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REACH

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

EC number: 401-990-0 | CAS number: 106990-43-6 CHIMASSORB 119; LOWILITE 19

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

According to the harmonized classification (EC No 1272/2008), the test substance will be classified in a weight of evidence approach as Skin Sens Cat 1.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Version / remarks:

July 22, 2010

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

6 July 2012

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd / SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH, Kreuzelweg 53, NL-5961 NM Horst
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.6 g – 21.8 g (main test)
- Housing: 1 animal per cage (polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany), which contain PLEXX mouse tunnel (red, transparent) and nest building material Nestlets (Ancare, Typ NES 3600) (PLEXX b.v.; AB Elst, Netherlands).
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 45 – 65%
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Remarks:

Polyethylene glycol was used as the vehicle because good homogeneity of the preparation was achieved.

Concentration:

5%, 10%, 25% in PEG
The highest test-substance concentration that could be technically used was a 25% test-substance preparation.

No. of animals per dose:

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment with a comparable batch of the test substance was performed according to the recommendations given by OECD 429. The test-substance preparations at different concentrations were suspensions in polyethylene glycol. The highest test-substance concentration that could be technically used was a 25% test-substance preparation. At higher concentrations, an applicable formulation of the test item could not be achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item.
- Irritation: no relevant signs of local irritation (confirmed by determination of the ear weights and ear thickness measurements)
- Systemic toxicity: No signs of systemic toxicity were observed.

MAIN STUDY
- Randomization: Prior to first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions of Win Rando Version 3.2
- Form of application: epicutaneous
- Application volume: 25 µL per ear
- Site of application: dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), ca. 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.
- Terminal procedures: The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
o Determination of ear weight: Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
o Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
o Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 8 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter.
o Measurement of 3H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation counter.

EVALUATION OF RESULTS:
A test item is regarded as sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H-thymidine at least 3-fold or greater than that determined in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed endpoints (i.e. lymph node cell counts, lymph node weights, ear weights).
Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of ≥ 1.5 and ≥ 1.25, respectively.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations of the measured parameters were calculated per test
group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell
count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.
Statistical analysis to determine parameters including 3H-thymidine incorporation, cell count, lymph node weight and ear weight, the WILCOXON-Test was performed.

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

Test group / Remarks:

vehicle

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

1.5

Test group / Remarks:

5% test item in PEG

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

1.5

Test group / Remarks:

10% test item in PEG

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

1.06

Test group / Remarks:

25% test item in PEG

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
None of the applied test-substance concentrations induced biologically relevant increases
(increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) of 3H-thymidine
incorporation into the cells from the auricular lymph nodes. The 10% concentration caused a statistically significant increase in 3H-thymidine incorporation.
Concomitantly, none of the applied test-substance concentrations induced biologically relevant or statistically significant responses (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.
In addition, no relevant or statistically significant increases in lymph node weights were noted at all concentrations.

DETAILS ON STIMULATION INDEX CALCULATION
Mean values and standard deviations of the measured parameters were calculated per test
group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell
count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.

EC3 CALCULATION
If applicable, the EC (estimated concentration) leading to the respective SI values were
calculated by linear or semi-logarithmical regression between the data points directly below
and above the SI if possible or by using the two nearest points below or above the SI.

CLINICAL OBSERVATIONS:
- Ear weights: The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights, demonstrating the absence of ear skin irritation.
- No signs of systemic toxicity were noticed in all animals during general observation.
- No local findings were observed during the observation period.

BODY WEIGHTS
The expected body weight gain was generally observed during the study

^{Table 1: Stimulation indices}

Test group	Treatment	³H-thymidine incorporation Stimulation Index¹	Cell Count Stimulation Index¹	Lymph Node Weight Stimulation Index¹	Ear Weight Stimulation Index¹
1	vehicle polyethylene glycol	1.00	1.00	1.00	1.00
2	5% in polyethylene glycol	1.50	1.05	1.17	1.01
3	10% in polyethylene glycol	1.50 #	1.09	1.14	1.01
4	25% in polyethylene glycol	1.06	0.87	0.95	0.96

¹vs. mean of test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Interpretation of results:

GHS criteria not met

Conclusions:

Thus, it is concluded that the test item in Polyethylene does not exhibit a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test item in Polyethylene was assessed using the radioactive Local Lymph Node Assay.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 5%, 10% and 25% (w/w) concentrations of the test substance in polyethylene glycol or with the vehicle alone. The 25% concentration was the maximum technically applicable concentration. Each test animal was treated with 25 μL per ear of the appropriate test-substance concentration or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, ca. 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

In addition, no relevant or statistically significant increases in lymph node weights were noted at all concentrations. The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights, demonstrating the absence of ear skin irritation.

Thus, it is concluded that the test item in Polyethylene does not exhibit a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Version / remarks:

July 22, 2010

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Nº 640/2012 of 6 July 2012

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd / SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH, Kreuzelweg 53, NL-5961 NM Horst
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.5 g – 22.2 g (main test)
- Housing: 1 animal per cage (polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany), which contains PLEXX mouse tunnel (red, transparent) and nest building material Nestlets (Ancare, Typ NES 3600) (PLEXX b.v.; AB Elst, Netherlands).
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 45 – 65%
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Remarks:

Polyethylene glycol was used as the vehicle because good homogeneity of the preparation was achieved.

Concentration:

5%, 10%, 25% in PEG
The highest test-substance concentration that could be technically used was a 25% test-substance preparation.

No. of animals per dose:

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment with a comparable batch of the test substance was performed according to the recommendations given by OECD 429. The test-substance preparations at different concentrations were suspensions in polyethylene glycol. The highest test-substance concentration that could be technically used was a 25% test-substance preparation. At higher concentrations, an applicable formulation of the test item could not be achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item.
- Irritation: no relevant signs of local irritation (confirmed by determination of the ear weights and ear thickness measurements)
- Systemic toxicity: No signs of systemic toxicity were observed.

MAIN STUDY
- Randomization: Prior to first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions of Win Rando Version 3.2
- Form of application: epicutaneous
- Application volume: 25 µL per ear
- Site of application: dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), ca. 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.
- Terminal procedures: The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
o Determination of ear weight: Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
o Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
o Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 8 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter.
o Measurement of 3H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation counter.

EVALUATION OF RESULTS:
A test item is regarded as sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H-thymidine at least 3-fold or greater than that determined in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the
other assessed endpoints (i.e. lymph node cell counts, lymph node weights, ear weights).
Hereby, the thresholds used for assessment of cell counts and ear weights are represented by
stimulation indices (SI) of ≥ 1.5 and ≥ 1.25, respectively.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

Test group / Remarks:

vehicle

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

1.37

Test group / Remarks:

5% test item in PEG

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

1.45

Test group / Remarks:

10% test item in PEG

Parameter:

Remarks:

³H-thymidine incorporation Stimulation index

Value:

1.43

Test group / Remarks:

25% test item in PEG

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
None of the applied test-substance concentrations induced biologically relevant increases
(increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) of 3H-thymidine
incorporation into the cells from the auricular lymph nodes. The 25% concentration caused a statistically significant increase in 3H-thymidine incorporation.
Concomitantly, none of the applied test-substance concentrations induced biologically relevant or statistically significant responses (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.
In addition, no relevant or statistically significant increases in lymph node weights were noted at all concentrations.

DETAILS ON STIMULATION INDEX CALCULATION
Mean values and standard deviations of the measured parameters were calculated per test
group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell
count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.

EC3 CALCULATION
If applicable, the EC (estimated concentration) leading to the respective SI values were
calculated by linear or semi-logarithmical regression between the data points directly below
and above the SI if possible or by using the two nearest points below or above the SI.

CLINICAL OBSERVATIONS:
- Ear weights: The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights, demonstrating the absence of ear skin irritation.
- No signs of systemic toxicity were noticed in all animals during general observation.
- No local findings were observed during the observation period.

BODY WEIGHTS
The expected body weight gain was generally observed during the study

^{Table 1: Stimulation indices}

Test group	Treatment	³H-thymidine incorporation Stimulation Index¹	Cell Count Stimulation Index¹	Lymph Node Weight Stimulation Index¹	Ear Weight Stimulation Index¹
1	vehicle polyethylene glycol	1.00	1.00	1.00	1.00
2	5% in polyethylene glycol	1.37	1.18	1.20	0.98
3	10% in polyethylene glycol	1.45	1.14	1.19	1.00
4	25% in polyethylene glycol	1.43#	1.15	1.15	1.01

¹vs. mean of test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Interpretation of results:

GHS criteria not met

Conclusions:

it is concluded that the test item in Polypropylene does not exhibit a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test item in Polypropylene was assessed using the radioactive Local Lymph Node Assay.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 5%, 10% and 25% (w/w) concentrations of the test substance in polyethylene glycol or with the vehicle alone. The 25% concentration was the maximum technically applicable concentration. Each test animal was treated with 25 μL per ear of the appropriate concentration or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, ca. 20 μCi ³H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring ³H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation.

None of the applied test-substance concentrations induced biologically relevant increases (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) of ³H-thymidine incorporation into the cells from the auricular lymph nodes. The 25% concentration caused a statistically significant increase in ³H-thymidine incorporation.

Concomitantly, none of the applied test-substance concentrations induced biologically relevant or statistically significant responses (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.

Thus, it is concluded that the test item in Polypropylene does not exhibit a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

Reference 3

Endpoint:: skin sensitisation: in vitro
Type of information:: experimental study
Adequacy of study:: weight of evidence
Reliability:: 4 (not assignable)
Rationale for reliability incl. deficiencies:: other: This test has not been validated and is not regulatorily accepted. Borderline positive results (>6 genes induced = positive, <7 genes induced = negative) and inconclusive results of the separate experiments.
Remarks:: This test has not been validated yet and is therefore not yet regulatorily accepted;
Qualifier:: no guideline available
Principles of method if other than guideline:: - Principle of test: The Sens-IS assay uses 3D human reconstructed epidermis (EpiSkin) to investigate the gene expression of genes involved in sensitization. The expression profle of 61 genes divided in three sets were analyzed: one set of 23 genes related to the irritation process and the two other sets of genes, named "SENS-IS" and "ARE", with 21 and 17 biomarkers respectively, involved in skin sensitization.

- Short description of test conditions:
> Reference compounds: SLS 10% (Positive control for irritation and negative control for sensitization), TNBS 1M (Positive control for sensitization), DMSO (Negative control for irritation/sensitization)
> Preliminary test: Solubility Test
> Main test: SENS-IS assay

- Parameters analysed / observed:
> Samples were analyzed by the LightCycler 480 software using the second derivate maximum method. This method allows the calculation of the crossing point (Cp) of each sample defined as the number of cycles from which the fluorescence signal enters the exponential phase of the reaction. This value is dependent of the amount of the mRNA present in the sample. For each sample, the mRNA content for each gene of interest was normalized to the mean mRNA content of the 3 house-keeping genes. For each gene, the fold change in mRNA level over vehicle controls was calculated as followed:
deltaE = Expression level for the test item / Expression level for the vehicle controls
The endpoint values are the number of positive genes in each group.
GLP compliance:: no
Type of study:: other: Sens-IS assay
Justification for non-LLNA method:: The assay was conducted to proof whether the previous obtained LLNA results are artefacts or not.
If this assay is negative (i.e. it doesn’t show an increase in the expression of genes important during sensitization) this would be an indication that the test item is not a sensitizer.
The Sens-IS assay was not used as a stand-alone method, because in addition, a Buehler test was performed.
Details of test system:: other: non-transformed, human-derived epidermal keratinocytes (EpiSkin)
Details on the study design:: PRELIMINARY TEST: SOLUBILITY TEST
The solubility of the test item was assessed in phosphate buffered saline (PBS), olive oil (00), dimethylsulfoxide (DMSO) and dipropylene glycol (DPG) at a concentration of 10% and 50%, after
heating at 80°C. The solubility of the test item was assessed by visual inspection of each preparation.

MAIN TEST: SENS-IS ASSAY
The test item or its preparation (30 μL) were deposited on the epidermis surface and gently spread on the entire surface . After 15 minutes of exposure, the Episkin TM was rinsed with PBS and then incubated at 37°C for 6 hours. After incubation, reconstructed epidermis was removed from the inserts with forceps and placed in a cryotube for freezing in liquid nitrogen. The epidermis was then transferred in a tube. After addition of bromochloropropane, total RNA were purified using the miRNeasy extraction Kit according to the manufacturer's instructions (Qiagen, Courtaboeuf, France). RNA quality was assessed by measuring 260/280 absorbance ratio. The mRNA was then reversed as cDNA using SuperScript III Reverse Transcriptase kit and RNase inhibitor. After reverse transcription, quantitative gene expression was measured by qRT-PCR using a SYBRGreen® buffer and specific primers defined for the SENS-IS test. The housekeeping genes (Glucuronidase ß, ß2 microglobuline, and Nano « non-POU domain containing octamer-binding ») were analyzed in parallel.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3 replicates for each concentration
- Test chemical concentrations: 0.1, 1, 10, 25 % in olive oil and 100% (direct application of the unsolubilized powder)
- Exposure time: 15 min
- Incubation: 6 hours, 37°C

DATA EVALUATION
A test item is classified as irritant if at least 16/23 genes of the "IRRITATION" group are significantly overexpressed.
A test item is classified as sensitizer if at least 7/17 genes of the "ARE" group, and/or 7/21 genes in the "SENS-IS" group are significantly overexpressed.
Moreover, the results obtained with the different concentrations allow the classification of the test item according to the lowest concentration that gives a positive result (deltaE > 1.25). Thus, a test item is classified in:
- category 1A: strong to extreme skin sensitizer, when a positive result is obtained at
concentrations of 0.1 and/or 1 %,
- category 1 B: weak to moderate sensitizer, when a positive result is obtained at concentrations of 10 and/or 50%.

A test item is classified as a non-sensitizer when negative results are observed at 100% and at all other analyzed concentrations.
At least two independent experiments (repetitions) are performed in order to obtain two concordant conclusions. lf three repetitions should be performed for a given concentration, a majority of positive results (2 out of 3) must be obtained so that the final outcome is positive, otherwise the final outcome is negative.
Vehicle / solvent control:: other: olive oil, 25% (w/v) after heating at 85°C
Negative control:: other: DMSO (irritation/sensitization), SLS 10% (sensitization)
Positive control:: other: SLS 10% (irritation), TNBS 1M (sensitization)
Positive control results:: SLS at 5% was classified as irritant (number of overexpressed irritant genes > 15) and non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7.
TNBS at 1 M was classified as sensitizer in all experiments since more than 6 genes were overexpressed in at least one of the two groups of genes (SENS-IS or ARE).
Group:: other: Test item in 10, 1, 0.1% olive oil
Run / experiment:: other: Experiment 1
Parameter:: other: number of overexpressed genes
Remarks on result:: positive indication of skin sensitisation
Group:: other: test item in 10, 1, 0.1% olive oil
Run / experiment:: other: 2. Experiment
Parameter:: other: number of overexpressed genes
Remarks on result:: no indication of skin sensitisation
Group:: other: 100% of the test item and 25, 10, 1, 0.1% of the test item in olive oil
Run / experiment:: other: 3. Experiment
Parameter:: other: number of overexpressed genes
Remarks on result:: other: Positive outcome for 100% and 25%. Negative outcome for 10, 1, 0.1%.
Group:: test chemical
Run / experiment:: other: 4. Experiment
Parameter:: other: number of overexpressed genes
Remarks on result:: no indication of skin sensitisation
Group:: test chemical
Run / experiment:: other: 5. Experiment
Parameter:: other: number of overexpressed genes
Remarks on result:: no indication of skin sensitisation
Outcome of the prediction model:: other:
Interpretation of results:: study cannot be used for classification
Conclusions:: In conclusion, under the experimental conditions of this SENS-IS assay, the test item did not demonstrate an in vitro sensitizing potential when applied pure or diluted on the epidermis.
It should be mentioned that the SENS-IS method cannot be considered sufficient as stand alone to conclude on skin sensitization potential and should be completed by other in chemico or in vitro methods that can be combined with in silico information, according to the OECD recommendations.
Executive summary:: The objective of this study was to evaluate the capacity of the test item to induce the expression of specific irritation and sensitization biomarkers in a 3D-reconstructed epidermis model.
The results obtained for the positive and negative controls were within acceptance criteria defined in the Study Plan, except for DMSO in experiment 2SI20011.
Considering the number of over-expressed gene in the "SENS-IS" and "ARE" gene groups, the test item gave negative result (less than 7 genes induced) when it was tested at 0.1, 1 %, 10 and 25% (w/v) in olive oil in two out of three experiments. Moreover, negative results were obtained when the test compound was directly applied on the epidermis without solubilization in the solvent in two out of three experiments.
In conclusion, under the experimental conditions of this SENS-IS assay, the test item did not demonstrate an in vitro sensitizing potential when applied pure or diluted on the epidermis.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The purity of the test item used for the previous conducted LLNA study (2003) does not correspond to the purity status of the test item in 2019. In addition, the outcome of the test showed a strong sensitisation, but no conclusive dose-response.
Therefore two pre-LLNA tests (2019) with the actual test item composition were conducted. However, those were aborted due to the low water solubility of the test item. Further, no dose-response was observed and an irritation potential cannot be excluded.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, NL-5800 AN Venray
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]
- Age at study initiation: adult (not further specified)
- Weight at study initiation: not specified
- Housing: 2-3 animals per cage (macrolone cages (approx. 2280 cm2)), containing hay bricks (Granovit AG, 4303 Kaiseraugst, Switzerland) and hiding houses of red polycarbonate (PLEXX, The Netherlands).
- Diet (e.g. ad libitum): ad libitum. Pelleted diet "Altromin 3123' (Altromin, D-32791 Lage, Lippe)
- Water (e.g. ad libitum): ad libitum. Domestic quality drinking water being enriched with vitamin C and acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20±3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

50%(w/w)

Day(s)/duration:

6 days/duration (sum: 3 application = 15 days)

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

25% (w/w)

Day(s)/duration:

Day 27- Day30 (5 days)

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Main Study: N=20 (treated), N=10 (vehicle)
Prestudy: N=3 (treated)

Details on study design:

RANGE FINDING TESTS:
- N=3
- test concentrations in PEG (%w/w): 50, 25, 10, 5
- Day 0: 0,5 mL of the test item was applied for an exposure duration of 6-hours.
- Day1,2: Examination of the application sites after 24 and 48 hours after termination of exposure.
- Scoring: All animals, except one (Score 1 at 50%), showed an erythema score of 0.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours
- Test groups: N=20
- Control group: N=10
- Site: skin of the left flank
- Frequency of applications: 3
- Duration: dermal application site was examined 24 and 48 hours after the termination of exposure
- Concentrations: 50% (w/w)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 33
- Exposure period: 6 hours
- Test groups: N=20
- Control group: N=10
- Site: cranial part of the right flank for the application of the test item, while the vehicle was applied on the caudal part of the right flank.
- Concentrations: 25% (w/w)
- Evaluation (hr after challenge): 24 and 48 hours after the termination of exposure.

Positive control substance(s):

yes

Remarks:

The last positive control (PC) study with the reference material a-Hexylcinnamaldehyde, technical grade, 85 %, was conducted from Feb-2020 to Mar-2020. In the PC test group, 11 of 20 guinea pigs (55 %) reacted with slight to moderate skin reactions (eryth

Positive control results:

Refer to Table 2 in Section "Any other information on results incl. tables"

Reading:

1st reading

Hours after challenge:

Group:

test chemical

Dose level:

25%(w/w)

No. with + reactions:

Total no. in group:

Clinical observations:

no effects observed. erythema score:0

Reading:

1st reading

Hours after challenge:

Group:

negative control

Dose level:

50% (w/w)

No. with + reactions:

Total no. in group:

Clinical observations:

no effects observed. erythema score:0

Reading:

2nd reading

Hours after challenge:

Group:

test chemical

Dose level:

25% (w/w)

No. with + reactions:

Total no. in group:

Clinical observations:

no effects observed. erythema score:0

Reading:

2nd reading

Hours after challenge:

Group:

negative control

Dose level:

25%(w/w)

No. with + reactions:

Total no. in group:

Clinical observations:

no effects observed. erythema score:0

Table 2: Number and intensity grade of observed skin reactions to challenge treatments with the reference material and vehicle in study Lab. No. 04191.

fLab. No.:	04191	Challenge
Exp. period:	02/2020 - 03/2020	test item [50 % (w/w) a-Hexylcinnamaldehyde, technical grade, 85 %]			vehicle [PEG]
Type of study	group	24 hrs	48 hrs	sum	24 hrs	48 hrs	sum
Buehler test (OECD 406)	control group	1/10	1/10	1/10	0/10	0/10	0/10
	grade 0	9/10	9/10		10/10	10/10
	grade 1	1/10	1/10		0/10	0/10
	test group	10/20	10/20	11/20	0/20	0/20	0/20
	grade 0	10/20	10/20		20/20	20/20
	grade 1	6/20	5/20		0/20	0/20
	grade 2	4/20	5/20		0/20	0/20

Table 3: Observed skin reactions in the test group during the induction phase of the main test after the induction treatment with the 50 % (wlw) test item.

Test group	Animal No.	Skin reaction on the left flank 24 hours after the
	Animal No.	1. induction	2. induction	3. induction
	11	0	0	0
	12	0	0	0
	13	0	0	0
	14	0	0	0
	15	0	0	0
	16	0	0	0
	17	0	0	0
	18	0	0	0
	19	0	0	0
	20	0	0	0
	21	0	0	0
	22	0	0	0
	23	0	0	0
	24	0	0	0
	25	0	0	0
	26	0	0	0
	27	0	0	0
	28	0	0	0
	29	0	0	0
	30	0	0	0

Table 4: Observed skin reactions in the test group after the challenge treatment during the main test as well as data of body weight. Challenge treatment: right cranial test field = test item (25 % w/w), right caudal test field = PEG 400

test group	Animal No.	Skin reactions after				Body weight in g
		24 hours		48 hours		Day0	Day33
		right cranial	right caudal	right cranial	right caudal	Day0	Day33
	11	0	0	0	0	340	540
	12	0	0	0	0	340	561
	13	0	0	0	0	306	453
	14	0	0	0	0	315	525
	15	0	0	0	0	323	520
	16	0	0	0	0	315	539
	17	0	0	0	0	350	525
	18	0	0	0	0	342	546
	19	0	0	0	0	332	528
	20	0	0	0	0	315	541
	21	0	0	0	0	332	572
	22	0	0	0	0	320	512
	23	0	0	0	0	325	527
	24	0	0	0	0	312	486
	25	0	0	0	0	330	507
	26	0	0	0	0	330	537
	27	0	0	0	0	307	522
	28	0	0	0	0	311	531
	29	0	0	0	0	308	477
	30	0	0	0	0	301	473

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this Buehler test, the test item was not sensitizing to the skin.

Executive summary:

The dermal sensitizing potential of the test item was investigated by means of the Buehler test. The main study was performed on 30 guinea pigs in total. A control group of 10 animals and a test group of 20 animals were formed. The test concentrations for the main study were selected based on the results of a preliminary investigation. In the pre-test, the minimal irritant concentration for the induction phase was found to be a 50 % (w/w) test substance preparation, whereas the maximum non-irritant concentration for the challenge phase was determined to be a 25 % (w/w) test substance preparation in polyethylene glycol (PEG). The main study started with an induction phase including an occlusive patch topical application for 6 hours once a week for three consecutive weeks. The animals of the test group were induced with the 50 % (w/w) test item, whereas the animals of the control group were induced with the vehicle polyethylene glycol.

During the induction phase, none of the animals displayed skin reactions neither in the test group nor in the control group. Four weeks after the first induction, the 6-hour challenge procedure was conducted by an occlusive topical application of the test item on the right cranial flank of all animals. A vehicle exposure took place on the right caudal flank. The skin reactions were evaluated 24 hours and 48 hours after the challenge application. For the challenge, the test item was used in a concentration of 25 % (w/w) according to the agreement with the sponsor. None animal of the test group (0/20; 0 %) and none animal of the control group (0/10; 0 %) responded with skin reactions to the challenge treatment with the test item neither after 24 hours nor after 48 hours. Also, the challenge treatment with the vehicle did not cause skin reactions at any time point neither in the test group (0/20; 0 %) nor in the control group (0/10; 0 %). In agreement with the sponsor, a rechallenge was waived due to the unambiguous test result. A delayed contact sensitization in the guinea pig was excluded by absence of skin reactions in the test group. Based on the results of the non-adjuvant sensitization study described in this final report, the test item does not display a skin sensitizing potential.

Reference 5

Endpoint:: skin sensitisation: in vivo (LLNA)
Remarks:: Pretest with purified test material
Type of information:: experimental study
Adequacy of study:: weight of evidence
Reliability:: 3 (not reliable)
Rationale for reliability incl. deficiencies:: unsuitable test system
Remarks:: The result of the study is inconclusive, which is due to the low water solubility of the material.
Qualifier:: according to guideline
Guideline:: OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:: no
GLP compliance:: not specified
Type of study:: mouse local lymph node assay (LLNA)
Species:: mouse
Vehicle:: methyl ethyl ketone
Concentration:: 1, 5, 10% in MEK
No. of animals per dose:: 2
Details on study design:: PRE-SCREEN TESTS:
- Compound solubility: not specified
- Systemic toxicity: no effects
- Ear thickness measurements: yes
- Erythema scores: not examined
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 1
Test group / Remarks:: vehicle
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 4.06
Test group / Remarks:: 1% in MEK
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 4.26
Test group / Remarks:: 5% in MEK
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 4.45
Test group / Remarks:: 10% in MEK
Cellular proliferation data / Observations:: CELLULAR PROLIFERATION DATA
All applied test-substance concentrations induced biologically relevant increases
(increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) of 3H-thymidine
incorporation into the cells from the auricular lymph nodes.

Lymph node weights were comparable of the tested concentrations. No dose-response was noticed. Lymph node weight of the control was not provided.

CLINICAL OBSERVATIONS: no effects observed

BODY WEIGHTS: no effects observed

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The 10% concentration caused an ear sweeling at d5 up to 18%. No clear dose-response could be observed.
Interpretation of results:: study cannot be used for classification
Remarks:: The study was terminated after conducting the pretest. The result of the study is inconclusive, which is due to the low water solubility of the material.

Reference 6

Endpoint:: skin sensitisation: in vivo (LLNA)
Remarks:: Pretest with actual used (not purified) test material
Type of information:: experimental study
Adequacy of study:: weight of evidence
Reliability:: 3 (not reliable)
Rationale for reliability incl. deficiencies:: unsuitable test system
Remarks:: The result of the study is inconclusive, which is due to the low water solubility of the material.
Qualifier:: according to guideline
Guideline:: OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:: no
GLP compliance:: not specified
Type of study:: mouse local lymph node assay (LLNA)
Species:: mouse
Vehicle:: methyl ethyl ketone
Concentration:: 1, 5, 10% in MEK
No. of animals per dose:: 2
Details on study design:: PRE-SCREEN TESTS:
- Compound solubility: not specified
- Systemic toxicity: no effects
- Ear thickness measurements: yes
- Erythema scores: not examined
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 1
Test group / Remarks:: vehicle
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 4.54
Test group / Remarks:: 1% in MEK
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 4.13
Test group / Remarks:: 5% in MEK
Parameter:: SI
Remarks:: ³H-thymidine incorporation Stimulation index
Value:: 4.89
Test group / Remarks:: 10% in MEK
Cellular proliferation data / Observations:: CELLULAR PROLIFERATION DATA
All applied test-substance concentrations induced biologically relevant increases
(increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) of 3H-thymidine
incorporation into the cells from the auricular lymph nodes.

Lymph node weights were comparable of the tested concentrations. No dose-response was noticed. Lymph node weight of the control was not provided.

CLINICAL OBSERVATIONS: no effects observed

BODY WEIGHTS: no effects observed

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The 10% concentration caused an ear sweeling at d5 up to 20%. A dose-response is likely according to the results.
Interpretation of results:: study cannot be used for classification
Remarks:: The study was terminated after conducting the pretest. The result of the study is inconclusive, which is due to the low water solubility of the test material.

Reference 7

Endpoint:: skin sensitisation: in vivo (LLNA)
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: from 2003-04-2 until 2003-05-28
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)
GLP compliance:: yes
Remarks:: RCC Ltd
Type of study:: mouse local lymph node assay (LLNA)
Species:: mouse
Strain:: CBA
Sex:: female
Details on test animals and environmental conditions:: TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15.3 g - 21.1 g
- Housing: groups of four in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba 3433, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: Under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 2, 2003 To: May 28, 2003
Vehicle:: acetone/olive oil (4:1 v/v)
Concentration:: 2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8: in acetone:olive oil, 4:1, w/v); Groups 1 and 5 were treated with the vehicle (control groups)
No. of animals per dose:: 4
Details on study design:: RANGE FINDING TESTS:
- The highest non-irritant and technically applicable test item concentration was determined in a non-GLP pretest in two mice with concentrations of 1 %, 5%, 10% and 25 % (w/v). The test item in the main study was assayed at three consecutive concentrations. The top dose (10%) was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
- Name of test method: LLNA
- Criteria used to consider a positive response: regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8) (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. Two control groups of mice were treated with an equivalent volume of the relevant vehicle alone. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
- Administration of 3H-Methyl Thymidine: Five days after the first topical application, all mice were administered with 250 µl of 78.4µCi/ml 3HTdR (equal to 19.6 µCi 3HTdR) (for Groups 1-4) and 85.0 µCi/ml 3HTdR (equal to 21.3 µCi 3HTdR) (for Groups 5-8) by intravenous injection via a tail vein.
- Determination of incorporated 3HTDR: Approximately five hours after treatment with 3HTdR all mice were euthanized. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of “Ultima Gold” scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a P-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
- Interpretation of Data: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
1. First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the SI.
2. Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):: hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:: The mean values and standard deviations were calculated in the body weight tables.
Positive control results:: SI of 2.5, 3.7 and 9.7 were determined with the positive control at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v).
Parameter:: SI
Remarks on result:: other: In this study, SI of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). (See Table 1)
Parameter:: other: disintegrations per minute (DPM)
Remarks on result:: other: See Table 1
Interpretation of results:: Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:: In this study STIMULATION INDICES of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). The test item was therefore found to be a strong sensitizer.
Executive summary:: In order to study a possible contact allergenic potential of the test substance, a GLP-compliant local lymph node assay according to OECD guideline 429 was performed. Six groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8), (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. Two control groups of each four mice were treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labeled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ^H-methyl thymidine measured in a β-scintillation counter. No test item-related clinical signs were observed in any animals of the control groups (Groups 1 and 5), Group 2 (2.5 %), Group 3 (5 %), Group 4 (10 %) or Group 6 (2.5 %). On the third application day, a slight ear erythema was observed at both dosing sites in all mice of Group 7 (5 %) and Group 8 (10 %). On the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 8 (10 %). These signs persisted for the remainder of the in-life phase of the study. All treated animals survived the scheduled study period. In this study STIMULATION INDICES of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). As a result, the test item was found to be a strong sensitizer.

Reference 8

Endpoint:: skin sensitisation: in vivo (non-LLNA)
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: From May 13, 1986 to Jul. 18, 1986
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 406 (Skin Sensitisation)
GLP compliance:: yes
Remarks:: CIBA-GEIGY LIMITED (GU 2 Toxicology)
Type of study:: guinea pig maximisation test
Justification for non-LLNA method:: The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Species:: guinea pig
Strain:: other: Pirbright White Strain
Sex:: male/female
Details on test animals and environmental conditions:: TEST ANIMALS
- Source: CIBA-GEIGY LTD. Tierfarm, 4334 Sisseln, Switzerland.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: 320-463 g
- Housing: Housed individually in Macrolon cages (Type 3)
- Diet: guinea pig pellets - NAFAG No.846, ad libitum
- Water: ad libitum, fresh water
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
Route:: intradermal
Vehicle:: other: sesame oil
Concentration / amount:: 1%
Route:: epicutaneous, occlusive
Vehicle:: other: vaseline
Concentration / amount:: Approximately 0.4 g paste of 10% test substance in vaseline
No.:: #1
Route:: epicutaneous, occlusive
Vehicle:: other: vaseline
Concentration / amount:: Approximately 0.2 g paste of 0.3% test substance in vaseline.
No. of animals per dose:: 10/sex/dose
Details on study design:: RANGE FINDING TESTS:
The concentration of the test compound for the induction and challenge periods were determined on separate animals.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Test groups: 1) Three pairs of intradermal application (0.1 ml per injection): 1:1 mixture of adjuvant and saline, test compound in sesame oil, and test compound in the adjuvant saline mixture; 1% of the test compound in the sesame oil and adjuvant mixture. 2) Epicutaneous application: 1 week after intradermal application, with approximately 0.4 g of the 10 % test compound in vaseline for 48 h under occlusive condition, patch 2x4 cm
- Control group: Treated with adjuvant and the vehicle during induction period
- Site: Neck of the animals

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after the epicutaneous induction application
- Exposure period: 24 hours- Test groups: test compound in vaseline
- Control group: treated with the vehicle as well as with the test compound (at least 10 animals) to control the maximal subirritant concentration of the test compound in adjuvant treated.animals.
- Site: Flank of the animals
- Concentrations: Approximately 0.2 g paste of 0.3% test compound in vaseline
- Evaluation (hr after challenge): 24 and 48 hours after removing the dressings

OTHER:
Twenty four hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings. The sensitizing potential was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.
Challenge controls:: Adjuvant treated control group was challenged with the test compound and the vehicle.
Positive control results:: The sensitivity of the strain is checked every six months with paraphenylene-diamine or potassium-dichromate.
Reading:: 1st reading
Hours after challenge:: 24
Group:: test chemical
Dose level:: 0.2 g paste of 0.3% test compound
No. with + reactions:: 15
Total no. in group:: 20
Reading:: 1st reading
Hours after challenge:: 24
Group:: test chemical
Dose level:: vehicle reated
No. with + reactions:: 0
Total no. in group:: 20
Reading:: 2nd reading
Hours after challenge:: 48
Group:: test chemical
Dose level:: 0.2 g paste of 0.3% test compound
No. with + reactions:: 12
Total no. in group:: 20
Reading:: 2nd reading
Hours after challenge:: 48
Group:: test chemical
Dose level:: vehicle treated
No. with + reactions:: 0
Total no. in group:: 20
Reading:: 1st reading
Hours after challenge:: 24
Group:: negative control
Dose level:: 0.2 g paste of 0.3% test compound
No. with + reactions:: 0
Total no. in group:: 10
Reading:: 1st reading
Hours after challenge:: 24
Group:: negative control
Dose level:: vehicle treated
No. with + reactions:: 0
Total no. in group:: 20
Reading:: 2nd reading
Hours after challenge:: 48
Group:: negative control
Dose level:: 0.2 g paste of 0.3% test compound
No. with + reactions:: 0
Total no. in group:: 10
Reading:: 2nd reading
Hours after challenge:: 48
Group:: negative control
Dose level:: vehicle treated
No. with + reactions:: 0
Total no. in group:: 20
Interpretation of results:: Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:: Based on the results of this study and other the conditions employed, the test article is regarded as a skin sensitizer.
Executive summary:: In a guinea pig maximization test according to OECD guideline No. 406, 10 male and 10 female animals were first induced and then challenged with the test article to investigate its sensitization potential. Induction was a two-stage operation: First, three pairs of intradermal injections (1 % test substance in sesame oil) were made into the neck of the animals with a 1:1 mixture (v/v) of adjuvant/physiological saline, the test substance in sesame oil, and the test substance in a 1:1 mixture (v/v) of adjuvant/physiological saline. One week later, the test article was incorporated in vaseline at a concentration of 10% (approx. 0.4 g) and applied on a filter paper patch to the neck of the animals (occlusive administration for 48 hours). Two weeks after the epidermal induction application the animals were tested on the flank with 0.3 % test substance in Vaseline (approx. 0.2 g) and the vehicle alone (24 h occlusive application). Twenty four hours after removing the dressings the challenge reactions were graded according the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings. A control group (20 m/20 f) was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound to control the maximal subirritant concentration of the test compound in adjuvant treated animals. Under the experimental conditions employed, 60% to 75% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings. Therefore, under the experimental conditions of this study the test substance is classified as a strong sensitizer when topically applied to albino guinea pigs.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Weight-of-Evidence Approach:

For the following discussion on the sensitization potential of the test item, the GPMT study, which was conducted according to guideline OECD 406 (Ciba-Geigy, 1986), is not considered as the specification of the test item (puritiy: 80%) differs from today’s specification (purity: 95%). All other studies mentioned in more detail below are evaluated in a weight of evidence approach.

There are three LLNA studies (RCC 2003, 2x BASF SE 2019) available showing strong positive results, independent from the purity grade of the test substance. The first LLNA assay gave clear evidence of the test item’s skin sensitizing potential, indicating the classification of the substance as sensitizer Category 1A. The other two LLNA studies were pre-studies and were conducted with the test substance of today’s specification and with the purified version of the test substance to exclude a sensitization potential of the impurities. Both studies had to be terminated due to solubility issues, but nevertheless indicated a strong sensitizing potential (Category 1A). In contrast, an available Buehler test (Frey Tox, 2020) indicates that the test substance is not a sensitizer and should therefore not be classified. This result is further supported by a non-guideline study (SensIS, EUROSAFE, 2021).

However, the chemical is an UV-filter that is contained in finished articles at very low concentrations. Therefore, Local Lymph node assays with the product (containing 1.15% of the test substance in polypropylene and 1% of the test substance in polyethylene) were conducted.

To further clear those difference and considering that the chemical is an UV-filter that is contained in finished articles at very low concentrations, two further Local Lymph node assays with milled polymer containing the test substance (% in PE or 1.15% in PP) were conducted. These assays could show that handling polymer articles containing the test substance is not hazardous to the consumer. Those assays were negative, so from a risk assessment perspective we show that handling of the test substance in plastic articles is safe. Since the classification of Skin Sens 1 will remain, it is ensured that the workers handling or producing the pure test substance or master batches are aware of the sensitization hazard and utilize proper personal protection equipment. However, due to the contradictory results, it is not possible to apply a subcategorization for the sensitization potential.

GPMT. Ciba-Geigy. 1986 (OECD 406): positive

In a GLP-compliant dermal sensitization study with the test item (80% purity) in sesame oil Pirbright White guinea pigs were tested using the guinea pig maximization test according to OECD guideline 406. In this study, the test group animals received 1 % of the test item for intradermal induction as well as 10 % of the test item for epicutaneous induction one week later. For intradermal induction, three pairs of injections (adjuvant/saline; test item in vehicle; test item in vehicle plus adjuvant/saline) were given. Epidermal challenge was performed by occlusive application for 24 h two weeks later (0.3% in vaseline). In 60% - 75% of the animals of the test group skin reactions were observed 24 and 48 hours after the removal of the dressings. Control animals did not show any reactions. Based on this result and under the conditions of this study, the test item is considered a skin sensitizer.

LLNA. RCC. 2003 (OECD 429): strong positive

In a Local Lymph Node Assay performed according to OECD guideline 429, CBA mice were treated by topical application with different test item concentrations of 2.5%, 5% and 10% in acetone:olive oil (4:1) once daily for three consecutive days. Five days after the first topical application, all mice were treated with ³H-Methyl Thymidine by intravenous injection. Approximately five hours after treatment the mice were sacrificed, the draining lymph nodes of each group were excised, pooled and the level of incorporated ³HTdR was measured. In this study, SI (stimulation index) of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v), respectively. As a result, the test substance is considered a strong skin sensitizer. An EC3 value could not be derived due to the severe reaction induced even at the lowest doses.

Pre-LLNA with regular test material. BASF SE. 2019 (OECD 429): positive (study aborted)

In a Local Lymph Node Pretest Assay, mice (N=2/dose) were treated with different test item concentrations of 1%, 5% and 10% in MEK. The intention of the study was to confirm the positive results from the LLNA assay conducted in 2003, but with today’s specification (95% purity; LLNA 2003: 91% purity). All applied concentrations induced increases of 3H-thymidine incorporation into the cells from auricular lymph nodes (SI >3). In addition, at concentration of 10% of the test item in MEK, ear swelling up to 20% on day 5 was observed. However, the study was terminated after conducting the pretest. The result of the study was inconclusive due to low water solubility of the test material.

Pre-LLNA with purified test material. BASF SE. 2019 (OECD 429): positive (study aborted)

In a second Local Lymph Node Pretest Assay, mice (N=2/dose) were also treated with different test item concentrations of 1%, 5% and 10% in MEK. However, for this pretest the test material was purified. The intention of the study was to exclude the possibility, that the sensitization potential observed was due to the impurities of the test material (98% purity). Results were comparable to the pretest conducted with the regular specification of the test item (95% purity). All applied concentrations induced increases of 3H-thymidine incorporation into the cells from auricular lymph nodes (SI >3). In addition, at concentration of 10% of the test item in MEK, ear swelling up to 18% on day 5 was observed. However, the study was terminated after conducting the pretest. The result of the study was inconclusive due to low water solubility of the test material.

Buehler Test. Frey Tox.2020 (OECD 429): negative

In a Buehler test performed according to OECD guideline 429 with Dunkin-Hartley Guinea pigs, the induction phase including an occlusive patch topical application was performed using 50% (w/w) of the test item for 6 hours once a week for three consecutive weeks. During the induction phase, none of the animals displayed skin reactions neither in the test group nor in the control group. Four weeks after the first induction, the 6-hour challenge procedure was conducted by an occlusive topical application of the test item (25% (w/w) on the right cranial flank of all animals. The skin reactions were evaluated 24 hours and 48 hours after the challenge application. None animal of the test group (0/20; 0 %) and none animal of the control group (0/10; 0 %) responded with skin reactions to the challenge treatment with the test item neither after 24 hours nor after 48 hours. Also, the challenge treatment with the vehicle did not cause skin reactions at any time point neither in the test group (0/20; 0 %) nor in the control group (0/10; 0 %). In agreement with the sponsor, a rechallenge was waived due to the unambiguous test result. A delayed contact sensitization in the guinea pig was excluded by absence of skin reactions in the test group. Based on the results of the non-adjuvant sensitization study described in this final report, the test does not display a skin sensitizing potential.

SensIS Assay.EUROSAFE. 2021 (no guideline): negative

The objective of this study was to evaluate the capacity of the test item (95% purity) to induce the expression of specific irritation and sensitization biomarkers in a 3D-reconstructed epidermis model. The results obtained for the positive and negative controls were within acceptance criteria defined in the Study Plan, except for DMSO in experiment 2SI20011. The test item is classified as sensitizer if at least 7/17 genes of the “ARE” group, and/or 7/21 genes in the “SENS-IS” group are significantly overexpressed. The test item is classified as a non-sensitizer when negative results are observed at 100% and at all other analyzed concentrations. Considering the number of over-expressed gene in the "SENS-IS" and "ARE" gene groups, the test item gave negative result (less than 7 genes induced) when it was tested at 0.1, 1 %, 10 and 25% (w/v) in olive oil in two out of three experiments. Moreover, negative results were obtained when the test compound was directly applied on the epidermis without solubilization in the solvent in two out of three experiments.

However, this test has not been validated yet and is therefore not yet regulatorily accepted. In the first experiment, the test item induced more than 6 genes in the “SENS-IS” gene group (indeed 9 genes) when tested at 0.1, 1 and 10% (w/v) in olive oil, whereas in the second experiment, the test item induced less than 7 genes in the “SENS-IS” and “ARE” gene groups (indeed: 2 and 4 genes) when tested at the mentioned concentrations. In the third experiment, the test item induced less than 7 genes in the “SENS-IS” and “ARE gene groups (indeed, 4,2 and 6 genes) when tested at 0.1, 1, 10% (w/v) in olive oil but it induced 7 genes in the “SENS-IS” gene group, when tested at 25% (w/v) in olive oil and at 100% (test item not solubilized). Positive results of the test item at 25% and 100% were not reproducible in the fourth and fifth experiment. In conclusion, under the experimental conditions of this SENS-IS assay, the test item did not demonstrate an in vitro sensitizing potential when applied pure or diluted on the epidermis.

LLNA Product test (test item in 1.15% PP). BASF SE. 2021 (OECD 429): negative

In a product test using the Local Lymph Node Assay performed according to OECD guideline 429, the skin sensitizing potential of the test item in 1.15% polypropylene was assessed with CBA mice treated with 5%, 10% and 25% (w/w) concentrations of the test substance once daily for three consecutive days. Three days after the last application, ca. 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed in all animals during general observation. None of the applied test-substance concentrations induced biologically relevant increases (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The 25% concentration caused a statistically significant increase in 3H-thymidine incorporation. Concomitantly, none of the applied test-substance concentrations induced biologically relevant or statistically significant responses (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, no relevant or statistically significant increases in lymph node weights were noted at all concentrations. The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights, demonstrating the absence of ear skin irritation. Thus, it is concluded that the test item in Polypropylene does not exhibit a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

LLNA Product test (test item in 1% PE). BASF SE. 2021 (OECD 429): negative

In a second product test using the Local Lymph Node Assay performed according to OECD guideline 429, the skin sensitizing potential of the test item in 1% polyethylene was assessed with CBA mice treated with 5%, 10% and 25% (w/w) concentrations of the test substance once daily for three consecutive days. Three days after the last application, ca. 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation. None of the applied test-substance concentrations induced biologically relevant increases (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The 10% concentration caused a statistically significant increase in 3H-thymidine incorporation. Concomitantly, none of the applied test-substance concentrations induced biologically relevant or statistically significant responses (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, no relevant or statistically significant increases in lymph node weights were noted at all concentrations. The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights, demonstrating the absence of ear skin irritation. Thus, it is concluded that the test item in Polyethylene does not exhibit a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:: no study available

Justification for classification or non-classification

According to the harmonized classification (EC No 1272/2008), the test substance will be classified in a weight of evidence approach as Skin Sens Cat 1.

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	Experiment 1 -Number of overexpressed genes
Gene group	SLS 5% Positive control for irritation and negative control for sensitization	TNBS 1 M Positive control for sensitization	DMSO 100% Negative control for irritation and sensitization
Irritation	23	4	1
SENS-IS	2	2	0
ARE	2	14	6
	Conclusion
Irritation	POSITIVE	NEGATIVE	NEGATIVE
Sensitization	NEGATIVE	POSITIVE	NEGATIVE
	Experiment 2- Number of overexpressed genes
Gene group	SLS 5% Positive control for irritation and negative control for sensitization	TNBS 1 M Positive control for sensitization	DMSO 100% Negative control for irritation and sensitization
Irritation	23	3	7
SENS-IS	3	3	4
ARE	2	12	0
	Conclusion
Irritation	POSITIVE	NEGATIVE	NEGATIVE
Sensitization	NEGATIVE	POSITIVE	NEGATIVE
	Experiment 3- Number of overexpressed genes
Gene group	SLS 5% Positive control for irritation and negative control for sensitization	TNBS 1 M Positive control for sensitization	DMSO 100% Negative control for irritation and sensitization
Irritation	20	3	2
SENS-IS	1	3	3
ARE	1	11	8
	Conclusion
Irritation	POSITIVE	NEGATIVE	NEGATIVE
Sensitization	NEGATIVE	POSITIVE	POSITIVE
	Experiment 4- Number of overexpressed genes
Gene group	SLS 5% Positive control for irritation and negative control for sensitization	TNBS 1 M Positive control for sensitization	DMSO 100% Negative control for irritation and sensitization
Irritation	22	1	2
SENS-IS	3	0	1
ARE	1	11	1
	Conclusion
Irritation	POSITIVE	NEGATIVE	NEGATIVE
Sensitization	NEGATIVE	POSITIVE	NEGATIVE
	Experiment 5- Number of overexpressed genes
Gene group	SLS 5% Positive control for irritation and negative control for sensitization	TNBS 1 M Positive control for sensitization	DMSO 100% Negative control for irritation and sensitization
Irritation	21	7	8
SENS-IS	6	1	2
ARE	5	15	1
	Conclusion
Irritation	POSITIVE	NEGATIVE	NEGATIVE
Sensitization	NEGATIVE	POSITIVE	NEGATIVE

	Experiment 1 -Number of overexpressed genes
Gene group	10% Olive oil		1% Olive oil		0.1% Olive oil
Irritation	3		1		4
SENS-IS	9		9		9
ARE	4		0		3
Cp value – HSP90AA1	18.7		19.0		19.3
	Conclusion
Sensitization	POSITIVE		POSITIVE		POSITIVE
	Experiment 2- Number of overexpressed genes
Gene group	10% Olive oil		1% Olive oil		0.1% Olive oil
Irritation	2		2		0
SENS-IS	2		4		2
ARE	0		1		1
Cp value – HSP90AA1	18.1		17.5		17.5
	Conclusion
Sensitization	NEGATIVE		NEGATIVE		NEGATIVE
	Experiment 3- Number of overexpressed genes
Gene group	100%*	25% Olive oil	10% Olive oil	1% Olive oil		0.1% Olive oil
Irritation	4	4	8	10		11
SENS-IS	7	7	4	2		6
ARE	3	4	4	3		4
Cp value – HSP90AA1	19.4	19.5	18.6	18.7		18.5
	Conclusion
Sensitization	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE		NEGATIVE
	Experiment 4- Number of overexpressed genes
Gene group	100%*	25% Olive oil
Irritation	3	9
SENS-IS	1	3
ARE	0	1
Cp value – HSP90AA1	18.4	18.1
	Conclusion
Sensitization	NEGATIVE	NEGATIVE
	Experiment 5- Number of overexpressed genes
Gene group	100%*	25% Olive oil
Irritation	7	9
SENS-IS	4	5
ARE	1	0
Cp value – HSP90AA1	19.0	19.3
	Conclusion
Sensitization	NEGATIVE	NEGATIVE

Test item concentration % (w/v)		Measurement DPM	Calculation			SI
Test item concentration % (w/v)		Measurement DPM	DPM-BG (a)	No. of lymph nodes	DPM per lymph node (b)	SI
--	BG I	3	--	--	--	--
--	BG II	1	--	--	--	--
--	CG 5 (c)	5497	5495	8	687	--
2.5	TG2	121387	121385	8	15173	22.1*
5	TG3	172678	172676	8	21585	31.4*
10	TG4	165859	165857	8	20732	30.2*
2.5	TG5	146953	146951	8	18369	26.7
5	TG6	190092	190090	8	23761	34.6
10	TG8	212233	212231	8	26529	38.6

	Test group (sensitized with test compound)		Control group (adjuvant-treated)
	After 24 h	After 48 h	After 24 h	After 48 h
Test compound	15/20	12/20	0/10	0/10
Vehicle control	0/20	0/20	0/20	0/20

Registration Dossier

Registration Dossier

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Diss Factsheets

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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Endpoint conclusion

Respiratory sensitisation

Endpoint conclusion

Justification for classification or non-classification

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