Ethyl 3,4-dihydroxybenzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2016 to 19 July 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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

Ethyl 3,4-dihydroxybenzoate

EC Number:

223-529-0

EC Name:

Ethyl 3,4-dihydroxybenzoate

Cas Number:

3943-89-3

Molecular formula:

C9H10O4

IUPAC Name:

ethyl 3,4-dihydroxybenzoate

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/J strain, inbred
- Source: Janvier, Le Genest-Saint-Isle, France
- Females: nulliparous and non-pregnant
- Microbiological status of animals, when known: SPF-Quality
- Age at study initiation: ca 10 weeks
- Weight at study initiation: 19.8-24.3 g
- Housing: group housed (dMakrolon MII type cages with a sheet of paper)
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water : tap water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no erythema (transparant /white staining of treatment area at 25 and 60% due to test substance remnants that does not prevent scoring)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25 and 69%

No. of animals per dose:

5 females/dose

Details on study design:

PRE-SCREEN TESTS: in 2 females at 25 and 60% during 3 days
- Compound solubility: maximum solubility was in N,N-dimethylformamide 60% was highest concentration possible
- Irritation: none observed
- Systemic toxicity: none observed
- Ear thickness measurements:no effects (checked at day 1, 3 and 6)
- Erythema scores: none (transparant staining observed)

MAIN STUDY: concentration 0, 10, 25 and 60% based on maximum solubility
treatment of the dorsal surface of the ears on 3 consequetive days, on day 6injection in tail vein of 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine, 5 hours thereafter excision of the dorsal lymphnodes (visual inspection), pooled lymphnodes were precessed for reactivity measurements that were performed on day 7 with Packard scintillation counter (2800TR).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI ≥ 3

TREATMENT PREPARATION AND ADMINISTRATION: prepared within 4 hours prior to each dosing. Homogeneity was assessed by visual inspection of the solutions.
The dorsal surface of both ears was topically treated (25 μL/ear) with test substance or vehicle

Positive control substance(s):

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

Results and discussion

Positive control results:

EC3 value 17.8%

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

SEM 0.4

Test group / Remarks:

vehicle control

Results 2

Key result

Parameter:

SI

Value:

9.1

Variability:

SEM 2.9

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

3.2

Variability:

SEM 1.0

Test group / Remarks:

25%

Results 4

Key result

Parameter:

SI

Value:

4.5

Variability:

SEM 1.9

Test group / Remarks:

60%

Any other information on results incl. tables

Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

group	TS^1 (%)	animal	Size nodes^2		DPM^3/ animal	mean DPM ± SEM^4			mean SI ± SEM
			left	right
1	0	1	n	n	448	327	±	83	1.0	±	0.4
		2	n	n	178
		3	n	n	593
		4	n	n	223
		5	n	n	193
2	10	6	++	+	1951	2963	±	582	9.1	±	2.9
		7	++	++	5031
		8	++	++	2615
		9	++	++	1867
		10	++	++	3351
3	25	11	++	++	851	1059	±	174	3.2	±	1.0
		12	++	++	477
		13	++	++	1332
		14	++	+	1412
		15	+++	++	1225
4	60	16	+	+	1331	1457	±	514	4.5	±	1.9
		17	+	+	3432
		18	+	+	921
		19	+	+	470
		20	+	+	1129

¹  TS = test item (% w/w).

²  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

³   DPM= Disintegrations per minute.

⁴   SEM = Standard Error of the Mean.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The study is a skin sensitizer. No EC3 value can be calculated as there is no dose-response relationship

Executive summary:

The substance was tested in an LLNA test at 0, 10, 25 and 60% in N,N-dimethylformamide. Female mice were treated (dorsal surface of both ears) during 3 days. On day 6 animals received radiolabled ³H- methylthymidine and after sacrifice the pooled lymphnodes per animal were assessed for incorporation of radiolabled thymidine. DPM values were increased (SI > 3) compared to vehicle controls at all 3 concentrations without a dose response relationship. The substance is therefore considered to be a skin sensitizer.