N,N'-bis[2-hydroxy-3-(2,2,3,3-tetrafluoropropoxy)propyl]-N,N,N',N'-tetramethylethane-1,2-diaminium dichloride

Skin sensitisation

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:

other: GLP study designed based on OECD Guideline 429, OPPTS 870.2600 and EEC Testing Methods Annex V Part B, B.42, 2004/73/EC L152, 2004

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

No impact on study validity.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

No impact on study validity.

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

No impact on study validity.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis[2-hydroxy-3-(2,2,3,3- tetrafluoropropoxy)propyl]-N,N,N',N'-tetramethylethane-1,2- diaminium dichloride

IUPAC Name:

N,N'-bis[2-hydroxy-3-(2,2,3,3- tetrafluoropropoxy)propyl]-N,N,N',N'-tetramethylethane-1,2- diaminium dichloride

Constituent 2

Reference substance name:

1,1,2,2,17,17,18,18-octafluoro-6,13-dihydroxy-8,8,11,11-tetramethyl-4,15-dioxa-8,11-diazaoctadecane-8,11-diium dichloride

EC Number:

700-737-4

Cas Number:

1220100-43-5

Molecular formula:

C18H34F8N2O4.2Cl

IUPAC Name:

1,1,2,2,17,17,18,18-octafluoro-6,13-dihydroxy-8,8,11,11-tetramethyl-4,15-dioxa-8,11-diazaoctadecane-8,11-diium dichloride

Details on test material:

Physical Description: White powder
Storage: Room temperature; desiccated

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test Animals Description

Species: Mouse
Strain/Substrain: CBA/J
Total Number: Main Study: 25
Gender: Female
Age Range: 9 weeks
Body Weight Range: 19 to 23 grams at the outset (Day 1) of the study.
Animal Source: Recognised supplier
Experimental History: Purpose-bred and experimentally naïve at the outset of the study.
Identification: Tail marked with an indelible marker.

Environmental Conditions

Lighting: 12 hours light/12 hours dark
Room Temperature: 23 to 27°C
Relative Humidity: 38 to 79 %

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic L92

Concentration:

10%, 25%, and 50% of the vehicle

No. of animals per dose:

Each group contained 5 females

Details on study design:

MAIN STUDY
- No. of exposures: 25
- Exposure period: 3 days
- Test groups: 1 to 5
- Control group: Vehicle (1), HCA (5)
- Site: Topically on the dorsal surface of both ears
- Frequency of applications: Once daily
- Duration: 3 consecutive days
- Concentrations: 10%, 25%, and 50% of the vehicle

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Data was manually collected except for the data generated by the scintillation counter (Beckman LS 6000 SC). The mean DPM for each group was calculated using SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response was that one or more concentrations of a test article elicited a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

The mean body weights and changes in body weight were also calculated and evaluated using SYSTAT.

Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM and body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett’s test was used to determine the degree of significance from the control means.

The data indicated that the test article was positive; therefore the EC3 was calculated using the formula:

EC3 = c+[(3-d)/(b-d)](a-c)

where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively.

Results and discussion

Positive control results:

The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 13.3. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p< 0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Exposure to the test article at concentrations of 10%, 25%, and 50% (w/v) resulted in stimulation indices of 0.6, 1.9 and 7.7, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Group 1 - Vehicle - DPM = 389.3 ± 58.4 Group 2 - 10 % - DPM = 214.6 ± 34.0 Group 3 - 25 % - DPM = 758.9 ± 91.8 Group 4 - 50 % - DPM = 2982.4 ± 379.2 Group 5 - Positive Control (HCA) - DPM = 5184.5 ± 551.1

Any other information on results incl. tables

There was no mortality and all animals appeared normal throughout the study.

No erythema or edema at the application site of any of the animals treated with vehicle, the positive control or the test article at 10%, 25%, and 50% (w/v) was observed. The ears of the mice treated with the positive control HCA appeared wet on Days 2 -6 and the ears of the mice treated with the test article at 50% appeared wet on Days 3 -6. There were no other findings.

The mean body weights of the mice treated with the test article on Days 1 and 6 showed no statistically significant differences. However, mice treated with 10 and 25% test substance, as well as 35% HCA showed statistically significant (p< 0.05, p< 0.01 and p< 0.05, respectively), but biologically irrelevant, increases in body weight changes when compared to the vehicle control group. Therefore, administration of the test article did not appear to exert overt toxicity.

At termination, enlarged lymph nodes relative to the vehicle-treated mice were observed in the HCA treated group and the test article group at 50%. The lymph nodes from the remaining test article treated mice and mice in the vehicle treated group were normal in size and appearance.

The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 13.3. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p< 0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

Exposure to the test article at concentrations of10%, 25%, and 50% (w/v) resulted in stimulation indices of 0.6, 1.9 and 7.7, respectively. Statistically significant differences were found between the groups treated with the test article at 10%, 25% and 50% and the vehicle control (p < 0.05, p < 0.01 and p < 0.001), respectively. The EC3 was calculated to be 29.7%.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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. Thus, a stimulation index above 3.0 is regarded as a positive response. Treatment with the test substance at a concentration of 50% (w/v) did result in stimulation indices greater than 3.0. Therefore, the test substance is considered to have skin sensitizing activity.

Executive summary:

A local lymph node assay study based on internationally accepted guidelines was carried out to determine if the test article would induce a hypersensitivity response in mice as measured by the proliferation of lymphocytes in the draining lymph nodes.

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. Thus, a stimulation index above 3.0 is regarded as a positive response. Treatment with the test substance at a concentration of 50% (w/v) did result in stimulation indices greater than 3.0. Therefore, the test substance is considered to have skin sensitizing activity. The EC3 was calculated to be 29.7%.