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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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 guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCrl (1st prestest and main study); CBA/CaOlaHsd (2nd and 3rd pre-test)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. (2nd and 3rd pre-test); Charles River UK (1st pre-test and main study)
- Age at study initiation: 9-10 weeks (main study); 8-10 weeks (pre-tests)
- Weight at study initiation: 17.1 - 20.5g
- Housing: group in Makrolon Type II / III with wire mesh top
- Diet (e.g. ad libitum): Pelleted standard diet, ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-100% (acclimatisation); 45-65% (main study)
- Photoperiod (hrs dark / hrs light): 12h/12h

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
planned
0.5%, 1%, 2.5% (no signs of excessive local skin irritation were observed at 1% and 2.5% during a pre-test)
as verified by analysis:
0.5%, 0.6%, 2.5%
No. of animals per dose:
2 (pre-test)
5 (main study)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 100%
- Irritation: at 10%, 25%, 50%, 100% irritation and an increase in ear thickness was observed. Animals treated with 100% of the test substance were sacrificed on day 3 due to distinctly reduced spontaneous activity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with 25µl/ear/day, was spread over the entire dorsal surface (app. 8 mm in diameter) of each ear once daily for three consecutive days. Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline (PBS) containing 20.4 μCi of 3HTdR (equivalent to approximately 81.5 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Criteria used to consider a positive response:
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:
The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1 v/v) was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
One-Way-ANOVA. In case of significant results, multiple comparisons were performed with the Dunnett test and the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers
Positive control results:
The periodic positive control was performed in August 2011
SI 5% = 1.35; 10% = 2.18; 25% = 8.08
The results are within the historical control data (10 experiments 2009 - 2010
Parameter:
SI
Remarks on result:
other: 0.5% = 1.44 0.6% = 1.87 2.5% = 4.27 EC3 = 1.5%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: control = 412.0 0.5% = 592.8* 0.6% = 769.6* 2.5% = 1757.8* *: statistically significant increase vs. control group (p<0.05)

Lymph node weight and cell counts were statistically increased in the highest dose (2.5%)

Index LN weights                  LN Cell Count Index

0.5%       1.1                                      1.1

0.6%       1.1                                      1.0

2.5%       1.5                                      1.63

Ear weights were significantly increased in the highest dose (2.5%) - An influence of the irritant property of the test substance on the result of the study cannot be excluded

Ear weight              Index compared to control

0.5%       26.4                            1.0

1.0%       27.2                            1.1

2.5%       30.7*                          1.2

calculated EC3 value:

EC3 = (a-c) [(3-d)/(b-d)] + c = 1.7 % (w/w)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

No symptoms of systemic or local toxicity were observed.

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test item was a skin sensitiser under the test conditions of this study.
Executive summary:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone:olive oil (4+1 v/v). For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 0.6% and 2.5% (w/w). 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 three pre-experiments).

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. However, a statistically significant increase in ear weight was observed in the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice (see Ref. 9) was exceeded in this group (index of 1.2).

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.44, 1.87, and 4.27 were determined with the test item at concentrations of 0.5%, 0.6% and 2.5% (w/w) in acetone:olive oil (4+1 v/v), respectively. A dose response was observed. The EC3 value calculated was 1.5 % (w/w).

A statistically significant increase in DPM value was observed in all test item treated groups in comparison to the vehicle control group (p<0.05), in the mid and high dose group, this increase was also considered as biologically relevant. Furthermore, a statistically significant and biologically relevant increase in lymph node weight and cell count was determined in the highest dose group and the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in this group (index of 1.63). Based on the above mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded. Nevertheless, on the basis of the present data, the test item has to be classified as a sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

An LLNA according to OECD 429 and GLP was perfomed using concentrations of 0.5%, 0.6%, and 2.5% of propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- in acetone:olive oil (4 +1 v/v). In three pre-experiments 2.5% was determined to be the highest concentration were no excessive local skin irritation was observed. 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. However, a statistically significant increase in ear weight was observed in the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice was exceeded in this group (index of 1.2). Thus an influence from irritative properties of propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- on the result cannot be excluded.

The stimulation indices (S.I.) were 1.44, 1.87, and 4.27 for 0.5%, 0.6%, 2.5% respectively. The highest concentration exceeded an S.I. of 3, the threshold value above which a substance is regarded as a sensitizer. The EC3 value was calculated to be 1.5 % (w/w).

A statistically significant increase in DPM value was observed in all test item treated groups in comparison to the vehicle control group (p<0.05), in the mid and high dose group, this increase was also considered as biologically relevant. Furthermore, a statistically significant and biologically relevant increase in lymph node weight and cell count was determined in the highest dose group and the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in this group (index of 1.63).

Based on the above mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded. Nevertheless, on the basis of the present data, the test item has to be classified as a sensitiser.


Migrated from Short description of key information:
Mouse (LLNA): sensitizing (BASF SE 2011 acc. to OECD 429 and GLP)

Respiratory sensitisation

Endpoint conclusion
Additional information:
Migrated from Short description of key information:
no data available

Justification for classification or non-classification

In the LLNA propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- was demonstrated to be a skin sensitizer due to a dose dependent increase in lymphocyte proliferation and an S.I. value >3 at the highest concentration tested. The EC 3 value was calculated to be 1.5%, which is below the threshold of 2% for classification as Cat. 1A according to GHS-criteria.Though an influence of irritation on lymphocyte proliferation cannot be excluded, it is unlikely that they account for all effects observed, so that the unbiased EC3 would be above 2%. Thus, the effects justify classification as a sensitizer cat 1A according to CLP/GHS-EU and with R43 according to 67/548/EEC.

There are no data available for classification of propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- as a sensitizer of the respiratory tract.