Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-03 till 2009-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40849/A TE
- Substance type: reactive dyestuff for cellulose fibers
- Physical state: blue powder
- Analytical purity: 76.9% of all colored components
- Lot/batch No.: TZ 5463 / BOP 03-09
- Expiration date of the lot/batch: 2014-06-30
- Storage condition of test material: At room temperature at about 20 °C

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: Mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 18.9 - 22.9 g
- Housing: Single caging.
- Diet : pelleted standard diet, ad libitum
- Water: tap water, ad libitum,
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 + 2°C
- Humidity (%): relative humidity 45-70%
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m
- bedding: granulated soft wood bedding

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
The highest test item concentration, which can be technically used was a 20 % solution in dimethylformamide.
First pre-test two mice were treated with concentrations of 10 and 20 %
Second pre-test two mice were treated with concentrations of 2.5 and 5 %
The test item in the main study was assayed at I, 2.5, and 5%.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 20 % solution in dimethylformamide.
In the first pre-test two mice were treated with concentrations of 10 and 20 % each on three consecutive days. Within1 hour after the first and second and 24 hours after the third application both animals showed reduced spontaneous activity and eyelid closure.

In the second pre-test two mice were treated with concentrations of 2.5 and 5 % each on three consecutive days. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation: Local Lymph Node Assay (adopted 24 April 2002).
- Criteria used to consider a positive response: In order to study a possible allergenic potential of FAT 40849/A TE, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and 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 -scintillation counter.

TREATMENT PREPARATION AND ADMINISTRATION:Redness of the ear skin could not be observed, due to the colour of the test item. The test item in the main study was assayed at I, 2.5, and 5%. 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.

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 1, 2.5, and 5% (w/v) in dimethylformamide. The application volume, 25 μl, was spread over the entire dorsal surface (Ø ~ 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 GE Healthcare (GE Healthcare 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 81.7 μCi/ml 3HTdR (corresponds to 20.4 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 Pentobarbital-Natrium (Release_, WDT, D-30827 Garbsen).
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 plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, D-63110 Rodgau) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β- 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)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 1%: 6.15 2.5% 13.29 5% 15.76
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group: 486.2 1%: 2991.9 2.5% 6461.2 5% 7660.1

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: dimethylformamide

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

 Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

23

---

---

---

---

---

BG II

32

---

---

---

---

---

1

3917

3890

8

486.2

 

1

2

23963

 23936

8

2991.9

6.15

2.5

3

51717

51690

8

6461.2

13.29

5

4

61308

 61281

8

7660.1

15.76

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 could not be calculated, since all S.I. are above 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. Due to the colour of the test item, redness of the ear

skin could not be observed.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test item FAT 40849/A TE was found to be a skin sensitiser under the described
conditions.
Executive summary:

In order to study a possible contact allergenic potential of FAT 40849/A TE, three groups each of four female mice were treated daily with the test item at concentrations of 1, 2.5, and 5% (w/v) 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 aβ-scintillation counter.

 

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Due to the colour of the test item, redness of the ear skin could not be 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 6.15, 13.29, and 15.76 were determined with the test item at concentrations of 1, 2.5, and 5% in dimethylformamide. The EC3 value could not be calculated, since all S.I. are above 3.

 

Conclusion

The test item FAT 40849/A TE was found to be a skin sensitiser under the described conditions.