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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 November 2015 to 18 January 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacryloamidopropyl-pentamethyl-1,3-propylene-2-ol-ammonium dichloride
EC Number:
Cas Number:
Molecular formula:
C15 H33 N3 O2 .2Cl
Methacryloamidopropyl-pentamethyl-1,3-propylene-2-ol-ammonium dichloride
Test material form:
solid - liquid: aqueous solution
Details on test material:
- State of aggregation: N/A
- Particle size distribution: N/A
- Mass median aerodynamic diameter (MMAD): N/A
- Geometric standard deviation (GSD): N/A
- Shape of particles: N/A
- Surface area of particles: N/A
- Crystal structure: N/A
- Coating: N/A
- Surface properties: N/A
- Density: 1.135
- Moisture content: N/A
- Residual solvent: N/A
- Activation: N/A
- Stabilisation: N/A
- Other:
Specific details on test material used for the study:
- Batch No.of test material: CY50561001
- Expiration date of the batch: 24/04/2016
- Purity test date: 65.4%

- Storage condition of test material: Room temperature 15-25°C, below 70 RH%
- Solubility and stability of the test substance in the solvent/vehicle: soluble in 1% aqueous Pluronic® PE9200 solution (1% Pluronic). Stability not checked

- Treatment of test material prior to testing: Formulations were prepared on weight: volume basis as % (w/v), and were considered to be stable for this short period. Purity conversion was applied in the study (for this purpose, the 65.4% purity value of the provided substance was taken into consideration).
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

In vivo test system

Test animals

other: CBA/CaOlaHsd mice
Details on test animals and environmental conditions:
- Source: Harlan Laboratories S.r.l.
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 10 weeks old (age-matched, within one week)
- Weight at study initiation:19.5 – 21.3 g
- Housing: Group caging / mice were provided with glass tunnel-tubes (Cage type: Type II. polypropylene / polycarbonate)
- Diet (e.g. ad libitum): Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” ad libitum.
- Water (e.g. ad libitum): Animals received tap water from the municipal supply from 500 mL bottle, ad libitum.
- Acclimation period: 21 days
- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian.

- Temperature (°C): 19.4-25.9°C
- Humidity (%): 22-73 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 25 November 2015 To: 01 December 2015

Study design: in vivo (LLNA)

other: Pluronic® PE9200
1% aqueous solution
No. of animals per dose:
4 animals / group
Details on study design:
- Compound solubility: DMF was selected as vehicle in the preliminary experiment. In the preliminary experiment the test material (65.4% dilution in water), and a 50% dilution of this in DMF were evaluated (2 animals/dose).
- Irritation: no signs of local irritation were observed in the preliminary test.
- Systemic toxicity: no systemic toxicity were observed in the preliminary test
- Ear thickness measurements: Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. Slightly increased ear thickness values were detected in some cases on Days 3 and/or Day 6; but the mean values were well below the irritant response.
- Erythema scores: No erythema was observed for any experimental animals in the preliminary experiment.


- Name of test method: The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.
- Criteria used to consider a positive response:
Stimulation index (SI = DPN (disintegrations per minute divided by the number of lymph nodes) value of a treated group divided by the DPN value of the negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- 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.

Based on the results of the preliminary study, the undiluted test item (i.e. 65.4%) was acceptable as top dose for the main test. Two additional groups of 4 animals each were treated at the test concentration of 50% and 25% respectively in 1% Pluronic.

Topical application:
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

1) Clinical Observations:
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed at least twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

2) Measurement of Body Weight:
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

3) Ear Thickness Measurements:
In the preliminary experiment, both ears of each mouse were observed for erythema and scored according to the guideline's criteria. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6 in the preliminary experiment. Additional quantification of the ear thickness in the preliminary experiment was also performed by ear punch weight determination on Day 6 after the euthanasia of the experimental animals.

1) 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 5 hours (± 30 minutes).

2) Removal and Preparation of Draining Auricular Lymph Nodes:
Five hours (± 30 minutes) 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.

3) 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 minutes at 4 °C. 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.

4) 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 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes 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-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (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.

DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
No statistics performed.

Results and discussion

Positive control results:
The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (1% Pluronic) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 4.8) was noted for HCA in the main experiment. This value confirmed the appropriate performance of the assay.

In vivo (LLNA)

Key result
< 3
Cellular proliferation data / Observations:
Larger / slightly larger than normal lymph nodes were observed in the positive control group, while the appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated groups (subjective judgement by analogy with observations of former experiments).

The stimulation index values were 1.7, 2.3 and 1.2 at concentrations of 65.4% (undiluted), 50% and 25% (w/v), respectively.

EC3 CALCULATION: No calculated (SI < 3 in all concentrations tested)

No mortality or signs of systemic toxicity was observed during the main test. There were no visual indications of any irritancy at the site of application.

No treatment related effect on body weight was detected in the main test. Body weight loss (>5%) was detected for one animal in the negative control group, for one animal in the undiluted (i.e. 65.4% w/v) dose group, and for one animal in the 50% (w/v) dose group. However, based on the results of other animals in those groups, these facts were considered as animal variability.

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups:

Test Group Name

Measured DPM / group


Number of lymph nodes


Stimulation Index

Background (5% (w/v) TCA)







Negative (vehicle) control (1% Pluronic)






Diquat undiluted (i.e. 65.4% w/v)






Diquat 50% (w/v) in 1% Pluronic






Diquat 25% (w/v) in 1% Pluronic






Positive control (25% (w/v) HCA in 1% Pluronic)






Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In conclusion, under the conditions of the present assay, Diquat as a 65.4% aqueous formulation and 2 dilutions, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of Diquat following dermal exposure. The study was performed with vertebrate animals as no regulatory in vitro alternative was available for classification. The minimum number of animals was used corresponding to the regulatory guidelines being followed.

Diquat was provided as a 65.4% (w/v) aqueous formulation. Based on the results of the preliminary solubility / compatibility tests and on the recommendations of the OECD Guideline No. 429, the test item was tested as supplied or formulated in N,N-Dimethylformamide (abbreviated as DMF) or 1% aqueous Pluronic® PE9200 solution (1% Pluronic). Due to the physical form sent by the Sponsor, the highest achievable test item concentration was 65.4% (w/v) (material as supplied).

In a Preliminary Irritation / Toxicity Test performed in CBA/CaOlaHsd mice, the 65.4% (supplied test item) was not irritant and was hence selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received Diquat at 65.4% (w/v) concentration (undiluted test item) or formulated in 1% Pluronic at 50 and 25% (w/v) concentrations,

- the negative control group received the vehicle (1% Pluronic),

- the positive control group received 25% (w/v) HCA (dissolved in 1% Pluronic).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No treatment related body weight loss was observed in the test item treated groups. There was no indication of any irritancy at the site of application.

The stimulation index values were 1.7, 2.3 and 1.2 at concentrations of 65.4% (supplied solution), 50% and 25% (w/v) concentrations, respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 4.8) was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, Diquat as a 65.4% aqueous formulation and 2 dilutions, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.