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Toxicological information

Skin sensitisation

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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:
guideline study
Justification for type of information:
data is from experimental reports

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
The purpose of the Local Lymph Node assay was to identify the contact allergenic potential of the test chemical when administered to the dorsum of both ear lobes of mice.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione
- Molecular formula : C16H8O2S2
- Molecular weight: 296.369 g/mol
- Smiles notation : O=C1c2c(S\C1=C1/Sc3c(cccc3)C1=O)cccc2
- InChl : 1S/C16H8O2S2/c17-13-9-5-1-3-7-11(9)19-15(13)16-14(18)10-6-2-4-8-12(10)20-16/h1-8H/b16-15-
- Substance type:Organic
- Physical state:Solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione
- Common Name: Thioindigo
- Molecular formula: C16H8O2S2
- Molecular weight: 296.369 g/mol
- Smiles notation: O=C1c2c(S\C1=C1/Sc3c(cccc3)C1=O)cccc2
- InChl : 1S/C16H8O2S2/c17-13-9-5-1-3-7-11(9)19-15(13)16-14(18)10-6-2-4-8-12(10)20-16/h1-8H/b16-15-
- Substance type: Organic
- Physical state: Solid
- Purity: >95%(w/w)
- Batch No.: AAFE 094505
- Stability: 0.1 mg/ml 4h in DMSO, 50 mg/ml 72h in DMSO
- Storage: At room temperature
- Expiration Date: October 21, 2015
- Manufacturing Date: October 21, 2005

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Details on test animals and environmental conditions

Test Animals:

- Source: Harlan Netherlands
- Sex: Females
- Age: 6 - 12 weeks (beginning of acclimatization)
- Identification: Single caging. The animals will be distributed into the test groups at random and identified by cage number.
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH)
- Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimatisation: Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.

Environmental Conditions:

- Temperature: 22 + 3°C
- Relative humidity: 30-79%
- Artificial light: 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
2.5, 5 and 10% (w/v) in DMSO
No. of animals per dose:
Number of animals for
the pre-test (non-GLP) 2 females
Number of animals for
the main study 16 females
Number of animals per group 4 females (nulliparous and non-pregnant)
Number of test groups 3
Number of control (vehicle) groups 1
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Warming and sonicating could not achieve a higher concentration. With other vehicles; e.g. acetone:olive oil (4:1), ethanol:water (7+3) (v/v), propylene glycol; a homogeneous suspension could not be achieved.
- Irritation: The treatment of mice with 1.25, 2.5, 5 and 10% test item in DMSO did not show any signs of severe local irritation or systemic toxicity
- Systemic toxicity:The treatment of mice with 1.25, 2.5, 5 and 10% test item in DMSO did not show any signs of severe local irritation or systemic toxicity
- Ear thickness measurements: no data available
- Erythema scores: Due to the discolouration by the test item redness at the ears caused by irritation could not be observed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA assay
- Criteria used to consider a positive response: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
The decision to select a STIMULATION INDEX (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Preparation
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMSO) was quantitatively added. The test item concentrations were prepared individually.
A non-GLP conform pre-test was conducted using two mice, test item concentrations of 1.25, 2.5, 5 and 10% (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application. Due to the discolouration by the test item redness at the ears caused by irritation could not be observed.
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2.5, 5 and 10% (w/v) in DMSO. The application volume, 25 μl, was spread over the entire dorsal surface (approx 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 μl of 81.3 μCi/ml 3HTdR (corresponds to 20.325 μCi 3HTdR per mouse) by intravenous injection via a tail vein.
Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were
prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the
lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
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:
The EC3 value was 6.7%

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
2.5%, 5% (w/v)
Remarks on result:
other: not sensitizing to skin
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10% w/v
Remarks on result:
other: not sensitizing
Cellular proliferation data / Observations:
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, recorded prior to the first application and prior to treatment
with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Calculations and results of Individual data

Vehicle: DMSO

 

Test item concentration% (w/v)

Group

Measurement DPM

Calculation

Results

 

DPM-BG

Number of lymph nodes*

DPM per lymph node **

SI

-

BG1

14.0

-

-

-

-

-

BG2

19.3

-

-

-

-

-

CG1

5914.7

5898.1

8

737.3

 

2.5

TG1

8844.1

8827.5

8

1103.4

1.5

5

TG2

9019.1

9002.4

8

1125.3

1.5

10

TG3

9663.9

9647.3

8

1205.9

1.6

BG = Background (1ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I = Stimulation Index

*= The mean value was taken from the figures BG1 and BG2

**= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the lymph nodes pooled

EC3 value could not be calculated as the S.I values were less than 3.

Results of the Positive Control

Positive control substance: alpha-hexylcinnamaldehyde

Vehicle: acetone:olive oil, 4:1(v/v)

Test item concentration% (w/v)

Group

Measurement DPM

Calculation

Results

 

DPM-BG

Number of lymph nodes*

DPM per lymph node **

SI

-

BG1

16.02

-

-

-

-

-

BG2

17.55

-

-

-

-

-

CG1

5083.65

5066.87

8

633.4

 

5

TG2

11655.70

11638.92

8

1457.0

2.30

10

TG3

21860.70

21843.92

8

2732.6

4.31

25

TG4

56809.20

56792.42

8

7101.2

11.21

 

 BG = Background (1ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I = Stimulation Index

*= The mean value was taken from the figures BG1 and BG2

**= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the lymph nodes pooled

Test group

Test item concentration% (w/v)

S.I

Group 3

5(a)

2.30(b)

Group 4

10(c)

4.31(d)

EC3= (a-c)[(3-d)/(b-d)]+c = 6.7% (w/v)

 

Applicant's summary and conclusion

Interpretation of results:
other: not sensitizing
Conclusions:
Stimulation Indices of 1.5, 1.5 and 1.6 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in DMSO. EC3 value could not be calculated as the S.I values were less than 3.
Hence, the test chemical can be considered to be not sensitizing to skin.
Executive summary:

The purpose of the Local Lymph Node assay was to identify the contact allergenic potential of the test chemical when administered to the dorsum of both ear lobes of mice. The study was performed according to OECD 429 Guidelines.

Three groups each of four female CBA/CaOlaHsd mice were treated daily with the test item at concentrations of 2.5, 5 and 10% (w/v) in DMSO 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 (DMSO) 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 of 3H-methyl thymidine measured in a beta-scintillation counter. In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:

1. Mortality / Viability once daily (week day) from experimental start to necropsy.

2. Body weights prior to the first application and prior to treatment with 3HTdR.

3. Clinical signs (local / systemic) at 1 – 2 hours after each application. Especially the treatment sites were observed carefully.

A test item was 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 of 3HTdR 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.

Stimulation Indices of 1.5, 1.5 and 1.6 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in DMSO. EC3 value could not be calculated as the S.I values were less than 3.

Hence, the test chemical can be considered to be not sensitizing to skin.