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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

276-541-3

EC Name:

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

Cas Number:

72252-61-0

Molecular formula:

C16H10CuN4O8S2.2Na

IUPAC Name:

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

Test material form:

solid: nanoform

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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.

Results and discussion

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.

In vivo (LLNA)

Results

Results 1

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

Results 2

Key result

Parameter:

SI

Value:

0.5

Test group / Remarks:

Stimulation with a concentration of 6.25%

Results 3

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Stimulation with a concentration of 12.5%

Results 4

Key result

Parameter:

SI

Value:

3.2

Test group / Remarks:

Stimulation with a concentration of 25%

Results 5

Key result

Parameter:

EC3

Value:

23.64

Remarks on result:

other:

Remarks:

calculated EC3 = 23.64%

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.