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Description of key information

Skin sensitisation in vivo, Local Lymph Node assay (OECD 429): not sensitising

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
Study period:
19 July - 15 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovak National Accreditation Service, Karloveska 63, 840 00 Bratislava 4, Slovak Republic
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: AnLab Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19.57 - 23.56 g
- Housing: 5 animals per cage
- Bedding: Lignocel S3/4, Lufa - ITL GmbH, Germany
- Diet: ssniff (Spezialdiäten GmbH, Germany), ad libitum, each day approximately at the same time
- Water: tap water, ad libitum
- Acclimation period: 6 days
- Indication of any skin lesions: the health condition was examined by a veterinarian before initiation of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 - 60
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To: 28 July - 15 Aug 2017
Vehicle:
dimethyl sulphoxide
Concentration:
Pre-screen test: 50 and 100%
Main study: 10, 25 and 50%
No. of animals per dose:
Pre-screen test: 2
Main study: 5 (negative and positive controls), 5 (in test groups)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test item was insoluble in the vehicle; therefore, a homogeneous suspension was obtained.
- Irritation: no signs of local irritation at the application site were observed
- Systemic toxicity: at 100% the test item caused apathy, marked loss of body weight, no intake of food and water in 1/2 animals while at 50% no changes in all monitored parameters were observed
- Ear thickness measurements: no effects on ear thickness were observed in any animal at all readings (Days 1, 3 and 6)
- Erythema scores: 0 in all animals at all observations (Day 1 - Day 6)
- Overall results: The erythema score and the lack of increase in ear thickness did not meet the criteria which are considered as signs for excessive local skin irritation (erythema score ≥ 3; ear thickness increase ≥25%).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 125I-iododeoxy uridine incorporation determined by γ-counting

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of the appropriate dilution of the test item was applied to the entire dorsal surface of each ear of each mouse. The application was repeated on days 2 and 3. Local irritation reactions as well as clinical signs of systemic toxicity were assessed daily. Body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals. On day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 2 µCi of 125I-iododeoxy uridine and 1x10E-5 M fluorodeoxy uridine was made into the tail vein of each experimental mouse. Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach). Cell suspension of lymph node cells from pooled treatment groups was prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged by 600 g at 4 °C for 10 min. The suspensions of cells were precipitated with 5% trichloroacetic acid (TCA) at 4 °C for 18 - 20 h. Pellets were centrifuged by 2000 g at 4 °C for 5 min., re-suspended in 1 mL TCA and transferred to gamma counting tubes for 125I-counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Standard deviation (S.D.) values and t-test values of body weights and ear thickness were calculated.
Positive control results:
The positive control substance (25% Hexylcinnamic aldehyde in DMSO) induced a positive reaction in the animals of the positive control group. The Stimulation Index (SI) of the pooled group was 5.69, well above the threshold of 3 indicating a positive response. In addition, the lymph node weight of control group and positive control group animals were 0.0300 g and 0.0741 g, respectively, further highlighting the positive response.
Parameter:
SI
Value:
2.3
Test group / Remarks:
10% test item in DMSO
Parameter:
SI
Value:
2.34
Test group / Remarks:
25% test item in DMSO
Parameter:
SI
Value:
2.32
Test group / Remarks:
50% test item in DMSO
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
In comparison with the control group, an increase of the pooled lymph node weights was recorded at all used concentrations. The pooled lymph node weights of treated groups were 0.0372 g, 0.0463 g and 0.0367 g for 10%, 25% and 50% concentration of tested item, respectively. The lymph node weight of the control group and the positive control group were 0.0300 g and 0.0741 g, respectively.

Incorporation of 125I-iododeoxy uridine was measured by 125I-counting with an Automatic Gamma Counter Wizard2 (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/animal. DPM were calculated according to formula:

DPM = CPM / absolute detector efficiency

The absolute detector efficiency = 66.73% for the detector used.

The DPM values for the three treated groups were 3828 (10%), 3900 (25%) and 3857 (50%).

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index (SI) was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM of the vehicle control group, yielding a mean SI. A substance is regarded as sensitiser in the LLNA test if SI ≥ 3.

The SI values for the three treated groups were 2.30 (10%), 2.34 (25%) and 2.32 (50%).

EC3 CALCULATION
The EC3 value is determined by linear interpolation of points on dose-response curve, immediately above and below of the SI value, according to the equation: EC3=c+[(3-d)/(b-d)]x(a-c)

a: higher concentration,
b: SI of higher concentration,
c: lower concentration,
d: SI of lower concentration

If all points are below the stimulation index of three, no EC3 value can be calculated. Therefore, an EC3 value was not calculated.

CLINICAL OBSERVATIONS AND BODY WEIGHTS
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs in any dose group. Body weights were measured prior to the first treatment and at scheduled sacrifice. A minor increase of the mean body weight was observed in the group treated at 10% concentration. In groups treated at 25 and 50% a minor decrease of mean body weights was observed. The increase/decrease was not statistically significant.

Table 1: Ear thickness (mm) ((2Z)-4-(octadecylamino)-4-oxo-2-butenoic acid 100%)

Mouse no.

Day 1 (pre dose)

Day 3

Day 6

1

0.17

0.18

0.18

0.17

0.18

0.19

2

0.20

0.20

0.20

0.19

0.19

0.20

Mean

0.188

0.185

0.190

S.D.

0.015

0.013

0.008

  

Table 2: Ear thickness (mm) ((2Z)-4-(octadecylamino)-4-oxo-2-butenoic acid 50%)

Mouse no.

Day 1 (pre dose)

Day 3

Day 6

1

0.21

0.20

0.21

0.20

0.21

0.21

2

0.22

0.23

0.22

0.23

0.22

0.22

Mean

0.215

0.215

0.215

S.D.

0.013

0.013

0.006

 

Table 3: Lymph node weights, DPM, SI, EC3 values

 

Lymph node weight (g)

Number of lymph nodes

DPM

SI

EC3 (%)

Control

0.0300

10

1666

-

 

 

-

Positive Control

0.0741

10

8707

5.69*

(2Z)-4-(octadecylamino)- 4-oxo2-butenoic acid

 

10%

0.0372

10

3828

2.30

25%

0.0463

10

3900

2.34

50%

0.0367

10

3857

2.32

*Calculated with corresponding control (value of 1529 DPM)

Table 4: Historical control data for positive controls

Date

Study No.

DPM - negative control (vehicle)

Acetone/Olive oil, 4:1 (v/v)              DMSO

DPM - positive control

Stimulation Index

06/2017

600386410

 

1575

12996

8.25

04/2017

600383610

984

 

8590

8.37

11/2016

6003584100

 

1040

6189

5.95

10/2016

600358130

 

469

3961

8.44

09/2016

600349510

777

 

4355

5.60

07/2016

600342910

1014

 

7601

7.49

04/2016

600333730

1093

 

6811

6.23

08/2015

600292090

 

313

3925

12.54

03/2014

6500000060250

1184.99

 

5055.63

4.27

01/2013

600170570

476.85

 

1887.81

3.96

08/2010

600079020

772.02

 

3915.76

5.07

06/2010

600071910

1067.21

 

4760.68

4.46

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising properties of the test substance were tested in a mouse Local Lymph Node Assay (LLNA), according to OECD 429 under GLP conditions (hameln, 2017c). A pre-screen test was conducted to determine the appropriate dose level. No signs of local irritation at the application site were observed, but signs of systemic toxicity (apathy, marked loss of body weight, no intake of food and water) were observed in one mouse after administration of 100% concentration of the test substance. In the main study 25 µL of the appropriate dilution (10, 25 and 50%) of the test item in DMSO was applied to the entire dorsal surface of each ear of each female CBA/CA mouse (5 mice per dose group). In comparison with the control group, an increase of the pooled lymph node weights was recorded at all applied concentrations. The pooled lymph node weights were 0.0372 g for the 10% concentration group, 0.0463 g for the 25% concentration group and 0.0367 g for the 50% concentration group. The lymph node weights of the control group and the positive control group were 0.0300 g and 0.0741 g, respectively. Cellular proliferation was evaluated by determination of the stimulation index (SI). The SI were 2.3; 2.34 and 2.32 for the test substance concentrations of 10, 25 and 100%, respectively. Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs in any dose group. Body weights were measured prior to the first treatment and at scheduled sacrifice. A minor increase of the mean body weight was observed in the group treated at 10% concentration. In groups treated at 25 and 50% a minor decrease of mean body weights was observed. The increase/decrease was not statistically significant. Under conditions of the LLNA none of the test item dilutions had a SI ≥3, accordingly the test substance was not a skin sensitiser.

 

 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Justification for selection of respiratory sensitisation endpoint: Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on skin sensitisation of the registered substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are, therefore, conclusive but not sufficient for classification. No data regarding respiratory sensitisation are available.