2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, trisodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, trisodium salt

EC Number:

612-028-6

Cas Number:

607724-47-0

Molecular formula:

C26H22N5Na3O16S5

IUPAC Name:

2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, trisodium salt

Test material form:

solid: particulate/powder

Details on test material:

1 MATERIALS AND METHODS
1.2 Test Item
Designation in Test Facility: 17031402G
Date of Receipt: 14. Mar. 2017
Condition at Receipt Room temperature, in proper conditions
1.2.1 Specification.
Name Blendazol Red Blendwell
Batch no. E 328
Appearance Dark Red Powder
Composition 2-Naphtalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, triso dium salt
Purity 95 % by HPLC
Homogeneity homogeneous
Expiry date 17. Feb. 2019
Storage Room Temperature: (20 ± 5°C); Keep away from humidity
CAS No. 607724-47-0
EINECS-No. 612-028-6
Chemical Class not stated
Stability H2O: 96h; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown
Solubility H2O: to be determined*; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown


* Will be determined in another GLP-study at LAUS GmbH and will be stated in the final report, this
This information is not provided by the sponsor but determined at LAUS GmbH.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:AnLab Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: free
- Age at study initiation: 8-10 weeks
- Weight at study initiation:
- Housing:
The animals were housed in polypropylene cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 3°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle. The sanitation was performed according to standard operation procedures
- Diet (e.g. ad libitum):
A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time.
- Water (e.g. ad libitum): tap water for human consumption
- Acclimation period: 6 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°
- Humidity (%): within the range of 50 - 60 %.
- Air changes (per hr): +/-3°C
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle
- IN-LIFE DATES: From: To: 24/05/2017 TO 20/06/2017

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Lot number : SZBG 1310V from Honeywell, Germany

Concentration:

25%; 50% and 100%,

No. of animals per dose:

2 mice/group per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was insoluble in the vehicle; therefore, a homogeneous suspension was obtained.
- Irritation: no
- Systemic toxicity: no
The test item was administered in two mice at concentration of 100%. After the test item application, no signs of local irritation at the application site or systemic toxicity were observed

On day 1 (pre dose), day 3 and day 6 the ear thickness was measured
- Ear thickness measurements: the increase in ear thickness did not meet the criteria
- Erythema scores: 0

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet
redness) to eschar formation preventing grading of erythema 4


TREATMENT PREPARATION AND ADMINISTRATION:
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6:
The body weight of each animal was recorded. 250µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).

Positive control substance(s):

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

Statistics:

Number of animals:
5 females – control (vehicle)
5 females – positive control
15 females – test item
4 females - pre-screen test plus spare animals

Results and discussion

Positive control results:

Lymph node weight (g) Number of lymph nodes DPM SI

Positive Control 0.0699 10 12996 8.25

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

ca. 42

Test group / Remarks:

25% 50% & 100%

Results 2

Key result

Parameter:

SI

Value:

ca. 2.08

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

ca. 3.43

Test group / Remarks:

50%

Results 4

Key result

Parameter:

SI

Value:

ca. 5.09

Test group / Remarks:

100%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA :
In comparison with the control group, an increase of the pooled lymph node weights was recorded at all used concentrations. The pooled lymph node weights of treated groups were 0.0476g for 25% concentration, 0.0516g for 50% concentration and 0.0606g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0311g and 0.0699g, respectively. The DPM values for the three treated groups were 3269 (25%), 5406 (50%) and 8021 (100%), respectively. The SI values for the three treated groups were 2.08 (25%), 3.43 (50%) and 5.09 (100%), respectively. The EC3 value is calculated to be 42.0%.

DETAILS ON STIMULATION INDEX CALCULATION :

The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI.

EC3 CALCULATION

EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation:
EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration
d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.

CLINICAL OBSERVATIONS:
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. A minor increase of body weight was observed in all treated groups. The increase was not statistically significant.

Any other information on results incl. tables

1.1.1       Lymph Node Proliferation

In comparison with the control group, an increase of the pooled lymph node weights was recorded at all used concentrations. The pooled lymph node weights of treated groups were 0.0476g for 25% concentration, 0.0516g for 50% concentration and 0.0606g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0311g and 0.0699g, respectively. The DPM values for the three treated groups were 3269 (25%), 5406 (50%) and 8021 (100%), respectively. The SI values for the three treated groups were 2.08 (25%), 3.43 (50%) and 5.09 (100%), respectively. The EC3 value is calculated to be 42.0%. 

The mean Lymph node weight, DPM, SI, EC3 values.

	Lymph node	Number of
	weight (g)	lymph nodes	DPM	SI	EC3 (%)
Control	0.0311	10	1575	-	42.0
Positive Control	0.0699	10	12996	8.25
Blendazol Red Blendwell 25%	0.0476	10	3269	2.08
Blendazol Red Blendwell50%	0.0516	10	5406	3.43
Blendazol Red Blendwell   100%	0.0606	10	8021	5.09

 

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

These results demonstrate that Blendazol Red Blendwell is a potential skin sensitizer under the test conditions of this study.

Executive summary:

The skin sensitization potential of Blendazol Red Blendwell was evaluated by LLNA method, which was developed based on scientific understanding of the immunological mechanism of skin sensitization.

In our experiments, the test item was applied over three consecutive days, at three concentrations. All animals survived throughout the test period without showing any clinical signs.In comparison with the control group, the increase in lymph node weight was observed in all treated groups. The increase of lymph node weight was dose dependent. A similar trend was registered in the evaluation of DPM of the lymph nodes.The EC3 value is calculated to be 42.0%. As the SI for some treatment dose group is ≥3 and a clear dose response is observed, the test item is regarded as a potential skin sensitizer (2, 11).

These results demonstrate that Blendazol Red Blendwell is a potential skin sensitizer under the test conditions of this study.