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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:
2018-02-07 to 2018-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
updated 06 July 2012
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Fatty acids, C14-18 and C16-18-unsatd., ammonium salts
EC Number:
271-685-3
EC Name:
Fatty acids, C14-18 and C16-18-unsatd., ammonium salts
Cas Number:
68604-33-1
Molecular formula:
C14H28O2.H3N / C16H32O2.H3N / C18H36O2.H3N / C18H34O2.H3N / C18H32O2.H3N / C18H30O2.H3N / C16H30O2.H3N .
IUPAC Name:
Fatty acids, C14-18 and C16-18-unsatd., ammonium salts
Test material form:
other: paste-like
Details on test material:
Identification: Fatty acids, C14-18 and C16-18-unsatd., ammonium salts.
Batch: 2017-30 2022-30 01.
Purity: 85.3% (contains 14.7% water).
Physical state, appearance: Yellow-brown paste.
Expiry Date: July 2022.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Test system: Mice, CBA/CaOlaHsd.
- Source: Mice, Envigo RMS B.V., Inc; Postbus 6174; 5960 AD Horst / The Netherlands.
- Number of animals for the pre-tests: 4 females (2 for each pre-test).
- Number of animals for the main study: 16 females.
- Number of animals per group: 4 females (nulliparous and non-pregnant).
- Number of test groups: 3 .
- Number of control (vehicle) groups: 1.
- Age (beginning of treatment): 1st pre-test: 10 - 11 weeks; 2nd pre-test: 11 - 12 weeks; Main study: 8 - 9 weeks.
- Body weight at study initiation: 1st pre-test: 20.7g and 19.2g; 2nd pre-test: 21.2g and 22.6g; Main study: 18.4g (#1), 16.4g(#2), 19.2g(#3), 18.1g(#4), 19.0g(#5), 18.9g(#6), 20.0g(#7), 20.0g(#8), 19.3g(#9), 18.5g(#10), 18.0g(#11), 17.7g(#12), 19.0g(#13), 17.0g(#14), 18.3g(#15) and 17.8g(#16).
- Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
- Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Housing: group.
- Bedding: granulated soft wood bedding.
- Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum.
- Water: tap water, ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C.
- relative humidity approx. 45-65%.
- Photoperiod (hrs dark / hrs light): 12h/12h (artificial light 6.00 a.m. - 6.00 p.m.).

Study design: in vivo (LLNA)

Vehicle:
other: Ethanol/water (7+3 v/v). Ethanol: Purity: ≥99.9%; Water: Sterile.
Concentration:
0% (Control Group); 2.5%(Low Dose); 5%(Mid Dose) and 10%(High Dose) [concentrations as determined in a pre-experiment].
No. of animals per dose:
4
Details on study design:
Test Item Preparation:
1. Vehicle and Dose Selection:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50% solution in ethanol/water (7+3, v/v). Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of systemic toxicity. The animals showed an erythema of the ear skin (Score 1 to 2). Additionally, the ears were visibly swollen, showed scaly skin and eschar formation. The animal treated with 50% test item concentration also showed slight erythema of the scalp and both animals had increased ear thickness and weight.
Therefore, a second pre-test was performed using test item concentrations of 5 and 10%. At the tested concentrations the animals did not show any signs of systemic toxicity. The animals showed an erythema of the ear skin (Score 1 to 2, see Appendix 1 for details). Additionally, the animal treated with 10% test item concentration showed scaly ears.
Thus, the test item in the main study was assayed at 2.5, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.
2. Test Item Preparation:
The test item was placed into an appropriate container on a tared balance and ethanol/water (7+3, v/v) was added.
The different test item concentrations were prepared individually.
The preparations were made freshly before each dosing occasion.

Test Item Administration:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in ethanol/water (7+3, v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine:
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 21.2 µCi of 3H-methyl thymidine (equivalent to 84.8 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Preparation of Single Cell Suspensions:
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR):
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Observations:
Clinical Observations: All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
Determination of Ear Thickness: In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
Determination of ear weights: In the pre-test, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.
Determination of Body Weights: The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Interpretation of raw data:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
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.

General Calculations:
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2017.
Test item concentration 10%: S.I. 2.77; Test item concentration 25%: S.I. 5.70; EC3 = 11.2% (w/v).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
Test item concentration 0% (Group 1)
Parameter:
SI
Value:
0.99
Test group / Remarks:
Test item concentration 2.5% (Group 2)
Parameter:
SI
Value:
0.8
Test group / Remarks:
Test item concentration 5% (Group 3)
Parameter:
SI
Value:
1.21
Test group / Remarks:
Test item concentration 10% (Group 4)
Cellular proliferation data / Observations:
Calculation of the EC3 value:
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Viability / Mortality:
No deaths occurred during the study period.

Clinical Signs:
No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. From day 1 to 4, the animals treated with a test item concentration of 5 and 10% showed an erythema of the ear skin (Score 1, for details see Appendix 3). Animals treated with 2.5% test item concentration did not show any signs of local skin irritation.

Body Weights:
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Calculation and Results of Individual Data

Test item
concentration
% 
Group Measurement
DPM
Calculation Result
DPM-BG a) number of
lymph nodes
DPM per
lymph node b)
S.I.
--- BG I 13 --- --- --- ---
--- BG II 16 --- --- --- ---
0 1 4759 4744.5 8 593.1 1
2.5 2 4735 4720.5 8 590.1 0.99
5 3 3808 3793.5 8 474.2 0.8
10 4 5764 5749.5 8 718.7 1.21

Vehicle: ethanol/water (7+3, v/v)

1 = Control Group; 2-4  = Test Group.

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

Pre-Test 1: Body Weights:

Animal
No.
Concentration
%
Body Weight (g)
prior
1st Application
prior
to Sacrifice (Day 6)
Difference
Day 1 to Day 6
Difference
%
1 25 20.7 22.1 1.4 6.8
2 50 19.2 19.4 0.2 1.0

Pre-Test 1: Ear Thickness:

Animal
No.
Conc.
%
Ear Thickness
prior to 1st Application
(µm)
prior to 3rd Application
(µm)
prior to Necropsy
(µm)
Right
Ear
Left
Ear
Mean Right
Ear
Left
Ear
Mean Right
Ear
Left
Ear
Mean
1 25 240 225 232.5 275 270 272.5 390 410 400
2 50 240 230 235 305 290 297.5 410 390 400

Animal
No.
Difference
Day 1 to Day 3 (µm)
Ear Swelling
Day 3 (%)
Difference
Day 1 to Day 6 (µm)
Ear Swelling
Day 6 (%)
1 40 17.2 167.5 70
2 62.5 26.6 165 70.2

Pre-Test 1: Ear Weights:

Animal
No.
Concentration
%
Ear Weights after Necropsy
(mg per animal)
% Increase Compared
to Vehicle Values
1 25 41 56.5
2 50 41 56.5

Mean of historical controls (ethanol/water (7+3, v/v)): 26.2 mg/ animal)

Pre-Test 1: Ear Erythema:

Animal
No.
Score
within 1 h after
1. appl.
24 h
after
1. appl.
within 1 h after
2. appl.
24 h
after
2. appl.
within 1 h after
3. appl.
24 h
after
3. appl.
Day 5 Day 6
1 / 1 1 1 2 /*°#
2 1 1 2 2*°+ 2*°+   /*°#

*visible swelling of ears   °scaly ears   +slight erythema of scalp   #strong eschar formation

Score: / = No visible erythema; 1 = Very slight erythema; 2 = Well defined erythema; 3 = Moderate to severe erythema; 4 = Severe erythema to formation of eschar which prevents grading of erythema.

Pre-Test 2: Body Weights:

Animal
No.
Concentration
%
Body Weight (g)
prior
1st Application
prior
to Sacrifice (Day 6)
Difference
Day 1 to Day 6
Difference
%
1 5 21.2 21.3 0.1 0.5
2 10 22.6 22 -0.6 -2.7

Pre-Test 2: Ear Thickness:

Animal
No.
Conc.
%
Ear Thickness
prior to 1st Application
(µm)
prior to 3rd Application
(µm)
prior to Necropsy
(µm)
Right
Ear
Left
Ear
Mean Right
Ear
Left
Ear
Mean Right
Ear
Left
Ear
Mean
1 5 230 240 235.0 240 240 240.0 240 245 242.5
2 10 230 230 230.0 240 235 237.5 235 235 235.0

Animal
No.
Difference
Day 1 to Day 3 (µm)
Ear Swelling
Day 3 (%)
Difference
Day 1 to Day 6 (µm)
Ear Swelling
Day 6 (%)
1 5 2.1 7.5 3.2
2 7.5 3.3 5 2.2

Pre-Test 2: Ear Weights:

Animal
No.
Concentration
%
Ear Weights after Necropsy
(mg per animal)
% Increase Compared
to Vehicle Values
1 5 27.71 5.8
2 10 29.48 12.5

Mean of historical controls (ethanol/water (7+3, v/v)): 26.2 mg/ animal)

Pre-Test 2: Ear Erythema:

Animal
No.
Score
within 1 h after
1. appl.
24 h
after
1. appl.
within 1 h after
2. appl.
24 h
after
2. appl.
within 1 h after
3. appl.
24 h
after
3. appl.
Day 5 Day 6
1 1 / 1 1 2 1 / /
2 1 / 1 1* 2 1* /* /*

*scaly ears

Score: / = No visible erythema; 1 = Very slight erythema; 2 = Well defined erythema; 3 = Moderate to severe erythema; 4 = Severe erythema to formation of eschar which prevents grading of erythema.

Body Weights in the Main Experiment: Individual animal weights at the start of the experiment:

Dose Group Animal
no.
Initial
Weight (g)
Mean  SD Range
Negative Control
EtOH 70%
1 18.4 18.0 +/- 1.2 19.2 - 16.4
2 16.4
3 19.2
4 18.1
Test item
Dose: 2,5%
5 19.0 19.5 +/- 0.6 20.0 - 18.9
6 18.9
7 20.0
8 20.0
Test item
Dose: 5%
9 19.3 18.4 +/- 0.7 19.3 - 17.7
10 18.5
11 18.0
12 17.7
Test item
Dose: 10%
13 19.0 18.0 +/- 0.8 19.0 - 17.0
14 17.0
15 18.3
16 17.8
Summary 18.5 +/- 1.0 16.4 - 20.0

Body Weights in the Main Experiment: Individual animal weights prior administration of 3H-methyl thymidine:

Dose Group Animal
no.
Initial
Weight (g)
Mean  SD Range
Negative Control
EtOH 70%
1 18.4 17.8 +/- 1.0 18.6 - 16.4
2 16.4
3 18.6
4 17.9
Test item
Dose: 2,5%
5 18.8 19.5 +/- 0.9 20.7 - 18.8
6 18.9
7 20.7
8 19.6
Test item
Dose: 5%
9 19.0 18.5 +/- 0.4 19.0 - 18.2
10 18.3
11 18.2
12 18.4
Test item
Dose: 10%
13 19.0 18.4 +/- 0.6 19.0 - 17.6
14 17.6
15 18.3
16 18.6
Summary 18.5 +/- 0.9 16.4 - 20.7

Observations in the Main Experiment: Ear Erythema:

Animal
No.
Score
Within 1 h prior to
1. appl. (day 1)
Within 1 h after
1. appl. (day 1)
24 h after
1. appl. (day 2)
Within 1 h after
2. appl. (day 2)
24 h after
2. appl. (day 3)
Within 1 h after
3. appl. (day 3)
24 h after
3. appl. (day 4)
Day 5 Day 6
1 / / / / / / / / /
2 / / / / / / / / /
3 / / / / / / / / /
4 / / / / / / / / /
5 / / / / / / / / /
6 / / / / / / / / /
7 / / / / / / / / /
8 / / / / / / / / /
9 / 1 / / / 1 / / /
10 / 1 / / / 1 / / /
11 / 1 / / / 1 / / /
12 / 1 / / / 1 / / /
13 / 1 1 1 1 1 1 / /
14 / 1 1 1 1 1 1 / /
15 / 1 1 1 1 1 1 / /
16 / 1 1 1 1 1 1 / /

Score: / = No visible erythema; 1 = Very slight erythema; 2 = Well defined erythema; 3 = Moderate to severe erythema; 4 = Severe erythema to formation of eschar which prevents grading of erythema.

Results of the GLP Positive Control:

Experiment performed in October 2017 (Envigo study number 1868000). Positive control substance: α-Hexylcinnamaldehyde:

Test item
concentration
% 
Group Measurement
DPM
Calculation Result
DPM-BG a) number of
lymph nodes
DPM per
lymph node b)
S.I.
--- BG I 14 --- --- --- ---
--- BG II 20 --- --- --- ---
0 1 8827 8810 8 1101.2 1.00
5 2 9403 9386 8 1173.2 1.07
10 3 24442 24425 8 3053.1 2.77
25 4 50225 50208 8 6276 5.70

Vehicle: acetone:olive oil (4+1, v/v)

1 = Control Group; 2-4  = Test Group.

a)  = The value was taken from the figure BG

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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Fatty acids, C14-18 and C16-18-unsatd., ammonium salts was not a skin sensitiser under the test conditions of this study.
The substance "Fatty acids, C14-18 and C16-18-unsatd., ammonium salts" is not considered to be classified for skin sensitisation.
Executive summary:

In the study the test item Fatty acids, C14-18 and C16-18-unsatd., ammonium salts formulated in ethanol/water (7+3, v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 1 to 4, the animals treated witha test item concentration of5 and 10% showed an erythema of the ear skin (Score 1, for details see Appendix 3). Animals treated with 2.5% test item concentration did not show any signs of local skin irritation.

In this study Stimulation Indices (S.I.) of 0.99, 0.80, and 1.21 were determined with the test item at concentrations of 2.5, 5, and 10% in ethanol/water (7+3, v/v), respectively.

The test item Fatty acids, C14-18 and C16-18-unsatd., ammonium salts was not a skin sensitiser under the test conditions of this study.