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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-09-12 to 2007-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with no deviations and performed in accordance with GLP regulations
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
-
EC Number:
451-530-8
EC Name:
-
Cas Number:
736150-63-3
Molecular formula:
Not applicable
IUPAC Name:
Not allocated
Details on test material:
Name of test material (as cited in study report): TS-ED 532 (Batch D)
- Lot/batch No.: batch 4010534806
- Expiration date of the lot/batch: 2009-02-06

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: mouse (CBA/CaOlaHsd strain)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 19.5-19.9 g
- Housing: single in makrolon Type 1 , with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-71%
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
TS-ED 532 was used in concentrations of 10, 25, and 50 % (w/v).

TS-ED 532 was placed into a volumetric flask on a tared balance and the vehicle (acetone:olive oil (4+1) (v/v)) was quantitatively added. Based on the results from an initial solubility experiment the highest TS-ED 532 concentration was a 50%. The weight/volume dilutions were prepared by serial dilution. Homogeneity of TS-ED 532 in the vehicle was maintained during treatment with a magnetic stirrer. The preparations were made freshly before each dosing occasion.
No. of animals per dose:
Number of animals in test groups: 5
Number of animals in negative control group: 5
Number of animals in positive control group: 5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The data of a preliminary solubility experiment showed that the highest TS-ED 532 concentration that could be
technically achieved was a 50% solution.
- Irritation: NA
- Lymph node proliferation response: NA

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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.

TREATMENT PREPARATION AND ADMINISTRATION:
Mice were treated daily for three consecutive days with 25 μL of the appropriate concentration of TS-ED 532 on the dorsal surface of each ear (approximately 8 mm in diameter). Five days after the 1st topical application, all mice were administered 250 μL of 81.3 μCi/mL 3HTdR (approx. 20.3 μCi 3HTdR/mouse) by intravenous injection via a tail vein. Approximately 5 hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Ketamin/Xylazin/Midazolam.

The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um 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 and thoroughly mixed. The level of 3HTdR incorporation was then measured in a scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The 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 ANOVA (Dunnett-test) was conducted to assess whether the difference between TS-ED 532 test groups and negative control (vehicle) group was statistically significant.

Statistical significance: p < 0.05.

Results and discussion

Positive control results:
Positive and vehicle controls were within the historical range ensuring the validity of the test. With the positive control an S.I. value of 5.42 was obtained at a concentration of 25% (See Table 1 below).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI: 1.00 (Control group). Si: 5.42 (Positive control group) SI: 2.19 (10 % (w/v)). SI: 1.99 (25 % (w/v)). SI: 2.20 ( 50 % (w/v)).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 1 below

Any other information on results incl. tables

The EC 3 value for TS-ED 532 could not be calculated, since all SI values were below 3.

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the mice and no systemic findings were observed during the study period. The body weights of the mice throughout the study were within the range commonly recorded for this strain and age.

Table 1 Calculations and results

TS-ED 532 concentration

Measurement
DPM

Calculation

Result

Calculation

DPM
per
lymph node
b)

Result

% (w/v)

Group

DPM-BG per animal
(2 lymph nodes)
a)

S.I.*

number
of
lymph nodes

S.I.

---

BG I

19

---

---

---

---

---

---

BG II

19

---

---

---

---

---

---

CG 1_1

1745

1726

---

---

---

---

---

CG 1_2

3523

3504

---

---

---

---

---

CG 1_3

2358

2339

---

---

---

---

---

CG 1_4

1550

1531

---

---

---

---

---

CG 1_5

2281

2262

---

10

1136.2

1.00

25

PG 1_1

6502

6483

2.9

---

---

---

25

PG 1_2

13972

13953

6.1

---

---

---

25

PG 1_3

14652

14633

6.4

---

---

---

25

PG 1_4

13704

13685

6.0

---

---

---

25

PG 1_5

12792

12773

5.6

10

6152.7s

5.42

10

TG 1_1

4419

4400

1.9

---

---

---

10

TG 1_2

8958

8939

3.9

---

---

---

10

TG 1_3

4475

4456

2.0

---

---

---

10

TG 1_4

2353

2334

1.0

---

---

---

10

TG 1_5

4764

4745

2.1

10

2487.4

2.19

25

TG 2_1

2426

2407

1.1

---

---

---

25

TG 2_2

5273

5254

2.3

---

---

---

25

TG 2_3

5232

5213

2.3

---

---

---

25

TG 2_4

3736

3717

1.6

---

---

---

25

TG 2_5

6020

6001

2.6

10

2259.2

1.99

50

TG 3_1

4947

4928

2.2

---

---

---

50

TG 3_2

7548

7529

3.3

---

---

---

50

TG 3_3

3894

3875

1.7

---

---

---

50

TG 3_4

4660

4641

2.0

---

---

---

50

TG 3_5

4050

4031

1.8

10

2500.4

2.20

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

CG =  Control Group

PG =  Positive Control Group

TG=  Test Group

S.I. =  Stimulation Index

*    =  mean net DPM value per lymph node of the mouse divided by the mean net DPM value of the vehicle control group

a)   =  The mean of the background value (BGI and BGII) was subtracted from the value.

b)    =  DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a group by number of lymph nodes taken from that group

S Mean DPM value for the group was according to the Dunnett test significantly higher than corresponding control value. The p value for the analysis was <0.05.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
TS-ED 532 was not a skin sensitiser in the LLNA study using concentrations of 10, 25 and 50%
Executive summary:

The dermal sensitization of TS-ED 532 was determined using the local lymph node assay in accordance with OECD Guideline for Testing of Chemicals 429. Groups of 5 female mice were dosed at: 0% (control), 10%, 25% and 50% solutions of TS-ED 532 diluted with a 4:1 mixture of acetone to olive oil.

No deaths occurred during the study period. No symptoms of local effects at the ears of the mice and no systemic findings were observed during the study period. The body weight of the mice throughout the study was within the range commonly recorded for this strain and age.

Stimulation indexes (SIs) for the 10% dose group ranged from 1.0 to 3.9 with a mean of 2.19. For the 25% dose group, the SIs ranged from 1.1 to 2.6 with a mean of 1.99. For the 50% dose group, the SIs ranged from 1.7 to 3.3 with a mean of 2.20. The SIs for the positive control ranged from 2.9 to 6.4, with a mean of 5.42. The EC3 value could not be calculated for TS-ED 532, since none of the tested concentrations induced an SI greater than 3.

None of the tested TS-ED 532 concentrations induced a statistically significant increase in DPM values when compared to the control. The positive control did cause a statistically significant increase which demonstrates the reliability and sensitivity of this assay to detect skin sensitization potential. The stimulation index is <3 which indicate that TS-ED 532 does not have the potential to cause skin sensitization.

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