Registration Dossier

Administrative data

Description of key information

IN vitro assessment of keratinocyte activation, DPRA and a LLNA

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
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


Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2, 20, 50%
No. of animals per dose:
5
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)
Statistics:
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.
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.
Key result
Parameter:
EC3
Value:
8.56
Parameter:
SI
Value:
2.02
Test group / Remarks:
2% dose group
Parameter:
SI
Value:
4.71
Test group / Remarks:
20% dose group
Parameter:
SI
Value:
7.22
Test group / Remarks:
50% dose group
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
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.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on study design:
Cell line used: HaCaT cells
obtained from Givaudan and maintained following protocols outlined in the supplier’s standard operating procedures and publications (Emter et al., 2010; Natsch et al., 2011). To determine sensitization potential, keratinocytes were incubated with each test chemical for about 48 hours at approximately 37ºC. At the end of incubation, luciferase induction and cell viability was determined.

Media:
The maintenance medium for the HaCaT cell line was prepared by supplementing Dulbecco’s Modified Eagle Medium (D-MEM) media (Gibco) with 9.1% fetal bovine serum (FBS) and either with (DMEM9.1(+)) or without (DMEM9.1(-)) geneticin (antibiotic; final concentration 500 µg/ml). Treatment medium consisted of D-MEM with 1% FBS and no geneticin (DMEM1(-)). All medium prepared was stored at approximately 4°C and used within 28 days.

Preparation of the controls and treatment solutions:
DMSO was used as the solvent for the test material and the control treatment. Cinnamic aldehyde (CA, CAS # 104-55-2) was used as the positive control. CA was prepared at a concentration of 6.4 mM in DMSO and further diluted to 64 µM, 32 µM, 16 µM, 8 µM, and 4 µM in culture medium. Stock solutions of the test material were prepared fresh in DMSO, at an initial concentration of 200 mM. All stock solutions were further serial diluted in DMSO to obtain a “100X master plate” consisting of each test material at twelve consecutive two-fold dilutions (ranging from 0.098 to 200 mM). These stocks were further diluted in the assay procedure as outlined below to result in the testing of a concentration range of 0.98 to 2000 µM in the final assay.

Luciferase and Cytotoxicity assays:
Frozen HaCaT cells (approximately -150ºC) were thawed in a water bath at approximately 37ºC, resuspended in DMEM9.1(-), and were pelleted by centrifugation at 125 g for 5 minutes at room temperature. The cell pellet was resuspended in DMEM9.1(-), seeded in a flask, and maintained at about 37oC with 5% CO2. After reaching 80-90% confluency, the HaCaT cells were washed twice with Dulbecco’s Phosphate-Buffered Saline (DPBS), trypsinized, and incubated at approximately 37ºC for about 7 minutes. Detached cells were resuspended in DMEM9.1(-) and centrifuged at 125 g for 5 minutes. The resulting pellet was resuspended and hereafter maintained in DMEM9.1(+).
Cell Seeding For Testing
• Cells at 80-90% confluency were washed twice with DPBS, harvested as described above, re-suspended in DMEM9.1(-), and the cell density was adjusted to approximately 80,000 cells/ml.
• 125 µl of the cell-suspension was distributed to each well in a 96-well plate (approximately10,000 cells/well).
• Each 96 well plate consisted of EPON™ Resin CS-337 at twelve different concentrations, six negative control wells containing 1% DMSO, five wells containing the positive control CA at five different concentrations, and one well which is blank containing no cells (one well each/plate).
• In each experiment, three parallel plates for luciferase assay (solid white plate) and two plates for cytotoxicity assessment (transparent plate) were prepared as above.
• The plated cells were grown for about 24 h at approximately 37ºC and 5% CO2.

Treatment Regimen
• Following the ~24 h incubation, the medium was aspirated and replaced with 150 µl of DMEM1(-).
• The 100X stock master plate was diluted 25-fold (10 µl of test chemical solution from master plate + 240 µl of DMEM1(-)) in a fresh 96 well plate (4X master plate).
• 50 µl of solution from the resulting 4X master plate was transferred to each replicate plate already containing the keratinocytes and 150 µl of DMEM1(-) (ranging from 0.98 to 2000 µM in culture medium).
• All plates were covered with sealing tape (STR-SEAL-PLT, EXCEL Sci, Omaha, Nebraska) and incubated at approximately 37ºC for an additional ~48 h.
Luciferase Measurements
• At the end of the 48 h incubation, the supernatant from the 96 well plates was aspirated, washed once with DPBS and cells in each well was incubated with 20 µl of passive lysis buffer (Promega Corp., Madison, Wisconsin) on an orbital shaker at room temperature for
20 min.
• The plates with the cell lysate were read (relative luminescence units; RLU) in the luminometer using the following program:
i. 50 µl of the luciferase substrate was added to each well
ii. waited for 1 second and integrated the luciferase activity for 2 sec
• Luciferase induction for the chemicals was calculated using the following approach:
i. (RLUFG) – (RLUBG) = BG corrected (RLU)
ii. (BG corrected (RLU) of each chemical containing well)/( average BG corrected (RLU) of six negative control wells) = Luciferase induction
RLUFG = Foreground Relative luciferase units
RLUBG = Background Relative luciferase units (no cells blank)
Cytotoxicity Assessment
• For the cell viability assay plates, the medium was aspirated and replaced with 200 µl of DPBS and 27 µl of Thiazolyl blue tetrazolium bromide (MTT) reagent (5 mg/ml in DPBS). The plate was covered with sealing tape and was incubated at approximately 37ºC for 4 hours.
• Following incubation, the supernatant was aspirated and 200 µl of DMSO was added to each well. Following thorough mixing by repeated pipetting, the cell lysate was transferred to a new clear 96 well plate and absorbance was quantified at 600 and 630 nm. Cell viability for the cells was calculated using the following method:
i. (Abs600) – (AbsBG) = BG corrected (Abs600)
ii. (Abs630) – (AbsBG) = BG corrected (Abs630)
iii. BG corrected (Abs600) - BG corrected (Abs630) = (Abs600 - 630)
iv. ((Abs600 - 630) of each chemical containing well)/( average (Abs600 - 630) of six negative control wells) *100 = % viability
Abs600 = Foreground absorbance measured at 600 nm
Abs630 = Foreground absorbance measured at 630 nm

ii. waited for 1 second and integrated the luciferase activity for 2 sec
• Luciferase induction for the chemicals was calculated using the following approach:
i. (RLUFG) – (RLUBG) = BG corrected (RLU)
ii. (BG corrected (RLU) of each chemical containing well)/( average BG corrected (RLU) of six negative control wells) = Luciferase induction
RLUFG = Foreground Relative luciferase units
RLUBG = Background Relative luciferase units (no cells blank)
Cytotoxicity Assessment
• For the cell viability assay plates, the medium was aspirated and replaced with 200 µl of DPBS and 27 µl of Thiazolyl blue tetrazolium bromide (MTT) reagent (5 mg/ml in DPBS). The plate was covered with sealing tape and was incubated at approximately 37ºC for 4 hours.
• Following incubation, the supernatant was aspirated and 200 µl of DMSO was added to each well. Following thorough mixing by repeated pipetting, the cell lysate was transferred to a new clear 96 well plate and absorbance was quantified at 600 and 630 nm. Cell viability for the cells was calculated using the following method:
i. (Abs600) – (AbsBG) = BG corrected (Abs600)
ii. (Abs630) – (AbsBG) = BG corrected (Abs630)
iii. BG corrected (Abs600) - BG corrected (Abs630) = (Abs600 - 630)
iv. ((Abs600 - 630) of each chemical containing well)/( average (Abs600 - 630) of six negative control wells) *100 = % viability
Abs600 = Foreground absorbance measured at 600 nm
Abs630 = Foreground absorbance measured at 630 nm
AbsBG = Background absorbance (of no cells blank)

Acceptance Criteria
Cinnamic aldehyde (CA, positive control) was considered positive when the gene induction by CA was above the threshold of 1.5-fold in at least one dose level and cell viability at that dose was greater than 70%.
Maximum luciferase induction (Imax) and EC 1.5 (test material concentration at which luciferase induction was greater than 1.5 fold) was calculated for CA. The assay was acceptable only if at least one of the two following criteria were fulfilled:
• Average luciferase induction in the two replicates for CA at 64 µM was between 2 and 8.
• The EC 1.5 was between 7.5 µM and 30 µM.
If only one criterion is fulfilled, the dose-response of CA was carefully checked to decide on acceptability (Natsch et al., 2011).
The average variability in the 6 solvent control wells of each of the two parallel test plate should be below 20%. If the variability was higher, the assay was deemed unreliable and the results were discarded.
The results for these acceptable criteria were reported along with the test results. Final interpretation of the assay acceptability was based on the above criteria and expert judgment.

Test material data reporting and interpretation:
For the test chemical, Imax, EC 1.5, and cell viability were calculated as described above. A chemical was reported as positive if:
• Luciferase induction (Imax) was greater than 1.5-fold and EC 1.5 is below 1000 µM.
• At EC 1.5, the cellular viability was above 70%.
• There was an apparent overall dose-response for luciferase induction.
Final interpretation of the test material results were based on the above criteria as well as expert judgment.



















Positive control results:
In the three independent replicates, the positive control compound, cinnamic aldehyde, exhibited a dose-dependent increase in luciferase activity with EC 1.5 values of 21.93, 14.24, and 10.93 µM, respectively. The relative cell viability at EC 1.5 was greater than 70%. In replicate 1, 2, and 3, cinnamic aldehyde exhibited a maximum luciferase induction (Imax) of 2.41-, 4.32-, and 4.44-fold, relative to the vehicle control. In addition, the average variability in the solvent control wells was below the acceptable 20% in all three replicates, thereby demonstrating appropriate assay responsiveness.
Key result
Parameter:
other: EC 1.5 (micro molar)
Run / experiment:
1
Value:
274.12
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5 (micro molar)
Run / experiment:
2
Value:
6.09
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5 (micromolar)
Run / experiment:
3
Value:
110.02
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone was tested at twelve concentrations ranging from 1 to 2000 µM in three independent assay replicates.
In replicate 1, 2, and 3 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone exhibited a maximum luciferase induction (Imax) of 2.56-, 3.66-, and 2.32-fold, relative to the vehicle control (Figure 1A, 1B, 1C). The EC 1.5 values in replicates 1, 2, and 3 were 274.17, 6.09, and 110.02 μM, respectively. Therefore in all three replicates, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone induced luciferase activity above the threshold 1.5-fold at non-cytotoxic concentrations.
Therefore, based on the findings of this study, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone was considered positive for skin sensitization potential in the in vitro KeratinoSens assay.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The results from this study indicate that 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone is positive in the in vitro KeratinoSens assay and therefore is predicted to have skin sensitization potential.
Executive summary:

1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (2,4-Dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one 4-((4-C7-17-branched alkylphenyl)azo) derivatives) was evaluated for skin sensitization potential in anin vitro KeratinoSens assay. In this study the KeratinoSens cells were exposed to a vehicle control (1% DMSO), positive control (cinnamic aldehyde) at five concentrations (4 – 64µM), and 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone at 12 concentrations (0.98 – 2000µM). Following 48 hours of exposure, the cell viability and luciferase activity were measured in treated and control cells. The test material was considered a sensitizer if relative luciferase activity was greater than 1.5-fold (EC 1.5 at concentration < 1000µM) and cell viability at EC 1.5 was greater than 70%. The positive control treated cells exhibited luciferase induction within the designated parameters and met all requirements for a viable assay. The relative luciferase activity of 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone was greater than 1.5-fold at 56.84 µM and cell viability at this concentration was greater than 70%. Therefore, under the conditions of this study, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone is predicted to have skin sensitization potential.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay
Details on study design:
see below
Positive control results:
Triplicate analysis of the positive control, cinnamaldehyde, incubated with cysteine-containing peptide had measured concentrations of 0.1629, 0.1586, and 0.1637 mM with an overall average of 0.1617 mM. Triplicate analysis of cinnamaldehyde incubated with lysine-containing peptide had measured concentrations of 0.3315, 0.3102, and 0.3316 mM with an overall average of 0.3244 mM. Average percent depletion values for cysteine and lysine were 68.9% and 36.0%, respectively. . It should be noticed that the positive control lysine peptide depletion value was 36.0%, which was lower than the OECD guideline value (40.2-69.4%). However, as the test material was classified as a sensitizer with moderate reactivity according to the mean cysteine and lysine peptide % depletion value from the current study, therefore, this lower positive lysine depletion value should not impact the study conclusion for the test material.
Key result
Parameter:
other: Cysteine depletion (%)
Run / experiment:
mean
Value:
27.9
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: Lysine depletion (%)
Run / experiment:
mean
Value:
19.6
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The current study results showed that the average percentage depletion values of cysteine and lysine peptide for the test material were 27.9% and 19.6%, respectively, with a mean percent depletion of 23.7%. Based on the test guideline (OECD 442C) and the mean peptide depletion of 23.7%, the test material is considered to have sensitization potential with moderate reactivity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

QSAR assessment of this substance identified the potential for skin sensitisation due to the presence of structural alerts. A Keratinosense assay and a DPRA were performed. These assays confirmed the potential for skin sensitisation, however it was not possible to assess the potential potency using these data. Consequently a LLNA was performed to provide the definitive answer on sensitising potential and potency.

The substance was positive for skin sensitising potential in the LLNA with an EC3 value of 8.56%. This substance is therefore a weak to moderate skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The EC3 value from the LLNA was 8.56% therefore the criteria for classification as category 1B skin sensitiser are met.