Reaction products of diazotized 2-amino-4-(methylsulfonyl)-phenol with 2-(3-Chlorophenyl)-2,4-dihydro-5-methyl-3H-pyrazol-3-one and Methyl-7-hydroxy-1-naphthylcarbamate – and chromium (III) 2:1, salted by ammonium, sodium and acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant studies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

Reaction products of diazotized 2-amino-4-(methylsulfonyl)-phenol with 2-(3-Chlorophenyl)-2,4-dihydro-5-methyl-3H-pyrazol-3-one and Methyl-7-hydroxy-1-naphthylcarbamate – and chromium (III) 2:1, salted by ammonium, sodium and acid

EC Number:

936-609-1

Molecular formula:

C40H32CrN4O12S2-R+] . [C36H28ClCrN7O10S2-R+] . [C34H26Cl2CrN8O8S2-R+]; R+ = NH4+, Na+ or H+

IUPAC Name:

Reaction products of diazotized 2-amino-4-(methylsulfonyl)-phenol with 2-(3-Chlorophenyl)-2,4-dihydro-5-methyl-3H-pyrazol-3-one and Methyl-7-hydroxy-1-naphthylcarbamate – and chromium (III) 2:1, salted by ammonium, sodium and acid

Test material form:

other: solid

Details on test material:

Expiry date: 02 September 2012
purity: > 80% (see certificate of analysis)
Physical state: solid
Appearance: Dark greybrown solid
Storage: At room temperature
Homogeneity: The test substance appeared to be homogeneous.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., 5960 AD Horst, Netherlands
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 18.4 - 24. 6 g
- Housing: five animals per cage
- Diet: ad libitum
- Wate: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m

IN-LIFE DATES: From: 2011-06-29 - To: 2011-07-21

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1, 5, 10 and 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used was a 50% (w/w) suspension in dimethylformamide. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonicating, warming to 37°C).



MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: 3fold increase in stimulation index.

TREATMENT PREPARATION AND ADMINISTRATION: The test item was placed into an appropriate container on a tared balance and dimethylformamide was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied

Positive control substance(s):

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

Statistics:

For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and/or Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).

Results and discussion

Positive control results:

The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil (4:1 v/v) (compound listed in OECD 429 Guideline) using CBA/CaOlaHsd mice in May 2011. The EC3 was 10.4%

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: EC3 = 23% For details, see table

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see table

Any other information on results incl. tables

Stimulationsindex:
Test item concentration % (w/v)		Measurement	Calculation	Result	Calculation			Result
		DPM	DPM-BG per animal (2 lymph nodes)	S.I. *	Number of animals	Mean DPM per animal	SD	S.I.
---	BG I	14	---	---	---	---	---	---
---	BG II	15	---	---	---	---	---	---
NK	1	403	389	---	---	---	---	---
NK	2	801	787	---	---	---	---	---
NK	3	279	265	---	---	---	---	---
NK	4	181	167	---	---	---	---	---
NK	5	256	242	---	5	369.5	246.4	1.00
1%	6	272	258	0.7	---	---	---	---
1%	7	607	593	1.6	---	---	---	---
1%	8	559	545	1.5	---	---	---	---
1%	9	575	561	1.5	---	---	---	---
1%	10	305	291	0.8	5	449.1	161.2	1.22
5%	11	628	614	1.7	---	---	---	---
5%	12	965	951	2.6	---	---	---	---
5%	13	751	737	2.0	---	---	---	---
5%	14	855	841	2.3	---	---	---	---
5%	15	843	829	2.2	5	793.9	126.2	2.15
10%	16	1078	1064	2.9	---	---	---	---
10%	17	868	854	2.3	---	---	---	---
10%	18	1139	1125	3.0	---	---	---	---
10%	19	1001	987	2.7	---	---	---	---
10%	20	446	432	1.2	5	891.9	276.6	2.41
50%	21	883	869	2.4	---	---	---	---
50%	22	2440	2426	6.6	---	---	---	---
50%	23	1035	1021	2.8	---	---	---	---
50%	24	1323	1309	3.5	---	---	---	---
50%	25	2190	2176	5.9	5	1559.7	700.1	4.22

Animal No.	Ear weights after necropsy
	Ear weight (mg per animal)	Mean ear weight (mg)	SD	Index
1	23.85
2	24.06
3	23.38
4	23.16
5	23.17	23.52	0.41	1.00
6	26.17
7	24.04
8	24.62
9	23.92
10	23.74	24.50	0.99	1.04
11	22.19
12	31.91
13	26.17
14	26.67
15	27.31	26.85	3.47	1.14
16	26.14
17*	31.78
18	26.53
19	25.14
20	26.83	26.16	0.74	1.11
21	25.40
22	23.43
23	26.98
24	25.73
25	24.76	25.26	1.30	1.07
*value was found to be an outlier and excluded from calculation

Animal No.	Lymph Node Weights after Sacrifice				Lymph Node Cell Counts after Sacrifice
	Lymph Node weight (mg per animal)	Mean Lymph Node weight (mg)	SD	Index	Lymph Node Cell Count (x10E06 per animal)	Mean Lymph Node Cell Count (x10E06 per animal)	SD	Index
1	4.26				8.04
2	6.30				12.74
3	5.96				11.85
4	5.64				7.90
5	6.32	5.70	0.85	1.00	9.17	9.94	2.23	1.00
6	5.24				9.06
7	5.81				14.78
8	4.92				9.57
9	7.07				12.82
10	4.29	5.47	1.05	0.96	10.65	11.38	2.39	1.14
11	5.16				12.12
12	6.99				19.03
13	5.69				17.04
14	5.31				14.33
15	6.83	6.00	0.86	1.05	14.80	15.46	2.65	1.56
16	5.68				14.75
17	6.86				18.36
18	6.82				17.10
19	6.54				17.28
20	5.93	6.37	0.53	1.12	9.64	15.43	3.49	1.55
21	6.32				13.19
22	9.04				21.32
23	7.58				16.19
24	7.77				17.19
25	8.52	7.85	1.04	1.38	16.08	16.79	2.94	1.69

Results of the range-finding test

At the tested concentrations of 25 and 50% the animals did not show any signs of systemic toxicity. On application day 1 and 2, evaluation of the ears for the presence of ear erythema was complicated by the colour of the test item. On study day 4 to 6, an erythema of the ear skin was observed in both treated animals (score 1 on day 4 and 5, score 2 on day 6). From day 2 onwards, the animals treated with the highest test item concentration showed hair loss in the region of the ears.

Furthermore, an increase in ear thickness exceeding the threshold value of 25% for excessive local skin irritation was measured in the animal treated with 25% test item concentration. The measured ear weight confirmed this observation. However, no relevant increase in ear thickness or ear weight was observed in the animal treated with 50% test item concentration.

Thus, the test item in the main study was assayed at concentrations of 1, 5, 10, and 50% (w/w).

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU