Registration Dossier

Administrative data

Description of key information

KeratinoSens (OECD TG 442D): negative

hCLAT (OECD TG 442E): positive

LLNA (OECD TG 429): negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Remarks:
OECD 442D
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 March to 6 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (4°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: 20 mg/ml in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none
Details on study design:
DESCRIPTION OF THE TEST SYSTEM
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection using a BMG Optima FLUOstar) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

METHOD OF ADMINISTRATION OF TEST ITEM
Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.

METHOD OF ADMINISTRATION OF REFERENCE ITEMS
Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).

EXPOSURE TIMES OF TEST ITEMS AND REFERENCE ITEMS
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.

NUMBER OF REPETITIONS
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT.

NEGATIVE CONTROL
DMSO, 99.7%

POSITIVE CONTROL
Cinnamic aldehyde, 99%

DATA ANALYSIS
The following parameters were calculated in the KeratinoSens test method:
- The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration of the test item and positive control.
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity).
- For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student’s t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
-The percentage of viability as compared to the negative control.

ASSAY ACCEPTANCE CRITERIA
Test results are acceptable if:
- The positive control (cinnamic aldehyde) produces positive results, i.e., the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations.
-The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet the following targets:
* Average induction in the three replicates for cinnamic aldehyde at 32 µM is between the XCellR8 historical range (currently 1.6 and 3)
* EC1.5 value for cinnamic aldehyde is between the XCellR8 historical range (currently 6 µM and 39 µM).

Note: At least one of these criteria must be met, otherwise the run is discarded. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
- CV% of blank values < 20%

INTERPRETATION OF RESULTS AND SKIN SENSITISATION PREDICTION MODEL
A test item is considered a sensitiser if the following conditions are met in 2 of 3 repetitions:
- The IMAX is higher than 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test).
- The cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
- The EC1.5 value is < 1000 µM or < 200 µg/mL for test chemicals with no defined MW.
- There is an apparent overall dose-response for luciferase induction (or a biphasic response).

Positive control results:
Induction (=1.5-fold):
1.518 at 8 µM
2.664 at 16 µM
5.019 at 32 µM
11.866 at 64 µM
20.861 at 128 µM
Key result
Positive controls validity:
valid
Remarks on result:
other: Negative

See attached additional information on results.

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro KeratinoSens test, conducted according to OECD test guideline 442D and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined negative.
Executive summary:

The in vitro KeratinoSens test, conducted according to OECD test guideline 442D and to GLP, was performed to determine skin sensitisation potential of Alfalfa, ext. by assessing keratinocytes activation.

After 48h exposure of cells to 12 concentrations of Alfalfa, ext. luciferase measurements and MTT viability test were performed.

The sensitisation potential of Alfalfa ext. was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value.

Alfalfa, ext. only caused luciferase induction above 1.5 in repetition 2, with an EC1.5 value below 0.098 µg/ml. Since no clear dose response was observed, this was classified as an inconclusive result for this repetition. In repetitions 1 and 3, the threshold for induction was not exceeded at any of the concentrations tested and, therefore, based on 2 out of 3 concordant results from independent experiments, the result for Alfalfa, ext. in this KeratinoSens test was negative.

The maximum induction for repetition 1 was observed at 200 µg/ml Alfalfa, ext. (1.238). For repetitions 2 and 3 the IMAX values of 3.139 and 0.969, respectively, were observed at 0.391 µg/ml.

All of the formal acceptance criteria of the tests were met except for acceptance criterion 2 (average induction of positive control at 32 µM). However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, results are considered as valid.

Therefore, in the in vitro test, conducted according to OECD test guideline 442D and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined negative for the second key event of the skin sensitisation Adverse Outcome Pathway.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 April to 14 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (4°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: 5000 µg/mL in culture medium
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none


Details on study design:
DESCRIPTION OF THE TEST SYSTEM
Human Cell Line Activation Test (h-CLAT) method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria.

EXPERIMENTAL DESIGN AND PROCEDURES OF XTT TEST
- After the cell seeding, 100 µL of the test item dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.
- At the end of the incubation period, 50 µL of the XTT labelling mixture (buffer solution and substrate solution, 1:100) were added to each well. The cells were incubated and subsequently transferred to a microplate reader. The absorbance was measured at 450 nm (reference wavelength 690 nm)
- A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance (= viability in [%]) as compared to the solvent control is calculated using this formula:

Relative absorbance (%) =100 x (mean absorbance test item – absorbance chemical blank)/ (mean absorbance solvent control – absorbance chemical blank)

-To calculate the concentration of toxicant needed to reduce the relative absorbance to 75% of the solvent control (CV75) the following formula is used:
CV75 = Conc. >75 – (Conc. >75 – Conc. <75) x (% >75 -75)/ (% >75 - %<75), where

Conc. >75 = max. measured concentration with the % of solvent control >75%
Conc. <75 = min. measured concentration with the % of solvent control <75%
% >75 = relative absorbance at a) in %
% <75 = relative absorbance at b) in %

- Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. Since the CV75 could not be determined, a stock solution of the highest soluble test item concentration was prepared and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

EXPERIMENTAL DESIGN AND PROCEDURES OF h-CLAT TEST
- The test item was tested in four independent runs.
- For the test item exposure the highest soluble test item concentration of the XTT test (without precipitations) was used instead of 1.2 × CV75, since no CV75 could be determined. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
- Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
- The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 minutes) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 - 8 °C (on ice) for approx. 15 miutes. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
- All solutions were kept light protected at 2 - 8°C or on ice during the staining and analysis procedures.
- The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 minutes. at 2 - 8 °C (on ice).
- After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.
- Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
- The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal.
- Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

NUMBER OF REPETITIONS
- h-CLAT test: Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

MEDIUM CONTROLE
Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin).

POSITIVE CONTROL (h-CLAT)
DNCB (2,4-dinitrochlorobenzene) final concentration: 2 and 3 µg/mL, Purity = 99%) dissolved in DMSO.

SOLVENT CONTROLE FOR POSITIVE CONTROLE (h-CLAT)
DMSO in culture medium, final concentration 0.2%, Purity = 99%.

DATA ANALYSIS (h-CLAT)
The RFI was used as an indicator of CD86 and CD54 expression, and was calculated as follows for each concentration of every chemical:
RFI (%) = (MFI of test item treated cells) – (MFI of test item treated isotype control cells)/(MFI of solvent control cells) – (MFI of solvent isotype control cells) X 100

MFI = Geometric Mean Fluorescence Intensity (GeoMean)

The cell viability of the h-CLAT experiment is calculated as follows for each concentration of every chemical:
Cell viability (%) = mean cytotox of solvent control cells / mean cytotox of test item treated cells x 100
where the Mean cytotox is the mean of GeoMean(7-AAD) isotype control, GeoMean(7-AAD) CD54 and GeoMean(7-AAD) CD86.

ASSAY ACCEPTANCE CRITERIA

The XTT test is considered to be acceptable if it meets the following criteria:
- mean absorbance of the medium control is = 0.5
- mean viability of the solvent control is = 90% in comparison to the medium control
The following acceptance criteria should be met when using the h-CLAT method:
- Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
- In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 = 150% and CD54 = 200%).
- For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 = 150% and CD54 = 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
- For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of <90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is = 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 µg/mL in saline (or medium or other solvents/vehicles), 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).

SKIN SENSITISATION PREDICTION MODEL
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
- The RFI of CD86 is = 150% at any tested concentration (with cell viability = 50%);
- The RFI of CD54 is = 200% at any tested concentration (with cell viability = 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).
Key result
Parameter:
other: Dendritic cell activation potetial
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

See attached additional information on results.

Interpretation of results:
study cannot be used for classification
Conclusions:
In an in vitro Human Cell Line Activation test, conducted according to OECD test guideline 442E and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined positive.
Executive summary:

The in vitro Human Cell Line Activation test (h-CLAT) conducted according to OECD test guideline 442E and to GLP, was performed to determine skin sensitisation potential of Alfalfa, ext. by assessing dendritic cell activation.

Alfalfa, ext. dissolved in culture medium was administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) was determined by two XTT tests.

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (5000 µg/mL). Due to the lack of cytotoxicity, a mean CV75 value could not be calculated. In addition, a proliferative effect was observed in both XTT tests. Since precipitations were observed in the two highest tested concentrations, 1250 µg/mL was used for the h-CLAT runs as highest test item concentration.

The following concentrations of the test item were tested in the first and second main run: 349, 419, 502, 603, 723, 868, 1042 and 1250 µg/mL.

Due to observed high cytotoxicity in the first two runs, the test item concentrations were adjusted for the third and fourth run: 81, 97, 117, 140, 168, 202, 243 and 291 µg/mL (run three) and 56, 68, 81, 97, 117, 140, 168 and 202 µg/mL (run four).

The Relative fluorescence intensity (RFI) of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of the third run and the RFI of CD54 was equal or greater than 200% in at least one concentration of the fourth run. A dose response could be observed for CD54 in both runs. Therefore, the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 = 200% and CD86 = 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 = 200% and CD86 = 150%) and the cell viability was >50%. Except the CD86 RFI value of the positive control (2.0 µg/mL DNCB) in the fourth h-CLAT run did not exceed the positive criterion (CD86 = 150%). However, this was considered to be acceptable since the CD86 RFI value of the positive control (3.0 µg/mL DNCB) in the same h-CLAT run (run four) exceeded the positive criteria.

Therefore, in an in vitro test, conducted according to OECD test guideline 442E and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined positive for the third key event of the skin sensitisation Adverse Outcome Pathway.

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)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No 604/2012 Part B
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refrigerator (2-8°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: disolved in 1% Pluronic L92 in Elix water
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not abailable
- Age at study initiation: ca. 12 weeks
- Weight at study initiation: 20.3-26.4 g
- Housing: up to 5 animals in polycarbonate cages containing sterilized sawdust as bedding material
- Diet: pelleted rodent diet ad libitum
- Water: municipal tap-water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
- IN-LIFE DATES: From: To: not available
Vehicle:
other: 1% Pluronic L92 in Elix water
Concentration:
5, 10 and 20%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: 25 and 50% in the vehicle; 50% did not meet the secection criteria due to the variation in ear thickness during the observation period
- Irritation: very slight irritation was observed at 25%
- Systemic toxicity: no signs of systemic toxicity at 25% was noted
- Ear thickness measurements: 25%; Day 1: 0.220-0.230 mm (L), 0.215-0.35 mm (R); Day 3: 0.265-0.270 (L), 0.270 mm (R); Day 6: 0.250-0.255 mm (L), 0.250-0.265 mm (R)
- Erythema scores: 25%; Day 1 and 2: no erythema; Day 3-4: very slight erythema (barely perceptible); Day 5-6: no erythema, scaliness

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI = 3
EC3 value = 2%: sub-category 1A
EC3 value > 2%: sub-category 1B

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
The dorsal surface of both ears was topically treated (25 µL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic
stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
Positive control substance(s):
other: hexylcinnamaldehyde (historical data)
Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
25%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA :
Assessment of lymph node proliferation was not performed

DETAILS ON STIMULATION INDEX CALCULATION :
A Stimulation Index (SI) was 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.

EC3 CALCULATION :
An EC3 value of 14.3% was calculated using linear interpolation.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Interpretation of results:
GHS criteria not met
Conclusions:
In the Local Lymph Node Assay, conducted according to OECD test guideline 429 and to GLP, Alfalfa, ext. was not classified as a skin sensitizer according to the Regulation (EC) No 1272/2008.
Executive summary:

The Local Lymph Node Assay, conducted according to OECD test guideline 429 and to GLP, was performed to determine skin sensitisation potential of Alfalfa, ext. in mice.

Test item concentrations selected for the main study were based on the results of a pre-screen test. At 50% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values. Therefore this concentration did not meet the selection criteria. At a 25% test item concentration, no signs of systemic toxicity were noted and very slight irritation was observed. Variation in ear thickness during the

observation period were more than 25% from Day 1 pre-dose values for one ear of one animal on Day 3 only, which was considered to be an incidental finding. Therefore this concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (1% Pluronic L92 in Elix water). 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.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 416, 365 and 334 DPM, respectively. The mean DPM/animal value for the vehicle control group was 206 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 1.8 and 1.6, respectively.

Since there was no indication that the test item elicits a SI = 3 when tested up to 25%, Alfalfa, ext. 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 25%.

The six-month reliability check with alpha-hexylcinnamaldehyde indicated that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, Alfalfa, ext. would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In an in vitro KeratinoSens test (key event 2), conducted according to OECD test guideline 442D and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined negative. In an in vitro Human Cell Line Activation test (key event 3), conducted according to OECD test guideline 442E and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined positive.

In the murine Local Lymph Node Assay (key event 4), conducted according to OECD test guideline 429 and to GLP,  Alfalfa, ext. was not considered as a skin sensitizer.

Based on the available in vitro and in vivo data, Alfalfa, ext. is not classified as a skin sensitizer according to criteria set out in the Regulation (EC) No 1272/2008.