Disodium 4,5-dihydroxybenzene-1,3-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 20 Sep 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted in 2002

Deviations:

yes

Remarks:

Purity of the test item was not stated in the report

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Adopted in 2010

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

EC Number:

205-741-5

EC Name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

Cas Number:

149-45-1

Molecular formula:

C6H6O8S2.2Na

IUPAC Name:

disodium 4,5-dihydroxybenzene-1,3-disulphonate

Test material form:

solid: particulate/powder

Remarks:

white powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Labouratories, Bar Harbour, Maine, USA
- Females: yes
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 17-25 g (range)
- Housing: individually, no further details in the report
- Diet: Certified Rodent Chow 7012C, ad libitum
- Water: tap water via water bottles, ad libitum
- Acclimation period: at least 6 days
- Indication of any skin lesions: only suitable animals, as determined by veterinary staff, were assigned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.25 ± 5.55
- Humidity (%): 29-49
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

other: 30:70 ethanol:water

Concentration:

1, 5, and 15% (w/v)

No. of animals per dose:

5

Details on study design:

JUSTIFICATION FOR DOSE LEVEL
The dose levels were specified by the sponsor and are documented in the raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: ³H-methyl thymidine determined by β-scintillation
- Criteria used to consider a positive response: A stimulation index ≥3.0 relative to the concurrent vehicle treated control, is regarded as a positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of 5 mice were treated on the dorsal surface of both ears once per day for 3 days with 25 µL (per ear) of 1, 5, or 15% (w/v) of test substance in vehicle, with the vehicle (30:70 ethanol:water), with the vehicle for the positive control (acetone:olive oil, 4:1 [AOO]), or with the positive control (HCA at 35% [v/v]). Local irritation reactions, mortality and systemic toxicity were assessed daily. On Day 6, the mice were injected intravenously (tail vein) with 20 µCi of ³H-methyl thymidine in 250 µL of sterile saline. Five hours later, the mice were euthanised and the draining auricular lymph nodes were removed. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8 °C. Pellets were recovered by centrifugation and were counted in a β-scintillation counter to determine the incorporation of ³H-methyl thymidine.

Positive control substance(s):

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

Statistics:

DPM data was analysed using SAS by log transformation of the data. Comparisons with the control group were based on the least significant difference criterion, with all tests conducted at a 1%, two-tailed probability level.
BW and change in BW means and SEM were calculated for each group. Evaluations were made by one way ANOVA using the F distribution to assess statistical significance, and where found, a Dunnett's test was used to determine the degree of significance from the control mean.

Results and discussion

Positive control results:

Exposure to the positive control, 35% HCA (v/v) in the AOO vehicle, resulted in a stimulation index (SI) of 8.3 indicating a positive response. This response was also statistically significant when compared to the AOO vehicle control group (p = 0.0001). This SI value showed the effectiveness of the positive control treatment in this assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.79

Variability:

pooled results per group

Test group / Remarks:

1% (w/v)

Results 2

Key result

Parameter:

SI

Value:

1.18

Variability:

pooled results per group

Test group / Remarks:

5% (w/v)

Results 3

Key result

Parameter:

SI

Value:

1.03

Variability:

pooled results per group

Test group / Remarks:

15% (w/v)

Results 4

Key result

Parameter:

SI

Value:

7.98

Variability:

pooled results per group

Test group / Remarks:

positive control

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX (SI) CALCULATION:
The SI ratio was calculated as the mean DPM value measured per dosage level of treated lymph nodes, over the mean DPM value measured for the control group: SI = Test/control ratio.

Group Concentration % (w/v) DPM Number of lymph nodes DPM/node Test/control (+) = positive
ratio (-) = negative

1 Vehicle control 230.504 5 46.101 NA NA Vehicle control
2 1 412.502 5 82.500 1.789 (-) Test substance
3 5 272.034 5 54.407 1.180 (-) Test substance
4 15 237.896 5 47.579 1.032 (-) Test substance
5 HCA (35% [v/v]) 1914.596 5 382.91 7.98 (+) Positive control

CLINICAL OBSERVATIONS:
There was no mortality and all animals appeared normal throughout the study. The application sites on the mice treated with the test substance at a concentration of 15% exhibited very slight erythema and very slight edema on Day 3.

BODY WEIGHTS:
BW and change in BW means were evaluated and no statistically significant differences were observed between any of the groups of treated animals.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008