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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:
From October 21, 2015 to October 27, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
PARAD substance 139
Specific details on test material used for the study:
- Name of test material (as cited in study report): PARAD Substance 139.
- Chemical name: Reaction product of 2-Hydroxyethyl methacrylate (HEMA) and phosphor pentoxide (P2O5).
- Batch No.: JBLB0008S.
- CAS No.: 1187441-10-6.
- Purity: 100%.
- Appearance: Clear colourless liquid.
- Manufacture date: February, 2014.
- Expiry date: February, 2016.
- Storage condition: Controlled room temperature (15 – 25ºC, below 70 RH%), protected from light.
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety. Irritant, causes serious eye damage.

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 S.r.l., San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy.
- Age at study initiation: 11 weeks old.
- Weight at study initiation: 19.1 - 20.4 g.
- Housing/ Enrichment: Group caging / mice were provided with glass tunnel-tubes.
- Cage type: Type II polypropylene / polycarbonate.
- Bedding: Lignocel 3/4-S Hygienic animal bedding.
- Source for bedding: J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) .
- Diet: ad libitum.
- Supplier of diet: ssniff spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany).
- Water: ad libitum.
- Acclimation period: 21 d.

ENVIRONMENTAL CONDITIONS
- Temperature: 17.9 – 25.9°C
- Humidity: 30 - 87%
- Air changes: 15 - 20 air exchanges per h
- Photoperiod: 12 h daily, from 6.00 a.m. to 6.00 p.m.
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Preliminary test- 25 and 50% in DMF
Main test- 0, 10, 25 and 50% in DMF
No. of animals per dose:
Preliminary test- 2 animals/dose
Main test- 4 animals/dose
Details on study design:
PRELIMINARY STUDY
A preliminary study was performed with the test substance concentrations 25 and 50% (w/v) in DMF. It was conducted in a similar experimental manner to the main study, but terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 h after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

MAIN STUDY
Based on results in preliminary test, 50% (w/v) dose was selected as top dose for the main test. The experimental groups and dose levels for the main experiment were:
1. Negative (vehicle) control (DMF)
2. 10% of test substance
3. 25% of test substance
4. 50% of test substance
5. Positive control (25% alpha-Hexylcinnamaldehyde solution in DMF)

Topical application: 25 µL of the appropriate formulation was applied using a pipette on the dorsal surface of each ear on Day 1, 2 and 3. No treatment was given on Day 4, 5 and 6.

Injection of tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5h (±30 min).

Removal and Preparation of Draining Auricular Lymph Nodes: 5h (±30 min) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate petri dishes containing a small amount (1 - 2 mL) of PBS to keep the nodes wet before processing.

Preparation of single cell suspension of lymph node cells: A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 min at 4ºC. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes

Determination of incorporated 3HTdR: After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 h) incubation at 2 - 8ºC, precipitates were centrifuged (approximately 190 x g for 10 min at 4ºC), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10 min measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per min (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.





Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A significant lymphoproliferative response (stimulation index value of 7.3) was noted for the positive control chemical, this result confirmed the validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
2.8
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
3.7
Test group / Remarks:
50%
Key result
Parameter:
EC3
Value:
30.6

Any other information on results incl. tables

PRELIMINARY IRRITATION/TOXICITY TEST:

No mortality or signs of systemic toxicity were observed. Slightly rigid ears were observed in the 50% (w/v) dose group on Day 4. No marked body weight loss (>5%) was detected on the mean body weight values of the groups; however one animal showed significant loss of body weight (5.1%) in the 50% (w/v) dose group.

MAIN TEST:

Clinical observation: No mortality or signs of systemic toxicity were observed during the study.

Body weight measurement: No treatment related effects were observed on the body weight changes of the experimental animals.

Table1: DPM, DPN and Stimulation Index values for all groups

Test Group Name

Measured DPM / group

DPM (Disintegration per minute)

Number
of lymph nodes

DPN (DPM/No. of lymph nodes)

Stimulation Index

Background

36

-

-

-

-

(5% (w/v) TCA)

32

Negative (vehicle) control (DMF)

1864

1830.0

8

228.8

1.0

50% (w/v) in DMF

6733

6699.0

8

837.4

3.7

25% (w/v) in DMF

5128

5094.0

8

636.8

2.8

10% (w/v) in DMF

3049

3015.0

8

376.9

1.6

Positive control

(25% (w/v) HCA
in DMF)

13377

13343.0

8

1667.9

7.3

 

Applicant's summary and conclusion

Interpretation of results:
other: category 1B
Remarks:
as per CLP criteria
Conclusions:
Under the study conditions, the test substance was shown to have sensitisation potential (sensitizer) in a local lymph node assay.
Executive summary:

The skin sensitization potential of the test substance was evaluated in a mouse local lymph node assay, conducted according to OECD Guideline 429 and EU Method B42, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a preliminary irritation test. In the main study, animals were treated topically with 10, 25 and 50% w/v of test substance on Days 1, 2 and 3. Negative control animals were similarly treated with vehicle alone (N,N-dimethylformamide (DMF)) and positive control animals received 25% alpha-hexylcinnamaldehyde. On Day 6, tritiated thymidine (3HTdR) was injected intravenously. 5h (±30 min) after intravenous injection, the mice were euthanized by asphyxiation and draining auricular lymph nodes were excised. Single cell suspension of lymph node cells was prepared. 3HTdR incorporation was measured by β-scintillation counter. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. The EC3 value of the test substance (EC3 means the effective chemical concentration required for SI=3) was calculated by linear interpolation. No mortality or signs of systemic toxicity were observed during the study. No treatment related effects were observed on the body weight changes of the experimental animals. DPM for the experimental groups treated with test substance concentrations 10, 25 and 50% were 3,015, 5,094 and 6,699, respectively. The DPM values observed for the vehicle and positive control substance in this experiment were within the general historical control range. The stimulation index values determined were 1.6, 2.8 and 3.7 at concentrations of 10, 25 and 50% (w/v), respectively. These results show that the test substance at 50% concentration elicits a SI ≥3. The calculated EC3 value of test substance was 30.6% (w/v). Under the study conditions, the substance was shown to have sensitisation potential (sensitizer) in the local lymph node assay. (Váliczkó É, 2015).