Serine, 2-[(2,3,4-trihydroxyphenyl)methylene]hydrazide, hydrochloride (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-3-hydroxy-N'-[(E)-(2,3,4-trihydroxyphenyl)methylidene]propanehydrazide hydrochloride

EC Number:

940-411-0

Cas Number:

1353749-74-2

Molecular formula:

C10H14ClN3O5

IUPAC Name:

2-amino-3-hydroxy-N'-[(E)-(2,3,4-trihydroxyphenyl)methylidene]propanehydrazide hydrochloride

Details on test material:

- Purity: 100%

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 22.0 - 25.0 g
- Housing: All animals were housed in solid-bottom cages with appropriate bedding and nestlets toys as enrichment. During quarantine, animals were housed in groups of 5 or fewer. Animals were single housed for approximately 2 hours following each application of the vehicle, test
substance, or positive control to allow additional time for drying and/or absorption. Following the 2-hour single-housing period, animals were returned to their group housing status.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): approximate 12-hour light/dark

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 5, 25, 40 and 60%

No. of animals per dose:

5

Details on study design:

- Criteria used to consider a positive response: A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of
each experimental group by the mean dpm of the vehicle group. The decision process in regard to a positive response includes an SI of greater than or equal to 3.0 together with consideration of dose response and, where appropriate, statistical significance.

TREATMENT PREPARATION AND ADMINISTRATION: Twenty-five μL of vehicle, test substance preparation, or positive control were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 1-3) at concentrations listed above. Test days 4-5 were days of rest followed by intravenous injection of 20 μCi of ³H-thymidine in PBS per mouse on test day 6. Approximately 5 hours after the injection, animals were sacrificed by isoflurane anaesthesia followed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8ºC overnight. On test day 7, the single cell suspensions were counted on a beta counter and reported as disintegrations per minute (dpm).

Positive control substance(s):

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

Statistics:

See table below.

Results and discussion

Positive control results:

A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at all test concentrations. SIs of greater than 3.0 were observed at all test concentrations of the test substance. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e. SI = 3) for the test substance was calculated to be <0.1%.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table below.

Any other information on results incl. tables

Stimulation Index Data

			MEAN	S.D.	SI
GROUP	MATERIAL TESTED	n	(dpm)	(dpm)
1	0% Vehicle	5	737.80	231.08	N/A
2	5%	5	5632.40^#	2288.06	7.63
3	25%	5	6579.20^#	3278.45	8.92
4	40%	5	8472.60^#	1664.22	11.48
5	60%	5	9399.00^#	5208.87	12.74
6	25% Positive Control^a	5	4750.80	1087.41	6.44

a Data were not included in the statistical analysis of the test substance groups.

# Statistically significant increase in dpm data from vehicle at p < 0.01 by Jonckheere-Terpstra trend test.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The test substance is considered an extreme dermal sensitizer in mice.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitization response in mice using the local lymph node assay (LLNA). Five groups of 5 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle), 5%, 25%, 40%, or 60% on both ears. N,N-dimethylformamide (DMF) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in DMF as a positive control. On test day 6 of the assay, mice received ³H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle group.

Statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at all test concentrations. SIs of greater than 3.0 were observed at all test concentrations of the test substance. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was calculated to be <0.1%. A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with. Under the conditions of this study, the test substance produced a dermal sensitization response in mice.