Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

EC Number:

266-273-5

EC Name:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Cas Number:

66241-11-0

Molecular formula:

Molecular formula is not available.

IUPAC Name:

Reaction products of 2,4-dinitrophenol with polysulfide, leuco derivatives, sodium salt

Details on test material:

Identification: Leuco Sulfur Black 1
CAS No.: 66241-11-0
Description: Thixothrophic, black, wet paste
Batch No.: ESA0007595
Composition: Leuco Sulfur Black 1: 52.75 % (calculated by amount of by-compounds)
2,4-Dinitrochlorobenzene: < 20 ppm
2,4-Dinitrophenole: < 20 ppm
Sulfate: 970 ppm
Chloride: 750 ppm
Sodium: 1.3 % (w/w)
Water: 45.75 % (w/w) by Karl-Fischer titration
Stability of the test item Stable under storage conditions
Stability in solvent: > 4 h in water
Storage: At room temperature (range 20 ± 5 °C), light protected
Expiration Date: July 11, 2011

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., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 25 g
- Housing:single caging
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Laboratories GmbH, 33178 Borchen), ad libidum
- Water (e.g. ad libitum): tap water, (Gemeindewerke, 64380 Rossdorf), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness will be used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 10, and 25%

No. of animals per dose:

4

Details on study design:

To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 10 and 25% each on three consecutive days.
In the pre-test clinical signs were recorded within 1 hour and 24 ± 5 hours after each application as well as on day 7. At the tested concentrations theanimals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 5, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

Experiment performend in January 2009, 5, 10, and 25% alpha-Hexylcinnamaldehyde yielded a S.I. of 1.49, 4.17, and 4.90, respectively. The EC3 value calculated was 7.8%

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 1.4 (at 5 %), 1.1 (at 10 %), 1.6 (at 25 %)

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see table below

Any other information on results incl. tables

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	25	---	---	---	---
---	BG II	39	---	---	---	---
0	1	2735	2703	8	337.9	1.00
5	2	3869	3837	8	479.6	1.42
10	3	3067	3035	8	379.4	1.12
25	4	4386	4354	8	544.3	1.61

BG =  Background (1 ml 5% trichloroacetic acid) in duplicate 1=Control Group 2-4=Test Group S.I.=Stimulation Index

^a)=The mean value was taken from the figures BG I and BG II ^b)=Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value can not be calculated, since all S.I.´s are below 3.

 Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

 Body Weights

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item Leuco Sulfur Black 1 was not a skin sensitiser under the described conditions.

Executive summary:

The present study (Vogel 2009) analyses the sensitising potential of Leuco Sulfur Black 1

Three groups each of four female mice were treated daily with the test item at concentrations of 5, 10 and 25% (w/w) in dimethylformamide by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethylformamide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of toxicity were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.42, 1.12 and 1.61 were determined with the test item at concentrations of 5, 10 and 25% in dimethylformamide.

The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than 3.

The test item Leuco Sulfur Black 1 was not a skin sensitiser under the described conditions.