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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-08-26 till 2008-09-17
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of the test material (as cited in the study report: FAT 40842/A TE
- Substance type: coloring dye
- Physical state: solid, dark bluish green powder
- Analytical purity: 96%
- Lot/batch No.: Blau DRI 2098 Op 1/07
- Expiration date of the lot/batch: June 30, 2014
- Storage condition of test material: at room temperature at about 20 ºC

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 NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Weight: 20.4-21.8g
- Housing: single caging
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Laboratories GmbH, D-33178 Borchen), ad libitum
- Water (e.g. ad libitum): tap water (Gemeindewerke, D-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 were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C
- Humidity (%): 30-70%
- 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 20 % (w/w) in dimethylformamide, 25 µL aplication volume
- The highest test item concentration, which can be technically used was a 20 % suspension in dimethylformamide.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 20 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 5, 10 and 20 %. 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.
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:


TREATMENT PREPARATION AND ADMINISTRATION:
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:
- Experiment performed in July 2008, 5 % (w/v) alpha-Hexylcinnamaldehyde yielded a S.I. of 5.24, 25 % yielded a S.I. of 9.32
- Detailed data attachend to the study report

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: - below 3 for all tested concentrations, see table 1
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - see table 1

Any other information on results incl. tables

- Table 1: Calculation and Results of Individual Data

Test item concentration % (w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

295

---

---

---

---

---

BG II

23

---

---

---

---

---

1

4448

4289

8

536.1

 

5

2

7722

7563

8

945.4

1.76

10

3

4741

4582

8

572.8

1.07

20

4

12100

11941

8

1492.6

2.78

Vehicle: dimethylformamide

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.´s are below 3.

- Additional 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 (see below)

- Tables of Body Weights

Animal No.

Dose Group

Initial Weight (g)

weight prior to treatment with3HTdR (g)

Individual Mean

±SD

Individual

Mean±SD

1

1

19.8

22.2±1.9

19.8

21.8±1.5

2

1

24.5

23.2

3

1

21.9

22.6

4

1

22.4

21.6

5

2

23.1

22.3±0.9

20.8

21.8±0.9

6

2

22.9

23.0

7

2

21.1

21.9

8

2

22.2

21.6

9

3

19.9

19.6±0.8

20.6

20.4±1.2

10

3

20.5

21.9

11

3

19.0

20.0

12

3

18.9

19.1

13

4

20.6

20.7±0.4

20.3

20.9±1.4

14

4

20.5

20.9

15

4

21.3

22.8

16

4

20.3

19.4

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item FAT 40842/A TE was found to be not a skin sensitiser under the described test conditions.
Executive summary:

The present study (Honrvar 2008) analyzes the sensitizing potential of potential of FAT 40842/A TE.

Three groups each of four female mice were treated daily with the test item at concentrations of 5, 10 and 20 % 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 of 3H-methyl thymidine measured in a beta-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 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.

In this study Stimulation Indices of 1.76, 1.07 and 2.78 were determined with the test item at concentrations of 5, 10 and 20 % in dimethylformamide.

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

The test item FAT 40842/A TE was found to be not a skin sensitiser under the described conditions.

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