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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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Test System: Mice, CBA/CaOlaHsd
Rationale: Recognised at the recommended test system
Source: Harlan Laboratories B.V., The Netherlands
Identification: The animals were distributed into the test groups at random and identified by cage number. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimatisation: 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.
Husbandry: The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: Group
Cage Type: Makrolon Type II (pre-test), III (main study), with wire mesh top (EHRET GmbH, Germany)
Bedding: Granulated soft wood bedding (Rettenmaier & Sohne GmbH + Co, Germany)
Feed: Pelleted standard diet, ad libitum (Harlan Laboratories B.V, Germany)
Water: Tap water, ad libitum (Gemeindewerke, Germany)
Environment: Temperature: 22°C ± 2°C. Relative Humidity: 45-65% (acclimation period) and 45-65% (pre-test and main study). Artificial light 6.00 am - 6.00 pm. Air Changes: About 10/hour.
Age (beginning of treatment): Pre-test: 9-10 weeks. Main study: 8-9 weeks

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
Test item concentrations of 10, 25 and 50% (w/w) were used.
No. of animals per dose:
5
Details on study design:
Number of animals for the pre-test: 2 females
Number of animlas for the main study: 20 females
Number of animals per group: 5 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle groups): 1
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 (as provided in the report), for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation). A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and the negative control group.

Results and discussion

Positive control results:
The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil(4:1 v/v) using CBA/CaOlaHsd mice. The results provided in the test report show that the positive control substance gave a Stimulation Index of between 3.73 and 10.77. This shows the positive control substance to be a skin sensitiser and confirms the sensitivity and reliability of the experimental technique.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.28
Test group / Remarks:
50%
Parameter:
SI
Value:
1.58
Test group / Remarks:
25%
Parameter:
SI
Value:
0.88
Test group / Remarks:
10%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM per animal (Determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group by the number of animals in that group (5 animals)). Vehicle (methyl ethyl ketone): 482.9 10% Pluriol BP 40E: 425.5 25% Pluriol BP 40E: 762.5 50% Pluriol BP 40E: 1101.1

Any other information on results incl. tables

Test Item Concentration (% w/w)

Group No.

Animal No.

DPM Values Measured

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

S. I.(b)

-

-

BG I

18

-

-

-

-

BG II

19

-

-

0

1

1

201

183

 

2

435

417

 

3

287

269

 

4(c)

1267

1249

 

5

317

299

 

10

2

6

230

212

0.4

7

611

593

1.2

8

310

292

0.6

9

682

664

1.4

10

387

369

0.8

25

3

11

441

423

0.9

12

483

465

1.0

13

746

728

1.5

14

966

948

2.0

15

1269

1251

2.6

50

4

16

917

899

1.9

17

1266

1248

2.6

18

1484

1466

3.0

19

1359

1341

2.8

20

572

554

1.1

 

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

S.I. = Stimulation Index (values of the test item groups related to the mean value of the control group)

(a) = Values corrected for mean background levels (BG I and BG II)

(b) = Stimulation Indices relative to the mean of the control group (Group 1, animal no. 1-5)

(c) = The value for animal 4 was identified as an outlier but was not excluded from the calculation, since the value is well within the range of the historical vehicle control data

Test item concentration

Group Calculation

Mean DPM per animal(a); (c)

SD(b)

S.I.

Vehicle (methyl ethyl ketone)

482.9

436.1

1.00

10% Pluriol BP 40E

425.5

194.6

0.88

25% Pluriol BP 40E

762.5

345.7

1.58

50% Pluriol BP 40E

1101.1*

371.6

2.28

(a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

(b) SD = Standard Deviation

(c) The DPM value for animal 4 was identified as an outlier but not excluded from the calculation

* Statistically significant increase vs. control group (p <0.05)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study Stimulation Indices (S.I.) of 0.88, 1.58 and 2.28 were determined with the test item at concentrations of 10, 25 and 50% (w/w), respectively. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentrations results in an S.I. of 3 or more. Therefore, the test item Pluriol BP 40E was not considered to be a skin sensitiser under the test conditions of this study.
Executive summary:

The skin sensitisation potential of the test substance was determined in accordance with OECD Guideline for Testing of Chemicals 429. The skin sensitisation of the test substance was measured using the Local Lymph Node Assay (LLNA) in mice. The LLNA was performed using test item concentrations of 10, 25 and 50% (w/w), prepared in a vehicle of methyl ethyl ketone. The highest concentration tested was the highest concentration that could be technically used and applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study, and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 0.88, 1.58 and 2.28 were determined with the test item at concentrations of 10, 25 and 50% (w/w), respectively. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentrations results in an S.I. of 3 or more. Therefore, the test item Pluriol BP 40E was not considered to be a skin sensitiser under the test conditions of this study.