Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper (2+) pentasodium 2-[2-{2-amino-5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxyphenyl}diazen-1-yl]-5-({3-carboxy-4-[2-{5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxy-2-oxidophenyl}diazen-1-yl]phenyl}methyl)benzoate and sodium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP Compliance Certificate of the Testing Laboratory attached in Annex 1 page 24 of the report

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

EC Number:

948-009-7

Molecular formula:

Not applicable for a multi-constituent substance

IUPAC Name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

Test material form:

solid: particulate/powder

Details on test material:

Acid brown 161 batch no. ID151860 CAS no. 85338-16-5
Composition of the muti-constituent substance is included in the report as an extract of the analytical report.

Specific details on test material used for the study:

Acid brown 161 batch no. ID151860, CAS no. 85338-16-5, Einecs Mo. 286-689-0.
Appearance: Dark brown powder. Purity and composition described in the attached "Extract of the analytical report".

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

29 females of 9-11 weeks, female animals were non-pregnant and nulliparous were used. The health condition of animals was examined by a veterinarian before initiation of the study. The animals were acclimatated in identical conditiona as during the experiment for 5 days prior to start the treatment. The acclimatation was according to standard operation procedures.
The animals were housed in IVC polycarbonate cages (5 animals per cage) suspended in stainless steel racks, in a room equipped with central air-conditioning. The room temperature was wihing the range of 22+-3ºC, relative humidity was within the range 50-60%.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

1000 mg/mL

No. of animals per dose:

5 animals per dose

Details on study design:

Dose preparation: 1000 mg was mixed with the vehicle up to a volume of 1 mL (1000 mg/mL).
This was the highest achievable concentration (100% w/v). The lower concentrations were prepared by mixing of 500 mg and 250 mg with vehicle up to 1 mL –giving 50, 25% w/v concentration. The dose preparation data are listed in the raw data. The preparations were made freshly before each dosing occasion.
Dose Administration
25 µl of the test item was applied to the dorsum of each ear. The α−Hexylcinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

Individual body weights - Positive control
Mouse no. Initial body weight Terminal body weight
1 18.58 19.29
2 17.26 17.86
3 18.47 19.24
4 17.95 18.40
5 18.93 19.43
Mean 18.24 18.84*
S.D. 0.649 0.683
* p < 0.005

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 0.84

Test group / Remarks:

Acid Brown 161 25%

Results 2

Key result

Parameter:

SI

Value:

ca. 1.29

Test group / Remarks:

Acid Brown 161 50%

Results 3

Key result

Parameter:

SI

Value:

ca. 0.75

Test group / Remarks:

Acid Brown 161 100%

Cellular proliferation data / Observations:

In comparison with the control group, an increase of the pooled lymph node weights was not observed. The pooled lymph node weights of treated groups were 0.0326g for 25% concentration, 0.0357g for 50% concentration and 0.0249g for 100% concentration of test item. The lymph node weight of control group and positive control group were 0.0387g and 0.0643g, respectively. The DPM values for the three treated groups were 1667 (25%), 2557 (50%) and 1483 (100%), respectively. The SI values for the three treated groups were 0.84 (25%), 1.29 (50%) and 0.75 (100%), respectively.
The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. was not
greater than the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitizing potential of Acid Brown 161 was assessed using the murine local lymph node assay.
Based on the results of this study, Acid Brown 161 is not considered a skin sensitizer under the condition of this LLNA study.

Executive summary:

The skin sensitization potential of Acid Brown 161 was evaluated by LLNA method, which basic underlying principle is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application.

In the present study, the test item was applied to the dorsum of each ear of five female mice (CBA/Ca) per group over three consecutive days, at three concentrations. All animals survived throughout the test period without showing any clinical signs of toxicity. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance as a sensitizer.

Therefore, it was not possible to calculate an EC3 value.

These results demonstrate that the test item Acid Brown 161 was not a skin sensitizer under the test conditions of this study.