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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:
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
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
liquid: viscous

In vivo test system

Test animals

Details on test animals and environmental conditions:
Test System : Mice
Species (strain) : Mus musculus (CBA/J)
Animal Source : Animal Breeding Facility, Jai Research Foundation
Number of Animals Used : 12 (2 mice/group) for screening study
and 25 (5 mice/group) for main study
Sex : Females (nulliparous and non- pregnant)
Initial Body Weight (g)
On Day 1 (Main Study) : Minimum: 18.2, Maximum: 23.2
Age (Main Study) : 8 to 10 weeks old at the initiation of treatment

Mice were received into the experimental procedure room after veterinary examination for health condition and allowed to acclimatise to the laboratory conditions for a period of 6 days prior to commencement of dosing.

Caging : Mice were housed individually in solid floor polypropylene (size: approximately 290 mm x 220 mm x 140 mm) solid bottom cages which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). These cages had stainless steel top grills through which pellet feed and drinking water were provided; steam sterilized corn cob bedding was used and changed along with the cage at least twice a week. On test day 6, animals were housed in metabolic cages.
Water Bottle : Each cage was supplied with a polypropylene water bottle (capacity 300 mL) with a stainless steel nozzle.
Enrichment : Mice were provided with tunnels in each cage. Rack unit was rotated once in a week.
Room Sanitation : Each day, floor of experimental room was swept and all work tops and floor were mopped with a disinfectant solution (Dettol 2.5%).
This study complied with all applicable sections of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines and Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011).
The quality of feed and water is regularly monitored at Jai Research Foundation ; copies of the relevant certificates of analysis are kept in the study file. There were no known contaminants in the feed and water at levels that would have interfered with the experimental results obtained
Animal Room : BMR 30 [On test day 6, all animals were shifted to Room N° 414]
Temperature Range : 20 to 23 °C
Relative Humidity Range : 49 to 58%
Photoperiod : The photoperiod was 12 h artificial light and 12 h darkness, light hours being 06:00 – 18:00 h (photoperiod maintained through automatic timer).
Air Changes Rate : 16 air changes/hour
After acclimatisation animals were randomized into different groups using in-house developed, validated computer software.

The following activities were performed daily during the experimental period in BMR 30:
- animal observations
- providing feed
- floor sweeping and mopping
- recording room temperature and relative humidity

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
2, 20, 50%
No. of animals per dose:
Details on study design:
An irritation screening test was conducted to identify the highest dose that does not cause irritation or overt systemic toxicity. The study consisted of six groups of female mice (2 mice per group) that were treated with 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone at concentrations of 2.5%, 5%, 10%, 25%, 50% or 75% (w/v) in AOO (25 µL/ear) for three consecutive days (days 1, 2 and 3). The dose was gently spread evenly over the dorsal surface of the ear using the tip of the pipette. No treatment was made on days 4 and 5. Clinical observations were recorded daily during the experiment. Ear thickness of each animal was measured (apex of the pinna) using a micrometer (digital micrometer; serial N° 293.821) on days 1 (pre-dose), 3 and 6. Increases in ear swelling on days 3 and 6 were calculated for each animal relative to the thickness measurement taken on day 1.
Body weight was recorded on days 1 and 6 (prior to termination). The ears were evaluated daily for erythema according to the scoring system in the table below. Ear swelling and erythema were used to identify concentrations of the test item that produced irritation to the ears of mice. Previous studies have determined that chemicals with irritancy potential, but without sensitization potential, can produce a detectable proliferative response when administered at concentrations that induce excessive local irritation (Kimber et al., 1994 and ICCVAM, 1999). Excessive local skin irritation is indicated by an erythema score ≥3 on any day of measurement and/or an increase in ear thickness of ≥25% on day 3 or 6 relative to the measurement on day 1 (OECD Test Guideline 429, 24 July 2010). These data along with expert judgment are used in the selection of the final doses to be used in the LLNA.

The % ear swelling was calculated for each ear using the following equation:
% Ear swelling = (B – A) /A x 100% where:

A = ear thickness measurement on day 1 (mm x 10-2)
B = ear thickness measurement on day 3 or 6 (mm x 10-2)

Irritation to mouse ears is only relevant in the context of the LLNA and should not be interpreted as an indication of irritation potential in humans.

Prior to treatment, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Thirty healthy naive female mice without pre-existing ear irritation were selected and distributed into treatment groups (5 mice per group).

Three treatment groups (G22 to G24) were treated topically once daily for three consecutive days (days 1, 2 and 3) on the dorsal surface of both ears (25 L/ear) using a calibrated micropipette with 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone at concentrations of 2%, 20% or 50% (w/v) in AOO, respectively. The dose was gently spread evenly over the dorsal surface of the ear using the tip of the pipette.

Mice from the vehicle control group (G21) and positive control group (G25) were handled in the same manner but received 25 L/ear of vehicle (AOO) and or 25% a-Hexylcinnamaldehyde (v/v) in vehicle (AOO), respectively. No treatment was applied on days 4 and 5 for any group. All dosage preparations were freshly prepared on the day of application.

On day 6 (approximately 72 h after the last treatment), all mice from the vehicle control, positive control and all treatment groups were intravenously injected via the tail vein with 250 µL of sterile phosphate buffered saline (PBS) containing approximately 20 ± 1 µCi (740 KBq) of 3H-methyl thymidine (Lot N° 07/17).

Body weights of individual mice were recorded on the first day of dosing (day 1) and prior to administration of 3H-methyl thymidine (day 6). Group mean body weights were calculated.

Individual animals were observed carefully daily for clinical signs, local irritation at the site of application and systemic toxicity. All the observations were systematically recorded for individual mice. Local irritation responses were made as per criteria given in OECD 429, 2010; Section 22

On day 6, approximately 5 hours post-administration of 3H-methyl thymidine, all mice from the vehicle control, positive control and all treatment groups were euthanised by CO2 asphyxiation. The draining auricular lymph nodes from each individual mouse were excised and pooled in phosphate buffered saline.

The draining auricular lymph nodes of individual mice were collected in separate petri dishes containing phosphate buffered saline (PBS). A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregation through 200 to 210 µm-mesh stainless steel gauze with the plunger of the syringe and collected in a petri dish. The gauze was washed with PBS into the petri dish and a single cell suspension was transferred into a 15 mL graduated centrifuge tube. The single cell suspension was finally made up to 10 mL with PBS used to rinse the petri dish. The cell suspension was centrifuged approximately at 190 to 200 gn for 10 minutes in a centrifuge at 4 °C.

After centrifugation, the supernatant was removed by aspiration using a micropipette leaving 1 – 2 mL of supernatant above each pellet. Each pellet was gently agitated before making up to 10 mL with PBS; this procedure was repeated twice. After the final wash, the supernatant was removed leaving a minimal volume (approximate 0.5 mL) of supernatant above each pellet.

Each pellet was agitated before re-suspending with 3 mL of 5% trichloroacetic acid (TCA) and kept for precipitation of macromolecules in the refrigerator for approximately 18 hours. After incubation with 5% TCA at 4  1 °C, each precipitate was recovered by centrifugation (190 to 200 gn) for 10 minutes and the supernatant was removed.

The precipitate was re-suspended in 1 mL of 5% TCA. Each precipitate was transferred to a scintillation vial with 10 mL of scintillation fluid (Hionic flour) and thoroughly mixed. The vials were loaded into a β–scintillation counter and after minimum 30 minutes, 3H-TdR incorporation was measured. Background 3H-TdR level was measured into 1 mL aliquots of 5% TCA.

Incorporation of 3H-methyl thymidine was measured by β-scintillation counting as disintegrations per minute (DPM) for each mouse and expressed as DPM/mouse and DPM/group. The total radioactivity of each sample was counted using LSA (Liquid Scintillation Analyser) after 30 minutes of mixing with scintillation fluid and required quench corrections were made.

The quench curve was established using extended quench standards (PerkinElmer, average activity assay: 278700 dpm/std. ± 3%) of DPM β source. Computer constructed quench curve was derived from the above commercially available series of scaled standards which automatically converts Counts Per Minute (CPM) to DPM

LSA : PerkinElmer (TRI-CARB®3100 TR )
Detector : High Performance Photo Multiplier Tube (PMT)
Method : Conventional DPM
Counting Time : 10 minutes (2 sigma value of counting reaction ≈ 0.5%)
Counting Region : 0 – 18.6 keV
SNC DPM (standard) : 267700 DPM (3H)

The proliferate response of lymph nodes from each mouse was expressed as the number of radioactive DPM/ per mouse, calculated by subtracting out background DPM (measured in 1 mL of 5% TCA aliquot).

Stimulation Index (SI) = mean DPM of test group divided by mean DPM of solvent/vehicle control group

The DPM/mouse, along with an appropriate measure of inter-animal variability (i.e., mean ± standard deviation), were calculated for each test group and vehicle and positive control groups. Final results were expressed as the (SI) which is calculated as a ratio of the mean DPM of test group divided by mean DPM of vehicle control group. Any test item that produces a SI > 3 in the LLNA is considered “positive” for dermal sensitization potential (Kimber et al., 1994).

While a SI > 3 was originally developed empirically, a robust statistical evaluation indicated that it is an acceptable practical value for hazard identification (Basketter et al., 1999a). Furthermore, by determining EC3 values (estimated concentration resulting in a 3-fold SI), one can compare relative sensitization potency of chemicals and/or formulations (Basketter et al., 1999b). While a test material that produced a SI of > 3 in the LLNA should be considered “positive” for contact sensitization (Kimber et al., 1994), recent opinions have suggested circumstances in which the LLNA result and sensitization potential should be further considered in the context of additional scientific judgment (Ryan et al., 2000; Basketter et al., 1998; Basketter et al., 2006). Based on the EC3 values derived from the LLNA, it has been proposed that contact allergens can be categorized as weak (> 10% - < 100%), moderate (> 1% - < 10%), strong (> 0.1% - < 1%), or extreme (< 0.1%) (ECETOC, 2003).

The test item is regarded as a skin sensitizer when the SI for a dose group is  3 together with consideration of a dose-response relationship.

EC3 value (theoretical concentration resulting in a SI value of 3) is determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold (Basketter et al., 1999). The equation used for calculation of EC3 was:

EC3 = c + [(3 - d)/(b - d)] x (a - c)

Where, a = the lowest concentration giving stimulation index > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and pair-wise comparison made between the treatment and the vehicle control group. All the parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, Student's t-test was performed to calculate significance. As assumption of normality is not justifiable at either 1% or 5% level of significance, a logarithmic normality test was applied according to OECD guidance document number 19, to perform parametric test, log transformation applied to control and positive control data.

Results and discussion

Positive control results:
The SI of 10.11 obtained for the concurrent positive control, -Hexylcinnamaldehyde, showed greater than a three-fold increase over the control value indicating a clear positive response for this known weak sensitizer that confirmed the reliability of this test procedure.

In vivo (LLNA)

Resultsopen allclose all
Key result
Test group / Remarks:
2% dose group
Test group / Remarks:
20% dose group
Test group / Remarks:
50% dose group

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Based on the results of this study, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone is positive (moderate sensitiser) for dermal sensitization potential in the LLNA. Due to the EC3 value being >2%, it is considered as a moderate to weak sensitiser and requires classification as catgory 1B for Skin sensitising potential.