88	*
	125.00	15.15	21.96	20.25	19.12	3.54	*
	250.00	33.38	54.98	44.02	44.12	10.80	*
	500.00	13.96	23.03	16.66	17.88	4.66	*
	1000.00	7.60	9.19	8.41	8.40	0.80	*
	2000.00	1.52	1.11	0.70	1.11	0.41

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

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	1.25	1.21	1.11	1.19	0.07
	8.00	1.51	1.41	1.19	1.37	0.16
	16.00	1.51	1.71	1.29	1.50	0.21	*
	32.00	2.22	2.24	1.86	2.11	0.21	*
	64.00	3.60	3.90	3.36	3.62	0.27	*
Test Item	0.98	1.33	1.19	1.42	1.31	0.11
	1.95	1.31	1.30	2.01	1.54	0.41
	3.91	1.56	1.43	1.97	1.65	0.28	*
	7.81	2.10	1.93	2.29	2.10	0.18	*
	15.63	2.52	3.07	3.42	3.01	0.45	*
	31.25	4.31	4.72	3.72	4.25	0.50	*
	62.50	4.98	5.39	7.88	6.08	1.57	*
	125.00	21.91	28.08	21.96	23.98	3.55	*
	250.00	34.39	39.32	39.63	37.78	2.94	*
	500.00	16.50	14.42	16.15	15.69	1.11	*
	1000.00	8.86	6.65	6.56	7.36	1.30	*
	2000.00	0.83	0.18	0.30	0.44	0.34

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

Induction of Luciferase Activity – Overall Induction

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	1.14	1.19	1.17	0.04
	8.00	1.19	1.37	1.28	0.13
	16.00	1.35	1.50	1.43	0.11
	32.00	1.64	2.11	1.87	0.33
	64.00	2.55	3.62	3.08	0.76
Test Item	0.98	1.74	1.31	1.53	0.30
	1.95	1.74	1.54	1.64	0.14	*
	3.91	1.76	1.65	1.70	0.07	*
	7.81	2.35	2.10	2.23	0.17	*
	15.63	2.48	3.01	2.74	0.37	*
	31.25	3.87	4.25	4.06	0.27	*
	62.50	5.88	6.08	5.98	0.14	*
	125.00	19.12	23.98	21.55	3.44	*
	250.00	44.12	37.78	40.95	4.49	*
	500.00	17.88	15.69	16.78	1.55	*
	1000.00	8.40	7.36	7.88	0.74	*
	2000.00	1.11	0.44	0.77	0.48

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n/a	1.78	n/a	n/a
Imax	44.12	37.78	40.95	4.49
IC30[µM]	385.00	296.50	340.75	62.58
IC50[µM]	444.29	407.59	425.94	25.95

n/a – not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	9.6	pass	6.4	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	24.29	pass	15.89	pass
Induction PC at 64 µM	2.00 < x < 8.00	2.55	pass	3.62	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

 

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be considered as sensitiser. The data generated with this test 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:

In the present study Aluminium Fast Gold L dried was dissolved in dist. water and aBased on a molecular weight of 559.93 g/mol a stock solution of 200 mM was prepared. A factor of 1.24 was used to correct for the purity of the test item.

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 exposurecells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (I_max) induction of 44.12 was determined at a test item concentration of 250 µM. The corresponding cell viability was 115.5%. Since no luciferase induction < 1.5 with a viability > 70% was found, no EC_1.5value could be calculated. Cytotoxicity was observed in the concentration range 500 µM – 2000 µM. A medium precipitation was observed in the concentration 500 µM, strong precipitation was observed in the concentrations 1000 µM – 2000 µM.

In the second experiment, a max luciferase activity (I_max) induction of 37.78 was determined at a test item concentration of 250 µM. The corresponding cell viability was 78.4%. The lowest tested concentration with a significant luciferase induction > 1.5 (1.54) was found to be 1.95 µM. The corresponding cell viability was > 70% (108.1%). The calculated EC_1.5 was< 1000 µM (1.78 µM). Cytotoxicity was observed in the concentration range 500 µM – 2000 µM. A slight precipitation was observed in the concentration 250 µM, medium precipitation was observed in the concentrations 500 µM – 2000 µM.

The substance showed a tendency to reduce MTT, therefore, the cells were washed thoroughly. However, test item residues with potential to reduce MTT cannot be excluded. The high values of cell viability might be explained by this phenomenon. Furthermore, the high luciferase induction values in the higher test item concentrations could be indicating cell stress.

Although no EC_1.5 could be calculated in the first experiment because the lowest concentration already showed an induction > 1.5, the EC_1.5 must be < 1000 µM. All (other) criteria for sensitising potential are fulfilled. Furthermore, a dose response for luciferase activity induction was observed in the non-cytotoxic concentrations for each individual run as well as for an overall luciferase activity induction. Therefore, under the conditions of this study the test item might be considered as sensitiser.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-04 to 2017-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Details on the study design:

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.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Conclusions:

In the present study the test item was dissolved in 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 100.0 mg/mL to 0.78 mg/mL were prepared by a serial dilution of 1:2 resulting in final assay concentrations ranging from 1000.0 to 7.81 µg/mL. 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.
The test item showed a strong shift of the fluorescence signal compared to the solvent control and thus the cytotoxic effect of the test item cannot be evaluated. It was assumed, that the test item is a strong fluorescent test chemical emitting at the same wavelength as FITC and interferes with the flow cytometric detection.
Therefore, the study was stopped after the first dose finding experiment.

Executive summary:

Starting from 100 mg/mL (in 0.9% NaCl) 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 10⁶cells/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 10⁶cells/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.

The test item showed a strong shift of the fluorescence signal compared to the solvent control and thus the cytotoxic effect of the test item cannot be evaluated. It was assumed, that the test item is a strong fluorescent test chemical emitting at the same wavelength as FITC and interferes with the flow cytometric detection. Therefore, the study was stopped after the first dose finding experiment.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Justification for classification or non-classification

Due to the equivocal results of non-animal approches and the limited applicability of these test systems the LLNA was performed to adress skin sensitisation potential of the test item. Based on the results of LLNA and according to Commission Regulation (EU) No 286/2011 (CLP) as well as GHS (Globally Harmonized Classification System) the test item has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.