Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental: 13 Jan - 16 Feb 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted in compliance with GLP regulations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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):Tris-(2-ethylhexyl)amin O 2446
- Analytical purity: 99.7%
- Lot/batch No.: Behälter 407 v. 16.07.2009

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Netherlands
- Age at study initiation: 8 - 12 weeks
- Housing: single caging
- Diet: Pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst / Netherlands), ad libitum
- Water: Tap water (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 20 - 65 %
- Photoperiod (hrs dark / hrs light): 12/12 h

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pretest 1: 50, 100 %
Pretest 2: 10, 25 %
Main test: 5, 10, 20 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone:olive oil (4+1). Vortexing was necessary to formulate the test item.
- Irritation:
To determine the highest non-irritant test concentration, two pre-tests were performed. In the pre-tests clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 6. Furthermore, prior to the first application of the test item and before sacrifice the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schluechtern, Germany). Additionally, for both animals, the ears were punched after sacrifice at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance.
In the first pre-test two mice were treated with test item concentrations of 50% and 100% each on three consecutive days. About 24 hours after the third application slight redness of the ear skin and swelling of the ears was observed at both tested concentrations. Ear swelling was still observed on day 6 before sacrifice. These signs of local irritation were also confirmed by the ear thickness and ear weight measurements. The ear thickness measurement on day 6 showed an ear swelling of approximately 84% in comparison to the measurement on day 1 prior to treatment.
Thus, a second pre-test was performed using test item concentrations of 10% and 25%. Two mice were treated with test item at the mentioned concentrations each on three consecutive days. At the tested concentrations the animals did not show any signs of systemic toxicity. The animals did not show severe signs of local irritation as confirmed by the ear thickness and ear weight measurements. However, in the animal treated with 25% test item concentration, the measured ear weight was about 30% higher than in the animal treated with 10% test item concentration.
Therefore, upon request of the sponsor, the test item in the main study was assayed at 5, 10, and 20% (w/v). The top dose is the highest test item concentration that could be used without inducing systemic toxicity or excessive local irritation.

MAIN STUDY
- 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:
In order to study a possible allergenic potential of Tris-(2-ethylhexyl)amin O 2446, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed using an analytical balance. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch and were immediately pooled per animal and weighed. Single cell suspensions of lymph node cells were prepared from lymph nodes pooled per experimental group, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in ß-scintillation counter.
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. The ANOVA (Dunnett-test) was conducted on the ear and lymphe node weights to assess wether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statisitcal significance were considered together.

Results and discussion

Positive control results:
Experiment performed in November 2009.
Positive control substance: a-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1)
Test item concentrations: 0, 5, 10, 25 % (w/v)
S.I.: 1.0, 1.21, 2.09, 6.22
EC3 = 13.3 % (w/v)

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Stimulation Indices (S.I.) of 0.72, 0.84, and 2.91 were determined with the test item at concentrations of 5, 10, and 20% in acetone:olive oil (4+1), respectively. A statistically significant increase in lymph node weights was observed in the group treated with 20% test item concentration in comparison to the vehicle control group (p<0.001), however, this is not considered as biologically relevant as none of the S.I.s determined for the tested concentrations exceeded the threshold of 3. A statistically significant increase in ear weights was also observed in this group in comparison to the vehicle control group (p=0.009) indicating the irritation potential of the test item.

Any other information on results incl. tables

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

Number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BGI

15

---

---

---

---

---

BGII

20

---

---

---

---

0

1

4976

4959

8

619.8

 

5

2

3599

3582

8

447.7

0.72

10

3

4186

4169

8

521.1

0.84

20

4

14464

14447

8

1805.8

2.91

 

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

1    = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a)   = The mean value was taken from the figures BG I and BG II.

b)   = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

 

The EC3 value could not be calculated, since all S.I.´s are below 3.

  

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information