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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:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
LLNA assay was performed to determine the sensitization potential of the test chemical
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide
EC Number:
267-636-0
EC Name:
N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide
Cas Number:
67905-17-3
Molecular formula:
C22H16N2O4
IUPAC Name:
N-{4-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]phenyl}acetamide
Test material form:
solid
Details on test material:
- Name of test material : N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide
- Molecular formula): C22H16N2O4
- Molecular weight : 372.378 g/mol
- Smiles notation : O=C(Nc1ccc(Nc2c3c(c(O)cc2)C(=O)c2c(cccc2)C3=O)cc1)C
- InChl: 1S/C22H16N2O4/c1-12(25)23-13-6-8-14(9-7-13)24-17-10-11-18(26)20-19(17)21(27)15-4-2-3-5-16(15)22(20)28/h2-11,24,26H,1H3,(H,23,25)
- Substance type: Organic
- Physical state: Powder

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan, Netherlands
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]: yes, nulliparous and non-pregnant
- Microbiological status of animals, when known: no data available
- Age at study initiation:8 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: no data available
- Housing:Makrolon Type I, with wire mesh top with granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum,
- Acclimation period:Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.
- Indication of any skin lesions: no data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 + 3°C
- Humidity (%): 30-70%
- Air changes (per hr): no data available
- Photoperiod (hrs dark / hrs light):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)
No. of animals per dose:
4 females per group - Main test
2 females for the pre -test
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: insoluble in water
- Irritation:Due to the intense blue colour of the test item local irritation reactions such as ear redness could not be detected at 5.0 and 10 % (w/v).
- Systemic toxicity: no data available
- Ear thickness measurements: No swelling of the ears was observed at any of the animals.
- Erythema scores: no data available

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: 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:
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). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from Amersham International. Five days after the first topical application, all mice were administered with 250 μl of 79.4 μCi/ml 3HTdR (corresponds to 19.9 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 animal (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). In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability once daily (week day) from experimental start to necropsy. Body weights prior to the first application and prior to treatment with 3HTdR. Clinical signs (local / systemic) at 1 – 2 hours after each application. Especially the treatment sites were observed carefully.
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.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
EC3 = 8.9%(w/v)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
group 1
Remarks on result:
other: No sensitization observed
Key result
Parameter:
SI
Value:
2.3
Test group / Remarks:
group 2
Remarks on result:
other: no sensitization observed
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
group 3
Remarks on result:
other: No sensitization observed
Cellular proliferation data / Observations:
EC3 CALCULATION: The EC3 Value could not be calculated, since all SI´s are below 3.

CLINICAL OBSERVATIONS: No symptoms of systemic findings were observed during the study period. Due to the intense blue colour of the test item at 5 % and 10 % (w/v) local irritation reactions such as
ear redness could not be detected. No swelling of the ears was observed.

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.

Viability / Mortality: No deaths occurred during the study period

Any other information on results incl. tables

Table 2: Calculation and results of individual data

Vehicle used: DMSO

Test concentration

%

(w/v)

Group

Measurement

DPM

Calculation

results

DPM- BGa

Number of lymph nodes

DPM per lymph nodeb

SI

-

BG I

28.9

--

--

--

--

-

BG II

16.9

-

-

-

-

-

CGI

4397.9

4375.0

8

546.9

 

2.5

TG2

7936.9

7914.0

8

989.3

1.8

5

TG3

10087.4

10064.5

8

1258.1

2.3

10

TG4

8827.9

8805.0

8

1100.6

2.0

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

CG = control group

TG = test group

S.I = Stimulation Index

a- The mean value was taken from the figures of BG1 and BGII

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

Table 3: Results of the Positive Control Study

Test concentration

%

(w/v)

Group

Measurement

DPM

Calculation

results

DPM- BGa

Number of lymph nodes

DPM per lymph nodeb

SI

-

BG I

6.4

--

--

--

--

-

BG II

0.0

-

-

-

-

-

CGI

3722.6

37194

8

464.9

 

5

TG2

8529.3

8526.1

8

1065.8

2.29

10

TG3

11947.6

11944.4

8

1493.1

3.21

25

TG4

31380.2

31377.0

8

3922.1

8.44

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

CG = Control Group

TG = 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

Groups

Test item concentration%

(w/v)

S.I

Group 2

5.0(a)

2.29(b)

Group 3

10.0(c)

3.21(d)

                                                                EC3 = (a-c) [(3-d)/(b-d)] + c = 8.9 % (w/v)

 

EC3 = Estimated concentration for a S.I. of 3.

a,b,c,d = Co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Applicant's summary and conclusion

Interpretation of results:
other: not sensitizing
Conclusions:
Stimulation Indices of 1.8, 2.3, and 2.0 were determined with the test chemical at concentrations of 2.5, 5.0, and 10 % (w/v) in DMSO. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.No symptoms of systemic findings were observed during the study period.
Since, Stimulation Index of the test chemical were below 3, so test chemical can be regarded as not sensitizer to skin.
Executive summary:

LLNA assay was performed to determine the sensitization potential of the test chemical.The study was performed in a GLP Laboratory according to OECD 429 Guidelines.

The concentrations for the main study were obtained from a pre test conducted in 2 mice.

In a pre-test in two mice, test item concentrations of 1.25, 2.5, 5, and 10 % (w/v) were tested on one ear each. Due to the intense blue colour of the test item local irritation reactions such as ear redness could not be detected at 5.0 and 10 % (w/v). No swelling of the ears was observed at any of the animals.

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. Mortality / Viability once daily (week day) from experimental start to necropsy. Body weights prior to the first application and prior to treatment with 3HTdR. Clinical signs (local / systemic) at 1 – 2 hours after each application. Especially the treatment sites were observed carefully. The validation- / positive control study was performed with alpha-Hexylcinnamaldehyde in acetone:olive oil, 4:1 (v/v) using CBA/CaOlaHsd mice.

Stimulation Indices of 1.8, 2.3, and 2.0 were determined with the test chemical at concentrations of 2.5, 5.0, and 10 % (w/v) in DMSO. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

No symptoms of systemic findings were observed during the study period.

Since, Stimulation Index of the test chemical were below 3, so the test chemical can be regarded as not sensitizer to skin.