Registration Dossier

Administrative data

Description of key information

The sensitisation endpoint was assessed under the EC NONS scheme according to the M&K maximisation test. This study resulted in a weak (4/10) positive result which resulted initially in the substance being classified as a sensitiser.

The substance has been subsequently assessed, however, according to the in vivo assays, LLNA and Buehler sensitisation, in vitro assays, h-CLAT, Keratinosens and DPRA and also by QSAR according to the DEREK Nexus system.

All of these assays resulted in negative results suggesting that the positive result obtained by the M&K maximisation is due to the aggressive test method rather than a true effect of sensitisation. This is further supported by the negative results obtained by exposure of the substance to human volunteers where no effects due to the substance are noted.

On the basis of the entirety of the data, the substance is considered non-sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was performed between 05 July 1999 and 15 august 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Standards (Schedule 1, Good Laboratory Practice Regulations 1997 (SI 1997/654)).
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was conducted prior to the introduction of the use of LLNA as a mandatory starting study for the examination of skin sensitising potential for substances.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK- Age at study initiation: eight to twelve weeks- Weight at study initiation: 303 to 380 g- Housing: solid floor polypropylene cages- Diet: ad libitum- Water: ad libitum- Acclimation period: at least five daysENVIRONMENTAL CONDITIONS- Temperature (°C): 17 to 23°C- Humidity (%): 30 to 70%- Air changes (per hr): approximately fifteen changes per hour- Photoperiod (hrs dark / hrs light): controlled by time switch to give twelve hours continuous light and twelve hours darknessIN-LIFE DATES: From: Day 0 To: End of study
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
75%, 50%, 25% and 10% w/w in Arachis oil BP.
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
75%, 50%, 25% and 10% w/w in Arachis oil BP.
No. of animals per dose:
15
Details on study design:
RANGE FINDING TESTS: The concentration of test material to be used at each stage of the main study were determined by sighting tests in which groups of guines pigs were treated with various concentrations of the test material. MAIN STUDYA. INDUCTION EXPOSURE- No. of exposures: 3- Exposure period: not applicable as injections- Test groups: 10 guinea pigs used for the main study- Control group: 5 guines pigs used for the control- Site: shoulder region approximately 40 mm x 60 mm- Frequency of applications: not stated- Duration: 24 to 28 hours- Concentrations: 75%, 50%, 25% and 10% w/w in Arachis oil BP.B. CHALLENGE EXPOSURE- No. of exposures: 1- Day(s) of challenge:Day 7 - Exposure period: 48 hours- Test groups: 10 guinea pigs- Control group: 5 guinea pigs were used for the control group- Site: shoulder region- Concentrations: 75% w/w in Arachis oil BP- Evaluation (hr after challenge): 48 hoursOTHER:
Challenge controls:
Not stated in report
Positive control substance(s):
no
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% test material in arachis oil BP
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
2 animals showed signs of just eythem and 1 animal showed signs of both erythema and oedema
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75% test material in arachis oil BP. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: 2 animals showed signs of just eythem and 1 animal showed signs of both erythema and oedema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% test material in arachis oil BP
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
2 animals have shown mild signs of erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75% test material in arachis oil BP. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: 2 animals have shown mild signs of erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% test material in arachis oil
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
3 animals have shown signs of just erythema and 1 aimal has shown signs of erythema and oedema
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% test material in arachis oil. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: 3 animals have shown signs of just erythema and 1 aimal has shown signs of erythema and oedema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% test material in arachis oil
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
3 animals have shown signs of erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% test material in arachis oil. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: 3 animals have shown signs of erythema.
Reading:
1st reading
Hours after challenge:
1
Group:
positive control
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
2 animals showed signs of erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 1.0. Group: positive control. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: 2 animals showed signs of erythema .
Reading:
2nd reading
Hours after challenge:
24
Group:
positive control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no effects were noted
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: positive control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effects were noted.

Main study:

Discrete or patchy to intense erythema and swelling was noted at the intradermal induction sites of all test group animals at the 24 hour observation with discrete or patchy to moderate and confluent erythema at the 48 hour observation period.

Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of all control group animals at the 24 hour observation period with discrete or patchy erythema at the 48 hour observation period.

Skin Reactions:

Discrete or patchy erythema was noted at the topical induction sites of six test group animals at the 1 - hour observation period. No evidence of erythema or oedema was noted after this period. Also discrete or patchy erythema was noted at the topical induction sites of two control group animals at the 1 hour observation. No evidence of erythema or oedema was noted at the topical induction sites of the control group animals at the 24 hour observation period.

Results observed after the topical challenge:

75% w/w in Arachis oil BP

Positive skin responses (discrete or patchy erythema with or without very slight oedema) were noted at the challenge sites of three test group animals at the 24 hour observation period and two test group animals at the 48 -hour observation period.

No skin reactions were noted at the challenge sites of the control group animals at the 24 and 48 hour observation.

50% w/w in Arachis Oil BP

Positive skin responses (discrete or patchy to moderate and confluent erythema with or without very slight oedema) were noted at the challenge sites of four test group animals at the 24 observation and three test group animals at the 48 hour observation.

Body weight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period.

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test material was classified as a sensitiser according to EU labelling regulations.
Executive summary:

The study was performed in compliance with the OECD Guidelines for Testing of Chemical No. 406 "Skin Sensitization" and Method B6 of Commission Directive 96/54/EC (which constitutes Annex V of Council Directive 67/548/EEC).

Ten test and five control animals were used for the main study.

Based on the results of the sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal Induction : 5%w/v in Arachis oil BP

Topical Induction : 75% w/w in Arachis oil BP

Topical Challenge : 75% and 50% w/w in Arachis oil BP

The test material produced a 40% (4/10) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin. The test material was also classified as a sensitiser according to EU labelling regulations. The symbol "Xi", the indication of danger "irritant" and the risk phrase R 43 "may cause sensitisation by skin contact" are required.

The test material was classified as a sensitiser according to EU labelling regulations.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
8 April to 11 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been conducted recently by a GLP accredited laboratory, although GLP compliance is not claimed for the study
Principles of method if other than guideline:
the study design followed the principles of the Buehler sensitisation method with three induction of 10 guinea pigs one week apart followed by a challenge of the 28 days after the first induction and skin reaction recorded to determine the immunological dermal response.
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Japan SLC, Inc.- Age at study initiation: Five weeks old- Weight at study initiation: 300 to 341g- Housing: Suspended aluminum caging (32W x 48D x 33H cm, with metal drainboard, Tokiwa Kagaku Kikai Co., Ltd.)They were steam-disinfected to use, and exchanged once in 6 or 7 days.- Diet (e.g. ad libitum): Pellets for laboratory animals (LABO G STANDARD, Nosan Corporation) was provided ad libi¬tum except during induction and challenge treatment.- Water (e.g. ad libitum): ad libi¬tum - Acclimation period: 5 days quarantine and 2 days acclimatisationENVIRONMENTAL CONDITIONS- Temperature (°C): Measured temperatures 21.3 to 22.7oC- Humidity (%): Measured values: 48.0 to 67.3%- Air changes (per hr): 6 - 20 air changes/hour, all-freshed air provided- Photoperiod (hrs dark / hrs light): 12 hours light/day cycle, lighted from 7:00-19:00IN-LIFE DATES: From: 8 April 2008 To: 16 May 2008
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
Test sample for the induction phaseThe test sample was suspended in PG to prepare 50 w/v % test solution. Test sample for the challenge phase The test sample was suspended in PG to prepare 25 w/v % test solution.
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
Test sample for the induction phaseThe test sample was suspended in PG to prepare 50 w/v % test solution. Test sample for the challenge phase The test sample was suspended in PG to prepare 25 w/v % test solution.
No. of animals per dose:
10 test 5 control animals
Details on study design:
RANGE FINDING TESTS:MAIN STUDYA. INDUCTION EXPOSURE- No. of exposures: 3- Exposure period: 28 days- Test groups: 10 animals- Control group: 5 animals- Site: left flank- Frequency of applications: Days 1, 8 and 15- Duration: continued for 6 hours- Concentrations: 50 w/v %B. CHALLENGE EXPOSURE- No. of exposures: 1- Day(s) of challenge: 29- Exposure period: continued for 6 hours- Test groups: 10 animals- Control group: 5 animals- Site: right flank- Concentrations: 25 w/v %- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
Induction with Propylene Glycol only on the left flank and challenge of 25 w/v % test solution on the right flank
Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 %w/v
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 %w/v. No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%w/w
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%w/w. No with. + reactions: 0.0. Total no. in groups: 10.0.
Interpretation of results:
not sensitising
Remarks:
Migrated information25%w/wCriteria used for interpretation of results: EU
Conclusions:
The substance is considered to be not sensitising under the conditions of the study
Executive summary:

The sensitisation potential of the substance has bene assessed according to the Buehler method with three inductions separated by 1 week to the left flank of 10 test animals with 50% w/v solution in propylene glycol. 5 control animals were exposed to the same regimen with propylene glocol only. All animals were then exposed to 25 %w/v of the test sample in propylene glycol to the right flank 28 days after the first induction. The results were assessed and it was determined that the substance was not sensitising under the conditions of the test.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 25, 2017 to July 18, 20 17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, No.442B, July 22, 2010, "Skin Sensitization: Local
Lymph Node Assay: BrdU-ELISA"
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Specific details on test material used for the study:
No further details specified in the study report.
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Twenty six female CBA/J mice (SPF, 7 weeks old) were obtained from Charles River Laboratories Japan Atsugi Breeding Center. The CBA/J is a substrain of CBA/JN which is recommended in OECD TG 442B, and our testing facility has the LLNA historical control data of CBA/J.
At the receipt, since all animals showed good health conditions, they were received, weighed and assigned animal receipt numbers. The animals were quarantined and acclimatized for six days after the receipt and then the body weights were measured. Since no abnormalities were observed in clinical signs, excretions or body weight changes of any animals, the quarantine was completed and the animals were subsequently acclimatized. Two days before the first application of pre-screen test, five animals (receipt number 1-5) were housed individually and used for pre-screen test. The remaining 21 animals were further acclimatized, and two days before the first sensitization of the main study, the animal were weighed and were allocated to five groups (four animals per group) by body weight-stratified randomization. Any individual body weights were confirmed to be within the range of mean body weight ±20% at the group allocation. The remaining animal was excluded from the study. At the initiation of application, the animals were 8 weeks old in the pre-screen test and 9 weeks old in the main study.
The animals were housed ten animals or less per cage before the group allocation and then one animal per cage in the pre-screen test and four animals per cage in the main study. Clinical conditions and excretions were observed once or more daily.
The animals were identified by painting on the tail with red ink before group allocation and in the pre-screen test, and by painting with other colors after group allocation in the main study. Cages were identified by individual cards and racks were identified by indications of the study number, sex and test groups.

HOUSING CONDITIONS
The animals were housed in the barrier-system animal rooms (quarantine room number 2 during the quarantine period and animal room number 5 after the quarantine period) which were maintained at 21 to 25 °C, relative humidity at 40 to 70%, 10 to 15 air changes per hour and photoperiod of 12 hr light per day (light on at 7:00 and off at 19:00).
The animals were housed in polycarbonate cages (265W x426D x 150H mm, TOKIWA KAGAKU KIKAI) with softwood woodflakes (Sun-Flakes, lot number 170131, Charles River Laboratories Japan) before the group allocation and in po1ycarbonate cages (210W x 320D x 130H mm, CLEA Japan) with the woodflakes after the group allocation. Cages with woodflakes were changed at the completion of quarantine, group allocation and before carrying the animals from the animal room to the autopsy room. Stainless cage tops, water bottles and racks were changed at the group allocation.
Animals had free access to a pelleted diet (MF., lot number 170222, Oriental Yeast) and 3 to 5 ppm chlorinated water via water bottles. The chlorinated water was prepared by adding sodium hypochlorite (Purelox, OYALOX) to Hita City supply. Diets, woodflakes and housing materials were autoclaved at 121 °C for 30 min before using.
The information of the contaminants in diet and woodflakes was obtained from supplier (analyzed in Eurofins) and the results were confirmed to meet the requirements in CERI Hita.
Contaminants in drinking water were analyzed twice a year, and the analyzed data before the animal receipt were confirmed to meet the requirements in the water regulations of the "Ordinance on drinking water quality standards" (Ordinance Numbers 101 of Ministry of Health, Labour and Welfare).
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The test substance was dissolved in AOO, which is a recommended vehicle in the OECD TG 442B, at a concentration of 50.0 w/v%. In addition, the solution was visually stable at room temperature for four hours after preparation. Therefore, the AOO was selected as a vehicle.
No. of animals per dose:
Pre-screen test: 5 animals
Main study: 4 animals per dose group
Details on study design:
PRE-SCREEN TEST
Objective: The objective of the pre-screen test was to set the dosage for the main study.
Grouping: Five dose levels of the test substance were set as follow. Since the test substance was solid, the highest concentration was set at 50.0 w/v%. The lower concentrations were determined according to OECD TG 442B.
Preparation of vehicle: On the first application day, 10 mL of acetone and 2.5 mL of olive oil were mixed to prepare AOO.
Preparation of test substance formulation: On the first application day, 0.50009 g of the test substance was dissolved in AOO and filled up to 1 mL to make 50.0 w/v% solution. The lower concentration solutions were prepared by serial dilution method as follows. These solutions had been stored at the cold place (acceptable temperature: to 10°C) and used for 3 days.
Application: Twenty-five microliters of each solution was applied to the dorsum of each ear of the animals using a micropipette once a day for three consecution days.
Clinical observation: All the animals were observed more than once a day from the first application day (Day 1) to terminal day (Day 6). Erythema scoring was not performed since no erythema was observed.
Body weights: Body weights of all the animals were measured on Day 1 and Day 6 using an electric balance.
Ear thickness: Thicknesses of each ear were measured on Day 1 (before application), Day 3 and Day 6 using a digital caliper (Digimatic Caliper, Mitutoyo ). Thickness of each ear was measured duplicate on each day, and the mean values were calculated.
Handling of animals after examinations: All the animals used in the pre-screen test were euthanized by cervical dislocation on Day 6.
Handling of dead or moribund animals: No moribund state or death occurred.
Evaluation of result: Dosages which induce following signs are supposed to be excluded from the main study.
-excessive irritation (erythema score 2: 3 and/or the ear thickness increases 25% or more)
-severe systemic toxicity and/or 5% or more body weight decrease

MAIN STUDY
Dose setting
In the pre-screen test, no abnormal changes which suggested systemic toxicity or excessive irritation were noted in any animals. Therefore, the dosages of the main study were set at 50.0, 25.0 and 10.0 w/v%.
Grouping
The test substance, vehicle control (AOO) and positive control (25.0 w/v% HCA) groups were set in the main study. Four animals were used in each group.

Preparations of vehicle, test substance formulation, positive control substance solution and BrdU solution
Vehicle: On each sensitization day, 10 mL of acetone and 2.5 mL of olive oil were mixed to prepare AOO.
Test substance formulation: On each sensitization day, 0.500 g of test substance was dissolved in AOO and filled up to 1 mL to make 50.0 w/v% solution. The actual weights were 0.50033 g (Day 1), 0.50001 g (Day 2) and 0.50001 g (Day 3). The 25.0 and 10.0 w/v% solutions were prepared by the same method as pre-screen test.
Positive control substance solution: On the first sensitization day, 0.25020 g of HCA was dissolved in AOO and filled up to 1 mL to make a 25.0 w/v% HCA solution under a yellow light condition. The solution was subdivided into three air-tight containers and the containers were shaded and preserved in refrigerator (acceptable temperature: 1-10 °C).
BrdU solution: Two days before the administration, 0.20002 g of 5-bromo-2'-deoxyuridine (BrdU, lot number M5H0079, Nacalai Tesque) was dissolved in physiological saline (lot number M6D75, Otsuka Pharmaceutical Factory) by ultrasonication and filled up to 20 mL to make 10 mg/mL solution. The BrdU solution was filtrated by a sterilizing filter (DISMIC®-25AS, pore size: 0.20 11m, lot number: 510221CD, Toyo roshi kaisha) and was stored in a sterile container in a refrigerator (acceptable temperature: 1-10 °C) until administration. The preparation was conducted under a yellow light condition, and the sterilization and after procedures were conducted in a clean bench.

Sensitization
The vehicle, test substance formulations and positive control solution were applied in the same way as the pre-screen test.

BrdU administration
Approximately 48 hours after the final sensitization, 0.5 mL ofBrdU solution was administrated intraperitoneally to each animal using a syringe and a needle (Terumo).

Clinical observations
Clinical observations were performed in the same way as the pre-screen test except for the erythema scoring.

Body weights measurements
Body weights were measured in the same way as for pre-screen test. The mean and the standard deviation were calculated for each group on each measurement day.

Collection and weights measurement of auricular lymph nodes
Approximately 24 hours after the BrdU administration, the animals were euthanized by cervical dislocation and each auricular lymph node was taken. The lymph nodes were carefully dissected and trimmed of surrounding tissue and fat, and weighed both sides together. The mean value and the standard deviation of the lymph nodes weights were calculated for each group. The lymph nodes were stored individually in the biomedical freezer (acceptable temperature: -30 to -10 °C).

BrdU labelling index
The auricular lymph nodes were defrosted at room temperature, homogenized and suspended in physiological saline (lot number 6HOON, Otsuka Pharmaceutical Factory). This suspension was filtered by cell strainer (Becton, Dickinson and Company), and dispensed into 3 wells per animal of a 96 well microplate (Costar). The BrdU uptake quantity was measured by ELISA using a commercial kit (Cell Proliferation ELISA, BrdU colorimetric, lot number 17267000, Roche Diagnostics). Absorbance at 3 70 nm with a reference wavelength of 492 nm was measured using multidetection microplate reader (FLUOstar OPTIMA, BMG LABTECH). The mean value of the 3 wells was defined as BrdU labelling index.

Handling of dead or moribund animals
No death or moribund state occurred.

Evaluation of result
Calculation of stimulation index (SI)
The SI for individual animals of was calculated by dividing the BrdU labelling index for each animal by the mean value of BrdU labelling index for the vehicle control group. The SIs were rounded off to one decimal place, and mean SI of each group was calculated.
SI = BrdU labelling index for individual animal/Men value of BrdU labelling indices for 4 animals of the vehicle control group

Evaluation method
Test substance is regarded as a "sensitizer" when the SI of the test substance group is 2.0 or more, and is regarded as a "non-sensitizer" when the SI of the test substance group is less than 1.6. When the SI is between 1.6-1.9, dose-response relationship and statistical significance are considered to evaluate the sensitization potential of the test substance.
However in this study, the statistical analysis was not conducted since the Sis of the any test substance groups were less than 1.6.

Validity of study
The test is regarded as valid when the SI of the positive control group is 1.6 or more.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI of the positive control group was 2.6.
The mean lymph node weights of the positive control group were 9.8 mg.
The mean BrdU labelling indices of the positive control group was 0.507.
Key result
Parameter:
SI
Value:
>= 0.9 - <= 1
Key result
Parameter:
other: BrdU labelling indices
Value:
> 0.168 - < 0.196
Cellular proliferation data / Observations:
The mean lymph nodes weights of the vehicle control group were 4.8 mg and those of 50.0, 25.0 and 10.0 w/v% test substance groups were 4.8, 4.7 and 5.8 mg, respectively.

Clinical signs in the pre-screen test

Exp. Group

Animal No.

Observation period

Group

Substance name

Concentration

(w/v %)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Test substance

PX-200

2.50

1

-

-

-

-

-

-

5.00

2

-

-

-

-

-

-

10.0

3

-

-

-

-

-

-

25.0

4

-

-

-

-

-

-

50.0

5

-

-

-

-

-

-

-: no abnormalities detected

 

Body weights in the pre-screen test

Exp. Group

Animal No.

Body weight (g)

Group

Substance name

Concentration

(w/v %)

Day 1

Day 6a)

Test substance

PX-200

2.50

1

21.0

21.2     (101)

5.00

2

22.5

22.7     (101)

10.0

3

22.7

23.3     (103)

25.0

4

21.6

21.9     (101)

50.0

5

21.0

21.2     (101)

a)Figures in parentheses indicate percentages compare to the initial body weight (Day 1).

 

Thickness of auricle in the pre-screen test

Exp. Group

Animal No.

Thickness of auricle (mm)

Group

Substance name

Concentration

(w/v %)

Day 1

Day 3

Day 6

Left

Right

Average

Left

Right

Averagea)

Left

Right

Averagea)

Test substance

PX-200

2.50

1

0.18

0.19

0.19

0.17

0.18

0.18

(95)

0.16

0.18

0.17

(89)

5.00

2

0.18

0.17

0.18

0.17

0.18

0.18

(100)

0.17

0.19

0.18

(100)

10.0

3

0.15

0.18

0.17

0.16

0.17

0.17

(100)

0.17

0.18

0.18

(106)

25.0

4

0.14

0.17

0.16

0.16

0.17

0.17

(106)

0.17

0.16

0.17

(106)

50.0

5

0.16

0.17

0.17

0.16

0.17

0.17

(100)

0.18

0.17

0.18

(106)

a)Figures in parentheses indicate percentages compare to the initial thickness (Day 1).

 

Clinical signs in the main study

Exp. Group

Animal No.

Observation period

Group

Substance name

Concentration

(w/v %)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Vehicle control

AOO

 

1

-

-

-

-

-

-

2

-

-

-

-

-

-

3

-

-

-

-

-

-

4

-

-

-

-

-

-

Positive control

HCA

25.0

5

-

-

-

-

-

-

6

-

-

-

-

-

-

7

-

-

-

-

-

-

8

-

-

-

-

-

-

Test substance

PX-200

10.0

9

-

-

-

-

-

-

10

-

-

-

-

-

-

11

-

-

-

-

-

-

12

-

-

-

-

-

-

25.0

13

-

-

-

-

-

-

14

-

-

-

-

-

-

15

-

-

-

-

-

-

16

-

-

-

-

-

-

50.0

17

-

-

-

-

-

-

18

-

-

-

-

-

-

19

-

-

-

-

-

-

20

-

-

-

-

-

-

AOO: acetone:olive oil (4:1 v/v)

HCA: α-hexylcinnamaldehyde

-: no abnormalities detected

 

Body weights in the main study

Exp. Group

Animal No.

Body weights (g)

Group

Substance name

Concentration

(w/v %)

Day 1

Day 6

Individual

Mean ± S.D.

Individual

Mean ± S.D.

Vehicle control

AOO

 

1

21.2

21.3 ± 0.9

22.8

22.2 ± 0.9

2

22.3

22.5

3

20.2

20.8

4

21.4

22.6

Positive control

HCA

25.0

5

21.2

21.2 ± 1.0

20.9

21.6 ± 1.8

6

21.1

21.5

7

20.0

19.8

8

22.4

24.1

Test substance

PX-200

10.0

9

20.5

21.0 ± 1.6

21.1

21.7 ± 1.3

10

23.0

23.2

11

19.1

20.3

12

21.3

22.2

25.0

13

21.7

21.4 ± 1.1

21.6

22.1 ± 1.0

14

22.7

23.4

15

20.1

21.2

16

21.0

22.0

50.0

17

22.6

21.8 ± 1.0

22.3

21.3 ± 1.0

18

22.3

21.9

19

20.3

20.0

20

21.8

21.0

S.D.: standard deviation

AOO: acetone:olive oil (4:1 v/v)

HCA: α-hexylcinnamaldehyde

 

Lymph node weights in the main study

Exp. Group

Animal No.

Lymph node weights

(mg)

Group

Substance name

Concentration

(w/v %)

Individual

Mean ± S.D.

Vehicle control

AOO

 

1

4.8

4.8 ± 0.4

2

5.1

3

5.0

4

4.2

Positive control

HCA

25.0

5

9.1

9.8 ± 0.7

6

9.3

7

10.3

8

10.5

Test substance

PX-200

10.0

9

6.3

5.8 ± 0.6

10

5.7

11

5.0

12

6.2

25.0

13

3.5

4.7 ± 1.3

14

4.7

15

4.2

16

6.5

50.0

17

4.8

4.8 ± 0.3

18

5.1

19

4.9

20

4.5

S.D.: standard deviation

AOO: acetone:olive oil (4:1 v/v)

HCA: α-hexylcinnamaldehye

 

BrdU labelling indices and stimulation indices in the main study

Exp. Group

Animal No.

BrdU labelling indices

Simulation indices

Group

Substance name

Concentration

(w/v %)

Individual

Mean ± S.E.

Individual

Mean ± S.E.

Vehicle control

AOO

 

1

0.152

0.194 ± 0.024

0.8

1.0 ± 0.1

2

0.247

1.3

3

0.155

0.8

4

0.222

1.1

Positive control

HCA

25.0

5

0.613

0.507 ± 0.052

3.2

2.6 ± 0.3

6

0.425

2.2

7

0.577

3.0

8

0.411

2.1

Test substance

PX-200

10.0

9

0.174

0.196 ± 0.018

0.9

1.0 ± 0.1

10

0.234

1.2

11

0.216

1.1

12

0.159

0.8

25.0

13

0.187

0.192 ± 0.019

1.0

1.0 ± 0.1

14

0.189

1.0

15

0.151

0.8

16

0.241

1.2

50.0

17

0.156

0.168 ± 0.004

0.8

0.9 ± 0.1

18

0.168

0.9

19

0.169

0.9

20

0.77

0.9

S.E.: standard error

AOO: acetone:olive oil (4:1 v/v)

HCA: α-hexylcinnamaldehyde

Interpretation of results:
GHS criteria not met
Conclusions:
In the main study, no abnormal findings which suggested excessive irritation or severe systemic toxicity were observed in clinical signs or body weight changes in any treatment groups. Therefore, all the data obtained were used for the assessment of skin sensitization potential. As a result, the Sis of the 50.0, 25.0 and 10.0 w/v% were 0.9, 1.0 and 1.0, respectively: the Sis of any dose levels were less than 1.6.
Therefore, under the conditions tested, PX-200 was considered to be a non-sensitizer. As for the positive control group, the SI was 2.6 and thus the study was confirmed to be valid.
Executive summary:

Local lymph node assay: BrdU-ELISA was performed in accordance with OECD TG 442B, using female CBA/J mice (SPF), and the skin sensitization potential of PX-200 was assessed by stimulation index (SI).

 

In the pre-screen test, 50.0, 25.0, 10.0, 5.00 and 2.50 w/v% test substance solutions, prepared with acetone: olive oil ( 4:1 v/v, AOO), were applied to mice daily for three consecutive days (one animal per dose level), and clinical observations, body weights measurements and ear thickness measurements were conducted. As a result, no abnormal changes which suggested excessive irritation or systemic toxicity were detected. Thus, the dose levels at 50.0, 25.0 and 10.0 w/v % were set for the main study.

 

In the main study, in addition to three test substance groups, vehicle control group which was applied with AOO and positive control group which was applied with a-hexylcinnamaldehyde (HCA) were set.

Four animals each were applied the vehicle, 25.0 w/v % HCA solution or test substance formulations daily for three consecutive days, and clinical observations and body weights measurements were performed. Approximately 48 hours after the final sensitization, 5-bromo-2'deoxyuridine (BrdU) was administered. Approximately 24 hours later, auricular lymph nodes were collected and their BrdU uptake quantities were measured to calculate the SIs. During the main study, no abnormal changes which suggested excessive irritation or systemic toxicity were noted. Therefore, all the data obtained were subjected to the skin sensitization evaluation.

 

Consequently, the SIs of the 50.0, 25.0 and 10.0 w/v % were 0.9, 1.0 and 1.0, respectively: the SIs of any dose levels were lower than 1.6. Therefore, under the conditions tested, PX-200 was considered to be a non-sensitizer.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 December 2016 to 09 January 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))
Version / remarks:
OECD guideline No. 442C: in chemico skin sentitization: Direct Peptide Reactivity Assay (DPRA), adopted on 04 February 2015.
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. certificate)
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)
Specific details on test material used for the study:
No further details specified in the study report.
Details on study design:
DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as described below.

Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

Co-elution control samples preparation
For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

Reference control samples preparation
Reference control A and B samples: In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.

Reference control C samples: Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.

For the reference control C prepared with cysteine peptide: 50 μL of vehicle (acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.

For the reference control C prepared with lysine peptide: In parallel, 250 μL of vehicle (acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Test item samples preparation
For the reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of test item with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25 °C and protected from light before injection into the HPLC/UV system.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation.
Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature or at +4 °C and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
-one blank sample (peptide dilution buffer),
-one calibration curve injected at the beginning of the analytical batch,
-three reference control A samples,
-the co-elution control sample,
-three reference control B samples,
-reference control C samples (replicate 1),
-positive control sample (replicate 1),
-test item study sample (replicate 1).
The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
-three reference control B samples.
Key result
Parameter:
other: peptide reactivity
Value:
0.36
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.
Analysis of the chromatograms of the co-elution samples (Figures 1 and 4) indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:
-for the cysteine peptide, the mean depletion value was 0.59%,
-for the lysine peptide, the mean depletion value was 0.13%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.36%. Accordingly, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.
Since precipitate and/or phase separation (micelles) were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient confidence.

Percent peptide depletion for the test item samples

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE DEPLETION IN SAMPLES SPIKED WITH A SOLUTION AT 100mM OF PX-200

Sample number

Cysteine peptide

Lysine peptide

Mean depletion rate (%) of PX-200

Depletion classification

Peak area

(μV/sec)

% depletion

Peak area

(μV/sec)

% depletion

1

2

3

2596654

2593066

2601841

0.61

0.75

0.41

2085878

2088116

2053145

0.00*

0.00*

0.38

Mean

SD

% CV

-

-

-

0.59

0.17

28.5

-

-

-

0.13

0.22

173.2

0.36

No reactivity/ minimal reactivity

Precipitate:

Yes

Yes

Micelle

No

No

 

 

*: Value set to 0 due to negative depletion

-: not applicable

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE CONCENTRATION IN REFERENCE CONTROL C SAMPLES PREPARED IN ACETONITRILE

 

Cysteine peptide

Lysine peptide

Sample number

Peak Area

(μV/sec)

Concentration

(mM)

%Dev

Peak Area

(μV/sec)

Concentration (mM)

%Dev

1

2

3

2619032

2613119

2605814

0.499

0.498

0.497

(-0.1)

(-0.3)

(-0.6)

2065090

2060532

2057403

0.498

0.497

0.496

(-0.4)

(-0.6)

(-0.7)

Mean

SD

% CV

2612655

-

-

0.498

0.001

0.3

(-0.4)

-

-

2061008

-

-

0.497

0.001

0.2

(-0.6)

-

-

 

DETERMINATION OF % INTERFERENCE DUE TO CO-ELUTION OF PX-200 WITH CYSTEINE OR LYSINE PEPTIDES

 

Peak detected at the cysteine retention time

Peak detected at the lysine retention time

Sample number

Peak Area

(μV/sec)

% Interference

Peak Area

(μV/sec)

% Interference

1

0

(0.0)

0

(0.0)

Precipitate:

Yes

Yes

Micelle

No

Yes

 

Percent peptide depletion for the positive control samples

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE DEPLETION IN SAMPLES SPIKED WITH A SOLUTION AT 100 mM OF CINNAMALDEHYDE

Sample number

Cysteine peptide

Lysine peptide

Mean depletion rate (%) of Cinnamaldehyde

Depletion classification

Peak Area

(μV/sec)

% depletion

Peak Area

(μV/sec)

% depletion

1

2

3

728662

751271

743516

72.11

71.24

71.54

913821

922619

962281

55.66

55.23

53.31

Mean

SD
% CV

-

-

-

71.63

0.44

0.6

-

-

-

54.74

1.25

2.3

63.18

High reactivity

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE CONCENTRATION IN REFERENCE CONTROL C SAMPLES PREPARED IN ACETONITRILE

 

Cysteine peptide

Lysine peptide

Sample number

Peak Area

(μV/sec)

Concentration

(mM)

%Dev

Peak Area

(μV/sec)

Concentration (mM)

%Dev

1

2

3

2619032

2613119

2605814

0.499

0.498

0.497

(-0.1)

(-0.3)

(-0.6)

2065090

2060532

2057403

0.498

0.497

0.496

(-0.4)

(-0.6)

(-0.7)

Mean

SD

% CV

2612655

-

-

0.498

0.001

0.3

(-0.4)

-

-

2061008

-

-

0.497

0.001

0.2

(-0.6)

-

-

-: Not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the study, the test item PX-200, was considered to have no/minimal peptide reactivity, though with limitations due to test item precipitation or phase separation. The test item is considered negative in the DPRA assay.
Executive summary:

The objective of the study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

 

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

Results

The test item was dissolved at 100 mM in acetonitrile.

 

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation.

-for the cysteine peptide, the mean depletion value was 0.59%,

-for the lysine peptide, the mean depletion value was 0.13%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 0.36%. Accordingly, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

 

Since precipitate and/or phase separation (micelles) were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient confidence.

 

Conclusion

Under the experimental conditions of this study, the test item PX-200, was considered to have no/minimal peptide reactivity, though with limitations due to test item precipitation or phase separation. The test item is considered negative in the DPRA assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: OECD guideline 442E: In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)
Version / remarks:
OECD guideline 442E: "In vitro skin sensitization: human Cell Line Activation Test (h-CLAT), 29 July 2016",
Deviations:
yes
Remarks:
only one dose-range finding assay was performed and only four concentrations were tested.
GLP compliance:
no
Type of study:
other: human Cell Line Activation Test (h-CLAT)
Specific details on test material used for the study:
No further details specified in the study report.
Details on study design:
Test systems: THP-1 cell line.
The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient. The THP-1 cell line was obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France).
The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container. Cells were grown using general culture procedures. They were cultured in complete RPMI (cRPMI) medium and maintained in a humidified incubator set at 37 °C, 5% CO2. The complete culture medium (cRPMI) was composed of RPMI 1640 with 10% FBS, 0.05 mM 2-mercaptoethanol and with penicillin and streptomycin.
Cell viability was checked using trypan blue.

Vehicle control: Dimethylsulfoxide (DMSO).
This vehicle was used as the vehicle control, and was applied to cells at a concentration of 1% in culture medium.

Negative control: Dimethylsulfoxide (DMSO).
Since the negative control was replaced by a vehicle control, no negative control was used in the present study.

Positive control: 2,4-Dinitrochlorobenzene (DNCB).
DNCB was used formulated in pure DMSO and then diluted in cRPMI medium to reach a final DMSO concentration of 0.2% and a final DNCB concentration of 4 μg/mL.

Solubility assay:
The solubility of the test item was assessed visually for each preparation (particles, drops, cloudiness, non-miscible phases, etc). A preparation was deemed appropriate for cell treatment as long as it is qualified as a solution or stable dispersion (homogenous emulsion/suspension).
Saline (0.9% NaCl) and DMSO are the only vehicles allowed in the assay. The vehicle was chosen between these two in the order of preference, and in accordance with the steps described below.
First, the test item was dissolved in saline at 100 mg/mL. If the test item was soluble or gave a stable dispersion (homogenous emulsion/suspension) saline was used as a vehicle and the highest soluble concentration (HSC) was determined by testing greater concentrations as follows: 300 mg/mL → 500 mg/mL.
If not, the test item was dissolved at 500 mg/mL in DMSO. In case it was not soluble at 500 mg/mL in DMSO, the highest soluble concentration (HSC) was determined by testing lower concentrations in a common ratio of two (250 mg/mL → 125 mg/mL → continued if needed). Minimal possible concentration was 1 mg/mL in DMSO.

Test item preparation:
On the basis of solubility results, the test item was prepared in the selected vehicle.
Sonication for 5 minutes and vortexing for at least 15 minutes were used in order to allow the solubilization of the test item.
It was then diluted in treatment culture medium by serial dilutions, using a dilution factor of two (DRF assays) or 1.2 (main tests), to obtain a total of four concentrations.

h-CLAT assay:
The study was divided in two successive phases. First, a dose-range finding assay (DRF) was performed to assess test item toxicity and, if applicable, determine the CV75 i.e. the test item concentration that result in 75% cell viability compared to the vehicle control. Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in a minimum of two runs in the main tests to identify potential CD86 and CD54 upregulations.
Key result
Parameter:
other:
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Solubility assay:

Vehicle

Concentration

(mg/mL)

Aspect

Retained vehicle and maximum stock concentration

0.9% NaCl

100

Colorless heterogeneous solution with white particles *

No

DMSO

500

Whitish heterogeneous suspension with white particles *

No

250

Heterogeneous suspension

No

125

Limpid solution after 5 minutes sonication and 5 minutes vortexing

Yes

*: the aspect of these preparations did not change after 5 minutes of sonication.

 

Dose-range finding results

DRF

Run reference:

20161213

Concentration

(μg/mL)

Viability (%)

cRPMI

0.2% DMSO

31.25

62.50

125.00

250.00

96.68

98.11

97.12

97.71

96.35

95.62

 

Summary of the results:

Summary results of all runs and conclusion

Test item Name

Conc.

(μg/mL)

RFI for CD86

RFI for CD54

Viability (%)

Run conclusion

General conclusion

A

B

A

B

A

B

A

B

PX-200

144.68

173.61

208.33

250.00

75

63

72

67

82

93

81

86

122

117

131

117

73

82

67

64

95.5

95.3

95.0

94.5

94.8

95.6

95.2

95.7

N

N

Negative

N = run with negative outcome                        I = Invalidated run               Conc. = concentration

S = run with positive outcome                          Inc = Inconclusive run        RFI = Relative Fluorescence Index

No precipitate/emulsion was noted in the wells following treatment

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item PX-200 was negative in the h-CLAT assay.
Executive summary:

The objective of the study was to determine the ability of the test item to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method.

 

The design of this study was based on the OECD guideline 442E: "In vitro skin sensitization: human Cell Line Activation Test (h-CLAT), 29 July 2016", except that only one dose-range finding assay was performed and only four concentrations were tested.

 

Test systems: THP-1 cell line.

The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient. The THP-1 cell line was obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France).

The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container. Cells were grown using general culture procedures. They were cultured in complete RPMI (cRPMI) medium and maintained in a humidified incubator set at 37°C, 5% CO2. The complete culture medium (cRPMI) was composed of RPMI 1640 with 10% FBS, 0.05 mM 2-mercaptoethanol and with penicillin and streptomycin.

Cell viability was checked using trypan blue.

 

h-CLAT assay:

The study was divided in two successive phases. First, a dose-range finding assay (DRF) was performed to assess test item toxicity and, if applicable, determine the CV75 i.e. the test item concentration that result in 75% cell viability compared to the vehicle control. Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in a minimum of two runs in the main tests to identify potential CD86 and CD54 upregulations.

 

Under the experimental conditions of this study, the test item PX-200 was negative in the h-CLAT assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 November 2016 to 27 January 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)
Version / remarks:
OECD Guideline 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, adopted on February 2015.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
other: ARE-Nrf2 Luciferase Test
Specific details on test material used for the study:
No further details specified in the study report.
Details on study design:
STUDY DESIGN
The test item was tested in two independent runs using cells from a different passage number. The plates were processed as described below in the § Method.

Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water or treatment culture medium). Sonication for 5 minutes and heating up to 80 °C for 40 minutes were used in order to improve the solubility of the test item.
Since the test item was found soluble in DMSO at 200 mM after 5 minutes of sonication and 40 minutes of heating at 80 °C, this stock formulation was diluted in treatment culture medium to the final concentration of
2000 μM. Then, a visual inspection of the sample was performed to evaluate the presence of precipitate.

Method for a run of KeratinoSens assay
Cell seeding for testing
-Cells were grown using general culture procedures up to 80-90% confluence,
-the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
-cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
-after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.

Treatment
-After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium,
-from the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation,
-all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
-the plates were then incubated for 48 (± 2) hours at 37 °C, 5% CO2, 90% humidity.

Endpoint measurements
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate
-After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

Luminescence flash signal to evaluate induction signal - white plates
-After incubation, the supernatants from the white assay plates were discarded,
-the cells were washed once with D-PBS,
-a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking,
-the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- 50 μL of the luciferase substrate was added to each well,
- 1 second after this addition, the luciferase signal was integrated for 2 seconds.

Absorbance signal to evaluate the cytotoxicity - transparent plate
-For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
-a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
-the plates were covered with a sealing membrane and returned at 37 °C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
-at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
-the plates were covered with a sealing membrane and placed at 37 °C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
-after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.
Positive control results:
Second run
The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" was not fulfilled (i.e. Imax of 10.66). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.
Key result
Parameter:
other: cell viability
Value:
> 70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
No geometric mean IC30 or IC50 was calculated since the cell viability was > 70% in both runs.
The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative. Since precipitate was observed in the test item-treated wells at the end of the 48-hour treatment period, the luciferase activity may be underestimated. Therefore, the conclusion on the lack of activity cannot be drawn with sufficient confidence.
This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.

Evaluation of the viability (%) of cultures treated with the test item for each run

 

Concentration (μM)

PX-200

0.98

1.95

3.91

7.81

15.63

31.25

62.5

125

250

500

1000

2000

Viability (%) in Run 1

116

134

140

133

135

136

126

127

120

121

140

124

Viability (%) in Run 2

105

110

108

108

106

108

111

95

94

91

111

113

Mean Viability (%)

110

122

124

121

120

122

118

111

107

106

125

118

Geometric Mean (%)

110

122

123

120

120

121

118

110

107

105

125

118

SD

7

17

23

18

20

19

10

23

18

21

20

8

 

Gene induction values, Imax, IC30, IC50and EC1.5values, mean and SD values obtained after treatment with the test item in each run

 

Concentrations (μM)

PX-200

0.98

1.95

3.91

7.81

15.63

31.25

62.5

125

250

500

1000

2000

Induction values in Run 1

0.9

0.9

1.0

0.9

0.9

1.0

0.9

0.8

0.7

0.7

0.8

0.7

Induction values in Run 2

0.7

0.9

0.8

0.9

0.8

0.8

0.8

0.8

0.7

0.8

0.8

0.7

Mean Induction

0.8

0.9

0.9

0.9

0.8

0.9

0.9

0.8

0.7

0.8

0.8

0.7

SD

0.1

0.0

0.1

0.0

0.0

0.1

0.1

0.0

0.0

0.0

0.0

0.0

 

Imax and EC1.5results

PX-200

Imax

EC1.5

(μM)

IC50

(μM)

IC30

(μM)

Run 1

0.99

-

-

-

Run 2

0.88

-

-

-

Mean

0.94

n.r.

n.r.

n.r.

Geometric Mean

n.r.

-

-

-

SD

0.08

-

-

-

-: no data available

n.r.: not requested by the OECD Guideline

 

Evaluation of the viability (%) of cultures treated with the positive control for each run

 

Concentrations (μM)

Cinnamic aldehyde

4

8

16

32

64

Viability (%) in Run 1

112

127

116

132

144

Viability (%) in Run 2

93

112

118

126

75

Mean viability (%)

103

119

117

129

110

Geometric Mean (%)

102

119

117

129

104

SD

13

11

2

4

49

 

Gene induction values, Imax, IC30, IC50and EC1.5values obtained with the positive control for each run

 

Concentrations (μM)

Cinnamic aldehyde

4

8

16

32

64

Imax

EC1.5(μM)

IC50(μM)

IC30(μM)

Run 1

1.4

1.3

1.7

2.6

5.6

5.56

11.68

-

-

Run 2

1.1

1.4

1.9

2.8

10.7

10.66

9.50

-

-

Mean

1.3

1.4

1.8

2.7

8.1

8.11

n.r.

n.r.

n.r.

Geometric Mean

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

10.53

-

-

SD

0.2

0.1

0.1

0.2

3.6

3.60

1.54

-

-

-: no data available

n.r.: not requested by the OECD Guideline

 

Luminescence Values for the negative control wells and the %CV between these values for each run

Negative control

Luminescence reading

Mean

% CV

Run 1

Replicate 1

378866

422119

413546

335618

317716

365676

334680

14.88

Replicate 2

317060

259389

340164

259248

308682

365781

Replicate 3

309392

335692

392675

259223

292587

350804

Run 2

Replicate 1

346750

576196

588164

507216

532422

544442

472545

15.85

Replicate 2

444670

438847

468883

465063

542011

470636

Replicate 3

527643

507718

355248

363386

383899

442662

 

KERATINOSENS TEST

Historical Data

From 13 October 2015 to 13 October 2016

Control Item

Negative control

Positive control

Parameter

Mean RLU

% CV

EC1.5

Imax

n

28

28

28

28

Mean

469622.2

15.3

13.9

4.7

SD

302495.1

4.2

6.6

3.3

Lower CL 95%

352326.9

13.6

11.4

3.4

Upper CL 95%

586917.6

16.9

16.5

6.0

5thPercentile

181303.0

8.3

4.1

2.8

Median

326775.0

15.0

12.1

3.7

95thPercentile

1045640.0

22.5

22.6

15.1

Min

10496.0

8.3

2.8

2.6

Max

1093648.0

25.7

29.0

15.9

Mean – 2SD

/

6.8

0.8

/

Mean + 2SD

1074612.4

23.7

27.1

11.2

CL: Confidence limit

CV: Coefficient of Variation

EC1.5: Extrapolated concentration for a 1.5 fold luciferase gene induction

Imax: Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured

Min: Minimal value

Max: Maximal value

n: Number of values

RLU: Relative Luminescence Unit

SD: Standard deviation

/: not applicable, negative calculated value

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the study, the test item PX-200 was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor, though with limitations due to test item precipitation.
Executive summary:

The objective of this study was to evaluate the potential of the test item, PX-200, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.

Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

 

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37 °C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

 

Results

First and second runs

All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered as validated.

 

Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.

At these tested concentrations:

-a slight to strong precipitate was observed in treated wells at the end of the 48-hour treatment at concentrations ≥ 125 μM, in both runs,

-no noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70% in both runs), therefore no geometric mean IC30 or IC50 was calculated,

-no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations, in either run. Moreover, the Imax values were < 1.5.

 

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative. Since precipitate was observed in the test item-treated wells at the end of the 48-hour treatment period, the luciferase activity may be underestimated. Therefore, the conclusion on the lack of activity cannot be drawn with sufficient confidence.

 

This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.

 

Conclusion

Under the experimental conditions of this study, the test item PX-200 was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor, though with limitations due to test item precipitation.

Endpoint:
skin sensitisation: in chemico
Type of information:
calculation (if not (Q)SAR)
Remarks:
Derek Nexus
Adequacy of study:
supporting study
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Qualifier:
no guideline followed
Principles of method if other than guideline:
The Derek Nexus system has been designed for the qualitative prediction of the possible toxicity of chemicals. The predictions made by Derek Nexus are intended as an aid to toxicological assessment and, where appropriate, should be used in conjunction with other methods. The absence of Derek Nexus predictions for any given compound in no way implies that the compound is, or is expected to be, non-toxic.

PX-200 was analysed for skin sensitisation using Derek Nexus in a number of mammalian species. In addition, irritation endpoints were investigated using the same software.
GLP compliance:
no
Type of study:
other: qualitative prediction
Details on study design:
Knowledge base version: Derek KB 2015 2.0
Species: Escherichia coli, Salmonella typhimurium, bacterium, dog, guinea pig, hamster, human, mammal, monkey, mouse, primate, rabbit, rat and rodent
Endpoints searched: Skin sensitisation, Irritation.
Processing constraints: The option to perceive tautomers was selected.
Other effects / acceptance of results:
PX-200 contained no structural moieties commonly associated with skin sensitisation as identified in by Derek Nexus. Furthermore, no irritation alerts were triggered.
Using DEREK for windows (the predecessor of Derek Nexus) a review of a guinea pig archive data for compounds eliciting allergic contact dermatitis and a local lymph node assay (LLNA) data set was conducted. DEREK for windows correctly predicted 82.9% and 73% of the results from the guinea pig and LLNA data (Fedorowicz et al., 2005).
More recently it has been reported that, based on human datasets collected from publicly available data, Derek Nexus shows accuracy of 66-76%, sensitivity of 82-88% and specificity of 58-71% (Guesne et al., 2014). In a separate study, Derek Nexus showed an accuracy of 72.73% (Kumar et al., 2016).
Interpretation of results:
GHS criteria not met
Conclusions:
PX-200 was analysed for skin sensitisation using Derek Nexus in a number of mammalian species. In addition, irritation endpoints were investigated using the software.
PX-200 contained no structural moieties commonly associated with skin sensitisation as identified in by Derek Nexus. Furthermore, no irritation alerts were triggered by PX-200.
Executive summary:

PX-200 was analysed for skin sensitisation using Derek Nexus in a number of mammalian species. In addition, irritation endpoints were investigated using the same software.

PX-200 contained no structural moieties commonly associated with skin sensitisation as identified in by Derek Nexus. Furthermore, no irritation alerts were triggered by PX-200.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

It is considered that the conduct of three assays in vitro, as recommended in the OECD guidelines, is a robust examination of the sensitization potential of PX-200. These negative results are supported by the negative predictions in silico and by the negative results in the Buehler test in guinea pigs, further highlighting the very low potential for sensitization in vivo due to PX-200.

 

In addition, a Local Lymph Node Assay in mice and a study of skin sensitization potential in human volunteers were conducted and have negative results.

 

The only positive result in vivo was seen in a Magnusson Kligman test in guinea pigs. This test involves the intradermal injection of the test item, in conjunction with Freund’s Adjuvant, a potent immune-stimulator.

The positive result obtained is considered likely to be an effect of the exposure considerations of the M&K maximisation test and the negative responses received from the EU in vitro screening strategy and the in silico screening in addition to the negative responses from in vivo animal and human exposure are a true reflection of the sensitising potential for the substance.