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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:
13 March 2018 - 16 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2012
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx 9 weeks old
- Weight at study initiation: 19.5 to 24.8 g
- Housing: 5 animals together in polycarbonate cages containing sterilized sawdust as bedding material
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no; animals were checked before initiation of dosing and any animals considered unsuitable for use in the study were replaced.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24 (actual: 22 - 23)
- Humidity (%): 40-70 (actual: 41-51)
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 March 2018 - 16 April 2018

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Based on the results of a pre-screen test, the test item was tested in the main test in the following concentrations: 0%, 1%, 2% and 5% (w/w).
No. of animals per dose:
5 animals per treatment group
Details on study design:
PRE-SCREEN TESTS:
- Concentrations: inititally 50% and 100% and additionally 25%, 10%, 5% and 2%.
- Systemic toxicity: at 100%, 50%, 25% and 10%, variation in ear thickness were >25% from day 1 pre-dose values and/or clinical sign of systemic toxicity were noted. At 5% and 2%, no signs of systemic toxicity were noted and no to very slight irritation were observed. Therefore the 5% concentrations was selected as the highest concentration for the main study.

MAIN STUDY: Three groups of five animals were treated with one test item concentration per group (1, 2 and 5%). One group of five animals was treated with the vehicle.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: if the results indicate a SI = 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
- Induction (days 1, 2 and 3): both ears were topically treated with 25 µL test item per ear.
- Excision of the nodes (day 6): each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 µCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection and the draining lymph node of each ear was excised.
- Tissue processing for radioactivity (day 6): Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.
- Radioactivity measurements (day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

OBSERVATIONS:
- Mortality: twice daily
- Clinical observations: once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
- Bodyweight: individually on day 1 (predose) and 6 (prior to necropsy).

ANALYSIS:
A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.6
Test group / Remarks:
1% concentration
Parameter:
SI
Value:
1.6
Test group / Remarks:
2% concentration
Parameter:
SI
Value:
3
Test group / Remarks:
5% concentration
Key result
Parameter:
EC3
Value:
5
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA : see table 1
The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 1%, two animals treated at 2% and four animals treated at 5%, which were considered to be enlarged.

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 623, 590 and 1118 DPM, respectively. The mean DPM/animal value for the vehicle control group was 378 DPM. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.6, 1.6 and 3.0, respectively.

EC3 CALCULATION
The data showed a dose-response and an EC3 value of 5% was determined.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

OTHER
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Any other information on results incl. tables

Table 1 Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

group

TS1

(%)

animal

Size nodes2

DPM3/ animal

mean

DPM ± SEM4

mean

SI ± SEM

left

right

 

 

 

 

 

 

 

 

1

0

1

n

n

94

378

±

133

1.0

±

0.4

 

 

2

n

n

141

 

 

3

n

n

316

 

 

4

n

n

519

 

 

5

n

n

819

 

 

 

 

 

 

 

 

 

 

 

 

2

1

6

n

n

299

623

±

95

1.6

±

0.3

 

 

7

n

+

707

 

 

8

n

n

875

 

 

9

n

n

571

 

 

10

n

n

661

 

 

 

 

 

 

 

 

 

 

 

 

3

2

11

n

n

371

590

±

75

1.6

±

0.2

 

 

12

+

+

2786(5)

 

 

13

n

n

615

 

 

14

+

n

695

 

 

15

n

n

680

 

 

 

 

 

 

 

 

 

 

 

 

4

5

16

+

+

677

1118

±

192

3.0

±

0.5

 

 

17

+

+

657

 

 

18

+

+

1318

 

 

19

n

n

1616

 

 

20

n

+

1321

 

 

 

 

 

 

 

 

1  TS = test item (% w/w).

2  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

3    DPM= Disintegrations per minute.

4    SEM = Standard Error of the Mean.

5    Value not used for interpretation as this value was considered an outlier based on the Dixon’s Q-test. There

   were no other values identified as outliers within this study.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The results of an LLNA study, performed according to OECD guideline 429 and GLP principles, indicate that Tricyclodecanemonomethylol acrylate could elicit a SI = 3. An EC3 value of 5% was determined. Based on these results, the test substance is classified as Category 1B for skin sensitising properties according to GHS and Regulation (EC) 1272/2008.
Executive summary:

The objective of this study was to evaluate whether Tricyclodecanemonomethylol acrylate induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this study plan (TG OECD 429).

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 100%, 50%, 25% and 10% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At a 5% and 2% test item concentration, no signs of systemic toxicity were noted and no to very slight irritation were observed and therefore the 5% concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 1, 2 or 5% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 No irritation was observed in any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 1%, two animals treated at 2% and four animals treated at 5%, which were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 623, 590 and 1118 DPM, respectively. The mean DPM/animal value for the vehicle control group was 378 DPM. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.6, 1.6 and 3.0, respectively. These results indicate that the test item could elicit a SI = 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 5% was determined.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

The results of an LLNA study, performed according to OECD guideline 429 and GLP principles, indicate that Tricyclodecanemonomethylol acrylate could elicit a SI = 3. An EC3 value of 5% was determined. Based on these results, the test substance is classified as Category 1B for skin sensitising properties according to GHS and Regulation (EC) 1272/2008.