Tetrasodium 4-[[3,5-bis[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]-2,4(or 2,6)-dihydroxyphenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: experimental study on simillar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

IUPAC Name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

In vivo test system

Test animals

Species:

mouse

Strain:

other: BALB/CBYJICO

Sex:

female

Details on test animals and environmental conditions:

Source: Breeding farm VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
Number of animals:
Pilot experiment – 3 females
group: Exposed groups – 15 females (5 animals in three groups)
Positive control group – 5 females
Negative control group – 5 females
Total: 28 animals
Age: 8 to 10 weeks (at start of dosing)
Body weight range: 14.78 - 18.67 g (at start of dosing), in pilot experiment 14.96 - 17.59 g
Health examination: All animals were examined during the acclimatisation period
Acclimatisation: 7 days

Animal rooms: Monitored conditions, microbiologically defined background, according to internal SOP No.40
Room temperature: 22 ± 3 °C, permanently monitored
Relative humidity: 30 – 70 %, permanently monitored
Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am
Animal caging: Animals in groups in macrolon cages with sterilized softwood shavings
Water: Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law, Health Ministry
Diet: Pelleted standard diet for experimental animals ad libitum (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany).
Microbiological control and content of nutrients is performed according internal SOP No. 72.
Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10.

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 – mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol

Concentration:

50 % (v/v) correspond to 500 mg/ml
5 % (v/v) correspond to 50 mg/ml
0.5 % (v/v) correspond to 5 mg/ml

No. of animals per dose:

5 females per group also for the negative and positive controls.
In the pilot experiment only 1 femal were used.

Details on study design:

The test substance was administered to three animals to assess a possible systemic toxicity or high irritation of skin.
One animal per dose group was used in pilot experiment.
The test substance was administered in the form of suspension in DAE 433.
Concentrations of test substance in application form:
50% (w/v) = 500 mg/mL
5% (w/v) = 50 mg /mL
0.5% (w/v) =5 mg /mL

The pilot experiment was conducted under the conditions identical to the main study, except the assessment of lymph node proliferation. The appropriate suspensions of the test substance (50%, 5%, 0.5% w/v) was applied to three animals in volume 25 l to the dorsum of each ear once a day morning for 3 consecutive days. The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application. The application was performed very slowly by micropipette. The route of administration was the same as in the main study.
Both ears of each mouse were observed for erythema and scored and subsequently ear thickness was measured using digital thickness gauge.
Body weight was recorded before application and prior to termination (Day 6).
According to the results of pilot experiment the doses used in pilot experiment were chosen also for main study.

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed by applying the parametric test for testing whether all group samples originate from the same distribution and then the nonparametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons.

Results and discussion

Positive control results:

The positive control item Dinitrochlorobenzene (DNCB) as a contact allergen (concentration 0.5% (w/v) elicited the expected reaction pattern with significant increase in Stimulation Index of cell proliferation and of ear weight.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.68

Test group / Remarks:

dose 50%

Results 2

Parameter:

SI

Value:

0.85

Test group / Remarks:

dose 5%

Results 3

Parameter:

SI

Value:

0.66

Test group / Remarks:

dose 0.5 %

Results 4

Parameter:

other: disintegrations per minute (DPM)

Value:

279.31

Test group / Remarks:

dose 50%

Results 5

Parameter:

other: disintegrations per minute (DPM)

Value:

422.65

Test group / Remarks:

dose 5 %

Results 6

Parameter:

other: disintegrations per minute (DPM)

Value:

250.45

Test group / Remarks:

dose 0.5%

Any other information on results incl. tables

Main test:

Body Weight of Animals: Individual body weight of females before administration and before necropsy was relatively well balanced (result of random selection of animals into groups). Very slight reduction of body weight (in tenths of grams) was recorded only in one animal at the middle dose level. Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application. Body weight increment was lower in treated group at the middle dose level.

Mortality, Clinical Observations No animal died during the main study. No symptoms of toxicity and no erythema on application site were observed in all animals from the negative control group and all animals administered by the test substance. All animals in the positive control group showed symptoms caused by the application of DNCB: hyperaemia of skin with well defined erythema (see tab. No. 8) on application site, clonospasm and increased response to stimuli.

Cell Proliferation The value of DPM and SI for positive control group was increased. The SI was ≥ 3 (7.18) – the LLNA was efficient (see Table 9). The SI for the test groups treated by the test substance at the all dose levels was below the threshold, stimulation index (SI) is < 3.

Irritating Effect of the Test Substance:

In the positive control group, the weight of ear target was significantly increased against negative control group. No erythema of skin was observed during the clinical observation at all dose levels. Statistically significant increase of ear weight was recorded at the middle dose level. Residues of the test substance on the ears were visible during whole study so it could cause this weight increase.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Not sensitizer for skin

Executive summary:

Under the given test conditions, the animals exposed to the test substance does not elicit sensitising response in LLNA assay.