4-(2-phenylpropan-2-yl)phenyl 3-diazo-4-oxo-3,4-dihydronaphthalene-1-sulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 - 23 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

updated 06 July 2012

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(2-phenylpropan-2-yl)phenyl 4-oxo-3-(λ⁵-diazynylidene)-3,4-dihydronaphthalene-1-sulfonate

Cas Number:

23121-00-8

Molecular formula:

C25H20N2O4S

IUPAC Name:

4-(2-phenylpropan-2-yl)phenyl 4-oxo-3-(λ⁵-diazynylidene)-3,4-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: room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 12 weeks (pre-test), 11 - 12 weeks (main study)
- Weight at study initiation: 20.6 - 23.7 g (pre-screen test) (range), 18.1 - 24.3 g (main study) (range)
- Housing: 2 - 4 animals per cage, in Makrolon Type II (pre-screen test) / III (main study), with wire mesh top and granulated soft wood bedding
- Diet: 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 – 65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

(DMF)

Concentration:

10, 25 and 50%

No. of animals per dose:

1 female (pre-screen test)
4 females (main test)

Details on study design:

PRE-SCREEN TESTS:
Two concentrations (25 and 50% dissolved in DMF) were applied topically to the ears of each animal (one mouse 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 at least once daily. Furthermore, body weights and ear thickness measurements were performed (body weight: before initial application and on Day 6, ear thickness: before initial application, on Day 6 and on Day 3). None of the animals showed any signs of systemic toxicity. The ears were punched after sacrifice (Day 6) at the apical area using a biopsy punch (Ø 8 mm, corresponding to 0.5 cm²), pooled per animal and weighed using an analytical balance. The animal treated with 50% test item showed a very slight erythema of the ear skin (score 1), while the animal treated with 25% did not show any signs of signifcant irritation (indicated by an erythema score ≥ 3 and /or an increase of less 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: slight irritation (score 1) was observed in 1/2 animals
- Systemic toxicity: no systemic toxicity was observed
- Ear thickness measurements: yes (less than 25% increase in ear thickness was measured)
- Erythema scores: 1 (test animal treated with 50% of test item, 24 h after second application, fully reversible within 6 days)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine (3HTdR) incorporation, determined by ß-scintillation
- Criteria used to consider a positive response: SI ≥ 3

TREATMENT PREPARATION AND ADMINISTRATION:
25 μl of each dose formulation (10, 25 and 50%) or the vehicle alone were applied to the dorsal skin of each ear of each animal once a day for 3 consecutive days. On Day 6, 19.8 μCi 3H-methyl thymidine, contained in 250 μL of PBS (= 79.1 μCi/mL), was administered to each mouse via the tail vein. Approximately 5 h after administration, local lymph nodes were collected, pooled and separated by gentle mechanical disaggregation through a stainless steel gauze (200 μm mesh size). After washing two times in PBS the lymph node cells were treated with ~3 mL of 5% trichloroacetic acid (TCA) at ~4 °C for at least 18 h before the determination of the amount of 3H-methyl thymidine incorporation (as disintegrations per minutes (DPM)) on Day 7.

Positive control substance(s):

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

Statistics:

All calculations conducted on the DPM values were performed with "R" (language and environment for statistical computing and graphics).
The mean values and standard deviations were calculated for the body weights.

Results and discussion

Positive control results:

The positive control substance (hexyl cinnamic aldehyde) was tested in a separate experiment in April 2016 (Envigo study number 1764300) at 5, 10 and 25% in AOO (acetone:olive oil (4+1 v/v)). It induced a positive reaction at a concentration of 25%, determined by a DPM/lymph node of 8170.5 compared with 1042.8 DPM/lymph node in the vehicle control group, leading to a SI of 7.84.
The EC3 value was calculated to be 10.6% (w/v) (using the results of 10 and 25% hexyl cinnamic aldehyde).
The historical control data of the last 10 positive control experiments revealed SI Values between 3.7 and 17.6 for 25% in AOO.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.16

Test group / Remarks:

10% test group

Results 2

Key result

Parameter:

SI

Value:

1.47

Test group / Remarks:

25% test group

Results 3

Key result

Parameter:

SI

Value:

1.02

Test group / Remarks:

50% test group

Results 4

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

solvent control group (DMF)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
No significant lymphoproliferation (SI ≥ 3) was observed for the test substance at treatment concentrations of 10, 25 and 50% (w/v). (For details refer to Table 5)

DETAILS ON STIMULATION INDEX CALCULATION: SI = DPM/lymph node of a treated group divided by the DPM/lymph node of the respective negative/solvent control group. Before DPM/lymph node values were determined, mean scintillation-background DPM (in main test = 18.5) was subtracted from test and control raw data. Since the lymph nodes of the animals of a dose group were pooled, DPM/ lymph node was determined by dividing the measured value by the number of pooled lymph nodes (= 8).
Following values (DPM/ lymph node) were obtained: 754.3, 873.6, 1110.9 and 769.4 for the vehicle control of the test substance (DMF) and 10, 25 and 50% test groups, respectively. (For details refer to Table 5)

EC3 CALCULATION: Based on the obtained results no EC3 value of the test substance could be calculated.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No signs of local skin irritation (indicated by an erythema score ≥ 3) or any other local skin effect were observed in any treatment group.

BODY WEIGHTS:
Body weights changes were within the range expected for animals of this strain and age.(For details refer to Table 6)

Any other information on results incl. tables

Results of the Pre-screen test

Table 1: Body Weights

Animal No.	Concentration %	Body Weight (g)
		Prior to 1st application	Prior to Sacrifice (Day 6)	Difference Day 1 to Day 6	Difference %
1	25	20.6	22	1.4	6.8
2	50	23.7	22.6	-1.1	-4.6

Table 2: Ear Thickness

Animal No.	Concentration %	Ear Thickness
		Prior to 1st Application (μm)	Prior to 3rd Application (μm)	Prior to Necropsy (μm)	Difference Day 1 to Day 3	Ear Swelling Day 3 (%)	Difference Day 1 to Day 6 (μm)	Ear Swelling Day 6 (%)
		Mean (Right and Left Ear)
1	25	237.5	245.0	242.5	7.5	3.2	5	2.1
2	50	235.0	252.5	247.5	17.5	7.4	12.5	5.3

Table 3: Ear Weights

Animal No.	Concentration %	Ear Weights after Necropsy (mg per animal)	% Increase Compared to Vehicle Values
1	25	27.83	8.3
2	50	29.29	14

Mean of historical controls (DMF): 25.7 mg/animal

Table 4: Ear Erythema

Animal No.	Score
	within 1 h after 1. Appl.	24 h after 1. Appl.	within 1 h after 2. Appl.	24 h after 2. Appl.	within 1 h after 3. Appl.	24 h after 3. Appl.	Day 5	Day 6
1	0*	0	0*	0*	0*	0	0	0
2	0*	0	0*	1*	1*	1*	1*	0

Score:

0 = No visible erythema

1 = Very slight erythema

2 = Well defined erythema

3 = Moderate to severe erythema

4 = Severe erythema to formation of eschar which prevents grading of erythema

*substance residue

Table 5: Stimulation index in mice (main test)

Compound	Concentration [%]	DPM/ lymph node	Stimulation index	Judgement
DMF	100	754.3	1.00	-
Test item	10	873.6	1.16	Negative
	25	1110.9	1.47
	50	769.4	1.02
AOO*	100	1042.8	1.00	-
HCA*	10	2908.9	2.79	Negative
	25	8170.5	7.84	Positive

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

DMF = N,N -dimethylformamide

HCA = Hexyl cinnamic aldehyde

- = Not applicable

* = Performed in separate experiment

Table 6: Body weight (main test)

Compound	Concentration [%]	Animal ID No.	Body weight
			Day 1 [g]	Day 6 [g]
DMF	100	1	20.8	21.0
		2	20.5	21.8
		3	20.2	20.9
		4	20.8	21.3
		Mean ± SD	20.6 ± 0.3	21.3 ± 0.4
Test item	10	5	19.8	20.2
		6	21.1	21.7
		7	21.0	21.0
		8	19.8	21.7
		Mean ± SD	20.4 ± 0.7	21.0 ± 0.6
	25	9	18.6	20.0
		10	24.3	22.6
		11	20.1	20.8
		12	20.3	21.4
		Mean ± SD	20.8 ± 2.4	21.2 ± 1.1
	50	13	22.5	22.5
		14	19.9	20.9
		15	19.7	20.1
		16	18.1	19.1
		Mean ± SD	20.1 ± 1.8	20.7 ± 1.4

DMF = N,N -dimethylformamide

SD = Standard deviation

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