Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sokalan PG B62

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
Age: about 9 weeks.
Weight: 17 - 22 g at day 1.
Animals were group housed (5 per cage) upon receipt then individually housed prior to the initiation of dosing in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals".
Lighting: 12 hours light / 12 hours dark
Room Temperature: 25.6 to 26.7°C
Relative Humidity: 24 to 40%
Acclimation period: 5 days.
Food and water: ad libitum.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
0.1, 1, 5, and 10% (w/v) of the test substance
No. of animals per dose:
5
Details on study design:
Groups of 5 mice were treated on the dorsal surface of both ears once per day for 3 days. Application volume: 25 µl/ear.
Negative controls: dimethylsulfoxide for test substance; acetone/olive oil, 4:1 for positive control. On day 6, the mice were injected, i.v., with 20 µCi of 3H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5% trichloroacetic (TCA) and the pellets counted in a beta-scintillation counter to determine incorporation of 3H-thymidine.

Observations:
Daily observations of mortality/morbidity and clinical observations of the application sites. On day 1 and 6:body weight.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
DPM: stat. software package developed by SAS Institute Inc. (version 6.12). body weights: one way ANOVA, Dunnett's test

Results and discussion

Positive control results:
The positive control HCA, resulted in a stimulation index (SI) of 5.13 indicating a positive response (p = 0.0001) compared to the AOO vehicle control group.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Exposure to test substance at 0.1, 1.0, 5 or 10 % (w/v) resulted in stimulation indices of 1.25, 1.17, 1.96 and 1.64, respectively. No statistically differences were found between the groups treated with test substance and the DMSO vehicle control, except for the 5.0%-group (p = 0.0343). However, given of the low stimulation index and the absence of any dose response, this difference is not considered meaningful.

Any other information on results incl. tables

Group

Treatment

Dose

DPM (mean ± sem)

SI (test/control ratio)

1

DMSO

-

453 ± 47

-

2

GTS20024

0.1% (w/v)

566 ± 62

1.25

3

GTS20024

1% (w/v)

532 ± 67

1.17

 4  GTS20024

5%(w/v)

887 ± 288   1.96
 5  GTS20024  10% (w/v)  745 ± 149  1.64
 6  4:1 acetone:olive oil (V/V)(AOO)  -  313 ± 11  -
 7  HCA in AOO  35% (v/v)  1607 ± 202  5.13

There was no mortality and all animals appeared normal throughout the study. The application sites on the mice from the positive control group appeared wet on Days 2-4. The A00 treated animals also exhibiting wet ears on Day 4. There were no other findings.

At termination, the lymph nodes of the mice treated with the positive control were enlarged relative to the lymph nodes from the positive control vehicle treated mice but otherwise appeared normal. The lymph nodes from the mice in the vehicle treated groups and all the test article treated groups were normal in size and appearance.

Mean body weights at Day I and Day 6 and mean changes in body weights were evaluated. There were no statistically significant differences obsewed between any of the treatment groups. Therefore, the test articles did not appear to cause any overt toxicity. The positive control, 35% (vlv) HCA, resulted in a stimulation index (SI) of 5.13 indicating a positive response. This response was also statistically significant when compared to the A00 vehicle control group (p = 0.0001).

Exposure to 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate at 0.1 %, 1.0%, 5% or 10% (w/v) resulted in stimulation indices of 1.25, 1.17, 1.96 and 1.64, respectively. No statistically significant differences were found between the groups treated with the test substance at 0.1 %, 1.0% or 10% (w/v) and the DMSO vehicle control. A statistically significant difference was found between the group treated with the test substance at 5.0% (w/v) and the DMSO vehicle control (p = 0.0343).

However, given the low stimulation index and the absence of any dose response, this difference is not considered meaningful.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and is thus not regarded as skin sensitiser.
Executive summary:

In a LLNA skin sensitisation test according to OECD guideline 429 (Calvert Lab. 2006), groups of 5 CBNJ female mice were treated on the dorsal surface of both ears once per day for 3 days with 0.1 %, 1 %, 5% or 10% (w/v) of the test substance, with the vehicle (DMSO), with the vehicle for the positive control (acetone/olive oil, 4:1), or with the positive control (HCA at 35%). On Day 6, the mice were injected iv with 20 µCi of 3H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a beta-scintillation counter to determine incorporation of the 3-thymidine.

A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control.

There was no mortality and all animals appeared normal throughout the study. The application sites on the mice from the positive control group appeared wet on Days 2-4. The positive vehicle control animals also exhibiting wet ears on Day 4. There were no other findings. At termination, the lymph nodes of the mice treated with the positive control were enlarged relative to the lymph nodes from the positive control vehicle treated mice but otherwise appeared normal. The lymph nodes from the mice in the vehicle treated groups and all the test article treated groups were normal in size and appearance. Mean body weights at Day 1 and Day 6 and mean changes in body weights were evaluated. There were no statistically significant differences observed between any of the treatment groups. Therefore, the test articles did not appear to cause any overt toxicity. The positive control, 35% (vfv) HCA, resulted in a stimulation index (SI) of 5.1 indicating a positive response. This response was also statistically significant when compared to the A00 vehicle control group (p = 0.0001). Exposure to the test substance at 0.1 %, 1.0%, 5% or 10% (w/v) resulted in stimulation indices of 1.25, 1.17, 1.96 and 1.64, respectively. No statistically significant differences were found between the groups treated with the test substance at 0.1%, 1.0 % or 10% (w/v) and the DMSO vehicle control. A statistically significant difference was found between the 5.0% (w/v) group and the DMSO vehicle control (p = 0.0343). However, given the low stimulation index and the absence of any dose response, this difference is not considered meaningful.

Treatment with the test substance up to did not resulted in stimulation indices of 3 or greater, indicating a negative response.

Under our experimental conditions, the test substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.