Registration Dossier

Administrative data

Description of key information

Skin sensitisation studies are available for three substances within the TDI-I category. Local Lymph node assays (LLNA) are available for Diurea 8 (Sanders 2012), Tetraurea 2 (Sanders, 2011e), and reaction product of m-tolylidene diisocyanate, cyclohexylamine, cyclohex-1,2 -ylenediamine and (Z)-octadec-9 -enylamine (Toussaint 2009). Diurea 8 and Tetraurea 2 were considered to be non-sensitising under the conditions of the tests with a highest Stimulation Index of 1.24 and 1.13 recorded at the 5 % concentration in 1 % pluronic L92 in distilled water for Diurea 8 and Tetraurea 2, respectively. At the highest concentrations tested a Stimulation Index of 1.15 was reported for Diurea 8 and 1.02 for Tetraurea 2.

In the LLNA study conducted with reaction product of m-tolylidene diisocyanate, cyclohexylamine, cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine (Toussaint 2009) the highest Stimulation Index observed was 1.49 at the 50 % concentration in DMSO, the highest concentration tested. The study concluded that the substance was a senstiser using a Stimulation Index 1.4 as the basis for classification as a sensitiser. The study has been thoroughly reviewed in accordance with the ECHA guidance for non-guideline studies and has been deemed unreliable; therefore Keratino-Sens and U-Sens assays have been commissioned to review the applicability of the result. During the preliminary solubility work conducted prior to commencement of the Keratino-Sens assay, the laboratory concluded that the test item was not soluble, or able to form homogeneous suspensions, in the standard solvents used for this assay and therefore the test item was not suitable for this type of assay (Woutersen 2018). As the Keratino-Sens assay could not be performed, the U-SENS assay was also cancelled; the U-SENS assay is considered to be technically not feasible based on the results of the solubility test for the Keratino-Sens assay and the general structure of the substance. Additionally, if a U-SENS assay only is conducted, this would be the only in vitro result, which would not be sufficient to conclude whether the substance is a skin sensitiser or not. Therefore, no conclusion on skin sensitisation properties and potency can be drawn from alternative testing, and an LLNA study was commissioned (van Sas 2018). The LLNA conducted according to OECD 429, US EPA OPPTS 870.2600 and EU Method B.42 concluded that reaction product of m-tolylidene diisocyanate, cyclohexylamine, cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine was not a skin sensitiser (van Sas 2018).

Therefore, all of the TDI-I category members are not classified as skin sensitisers.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 20.1 to 24.7 g
- Housing: Group housed (5 females) in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24, daily mean temperature of 22°C
- Humidity (%): 42 to 61
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethylformamide
Concentration:
0, 1, 2, 5 % w/w
No. of animals per dose:
5 females
Details on study design:
- Vehicle choice: The vehicle, N,N-dimethylformamide, was selected on the basis of maximizing the solubility based on trial preparations performed at the laboratory.

PRE-SCREEN TESTS:
- Concentrations: 5, 10, 25 and 40 % w/w
- Method: The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two females per concentration were treated on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing and on Days 1 and 3, and 6.
- Ear thickness measurements, systemic toxicity and irritation: At a 40%, 25% and 10% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At a 5% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.

MAIN STUDY
- Name of test method: LLNA
- Criteria used to consider a positive response: SI > 3
- Treatment preparation: The dosing formulations were prepared daily, kept at room temperature and dosed within 4 hours after adding the vehicle to the test item. The concentrations were stirred with a magnetic stirrer immidiately prior to dosing.
- Treatment administration: The dorsal surface of both ears was topically treated with 25 microlitres/ear with the test item, at approximately the same time on each day on Days 1, 2 and 3. On Day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol 20%. The draining (auricular) lymph node of each ear was excised and the relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS and a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze. LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. The LNC were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day. On Day 7, precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter, with counting time to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first.
- Observations: Animals were observed for general health/mortality and moribundity twice daily. Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing) for reaction to dosing. Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy). Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Disintegrations Per Minute (DPM) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. Consideration is also given to the EC3 value (the estimated test item concentration that will give a SI =3).
Positive control results:
The SI values calculated for the item concentrations 5, 10 and 25% were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2%.
Parameter:
SI
Value:
1
Variability:
0.2
Test group / Remarks:
0 % w/w
Remarks on result:
other:
Remarks:
Mean SI value
Parameter:
SI
Value:
1.9
Variability:
0.2
Test group / Remarks:
1 % w/w
Remarks on result:
other:
Remarks:
Mean SI value
Parameter:
SI
Value:
0.6
Variability:
0.1
Test group / Remarks:
2 % w/w
Remarks on result:
other:
Remarks:
Mean SI value
Parameter:
SI
Value:
0.5
Variability:
0.1
Test group / Remarks:
5 % w/w
Remarks on result:
other:
Remarks:
Mean SI value
Cellular proliferation data / Observations:
- Skin Reactions / Irritation: The very slight erythema of the ears as shown by the animals of the highest dose group was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of test item treated animals between Days 2 and 5, which did not hamper scoring of the skin reactions.
- Systemic Toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopic Examination of the Lymph Nodes and Surrounding Area: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity Measurements and SI Values: Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 343, 103 and 82 DPM, respectively. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.9, 0.6 and 0.5, respectively.
- Vehicle control: The mean DPM/animal value for the vehicle control group was 178 DPM.
- Conclusion: Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 5%, the substance was not considered to be a skin sensitizer. It was established that the EC3 value exceeds 5% and, based on the absence of a dose response, it is not to be expected that higher concentrations would lead to an SI ≥ 3. Based on these results, the substance would not be regarded as a skin sensitizer.

Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

group

TS1

(%)

animal

Size nodes2

DPM3/ animal

mean

DPM ± SEM4

mean

SI ± SEM

left

right

 

 

 

 

 

 

 

 

1

0

1

n

n

137

178

±

29

1.0

±

0.2

 

 

2

n

n

230

 

 

3

n

n

153

 

 

4

n

n

109

 

 

5

n

n

261

 

 

 

 

 

 

 

 

 

 

 

 

2

2

6

n

n

352

343

±

37

1.9

±

0.2

 

 

7

n

n

305

 

 

8

n

n

315

 

 

9

n

n

479

 

 

10

n

n

262

 

 

 

 

 

 

 

 

 

 

 

 

3

5

11

n

n

80

103

±

20

0.6

±

0.1

 

 

12

n

n

82

 

 

13

n

n

83

 

 

14

n

n

182

 

 

15

n

n

87

 

 

 

 

 

 

 

 

 

 

 

 

4

10

16

n

n

107

82

±

21

0.5

±

0.1

 

 

17

n

n

26

 

 

18

n

n

111

 

 

19

n

n

37

 

 

20

n

n

129

 

 

 

 

 

 

 

 

1  TS = test item (% w/w).

2  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).

3    DPM= Disintegrations per minute.

4    SEM = Standard Error of the Mean.

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 5%, the substance was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 5%.
Executive summary:

The substance was administered to three experimental groups of five female mice at concentrations of 0 (vehicle control), 1, 2 or 5% w/w on three consecutive days, by open application on the ears. Three days after the last exposure, all animals were injected with 3H-methyl thymidine and, after five hours, the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, 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 auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. There was no indication that the test item elicits a SI ≥ 3 when tested up to 5% and, based on the absence of a dose response, it is not to be expected that higher concentrations would lead to an SI ≥ 3, so it was established that the EC3 value exceeds 5%. Therefore, the substance was not considered to be a skin sensitizer.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 29 November 2011 and 20 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
other: 1% pluronic L92 in distilled water
Concentration:
Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water
No. of animals per dose:
Groups of four mice were treated
Details on study design:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test item at a concentrations of 10% w/w in 1% pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.
3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None provided.
Positive control results:
Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.
Test Item: α Hexylcinnamaldehyde, tech., 85%
Project number: 41103440
Study dates: 01 December 2011 to 07 December 2011
Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as an emulsion in 1% pluronic L92 in distilled water at a concentration of 25% v/v. A further control group of five animals was treated with 1% pluronic L92 in distilled water alone.
Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in
1% pluronic L92 in distilled water Stimulation Index Result
25 7.86 Positive
Conclusion. α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Value:
0.95
Test group / Remarks:
Four animals were treated with 50 μl (25 μl per ear) of the test item as an emulsion/suspension in 1% pluronic L92 in distilled water at a concentrations of 2.5% w/w.
Remarks on result:
other: A stimulation index of less than 3 was recorded for test item at concentrations of 10%, 5% and 2.5% v/v in 1% pluronic L92 in distilled water. The stimulation index (SI) results are given in Table 2.
Parameter:
SI
Value:
1.24
Test group / Remarks:
Four animals were treated with 50 μl (25 μl per ear) of the test item as an emulsion/suspension in 1% pluronic L92 in distilled water at a concentrations of 5% w/w.
Remarks on result:
other: A stimulation index of less than 3 was recorded for test item at concentrations of 10%, 5% and 2.5% v/v in 1% pluronic L92 in distilled water. The stimulation index (SI) results are given in Table 2.
Parameter:
SI
Value:
1.15
Test group / Remarks:
Four animals were treated with 50 μl (25 μl per ear) of the test item as an emulsion/suspension in 1% pluronic L92 in distilled water at a concentrations of 10 % w/w.
Remarks on result:
other: A stimulation index of less than 3 was recorded for test item at concentrations of 10%, 5% and 2.5% v/v in 1% pluronic L92 in distilled water. The stimulation index (SI) results are given in Table 2.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

 Interpretation of Results

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node(disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non‑sensitiser".

The results were also evaluated according to the Classification, Labelling and Packaging Regulation and the Globally Harmonised System of Classification and Labelling of Chemicals.

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than25% increase in mean ear thicknesswere noted.

Based on this information the dose levels selected for the main test were10%,5% and2.5% w/win1% pluronic L92in distilled water.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in
1% pluronic L92in distilled water

Stimulation Index

Result

2.5

0.95

Negative

5

1.24

Negative

10

1.15

Negative

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 6.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in
1% pluronic L92in distilled water

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

10

S-1

17

18

0

0

0

0

0

0

0

0

0


0=      No signs of systemic toxicity

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration
(%
w/w) in
1% pluronic L92in distilled water

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

10

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration
(%
w/w) in
1% pluronic L92in distilled water

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

10

S-1

0.235

0.230

0.235

0.240

0.230

0.230

overall mean (mm)

0.233

0.238

0.230

overall mean ear thickness change (%)

na

2.151

-1.075


na=     Not applicable

Table 4              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
1% pluronic L92in distilled water

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

6123.09

765.39

na

na

2.5

5801.79

725.22

0.95

Negative

5

7582.02

947.75

1.24

Negative

10

7072.14

884.02

1.15

Negative


dpm=  Disintegrations per minute

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=      Stimulation Index of 3.0 or greater indicates a positive result

na =    Not applicable

Table 5              Individual Clinical Observations and Mortality Data

Concentration
(% w/w) in
1% pluronic L92in distilled water

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

2.5

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

5

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

10

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0


0=      No signs of systemic toxicity

Table 6              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
1% pluronic L92in distilled water

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

17

18

1

1-2

17

17

0

1-3

19

19

0

1-4

19

20

1

2.5

2-1

19

19

0

2-2

18

17

-1

2-3

18

18

0

2-4

19

19

0

5

3-1

17

17

0

3-2

20

20

0

3-3

18

19

1

3-4

18

17

-1

10

4-1

16

18

2

4-2

17

18

1

4-3

18

18

0

4-4

17

18

1

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: Classification, Labelling and Packaging Regulation or the Globally Harmonised Classification System.
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.
The test item did not meet the criteria for classification according to the Classification, Labelling and Packaging Regulation or the Globally Harmonised Classification System.
Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test item as an emulsion/suspension in 1% pluronic L92in distilled waterat concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with1% pluronic L92in distilled water alone.

Results. 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in
1% pluronic L92in distilled water

Stimulation Index

Result

2.5

0.95

Negative

5

1.24

Negative

10

1.15

Negative

Conclusion. 

The test item was considered to be a non-sensitiser under the conditions of the test.

The test item did not meet the criteria for classification according to the Classification, Labelling and Packaging Regulation or theGlobally Harmonised Classification System.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 04 October 2011 and 03 November 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
other: 1% pluronic L92 in distilled water
Concentration:
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water
No. of animals per dose:
Groups of four mice were treated
Details on study design:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test item at a maximum attainable concentration of 25% w/w in 1% pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None provided.
Positive control results:
Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.
Test Item: α Hexylcinnamaldehyde, tech., 85%
Project number: 0039/1151
Study dates: 11 June 2010 to 17 June 2010
Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a suspension in 1% pluronic L92 in distilled water at a concentration of 25% v/v. A further control group of five animals was treated with 1% pluronic L92 in distilled water alone.
Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in
1% pluronic L92 in distilled water Stimulation Index Result
25 6.77 Positive
Conclusion. α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Value:
1.13
Test group / Remarks:
Four animals, were treated with 50 μl (25 μl per ear) of the test item as an emulsion in 1% pluronic L92in distilled water at a concentration of 5% w/w.
Remarks on result:
other: A stimulation index of less than 3 was recorded for the test material at concentrations of 25%, 10% and 5% v/v in 1% pluronic L92 in distilled water. The stimulation index (SI) results are given in Table 2.
Parameter:
SI
Value:
1.01
Test group / Remarks:
Four animals, were treated with 50 μl (25 μl per ear) of the test item as an emulsion in 1% pluronic L92in distilled water at a concentration of 10% w/w.
Remarks on result:
other: A stimulation index of less than 3 was recorded for the test material at concentrations of 25%, 10% and 5% v/v in 1% pluronic L92 in distilled water. The stimulation index (SI) results are given in Table 2.
Parameter:
SI
Value:
1.02
Test group / Remarks:
Four animals, were treated with 50 μl (25 μl per ear) of the test item as an emulsion in 1% pluronic L92in distilled water at a concentration of 25% w/w.
Remarks on result:
other: A stimulation index of less than 3 was recorded for the test material at concentrations of 25%, 10% and 5% v/v in 1% pluronic L92 in distilled water. The stimulation index (SI) results are given in Table 2.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

 Interpretation of Results

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non‑sensitiser".

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thicknesswere noted.

Based on this information the dose levels selected for the main test were 25%,10% and5% w/w in1% pluronic L92 in distilled water.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in
1% pluronic L92in distilled water

Stimulation Index

Result

5

1.13

Negative

10

1.01

Negative

25

1.02

Negative

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 6.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in
1% pluronic L92in distilled water

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

25

S-1

18

19

0

0

0

0

0

0

0

0

0


0=      No signs of systemic toxicity

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration
(%
w/w) in
1% pluronic L92in distilled water

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

25

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3              Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration
(%
w/w) in
1% pluronic L92in distilled water

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

25

S-1

0.210

0.215

0.210

0.220

0.215

0.220

overall mean (mm)

0.213

0.215

0.218

overall mean ear thickness change (%)

na

1.176

2.353


na=     Not applicable

Table 4              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
1% pluronic L92in distilled water

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

8878.23

1109.78

na

na

5

10068.66

1258.58

1.13

Negative

10

8950.15

1118.77

1.01

Negative

25

9088.43

1136.05

1.02

Negative


dpm=  Disintegrations per minute

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=      Stimulation Index of 3.0 or greater indicates a positive result

na =    Not applicable

Table 5              Individual Clinical Observations and Mortality Data

Concentration
(% w/w) in
1% pluronic L92in distilled water

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

5

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

10

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

25

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0


0=      No signs of systemic toxicity

Table 6              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
1% pluronic L92in distilled water

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

18

17

-1

1-2

18

18

0

1-3

18

18

0

1-4

18

19

1

5

2-1

17

18

1

2-2

19

19

0

2-3

19

20

1

2-4

17

19

2

10

3-1

18

18

0

3-2

18

19

1

3-3

19

18

-1

3-4

18

18

0

25

4-1

18

19

1

4-2

18

18

0

4-3

20

20

0

4-4

18

19

1

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: The test item was considered to be a non-sensitiser under the conditions of the test.
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed tobe compatible withthe following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted22 July 2010)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test item as an emulsion in 1% pluronic L92in distilled water at concentrations of 25%,10% or 5% w/w. A further group of four animals was treated with1% pluronic L92in distilled water alone.

Results. 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in
1% pluronic L92in distilled water

Stimulation Index

Result

5

1.13

Negative

10

1.01

Negative

25

1.02

Negative

Conclusion. 

The test item was considered to be a non-sensitiser under the conditions of the test.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
yes
Remarks:
Study director concluded that the test item is not suitable to perform the test
GLP compliance:
yes
Remarks:
Full GLP study could not be conducted as the test item was not suitable to perform the test
Type of study:
other: KeratinoSens Assay
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).
Details on study design:
Solubility tests with DMSO, Milli-Q water and ethanol were performed based on visual assessment, prior to testing. The test item is not soluble in these standard solvents used for the KeratinoSens assay. In addition, a homogeneous suspension could not be obtained in these solvents. Therefore, it was concluded that the KeratinoSensTM assay could not be performed with the test item
Parameter:
other:
Remarks on result:
not determinable because of methodological limitations
Remarks:
not conducted as the test item was not suitable to perform this test

The test item is not soluble in the standard solvents used for the KeratinoSensTMassay, dimethyl sulfoxide, Milli-Q water or ethanol.

In addition, a homogeneous suspension could not be obtained in these solvents. Therefore, it is concluded that the KeratinoSensTMassay cannot be performed with the test item.

Interpretation of results:
study cannot be used for classification
Conclusions:
The test item is not soluble in the standard solvents used for the KeratinoSensTMassay, dimethyl sulfoxide, Milli-Q water or ethanol. In addition, a homogeneous suspension could not be obtained in these solvents. Therefore, it is concluded that the KeratinoSensTMassay cannot be performed with the test item.
Executive summary:

A KeratinoSensTMassay performed according to OECD TG 422D was proposed to be conducted with the test item. During the preliminary solubility work performed prior to the main study, the test item was found to not be soluble, or able to form a homogeneous suspension in, the standard solvents used for the test (dimethyl sulfoxide, Milli-Q water or ethanol). Therefore, it was concluded that the KeratinoSensTMassay could not be performed with the test item.

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

The study Toussaint (2009) is a non-guideline study and the ECHA guidelines for sensitisation testing (Chapter R7a ) (ECHA 2017) indicate that data obtained from non-guideline compliant tests or from refinements of a standard assay need to be further assessed. Additionally, it was noted that non-guideline test data are not referred to in the CLP Regulation with specific classification criteria. Information from such studies should only be used as supporting evidence which would normally require expert judgement in a weight of evidence approach and will generally not be considered adequate for classification on their own.

After the review of the study, although the study design is an acceptable one for assessment purposes, the dose concentrations selected for the LLNA itself are too high based on irritancy potential. Additionally, the choice of 1.4 for the SI threshold is based on validation data performed by BASF. The results of the irritation pre-screen showed ear thickness measurements >25% of the controls at the selected test concentrations, which under the current LLNA protocol is considered too irritating for testing. Consequently, our interpretation of this study is that, whilst it is acceptable for consideration, it is not regulatory compliant in its design (in accordance with the obsolete OECD 429 dated 24/04/2002), it is also not compliant with the current OECD 429 regarding the selection of dose concentrations (see Sections 18, 21 -23 of the updated OECD 429 guideline). Therefore, for these reasons this study is not used for the purposes of classification and a Keratino-Sens and U-Sens assay were commissioned for this substance. It was concluded that a Keratino-Sens or U-SENS assay could not be conducted with this substance (Woutersen 2018). Therefore, no conclusion on skin sensitization properties and potency can be drawn from alternative testing, and a LLNA study was commissioned (van Sas 2018). The results of the LLNA concluded that reaction product of m-tolylidene diisocyanate, cyclohexylamine, cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine is not a skin sensitiser.

The three reliable LLNA studies available for TDI-I category members (Sanders 2011e, Sanders 2012 and van Sas 2018) are deemed appropriate for read-across to the other TDI-I category members. PU TDI-I substance all contain the same core structure, with predominantly linear alkyl chains (C8 - C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-I structures are similar between all category members, and therefore organisms will be exposed to very similar structures, differing only by length of the alkyl chain or the presence of cyclic groups.

Justification for classification or non-classification

Reliable LLNA studies conducted with Diurea 8 (Sanders 2012), Tetraurea 2 (Sanders 2011e) and reaction product of m-tolylidene diisocyanate, cyclohexylamine, cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine showed that the Stimulation Index was < 3 at the highest concentrations tested. Therefore it was concluded that the test items are not sensitisers.