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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
Study period:
February 10, 2006 to February 17, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TPI 1618
- Substance type: Formaldehyde releaser
- Physical state: Colourless liquid
- Analytical purity: Releasable formaldehyde: 46.1%
- Composition of test material, percentage of components: Reaction product of ethylene glycol, urea and paraformaldehyde
- Lot/batch No.: 1100968
- Expiration date of the lot/batch: Ocxtober 2006
- Storage condition of test material: At room temperature, protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 18.6 – 22.4 g
- Housing: 4 to 6 animals housed in Macrolon cages Typ III
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-2
- Humidity (%): 55 +-15
- Photoperiod (12 hrs dark /12 hrs light):

IN-LIFE DATES: From: February 10, 2006 to February 17, 2006

Study design: in vivo (LLNA)

Vehicle:
other: Olive oil
Concentration:
Preparation of EUF (TPI 1618) doses
100 % EUF: Delivered undiluted EUF was used as 100 %.
80 % EUF/olive oil: 4 mL EUF were mixed in a glass tube with 1 mL olive oil.
50 % EUF/olive oil: 4 mL EUF were mixed in a glass tube with 4 mL olive oil.
25 % EUF/olive oil: 4 mL of 50 % EUF/olive oil solution were mixed in a glass tube with 4mL olive oil

Preparation of HCA doses
30 % HCA/AOO: 1.5 ml of HCA were mixed with 3.5 mL AOO in a glass tube.
10 % HCA/AOO: 0.5 ml of HCA were mixed with 4.5 mL AOO in a glass tube.
3 % HCA/AOO: 0.5 mL of 30 % HCA/AOO solution were mixed with 4.5 mL AOO in a glass tube

Preparation of formaldehyde doses
20 % formaldehyde/ 2.7 ml of 37 % formaldehyde solution were mixed with 2.3 mL AOO in a glass tube
10 % formaldehyde/ 2.5 ml of 20 % formaldehyde solution were mixed with 2.5 mL AOO in a glass tube.
0.1 % formaldehyde/: 100 µl of 10 % formaldehyde solution were mixed with 9.9 mL AOO in a glass tube.
No. of animals per dose:
HCA control: 3 groups with 6 animals each
Vehicle control: 2 groups with 6 animals each
Formaldehyde control: 3 groups with 4 animals each
Negative control: 1 group with 4 animals
Test item EUF: 4 groups with 6 animals each
Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA) according to OECD guideline 429 (2002).
- Criteria used to consider a positive response: Proliferation and ear weight index were calculated by dividing the means of cell counts and ear weights of the substance treated groups by the vehicle treated ones. A differentiation index (DI) was calculated according to the equation (max. index ear weight = 2.09, max. index proliferation = 5)

A substance is classified as a skin sensitizer if at one or more test concentrations it showed a DI greater than 1 and as skin irritating if it showed a DI between 0 and 1.


TREATMENT PREPARATION AND ADMINISTRATION:

Groups of mice were exposed topically on the dorsum of both ears to the test item for 3 consecutive days. Control groups were treated with reference compounds HCA, formaldehyde or vehicle (water, olive oil). Mice were killed and small pieces were stamped from each ear and weighed. The draining lymph nodes were excised, weighed and single cell suspensions were prepared. Cell counts of the LNC suspensions were measured. Changes of the lymph node cell counts correlated with the in vivo proliferation and sensitizing potency of the test item.
The skin-sensitizing substance alpha-hexylcinnamaldehyde (HCA) was used as reference item. TPI 1618 releases formaldehyde in aqueous solutions. Formaldehyde is a sensitizing substance and its effect in the LLNA had to be distinguished from the potency of the test item. Therefore, additional groups of animals were treated with formaldehyde.

HCA was tested as skin sensitizer by reason that it showed at least at one test concentration a differentiation index greater than 1.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other: Formaldehyde was tested as skin irritant due to the fact that it showed at two test concentrations a differentiation index between 0 and 1
Statistics:
Body weights, ear weights and cell counts of the treatment groups were compared to the relevant vehicle control. Mean and standard deviation of body weights, ear weights and cell counts of the treatment and control groups were calculated. Statistical analysis was performed by Dunnett test. Differences between treatment groups and vehicle control were considered as statistically significant at the level of p<0.05.

Results and discussion

Positive control results:
Positive control substance HCA showed at test concentration of 3 % a differentiation index smaller than 1. Concentrations of 10 % and 30 % HCA resulted in a differentiation index greater than 1.
In conclusion, HCA was tested as skin sensitizer by reason that it showed at least at one test concentration a differentiation index greater than 1.

In vivo (LLNA)

Results
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Tables 2-4

Any other information on results incl. tables

Table 2:     Calculation of the differentiation index (DI) for HCA (vehicle: AOO, group 04)

Group

treatment

Ear weight index

proliferation index

DI

assessment

01

3 % HCA

1.03

1.09

0.89

irritating

02

10 % HCA

1.04

1.18

1.23

sensitizing

03

30 % HCA

1.09

2.12

3.47

sensitizing

Table 3:     Calculation of the differentiation index (DI) for formaldehyde (vehicle: AOO, group 04)

Group

treatment

ear weight index

proliferation index

DI

assessment

06

0.1 % formaldehyde

1.04

0.95

-0.37

-

07

10 % formaldehyde

1.48

1.66

0.37

irritating

08

20 % formaldehyde

1.92

1.65

0.19

irritating

Table 4:     Calculation of the differentiation index (DI) for TPI 1618 (vehicle: olive oil, group 05)

Group

treatment

ear weight index

proliferation index

DI

assessment

10

25 % EUF

1.05

1.18

1.05

sensitizing

11

50 % EUF

1.04

1.26

1.79

sensitizing

12

80 % EUF

0.99

1.04

-2.13

-

13

100 % EUF

1.57

1.77

0.37

irritating

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
EUF (TPI 1618) had a sensitizing effect in a Local Lymph Node Assay in mice under the chosen test conditions and should thus be classified as sensitizer.
Executive summary:

A Local Lymph Node Assay (LLNA) was performed according to OECD guideline 429 (2002).

Principle: groups of mice were exposed topically on the dorsum of both ears to the test item for 3 consecutive days. Control groups were treated with reference compounds HCA, formaldehyde or vehicle (water, olive oil). Mice were killed and small pieces were stamped from each ear and weighed. The draining lymph nodes were excised, weighed and single cell suspensions were prepared. Cell counts of the LNC suspensions were measured. Changes of the lymph node cell counts correlated with the in vivo proliferation and sensitizing potency of the test item. The test item EUF (TPI 1618) showed acute skin sensitizing potency in female mice following topical treatment at concentrations of 25 and 50 % for 3 consecutive days in the local lymph node assay. The outcome of the LLNA indicates a skin sensitizing potency of EUF (TPI 1618).