1-Naphthalenesulfonic acid, 6-diazo-5,6-dihydro-5-oxo-, ester with phenyl(2,3,4-trihydroxyphenyl)methanone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Aug - 01 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

No justification for using different mouse strain than recommended in OECD 442B, individual housing instead of group-housing

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

adopted in July 2010

Deviations:

yes

Remarks:

No justification for using different mouse strain than recommended in OECD 442B, individual housing instead of group-housing

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-Naphthalenesulfonic acid, 6-diazo-5,6-dihydro-5-oxo-, ester with phenyl(2,3,4-trihydroxyphenyl)methanone

EC Number:

270-931-7

EC Name:

1-Naphthalenesulfonic acid, 6-diazo-5,6-dihydro-5-oxo-, ester with phenyl(2,3,4-trihydroxyphenyl)methanone

Cas Number:

68510-93-0

Molecular formula:

all potential esters of C10HH5O3N2SCl and C13H10O4

IUPAC Name:

3-benzoyl-2-hydroxy-6-({[5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalen-1-yl]sulfonyl}oxy)phenyl 5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalene-1-sulfonate; 4-benzoyl-2,3-bis({[5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalen-1-yl]sulfonyl}oxy)phenyl 5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalene-1-sulfonate; 4-benzoyl-2,3-dihydroxyphenyl 5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalene-1-sulfonate

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Pretest and Main test: 8 weeks
- Weight at study initiation: 18.4 - 21.8 g
- Housing: individual
- Diet: VRF1(P) (SDS Special Diets Services, Altrip, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 27 Aug To: 01 Sep 2014

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

(AOO)

Concentration:

10%, 25%, 50%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
In the pre-screening test, two concentrations (25 and 50% dissolved in AOO) were selected, and 25 μl of each dose formulation were applied to the ears of each animal (2 mice per concentration), once a day for 3 consecutive days. Animals were observed for clinical signs of systemic toxicity and local irritation at the application site. Furthermore, body weights and ear thickness measurements were performed (before initial application (Day 0) and on Day 5, ear thickness was additionally measured on Day 2). Additionally the ears were punched after sacrifice (Day 5) at the apical area using a biopsy punch and were immediately pooled per animal and weighed using an analytical balance. None of the animals showed any abnormalities or any signs of signifcant irritation (indicated by an erythema score ≥ 3 and /or an increase of more than 25% in ear thickness). No mortality was observed.
Based on these results and considering that 50% was the maximum attainable concentration, 50% (w/v) was selected as the highest test concentration for the main study. This concentration was expected not to induce systemic toxicity, nor to induce an increase in ear thickness exceeding 25% or to induce dermal erythema with a score of 3 or more.

- Compound solubility: 50% (maximum attainable concentration)
- Irritation: no
- Systemic toxicity: no
- Ear thickness measurements: yes, less than 25% increase in ear thickness
- Erythema scores: 0 (each test group, each time point)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The proliferative capacity of pooled lymph node cells was determined by the incorporation of BrdU measured in a photometer.
- Criteria used to consider a positive response:
BrdU incorporation in test item group at least 1.6-fold higher compared to control group (indicated by the Stimulation Index (SI))
Data compatible with a conventional dose response
Index, calculated for the lymph node weight and -cell count as well as for the ear weight (mean values of treated groups divided by mean values of vehicle control group), higher than 1.55 (for BALB/c mice) for the lymph node cell count and an increase in ear weight exceeding 25% (according to OECD 442B)

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the test item (10, 25, 50%), the vehicle alone (AOO) or the positive control (25% hexyl cinnamaldehyde) were applied topically to the entire dorsal surface of each ear of each mouse.The application was repeated on days 2 and 3; local irritation reactions were assessed. On day 5, 500 µl phosphate buffered saline (DPBS) containing 5 mg Bromodeoxyuridine (BrdU) were injected intraperitoneally. 24 hours later, the draining lymph nodes of each ear were excised and pooled per animal. A single cell suspension of lymph node cells was prepared from each mouse by gentle mechanical disaggregation through a 70 μm nylon mesh. The lymph node cells were re-suspended in DPBS. The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit (Roche Applied Science, Mannheim, Germany) After fixation and denaturation of the lymph node cells, anti BrdU antibody was added. After removal of the antibody the substrate solution was added. The chromogen formation was determined by the measurement of the absorbance at 370 nm (reference wavelength = 492 nm) (Absorbance-Reader SUNRISE, Tecan).

Positive control substance(s):

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

Statistics:

Mann-Whitney-U test for non-parametric comparison of BrdU values, ear weights, lymph node weights and lymph node cell counts

Results and discussion

Positive control results:

The positive control substance (25% hexyl cinnamic aldehyde (Batch number: MKBK3783V, SIGMA-ALDRICH, Taufkirchen, Germany) in AOO) induced a positive reaction, determined by a SI of 2.8. Very slight erythema and scaling could be observed. As expected, a statistical and biological relevant increase in lymph node weight and lymph node cell count was observed. Ear weight measurements revealed a statistical but not biological relevant increase.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

mean of 5 values

Value:

0.7

Test group / Remarks:

10 % test group

Results 2

Key result

Parameter:

SI

Remarks:

mean of 5 values

Value:

0.5

Test group / Remarks:

25 % test group

Results 3

Key result

Parameter:

SI

Remarks:

mean of 5 values

Value:

0.7

Test group / Remarks:

50 % test group

Results 4

Key result

Parameter:

SI

Remarks:

mean of 5 values

Value:

2.8

Test group / Remarks:

positive control group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: No significant lymphoproliferation (SI ≥ 1.6) was observed for the test item at treatment concentrations of 10, 25 and 50%.

DETAILS ON STIMULATION INDEX CALCULATION
The SI is derived by dividing the mean BrdU labeling index/mouse of each test item group or positive control group by the mean BrdU labeling index of the vehicle group.

CLINICAL OBSERVATIONS: No mortality or symptoms of systemic toxicity were observed in any treatment group. No signs of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group. No significant increase in lymph node weight, -cell count and ear weight was observed.

BODY WEIGHTS
Body weights changes were within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Table 1: Body weights (Pre-test)

Animal No.	Concentration %	Body Weight (g)
		Prior 1st application	Prior to Sacrifice (Day 5)	Difference Day 0 to Day 5	Difference %
1	25	18.5	17.6	-0.9	-4.9
2		20.2	19.2	-1	-5
3	50	20.3	19.6	-0.7	-3.4
4		19.9	19.5	-0.4	-2
5	AOO	19.9	19.5	-0.4	-2
6		20.9	20.2	-0.7	-3.3

AOO = Acetone: Olive oil (4:1 (v/v) mixture)

Table 2: Ear thickness (Pre-test)

Animal No.	Concentration %	Ear Thickness
		Prior to 1st Application (mm)	Prior to 3rd Application (mm)	Prior to Necropsy (mm)	Difference Day 1 to Day 2	Ear Swelling Day 2 (%)	Difference Day 1 to Day 5 (μm)	Ear Swelling Day 5 (%)
		Mean (Right and Left Ear)
1	25	0.22	0.23	0.23	0.01	4.55	0.01	4.55
2		0.23	0.23	0.23	0.01	2.22	0.01	2.22
3	50	0.22	0.24	0.26	0.02	6.82	0.04	18.18
4		0.24	0.24	0.24	0.00	0.00	0.00	0.00
5	AOO	0.23	0.24	0.23	0.01	4.44	0.01	2.22
6		0.23	0.23	0.23	0.00	0.00	0.00	0.00

Table 3: Ear weights (Pre-test)

Animal No.	Concentration %	Ear Weights after Necropsy (mg per animal)	Mean
1	25	27.8	27.7
2		27.5
3	50	29.6	29.8
4		30.0
5	AOO	28.4	28.6

Table 4: Stimulation index in mice (main test)

Compound	Concentration [%]	BrdU labeling Index	Mean BrdU labeling Index	Stimulation index (SI)	Mean SI
AOO	100	0.17	0.11	1.50	1
		0.078		0.70
		0.105		1.00
		0.111		1.00
		0.085		0.80
Test substance	10	0.06	0.078	0.5	0.7
		0.114		1
		0.086		0.8
		0.076		0.7
		0.053		0.5
	25	0.098	0.057	0.9	0.5
		0.051		0.5
		0.058		0.5
		0.05		0.5
		0.029		0.3
	50	0.063	0.072	0.6	0.7
		0.061		0.6
		0.092		0.8
		0.069		0.6
		0.072		0.7
HCA	25	0.316	0.310*	2.90	2.8
		0.309		2.8
		0.369		3.4
		0.231		2.1
		0.323		2.9

AOO = Acetone: Olive oil (4:1 (v/v) mixture)

HCA = Hexyl cinnamic aldehyde

* statistically significant increase vs. vehicle control group (p<0.05)

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

CLP: not classified