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Diss Factsheets

Administrative data

Description of key information

A number of skin sensitisation studies were included as part of NONS registrations. Buehler tests were conducted on A mixture of: 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl- 1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU10/A002/PU18; EC 406-530-2), and 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU12/A123; EC 406-690-3), a local lymph node assay (LLNA) was conducted with N,N''-(methylenedi-4,1-phenylene)bis [N'-octyl]urea (A124; EC 445-760-8), and a Guinea pig maximisation test was conducted with 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU12/A123; EC 406-690-3). No positive reactions for skin sensitisation were observed.


 


As part of an updated testing program, in vitro solubility assessments for skin sensitisation assays (hCLAT, keratinosens and DPRA) were conducted on:


- Reaction mass of 4,4'-methylenediphenyl diisocyanate and Amines, soya alkyl (A003; EC 905-837-3)


- Reaction product of MDI, Octadecylamine and Magnesium Hydroxide (PU05; EC 944-730-6)


- Reaction product of MDI and p-toluidine (PU07; EC 926-809-7)


- Reaction product of MDI, Octylamine and Cyclohexylamine (PU08; EC 926-119-6)


- Polyurea, produced by reacting diphenylmethane diisocyanate with octylamine and dodecyl amine (R03; EC 812-490-0) 


In the in vitro solubility assessments for skin sensitisation assays, all substances were deemed to be insoluble under the in vitro testing conditions and outside the applicability domains for the in vitro skin sensitisation assays.


 


Therefore, in vivo LLNA tests were conducted on:


- Reaction mass of 4,4'-methylenediphenyl diisocyanate and Amines, soya alkyl (A003; EC 905-837-3)


- Reaction product of MDI, Octadecylamine and Magnesium Hydroxide (PU05; EC 944-730-6)


- Reaction product of MDI and p-toluidine (PU07; EC 926-809-7)


- Reaction product of MDI, Octylamine and Cyclohexylamine (PU08; EC 926-119-6)


- Polyurea, produced by reacting diphenylmethane diisocyanate with octylamine and dodecyl amine (R03; EC 812-490-0)


Negative results were observed for all substances tested. Therefore, no sensitisation classification is required for MDI category members based on studies with category members indicating that they are non-sensitisers.

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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V (Buehler)
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Species:
guinea pig
Strain:
other: Himalayan albino
Sex:
not specified
Route:
other: Topical
Vehicle:
corn oil
Concentration / amount:
50% (w/w)
Day(s)/duration:
Not specified
Adequacy of induction:
not specified
Route:
other: Not reported
Vehicle:
corn oil
Concentration / amount:
25% w/w
Day(s)/duration:
Not specified
Adequacy of challenge:
not specified
No. of animals per dose:
Number of animals in test group: 20
Number of animals in negative control group: 10
Challenge controls:
Concentration of test material and vehicle used for each challenge: 25% (w/w) in corn oil
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
1
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 %
No. with + reactions:
1
Total no. in group:
10
Reading:
other: Not specified
Group:
positive control
Dose level:
Not specified
Remarks on result:
other: Positive control not specified

Maximum concentration not causing irritating effects in preliminary test: 50 %

Signs of irritation during induction: Three test group animals showed slight erythema after the last topical induction application.

Evidence of sensitisation of each challenge concentration: 0

Interpretation of results:
GHS criteria not met
Conclusions:
The test item exhibited no positive results in the challenge tests at a concentration of 25 % w/w in corn oil. Therefore, the test item is not classified for skin sensitisation.
Executive summary:

The test item was assessed for skin sensitisation at 50 % w/w suspension in corn oil at induction, and 25 % w/w suspension in corn for each challenge, following the Buehler method. Three test group animals showed slight erythema after the last topical induction application. However, no positive reactions were observed in the test item group at either assessment during the challenge. The test item is therefore not classified for skin sensitisation.

The study is a GLP compliant, guideline study and is suitable for assessment of this endpoint with restrictions.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
Version / remarks:
Modified Buehler test
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Species:
guinea pig
Strain:
other: Himalayan albino
Sex:
not specified
Route:
other: epicutaneous
Vehicle:
corn oil
Concentration / amount:
50% (w/w) suspension
Adequacy of induction:
other: Maximum concentration not causing irritating effects in preliminary test
No.:
#1
Route:
other: epicutaneous
Vehicle:
corn oil
Concentration / amount:
25% (w/w) suspension
Day(s)/duration:
Day 29
No.:
#2
Route:
other: epicutaneous
Vehicle:
corn oil
Concentration / amount:
5, 10 and 25% (w/w) suspensions on each animal
Day(s)/duration:
Day 36
No. of animals per dose:
Number of animals in test group: 20
Number of animals in negative control group: 10
Details on study design:
The use of 9 induction exposures is considered to be more rigorous than the Annex V method (see Buehler, Arch Dermat, 1965, vol. 91) and is therefore acceptable.
Positive control substance(s):
not specified
Positive control results:
Not reported
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
5
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
9
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
other: Not specified
Group:
positive control
Dose level:
Not specified
Remarks on result:
other: Positive control not specified

Signs of irritation during induction: 4/20 animals showed grade 1 erythema after the last induction exposure.

Evidence of sensitisation of each challenge concentration: 0 at all concentrations

Other observations: At first challenge, red spots were noted in 9 test animals. This minimal response is considered inconclusive evidence of sensitisation. A second challenge was therefore performed and no skin responses were seen. The overall conclusion is that the substance is not a skin sensitiser.

Interpretation of results:
GHS criteria not met
Conclusions:
At first challenge, red spots were noted in 9 test animals. This minimal response is considered inconclusive evidence of sensitisation. A second challenge was therefore performed and no skin responses were seen.
Executive summary:

The test item was assessed for skin sensitisation using Himalayan albino guinea pigs following EU Annex V guidelines. A modified Buehler study was conducted with 9 induction exposures administered every 2 or 3 days. At induction, 20 test animals received the test item in a 50% w/w suspension in corn oil and 10 test animals were designated as the negative control. The challenge exposure of 25% w/w suspension in corn oil was administered to the test group and negative control on Day 29. Red spots were noted in 9 test animals, which was considered inconclusive evidence of sensitisation. A second challenge was therefore performed with 5, 10 and 25 % w/w suspensions in corn oil administered to the test group and negative control on Day 36, in which no skin responses were seen. The overall conclusion is that the substance is not a skin sensitiser.

The study is a GLP compliant guideline experimental study acceptable with restrictions for assessment of this endpoint.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
other: Annex V
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Species:
guinea pig
Strain:
other: Himalayan spotted
Sex:
not specified
Route:
intradermal and epicutaneous
Vehicle:
petrolatum
Concentration / amount:
Intradermal: 1% w/w in ethanol
Topical: 15% w/w in vaseline
Adequacy of induction:
other: Maximum concentration not causing irritating effects in preliminary test: 10% (topical) and <1% (intradermal)
No.:
#1
Route:
other: epicutaneous
Vehicle:
petrolatum
Concentration / amount:
Topical: 0% w/w in vaseline
Topical: 10% w/w in vaseline
No. of animals per dose:
Number of animals in test group: 20
Number of animals in negative control group: 10
Positive control substance(s):
not specified
Positive control results:
Not reported
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
other: Not specified
Group:
positive control
Dose level:
Not specified
Remarks on result:
other: Positive control not specified

Signs of irritation during induction: Very slight erythema (grade 1) was recorded in 2 test animals 24 hours after topical application.

Evidence of sensitisation of each challenge concentration: None

Other observations: One control died during the study.

Interpretation of results:
GHS criteria not met
Conclusions:
No skin responses were seen at the challenge concentration.
Executive summary:

The test item was assessed for skin sensitisation using Himalayan spotted guinea pigs in a maximisation test following EU Annex V guidelines. At induction, 20 test animals received 1% test item w/w in ethanol intradermally and 15% test item w/w in vaseline topically and 10 test animals were designated as the negative control. The challenge exposure of 10% test item w/w in vaseline was administered topically to the test group and negative control. No skin responses were seen at the challenge concentration. The overall conclusion is that the substance is not a skin sensitiser.

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment of this endpoint.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
not specified
Vehicle:
dimethylformamide
Concentration:
0.25, 0.5, 1, 2.5 and 5%
The vehicle alone is administered to a control group
No. of animals per dose:
4
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
Not reported
Parameter:
SI
Value:
>= 1.17 - <= 1.21
Test group / Remarks:
0.25%
Remarks on result:
other: Range for all concentrations tested
Parameter:
SI
Value:
>= 1.17 - <= 1.21
Test group / Remarks:
0.5%
Remarks on result:
other: Range for all concentrations tested
Parameter:
SI
Value:
>= 1.17 - <= 1.21
Test group / Remarks:
1%
Remarks on result:
other: Range for all concentrations tested
Parameter:
SI
Value:
>= 1.17 - <= 1.21
Test group / Remarks:
2.5%
Remarks on result:
other: Range for all concentrations tested
Parameter:
SI
Value:
>= 1.17 - <= 1.21
Test group / Remarks:
5%
Remarks on result:
other: Range for all concentrations tested
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS:
Mortality and clinical signs: No mortality and no clinical signs were observed during the study.
Local irritation: No well-defined skin reaction is observed in the animals in the treated groups. A slight increase in ear thickness is noted in the animals that received the substance at a concentration of 5%, showing a slight irritating potential of the substance at this concentration.
Sensitizing potential: No lymphoproliferation was observed in the treated groups.
Under the experimental conditions of the test, the substance does not induce contact hypersensitivity.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the test, the substance does not induce contact hypersensitivity in an OECD 429 guideline study.
Executive summary:

The test item was assessed for skin sensitisation at 0.25, 0.5, 1, 2.5 and 5 % w/w suspensions in DMF alongside a control and positive reference substance in an OECD 429 guideline study. No mortality and no clinical signs were observed during the study and no well-defined skin reaction was observed in the animals in the treated groups. A slight increase in ear thickness was noted in the animals that received the substance at a concentration of 5%, showing a slight irritating potential of the substance at this concentration. However, no lymphoproliferation was observed in the treated groups and therefore under the experimental conditions of the test, the substance does not induce contact hypersensitivity.

 

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment of this endpoint.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21/09/2021 - 21/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
GLP compliance:
yes (incl. QA statement)
Type of study:
human Cell Line Activation Test (h-CLAT)
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442E study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 100 mg/mL (in saline or culture medium (RPMI 1640)) or 500 mg/mL in dimethyl sulfoxide (DMSO).

Pre-study solubility assessments were performed using dimethyl sulphoxide (DMSO), saline and culture medium. For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 502.59 mg/mL (culture medium), 505.35 mg/mL (saline) and 504.38 mg/mL (DMSO). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to 100 and 1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.
Vehicle / solvent control:
other: Saline, culture medium (RPMI 1640), DMSO
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in culture medium (RPMI-1640), saline or DMSO.

In culture medium (RPMI-1640), at the top concentration, it was noted that the test article was insoluble with the media sitting on top of it. At lower concentrations, a hazy suspension with a large mass of floating test article was noted.

In saline, it was noted that the test article was insoluble with the vehicle sitting on top of it, at all tested concentrations.

In DMSO, at the top concentration, it was noted that a thick off-white paste was formed. At lower concentrations, a hazy suspension with variably sized masses of floating test article was noted.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.
Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the the human Cell Line Activation Assay.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442E hCLAT test. The test article was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for the human Cell Line Activation Assay. It is concluded that the test article is outside the applicability domain for the human Cell Line Activation Assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21/09/2021 - 21/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442D study
Details on the study design:
The requirement of the OECD Test Guideline is that solubility will be assessed in dimethyl sulfoxide (DMSO), (sterile) water or culture medium with preference given to the first of the listed vehicles that produces a visually clear solution or a stable dispersion at a concentration of 200 mM, or for test articles with no defined molecular weight, a concentration of 40 mg/mL.

Pre-study solubility assessments were performed using dimethyl sulphoxide (DMSO), water and culture medium. For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 40.48 mg/mL (DMSO), 40.54 mg/mL (water) and 42.13 mg/mL (culture medium). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations: 30, 20, 10, 1 and 0.1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a suitable formulation for testing.
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in DMSO, water or culture medium (RPMI-1640).

In DMSO, at the top concentration, it was noted that a hazy white suspension with large masses of test article was formed. At 30, 20 and 10 mg/mL, it was noted that a hazy white suspension with many small masses of test article was formed. At 1 and 0.1 mg/mL, it was noted that a slightly hazy suspension with small floating masses of test article was formed.

In water, it was noted that the test article was insoluble with the vehicle sitting on top of it, at all tested concentrations.

In culture medium (RPMI-1640), at the top concentration, 30, 20 and 10 mg/mL, it was noted that a hazy suspension with test article floating on top, was formed. At 1 and 0.1 mg/mL, it was noted that a slightly hazy suspension with small particles of floating test article was formed.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.
Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the ARE-Nrf2 Luciferase Test.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442D Keratinosens test. The test article was insoluble in all suitable vehicles at sufficient concentration to meet the OECD Test Guidelines for the ARE-Nrf2 Luciferase Test Method. It is concluded that the test article is outside of the applicability domain for the ARE Nrf2 Luciferase Test Method.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16/11/2021 - 07/12/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 17 to 23 grams
- Housing: Animals were housed in groups of 5 during acclimitisation and in pairs during the main study
- Diet: 5LF2 EU Rodent Diet 14%, was provided ad libitum
- Water (e.g. ad libitum): Mains water was provided ad libitum via cage-mounted water bottles
- Acclimation period: 8 to 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 per hr
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Vehicle:
dimethylformamide
Concentration:
Preliminary screening study: 50% w/v in dimethylformamide
Main study: 0 (vehicle control), 10% w/v in dimethylformamide, 25% w/v in dimethylformamide and 50% w/v in dimethylformamide
No. of animals per dose:
Preliminary screening study: One female
Main study: Four female mice per group
Details on study design:
PRE-SCREEN TESTS:
The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in dimethylformamide) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and a score applied. Ear thickness measurements were performed using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Excessive local irritation was indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation. Death or signs of systemic toxicity/excessive irritation were not noted therefore, based on this information the dose levels selected for the main test were 10%, 25% and 50% w/v in dimethylformamide.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.

TREATMENT PREPARATION AND ADMINISTRATION: The test material was applied to the outer ear pinnae of mice via direct application (0.025 mL/pinna) using an automatic micro pipette. Mice were treated daily on test days 1, 2 and 3. Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article and were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of tritiated 3H-methyl thymidine.

On Day 6, a 20 μCi dose of 3HTdR was injected intravenously into each mouse. Approximately five hours later, the auricular lymph nodes were recovered from each animal. The lymph nodes collected were cut open and disaggregated. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible. After 5 minutes the pooled liquor was filtered into a second conical tube with any visible sediment remaining left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes, the supernatant was then discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes and the supernatant was again discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8 °C (nominal 4 °C). The following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added and analysis was conducted using a scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The DPM value for each test group was divided by the DPM for the control group to provide the Stimulation Index (SI) value for each test group.
Positive control results:
The historical positive control data confirms adequate performance of the assay using the positive control α hexylcinnamaldehyde.
Key result
Parameter:
SI
Value:
1.34
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.19
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.57
Test group / Remarks:
50%
Cellular proliferation data / Observations:
MORTALITY: All animals survived treatment with the test item.

CLINICAL OBSERVATIONS: There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50% w/v formulations of the test article.

BODY WEIGHTS: There was no indication of a treatment related effect on body weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The vehicle and test formulation application sites remained free of irritation.

Table 1: Group DPMs and Stimulation Index (SI)

Concentration (% w/v)  Group DPM  Stimulation Index (SI) 
Vehicle (dimethylformamide) 1098  NA 
10  1466  1.34
25  1312  1.19 
50  1723  1.57 

DPM: Disintegrations per minute

NA: Not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitisation potential of the test item was determined to be negative. The Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation. dimethylformamide
Executive summary:

The skin sensitization of the test item was investigated in a Local Lymph Node Assay following OECD Guideline 429 and Method B42 of Council Regulation (EC) No 440/2008. The test concentrations of 10, 25 and 50 % w/v in dimethylformamide were administered to three groups of four female mice on three consecutive days by topical application to the dorsal surface of each ear. Four vehicle control animals were similarly treated with Dimethylformamide. Three days after test item application each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of Tritiated 3H-methyl thymidine. After five hours, all animals were euthanized auricular lymph nodes were excised and prepared. After preparation, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.


 


All animals survived treatment with the test item and vehicle control and there were no clinical signs indicative of a systemic effect of treatment among mice or indication of a treatment related effect on body weight. The stimulation indices for the 10, 25 and 50% concentrations were determined to be 1.34, 1.19 and 1.57, respectively. Therefore, the Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29/09/2021 - 29/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
GLP compliance:
yes (incl. QA statement)
Type of study:
human Cell Line Activation Test (h-CLAT)
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442E study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 100 mg/mL (in saline or culture medium (RPMI 1640)) or 500 mg/mL in dimethyl sulfoxide (DMSO).

Solubility assessments of the test article were performed using culture medium, saline and DMSO.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 502.38 mg/mL (culture medium), 504.49 mg/mL (saline) or 500.34 mg/mL (DMSO). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations:
Culture medium: 200 and 1 mg/mL
Saline: 300 and 1 mg/mL
DMSO: 100 and 1 mg/mL.

Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.
Vehicle / solvent control:
other: Saline, culture medium (RPMI 1640), DMSO
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in culture medium (RPMI-1640), saline or DMSO.

In culture medium it was noted that insoluble test article was floating on top of the vehicle at all tested concentrations.

In saline it was noted that the test article was sitting on top of, or underneath, the vehicle at all tested concentrations.

In DMSO it was noted that the test article formed large insoluble particles at concentrations of 500.34 and 100 mg/mL. At 1 mg/mL it was noted that a suspension with small insoluble test article particles was formed.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.

Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the the human Cell Line Activation Assay.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442E hCLAT test. The test article was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for the human Cell Line Activation Assay. It is concluded that the test article is outside the applicability domain for the human Cell Line Activation Assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28/09/2021 - 28/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442D study
Details on the study design:
The requirement of the OECD Test Guideline is that solubility will be assessed in dimethyl sulfoxide (DMSO), (sterile) water or culture medium with preference given to the first of the listed vehicles that produces a visually clear solution or a stable dispersion at a concentration of 200 mM, or for test articles with no defined molecular weight, a concentration of 40 mg/mL.

Solubility assessments of the test article were performed using dimethyl sulfoxide (DMSO), (sterile) water and culture medium.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 40.56 mg/mL (DMSO), 43.11 mg/mL (water) and 42.28 mg/mL (culture medium). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations: 30, 20, 10 and 0.1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a suitable formulation for testing.
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in DMSO, water or culture medium (RPMI-1640).

In DMSO, it was noted that a hazy suspension with floating particles of test article was formed at all tested concentrations.

In water the test article was noted to be floating in the vehicle at concentrations of 43.11 and 0.1 mg/mL. At 30, 20 and 10 mg/mL a slightly hazy suspension with floating test article particles was observed.

In culture medium, it was noted that a slightly hazy suspension with floating masses or particles of test article, was formed at all tested concentrations.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.
Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the ARE-Nrf2 Luciferase Test.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442D Keratinosens test. The test article was insoluble in all suitable vehicles at sufficient concentration to meet the OECD Test Guidelines for the ARE-Nrf2 Luciferase Test Method. It is concluded that the test article is outside of the applicability domain for the ARE Nrf2 Luciferase Test Method.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23/11/2021 - 14/12/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 18 to 22 grams
- Housing: Animals were housed in groups of 5 during acclimitisation and in pairs during the main study
- Diet: 5LF2 EU Rodent Diet 14%, was provided ad libitum
- Water (e.g. ad libitum): Mains water was provided ad libitum via cage-mounted water bottles
- Acclimation period: 8 to 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 per hr
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Vehicle:
dimethylformamide
Concentration:
Preliminary screening study: 50% w/v in dimethylformamide
Main study: 0 (vehicle control), 10% w/v in dimethylformamide, 25% w/v in dimethylformamide and 50% w/v in dimethylformamide
No. of animals per dose:
Preliminary screening study: One female
Main study: Four female mice per group
Details on study design:
PRE-SCREEN TESTS:
The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in dimethylformamide) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and a score applied. Ear thickness measurements were performed using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Excessive local irritation was indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation. Death or signs of systemic toxicity/excessive irritation were not noted therefore, based on this information the dose levels selected for the main test were 10%, 25% and 50% w/v in dimethylformamide.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.

TREATMENT PREPARATION AND ADMINISTRATION: The test material was applied to the outer ear pinnae of mice via direct application (0.025 mL/pinna) using an automatic micro pipette. Mice were treated daily on test days 1, 2 and 3. Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article and were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of tritiated 3H-methyl thymidine.

On Day 6, a 20 μCi dose of 3HTdR was injected intravenously into each mouse. Approximately five hours later, the auricular lymph nodes were recovered from each animal. The lymph nodes collected were cut open and disaggregated. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible. After 5 minutes the pooled liquor was filtered into a second conical tube with any visible sediment remaining left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes, the supernatant was then discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes and the supernatant was again discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8 °C (nominal 4 °C). The following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added and analysis was conducted using a scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The DPM value for each test group was divided by the DPM for the control group to provide the Stimulation Index (SI) value for each test group.
Positive control results:
The historical positive control data confirms adequate performance of the assay using the positive control α hexylcinnamaldehyde.
Key result
Parameter:
SI
Value:
1.62
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
0.95
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
0.75
Test group / Remarks:
50%
Cellular proliferation data / Observations:
MORTALITY: All animals survived treatment with PU05.

CLINICAL OBSERVATIONS: There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50% w/v formulations of the test article.

BODY WEIGHTS: There was no indication of a treatment related effect on body weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The vehicle and test formulation application sites remained free of irritation.

Table: Group DPMs and Stimulation Index (SI)

Concentration (% w/v)  Group DPM  Stimulation Index (SI) 
Vehicle (dimethylformamide)  2111  NA 
10  3420  1.62 
25  2002  0.95 
50  1592  0.75 

DPM: Disintegrations per minute

NA: Not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitisation potential of the test item was determined to be negative. The Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.
Executive summary:

The skin sensitization of the test item was investigated in a Local Lymph Node Assay following OECD Guideline 429 and Method B42 of Council Regulation (EC) No 440/2008. The test concentrations of 10, 25 and 50 % w/v in dimethylformamide were administered to three groups of four female mice on three consecutive days by topical application to the dorsal surface of each ear. Four vehicle control animals were similarly treated with Dimethylformamide. Three days after test item application each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of Tritiated 3H-methyl thymidine. After five hours, all animals were euthanized auricular lymph nodes were excised and prepared. After preparation, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.


 


All animals survived treatment with the test item and vehicle control and there were no clinical signs indicative of a systemic effect of treatment among mice or indication of a treatment related effect on body weight. The stimulation indices for the 10, 25 and 50% concentrations were determined to be 1.62, 0.95 and 0.75, respectively. Therefore, the Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23/09/2021 - 23/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442C study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM. Particulate suspensions are not appropriate in this assay.

Solubility assessments of the test article were performed using acetonitrile, water, 1:1 mixture of water:acetonitrile, isopropanol, acetone and 1:1 mixture of acetone:acetonitrile.

As none of the above solvents were found to be suitable, solubilisation in DMSO followed by dilution in acetonitrile at a 1:9 and 1:1 DMSO:acetonitrile, were also tested.

The molecular weight of the test article is 464.57 g/mol, therefore a 100 mM solution is equivalent to 46.46 mg/mL.

For each vehicle tested, the test article was weighed out and made up to the appropriate volume to give a concentration of 46.46 mg/mL (equivalent to 100 mM).

Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.


Vehicle / solvent control:
other: acetonitrile, water, 1:1 mixture of water:acetonitrile, isopropanol, acetone, 1:1 mixture of acetone:acetonitrile, solubilisation in DMSO followed by dilution in acetonitrile at a 1:9 and 1:1 DMSO:acetonitrile
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in any of the tested vehicles.

At a concentration of 46.46 mg/mL, an opaque white suspension with the test article settling to the bottom was formed in acetonitrile, 1:1 mixture of water:acetonitrile, isopropanol, acetone, 1:1 mixture of acetone:acetonitrile, 1:9 mixture of DMSO:acetonitrile and 1:1 mixture of DMSO:acetonitrile. At the same concentration, a clear liquid with insoluble test article floating on top was produced in water.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.
Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the Direct Peptide Reactivity Assay.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442c Direct Peptide Reactivity Assay. The test article was insoluble in all suitable vehicles at sufficient concentration to meet the OECD Test Guidelines for the Direct Peptide Reactivity Assay. It is concluded that the test article is outside of the applicability domain for the Direct Peptide Reactivity Assay Test Method.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24/09/2021 - 24/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
GLP compliance:
yes (incl. QA statement)
Type of study:
human Cell Line Activation Test (h-CLAT)
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442E study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 100 mg/mL (in saline or culture medium (RPMI 1640)) or 500 mg/mL in dimethyl sulfoxide (DMSO).

Solubility assessments of the test article were performed using culture medium, saline and DMSO.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 500.15 mg/mL (culture medium), 503.28 mg/mL (saline) or 502.26 mg/mL (DMSO). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations:
Culture medium and DMSO: 200.00 and 1.00 mg/mL
Saline: 251.64 and 1.00 mg/mL

Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

Vehicle / solvent control:
other: Saline, culture medium (RPMI 1640), DMSO
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in culture medium (RPMI-1640), saline or DMSO.

In culture medium (RPMI-1640), at the top concentration and at 200 mg/mL, it was noted that the test article remained at the bottom of the volumetric, with the media sat on top. At a concentration of 1 mg/mL, the test article remained insoluble, floating on top of the media.

In saline, it was noted that the test article was very hydrophobic, with the test article surrounded by or floating on top of the vehicle, at all tested concentrations.

In DMSO, at the top concentration, it was noted that the test article soaked up the vehicle and solidified. At 200 and 1 mg/mL, it was noted that a white suspension with large masses of test article, was formed.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.


Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the the human Cell Line Activation Assay.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442E hCLAT test. The test article was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for the human Cell Line Activation Assay. It is concluded that the test article is outside the applicability domain for the human Cell Line Activation Assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24/09/2021 - 24/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442D study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 200 mM, or for test articles with no defined molecular weight, a concentration of 40 mg/mL.

Solubility assessments of the test article were performed using dimethyl sulfoxide (DMSO), (sterile) water and culture medium.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 94.22 mg/mL (DMSO), 94.40 mg/mL (water) and 95.14 mg/mL (culture medium). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations: 10 and 0.1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a suitable formulation for testing.

Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in DMSO, water or culture medium (RPMI-1640).

In DMSO, at the top concentration, it was noted that a thick white suspension with large masses of test article was formed. At 10 mg/mL, it was noted that a white suspension with large masses of test article was formed. At 0.1 mg/mL, it was noted that lots of insoluble test article particles were formed.

In water, it was noted that the test article was insoluble and floating on top of the vehicle, at all tested concentrations.

In culture medium (RPMI-1640), it was noted that the test article was insoluble and floating on top of the vehicle, at all tested concentrations

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.

Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the ARE-Nrf2 Luciferase Test.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442D Keratinosens test. The test article was insoluble in all suitable vehicles at sufficient concentration to meet the OECD Test Guidelines for the ARE-Nrf2 Luciferase Test Method. It is concluded that the test article is outside of the applicability domain for the ARE Nrf2 Luciferase Test Method.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/11/2021 - 24/11/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, Blackthorn, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 18 to 23 grams
- Housing: Animals were housed in groups of 5 during acclimitisation and in pairs during the main study
- Diet: 5LF2 EU Rodent Diet 14%, was provided ad libitum
- Water (e.g. ad libitum): Mains water was provided ad libitum via cage-mounted water bottles
- Acclimation period: 8 to 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 per hr
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Vehicle:
other: 80% v/v acetone in olive oil
Concentration:
Preliminary study: 10% w/v in 80% v/v acetone in olive oil
Main study: 0 (vehicle control), 2.5%, 5% and 10% w/v in 80% v/v acetone in olive oil
No. of animals per dose:
Preliminary screening study: One female
Main study: Four female mice per group
Details on study design:
PRE-SCREEN TESTS:
The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (10% w/v in 80% v/v acetone in olive oil) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and a score applied. Ear thickness measurements were performed using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Excessive local irritation was indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation. Death or signs of systemic toxicity/excessive irritation were not noted therefore, based on this information the dose levels selected for the main test were 2.5%, 5% and 10% w/v in 80% v/v acetone in olive oil.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.

TREATMENT PREPARATION AND ADMINISTRATION: The test material was applied to the outer ear pinnae of mice via direct application (0.025 mL/pinna) using an automatic micro pipette. Mice were treated daily on test days 1, 2 and 3. Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article and were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of tritiated 3H-methyl thymidine.

On Day 6, a 20 μCi dose of 3HTdR was injected intravenously into each mouse. Approximately five hours later, the auricular lymph nodes were recovered from each animal. The lymph nodes collected were cut open and disaggregated. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible. After 5 minutes the pooled liquor was filtered into a second conical tube with any visible sediment remaining left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes, the supernatant was then discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes and the supernatant was again discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8 °C (nominal 4 °C). The following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added and analysis was conducted using a scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The DPM value for each test group was divided by the DPM for the control group to provide the Stimulation Index (SI) value for each test group.
Positive control results:
The historical positive control data given confirms adequate performance of the assay.
Key result
Parameter:
SI
Test group / Remarks:
2.5% w/v in 80% v/v acetone in olive oil
Remarks on result:
not determinable because of methodological limitations
Remarks:
*Due to an unexpected death, only 3 animals remained at the scheduled necropsy/lymph node removal. The SI value for this group has not been reported.
Key result
Parameter:
SI
Value:
0.91
Test group / Remarks:
5% w/v in 80% v/v acetone in olive oil
Key result
Parameter:
SI
Value:
0.69
Test group / Remarks:
10% w/v in 80% v/v acetone in olive oil
Cellular proliferation data / Observations:
MORTALITY: A single animal died unexpectedly prior to the scheduled necropsy/lymph node removal, however this was not related to treatment with the test item.

CLINICAL OBSERVATIONS: There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 2.5, 5 or 10% w/v formulations of the test article.

BODY WEIGHTS: There was no indication of a treatment related effect on body weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The vehicle and test formulation application sites remained free of irritation. Greasy fur behind the ears and to the back of the neck were noted in all animals on Days 1 to 3 and also on Day 4 for animals in the vehicle control and 2.5 % in 80% v/v acetone in olive oil groups.

Table: Group DPMs and Stimulation Index (SI)

Concentration (% w/v)  Group DPM  Stimulation Index (SI) 
Vehicle (80% v/v acetone in olive oil)  1345  NA 
2.5  577  -* 
5  1221  0.91 
10  924  0.69 

DPM: Disintegrations per minute

NA: Not applicable

-*: Due to an unexpected death, only 3 animals for Group 2 remained at the scheduled necropsy/lymph node removal. The SI value for this group has not been reported.

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitisation potential of the test item was determined to be negative. The Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.

Executive summary:

The skin sensitization of the test item was investigated in a Local Lymph Node Assay following OECD Guideline 429 and Method B42 of Council Regulation (EC) No 440/2008. The test concentrations of 2.5, 5 and 10 % w/v in 80% v/v acetone in olive oil were administered to three groups of four female mice on three consecutive days by topical application to the dorsal surface of each ear. Four vehicle control animals were similarly treated with 80% v/v acetone in olive oil. . Three days after test item application each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of Tritiated 3H-methyl thymidine. After five hours, all animals were euthanized auricular lymph nodes were excised. After preparation, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

A single animal died unexpectedly at 2.5% w/v treatment group, however this was not related to treatment with the test item, but the stimulation indices could not be calculated. There were no clinical signs indicative of a systemic effect of treatment among mice or indication of a treatment related effect on body weight. The stimulation indices for the 5 % and 10 % w/v concentrations were determined to be 0.91 and 0.69, respectively. Therefore, the Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21/09/2021 - 21/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
GLP compliance:
yes (incl. QA statement)
Type of study:
human Cell Line Activation Test (h-CLAT)
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442E study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 100 mg/mL (in saline or culture medium (RPMI 1640)) or 500 mg/mL in dimethyl sulfoxide (DMSO).

Solubility assessments of the test article were performed using culture medium, saline and DMSO.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 504.87 mg/mL (culture medium), 502.42 mg/mL (saline) and 511.71 mg/mL (DMSO). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to 100 and 1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.


Vehicle / solvent control:
other: Saline, culture medium (RPMI 1640), DMSO
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in culture medium (RPMI-1640), saline or DMSO.

In culture medium (RPMI-1640), saline and DMSO, it was noted that large masses of insoluble test article were floating in the vehicle, at all concentrations.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.



Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the the human Cell Line Activation Assay.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442E hCLAT test. The test article was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for the human Cell Line Activation Assay. It is concluded that the test article is outside the applicability domain for the human Cell Line Activation Assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20/09/2021 - 20/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442D study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 200 mM, or for test articles with no defined molecular weight, a concentration of 40 mg/mL.

As the test article has no defined molecular weight, the target concentration was 40.0 mg/mL.

Solubility assessments of the test article were performed using dimethyl sulfoxide (DMSO), (sterile) water and culture medium.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 44.70 mg/mL (DMSO), 46.46 mg/mL (water) and 40.44 mg/mL (culture medium). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations: 30, 20, 10 and 0.1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a suitable formulation for testing.

Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in DMSO, water or culture medium where a single mass of test article was noted floating in each vehicle at every concentration assessed.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.


Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the ARE-Nrf2 Luciferase Test.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442D Keratinosens test. The test article was insoluble in all suitable vehicles at sufficient concentration to meet the OECD Test Guidelines for the ARE-Nrf2 Luciferase Test Method. It is concluded that the test article is outside of the applicability domain for the ARE Nrf2 Luciferase Test Method.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/11/2021 - 23/11/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 18 to 21 grams
- Housing: Animals were housed in groups of 5 during acclimitisation and in pairs during the main study
- Diet: 5LF2 EU Rodent Diet 14%, was provided ad libitum
- Water (e.g. ad libitum): Mains water was provided ad libitum, via cage-mounted water bottles
- Acclimation period: 8 to 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 per hr
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Vehicle:
other: 80% v/v acetone in olive oil
Concentration:
Preliminary study: 10% w/v in 80% v/v acetone in olive oil
Main study: 0 (vehicle control), 2.5%, 5% and 10% w/v in 80% v/v acetone in olive oil
No. of animals per dose:
Preliminary screening study: One female
Main study: Four female mice per group
Details on study design:
PRE-SCREEN TESTS:
The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (10% w/v in 80% v/v acetone in olive oil) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and a score applied. Ear thickness measurements were performed using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Excessive local irritation was indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation. Death or signs of systemic toxicity/excessive irritation were not noted therefore, based on this information the dose levels selected for the main test were 2.5%, 5% and 10% w/v in 80% v/v acetone in olive oil.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.

TREATMENT PREPARATION AND ADMINISTRATION: The test material was applied to the outer ear pinnae of mice via direct application (0.025 mL/pinna) using an automatic micro pipette. Mice were treated daily on test days 1, 2 and 3. Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article and were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of tritiated 3H-methyl thymidine.

On Day 6, a 20 μCi dose of 3HTdR was injected intravenously into each mouse. Approximately five hours later, the auricular lymph nodes were recovered from each animal. The lymph nodes collected were cut open and disaggregated. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible. After 5 minutes the pooled liquor was filtered into a second conical tube with any visible sediment remaining left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes, the supernatant was then discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes and the supernatant was again discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8 °C (nominal 4 °C). The following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added and analysis was conducted using a scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The DPM value for each test group was divided by the DPM for the control group to provide the Stimulation Index (SI) value for each test group.
Positive control results:
The historical positive control data confirms adequate performance of the assay using the positive control α hexylcinnamaldehyde.
Key result
Parameter:
SI
Value:
0.99
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
1.25
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.87
Test group / Remarks:
10%
Cellular proliferation data / Observations:
MORTALITY: All animals survived treatment with the test item

CLINICAL OBSERVATIONS: There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 2.5, 5 or 10% w/v formulations of the test article.

BODY WEIGHTS: There was no indication of a treatment related effect on body weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The vehicle and test formulation application sites remained free of irritation. Greasy fur behind the ears and to the back of the neck were noted in all animals on Days 1 to 3,on Day 4 for animals in the vehicle control and 5 % in 80% v/v acetone in olive oil groups and on Days 5 and 6 in the vehicle control group.

Table: Group DPMs and Stimulation Index (SI)

Concentration (% w/v)  Group DPM  Stimulation Index (SI) 
Vehicle (80% v/v acetone in olive oil)  1399  NA 
2.5  1382  0.99 
5  1750  1.25 
10  1220  0.87 

DPM: Disintegrations per minute

NA: Not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitisation potential of the test item was determined to be negative. The Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.
Executive summary:

The skin sensitization of the test item was investigated in a Local Lymph Node Assay following OECD Guideline 429 and Method B42 of Council Regulation (EC) No 440/2008. The test concentrations of 2.5, 5 and 10 % w/v in 80% v/v acetone in olive oil were administered to three groups of four female mice on three consecutive days by topical application to the dorsal surface of each ear. Four vehicle control animals were similarly treated with 80% v/v acetone in olive oil. Three days after test item application each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of Tritiated 3H-methyl thymidine. After five hours, all animals were euthanized auricular lymph nodes were excised. After preparation, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All animals survived treatment with the test item and vehicle control and there were no clinical signs indicative of a systemic effect of treatment among mice or indication of a treatment related effect on body weight .The stimulation indices for the 2.5, 5 and 10 % concentrations were determined to be 0.99, 1.25 amd 0.87, respectively. Therefore, the Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23/09/2021 - 23/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
GLP compliance:
yes (incl. QA statement)
Type of study:
human Cell Line Activation Test (h-CLAT)
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442E study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 100 mg/mL (in saline or culture medium (RPMI 1640)) or 500 mg/mL in dimethyl sulfoxide (DMSO).

Solubility assessments of the test article were performed using culture medium, saline and DMSO.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 500.75 mg/mL (culture medium), 502.79 mg/mL (saline) or 503.58 mg/mL (DMSO). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations: 100 and 1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.



Vehicle / solvent control:
other: Saline, culture medium (RPMI 1640), DMSO
Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in culture medium (RPMI-1640), saline or DMSO.

In culture medium (RPMI-1640), it was noted that a hazy suspension with floating test article particles was formed, at all tested concentrations.

In saline, it was noted that the vehicle remained clear, with floating test article particles, at all tested concentrations.

In DMSO, at the top concentration, it was noted that a thick beige paste was formed. At 100 and 1 mg/mL, it was noted that a hazy suspension with visible test article particles, was formed.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.




Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the the human Cell Line Activation Assay.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442E hCLAT test. The test article was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for the human Cell Line Activation Assay. It is concluded that the test article is outside the applicability domain for the human Cell Line Activation Assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22/09/2021 - 22/09/2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary solubility assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details of test system:
other: Preliminary solubility trial prior to conducting an OECD 442D study
Details on the study design:
The requirement of the OECD Test Guideline was that preference would be given to the first of the listed vehicles that produced a visually clear solution or a stable dispersion at a concentration of 200 mM, or for test articles with no defined molecular weight, a concentration of 40 mg/mL.

Solubility assessments of the test article were performed using dimethyl sulfoxide (DMSO), (sterile) water and culture medium.

For each vehicle, the test article was weighed out and made up to the appropriate volume to give a concentration of 42.55 mg/mL (DMSO), 43.67 mg/mL (water) and 42.20 mg/mL (culture medium). Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a solution.

As solubility was not achieved at the above concentrations, the preparations were subsequently diluted with the respective solvent to the following concentrations: 30, 20, 10 and 0.1 mg/mL. Warming at 37°C, vortex mixing and ultrasonication were used to try and achieve a suitable formulation for testing.

Key result
Remarks on result:
not determinable because of methodological limitations
Remarks:
No suitable formulation could be obtained in any vehicle
Other effects / acceptance of results:
Solubility data indicated that the test article was not directly soluble in DMSO, water or culture medium (RPMI-1640).

In DMSO, it was noted that a cloudy suspension with visible floating test article particles was formed, at all tested concentrations.

In water, it was noted that the test article was insoluble with the test article floating on top of the vehicle, at all tested concentrations.

In culture medium (RPMI-1640), at the top concentration, it was noted that the test article was insoluble with the test article floating on top of the vehicle. At 30, 20 and 10 mg/mL, it was noted that the vehicle became slightly cloudy with the test article floating on top of the vehicle. At 0.1 mg/mL, it was noted that solid test article was floating in the vehicle.

The test article was found not to be soluble in any of the above listed solvents at the concentrations stated in the OECD Test Guideline.



Interpretation of results:
study cannot be used for classification
Conclusions:
The skin sensitisation potential of the test item could not be determined and was outside of the applicability domain for the ARE-Nrf2 Luciferase Test.
Executive summary:

The skin sensitisation potential of the test item could not be determined because the substance was outside the applicability domain of the in vitro OECD 442D Keratinosens test. The test article was insoluble in all suitable vehicles at sufficient concentration to meet the OECD Test Guidelines for the ARE-Nrf2 Luciferase Test Method. It is concluded that the test article is outside of the applicability domain for the ARE Nrf2 Luciferase Test Method.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09/11/2021 - 30/11/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 18 to 22 grams
- Housing: Animals were housed in groups of 5 during acclimitisation and in pairs during the main study
- Diet: 5LF2 EU Rodent Diet 14%, was provided ad libitum
- Water (e.g. ad libitum): Mains water was provided ad libitum via cage-mounted water bottles
- Acclimation period: 8 to 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 per hr
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Vehicle:
propylene glycol
Concentration:
Preliminary screening study: 50% w/v in propylene glycol
Main study: 0 (vehicle control), 10% w/v in propylene glycol, 25% w/v in propylene glycol and 50% w/v in propylene glycol
No. of animals per dose:
Preliminary screening study: One female
Main study: Four female mice per group
Details on study design:
PRE-SCREEN TESTS:
The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in propylene glycol) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and a score applied. Ear thickness measurements were performed using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Excessive local irritation was indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation. Death or signs of systemic toxicity/excessive irritation were not noted therefore, based on this information the dose levels selected for the main test were 10%, 25% and 50% w/v in propylene glycol.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.

TREATMENT PREPARATION AND ADMINISTRATION: The test material was applied to the outer ear pinnae of mice via direct application (0.025 mL/pinna) using an automatic micro pipette. Mice were treated daily on test days 1, 2 and 3. Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article and were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of tritiated 3H-methyl thymidine.

On Day 6, a 20 μCi dose of 3HTdR was injected intravenously into each mouse. Approximately five hours later, the auricular lymph nodes were recovered from each animal. The lymph nodes collected were cut open and disaggregated. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible. After 5 minutes the pooled liquor was filtered into a second conical tube with any visible sediment remaining left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes, the supernatant was then discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes and the supernatant was again discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8 °C (nominal 4 °C). The following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added and analysis was conducted using a scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The DPM value for each test group was divided by the DPM for the control group to provide the Stimulation Index (SI) value for each test group.
Positive control results:
The historical positive control data confirms adequate performance of the assay using the positive control α hexylcinnamaldehyde.
Key result
Parameter:
SI
Value:
1.24
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
0.85
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.12
Test group / Remarks:
50%
Cellular proliferation data / Observations:
MORTALITY: All animals survived treatment with the test item.

CLINICAL OBSERVATIONS: There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50% w/v formulations of the test article.

BODY WEIGHTS: There was no indication of a treatment related effect on body weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The vehicle and test formulation application sites remained free of irritation. Greasy fur behind the ears and to the back of the neck were noted in all vehicle control animals on Day 1.

Table: Group DPMs and Stimulation Index (SI)

Concentration (% w/v)  Group DPM  Stimulation Index (SI) 
Vehicle (Propylene glycol) 1290  NA 
10  1599  1.24
25  1099  0.85 
50  1149  1.12 

DPM: Disintegrations per minute

NA: Not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitisation potential of the test item was determined to be negative. The Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.
Executive summary:

The skin sensitization of the test item was investigated in a Local Lymph Node Assay following OECD Guideline 429 and Method B42 of Council Regulation (EC) No 440/2008. The test concentrations of 10, 25 and 50 % w/v in propylene glycol were administered to three groups of four female mice on three consecutive days by topical application to the dorsal surface of each ear. Four vehicle control animals were similarly treated with propylene glycol. Three days after test item application each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of Tritiated 3H-methyl thymidine. After five hours, all animals were euthanized auricular lymph nodes were excised. After preparation, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

All animals survived treatment with the test item and vehicle control and there were no clinical signs indicative of a systemic effect of treatment among mice or indication of a treatment related effect on body weight. The stimulation indices for the 10, 25 and 50% concentrations were determined to be 1.24, 0.85 and 1.12, respectively. Therefore, the Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.

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

No sensitisation classification is required for MDI category members based on studies with category members indicating that they are non-sensitisers.