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Administrative data

Description of key information

Skin sensitisation in vivo (LLNA), Brunt (2018)

Under the conditions of this study, the test material was not-sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 June 2018 to 04 July 2018
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:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test material was formulated within 2 hours of being applied to the test system. It was assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation.
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were group housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days.
- The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: At least fifteen changes per hour.
- Photoperiod: 12 hours continuous light and 12 hours darkness.
Vehicle:
other: 1 % pluronic L92 in distilled water
Concentration:
25, 10 and 5 % w/w
No. of animals per dose:
Four
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: For the purpose of the study, the test material was freshly prepared as a suspension in 1 % pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing compared with the other vehicles examined in the Vehicle Determination. Other vehicles tested were: acetone/olive oil (4:1), dimethyl formamide, butanone, dimethyl sulphoxide, acetone, ethanol/distilled water (7:3) and propylene glycol.
- Preliminary Screening Test: As no toxicological information was available regarding the systemic toxicity of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test material at a concentration of 25 % w/w in 1 % pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse – Pooled Method
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
- Groups of four mice were treated with the test material at concentrations of 25, 10 or 5 % w/w in 1 % pluronic L92 in distilled water. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
- 3H-Methyl Thymidine Administration: Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 µL) of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

OBSERVATIONS
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
A group of five animals was treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85 % as an emulsion in 1 % pluronic L92 in distilled water at a concentration of 25 % v/v. A further control group of five animals was treated with 1 % pluronic L92 in distilled water alone.
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
25 % v/v positive control in 1 % pluronic L92 in distilled water had a Stimulation Index of 6.48 and was therefore considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
SI
Value:
1.13
Test group / Remarks:
5 % w/w in 1 % pluronic L92 in distilled water
Key result
Parameter:
SI
Value:
0.93
Test group / Remarks:
10 % w/w in 1 % pluronic L92 in distilled water
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
25 % w/w in 1 % pluronic L92 in distilled water
Cellular proliferation data / Observations:
PRELIMINARY SCREENING TEST
- No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted.
- Based on this information the dose levels selected for the main test were 25, 10 and 5 % w/w in 1 % pluronic L92 in distilled water.

CELLULAR PROLIFERATION DATA
- The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 5 % test material concentration SI = 1.13, 10 % test material concentration SI = 0.93 and 25 % test material concentration SI = 1.90. The SI values gave negative results at all concentrations tested.

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

BODY WEIGHTS
- Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Table 1: Results of the Main Test

Concentration (% w/w) in

1 % pluronic L92 in distilled water

Stimulation Index

Result

5

1.13

Negative

10

0.93

Negative

25

1.90

Negative

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material was not-sensitising.
Executive summary:

The potential of the test material to be sensitising to the skin was investigated in accordance with the standardised guidelines OECD 429 and EU Method B42, under GLP conditions.

A Local Lymph Node Assay was performed in the CBA/Ca strain of mouse with topical application of the test material to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25 % w/w of the test material in 1 % pluronic L92 in distilled water, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a suspension in 1 % pluronic L92 in distilled water at concentrations of 25, 10 or 5 % w/w. A further group of four animals was treated with 1 % pluronic L92 in distilled water alone.

A positive control study was performed to assess the sensitivity of the strain of mouse used at these laboratories using α- Hexylcinnamaldehyde. The SI was 6.48 and therefore α-Hexylcinnamaldehyde, tech., 85 % was considered to be a sensitiser under the conditions of the test.

The Stimulation Index of the test material expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 5 % test material concentration SI = 1.13, 10 % test material concentration SI = 0.93 and 25 % test material concentration SI = 1.90. The SI values gave negative results at all concentrations tested.

Under the conditions of this study, the test material was not-sensitising.

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

Skin Sensitisation In Vivo (LLNA), Brunt (2018)

The potential of the test material to be sensitising to the skin was investigated in accordance with the standardised guidelines OECD 429 and EU Method B42, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A Local Lymph Node Assay was performed in the CBA/Ca strain of mouse with topical application of the test material to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25 % w/w of the test material in 1 % pluronic L92 in distilled water, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a suspension in 1 % pluronic L92 in distilled water at concentrations of 25, 10 or 5 % w/w. A further group of four animals was treated with 1 % pluronic L92 in distilled water alone.

A positive control study was performed to assess the sensitivity of the strain of mouse used at these laboratories using α- Hexylcinnamaldehyde. The SI was 6.48 and therefore α-Hexylcinnamaldehyde, tech., 85 % was considered to be a sensitiser under the conditions of the test.

The Stimulation Index of the test material expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 5 % test material concentration SI = 1.13, 10 % test material concentration SI = 0.93 and 25 % test material concentration SI = 1.90. The SI values gave negative results at all concentrations tested.

Under the conditions of this study, the test material was not-sensitising.

Skin Sensitisation In Vitro

Soybean meal, protein extn. Residue (CAS No. 91081-84-4) is to be registered under Annex VII of the REACH regulation (1). Under Annex VII, an assessment of skin sensitisation potential is required in order to determine whether the substance is a skin sensitiser and if so, whether it can be presumed to have the potential to produce significant sensitisation in humans (Category 1A). Before any new tests are conducted, all available in vitro data, in vivo data, historical human data, data from valid (Q)SARS and data from structurally related substances (read-across approach) should firstly be assessed. If these data are inadequate for hazard and risk assessment, including classification and labelling, further testing should be considered in accordance with point 8.3 of Annex VII. The studies under points 8.3.1 and 8.3.2 of Annex VII do not have to be conducted if the substance is classified as skin corrosion (Category 1), is a strong acid (pH≤ 2.0) or base (pH ≥ 11.5) or is spontaneously flammable in air or in contact with water (2).  

Available Information on Soybean meal, protein extn. Residue (CAS No. 91081-84-4):

Information provided by the Sponsor indicates that Soybean meal, protein extn. Residue (CAS No. 91081-84-4) is a plant extract and a UVCB. A representative structure of the substance is not available and it has not been possible to identify individual components of the substance by analytical techniques. The log10 Pow of the substance at pH 3 has been determined to be in the range less than 0.3 to greater than 6.5. A literature search has been undertaken for the substance but no useful information was found with regard to skin sensitisation potential. In silico assessment is unsuitable for this UVCB substance due to the unknown composition.

The substance is not classified for skin corrosion (Category 1), is not a strong acid or base and is not spontaneously flammable in air or in contact with water. Hence, in accordance with point 8.3 of Annex VII an assessment of skin sensitisation potential is required.

Consideration of new skin sensitisation testing

According to point 8.3.1 of Annex VII, information from in vitro/in chemico test method(s) recognised according to Article 13(3), addressing each of the following key events of skin sensitisation is required:

(a) Molecular interaction with skin proteins (key event 1)

(b) Inflammatory response in keratinocytes (key event 2)

(c) Activation of dendritic cells (key event 3)

However, the tests do not have to be conducted if the available in vitro/in chemico test method(s) are not applicable for the substance or are not adequate for classification and risk assessment. In such a case, according to Point 8.3.2, an in vivo study shall be conducted, and this shall be an OECD 429 LLNA (3) (unless justification is provided to justify the use of a different test method).

Applicability of the in vitro/in chemico test methods for assessment of skin sensitisation potential

Key Event 1

The test method that is currently available for identifying whether a substance will interact (covalently bind) with skin proteins, thereby addressing key event 1 of the skin sensitisation Adverse Outcome Pathway (AOP),  is the OECD 442C In Chemico Direct Peptide Reactivity Assay (DPRA) (4). However, at present, the prediction model used with this test method cannot be used for complex mixtures of unknown composition or for UVCB substances, as the defined molar ratio of test chemical and peptide is required (Paragraph 11 of OECD 442C, 4 February 2015). Hence the test method is not applicable for the assessment of Soybean meal, protein extn. Residue (CAS No. 91081-84-4) .

Key Events 2 and 3

The currently available test guidelines/methods for addressing key events 2 and 3 of the skin sensitisation AOP are:

Key Event 2:

OECD 442D (25 June 2018) Key Event Based Test Guideline 442D In Vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation (5):

- The ARE Nrf2 Luciferase KeratinoSens™ test method

- The ARE Nrf2 Luciferase LuSens test method

Key Event 3:

OECD 442E (25 June 2018) Key Event Based Test Guideline In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for skin sensitisation (6).

- Human Cell Line Activation Test (h-CLAT)

- U937 Cell Line Activation test (U-SENS™)

- Interleukin-8 Reporter Gene assay (IL-8 Luc Assay)

For the KeratinoSens™ and Luciferase test methods, h-CLAT and IL-8 Luc Assay, it is stated in the test guideline that because of the limited metabolic capacity of the cell line used and because of the experimental conditions, pro-haptens and pre-haptens, in particular those with a slow oxidation rate, may provide negative results. On the other hand, based on a review of the  DPRA, KeratinoSens™ and h-CLAT test methods to detect pre and pro-haptens, it has been reported that skin metabolism was not likely to be a major consideration for assessing skin sensitisation potential and that sensitisers requiring activation could be identified correctly using one or more of these non-animal methods (7).

However, for a UVCB, such as Soybean meal, protein extn. Residue (CAS No. 91081-84-4), it is considered that negative results in these studies would have to be viewed with caution since the possibility of one or more unknown components of the substance being a pre and/or pro-hapten cannot be disregarded.

Whilst conduct of testing according to OECD 442D and OECD 442E might be technically possible for Soybean meal, protein extn. Residue (CAS No. 91081-84-4), the information that would be generated and its adequacy for classification and risk assessment has been considered. The possible combinations of outcomes of testing conducted according to these two guidelines are as follows:

- Positive results generated in both test methods: As none of the available in vitro test methods allow an assessment of skin sensitisation potency and there are no existing information/data to conclude on whether the substance requires classification as Category 1A, an OECD 429  LLNA would be required.

- A positive result from only one of the test methods: This would be an inconclusive outcome and in the absence of other information on skin sensitisation potential, an OECD 429 LLNA would be required to conclude on classification.

- One or both test methods found to be non-applicable to the substance: In the absence of other information on skin sensitisation potential, an OECD 429 LLNA would be required.

- Negative results generated in both test methods: For non-UVCB substances the results could potentially be used within a weight-of-evidence assessment to conclude that the substance does not require classification for skin sensitisation (applying the ‘2 out of 3’ prediction model). However, as it is not possible to conclude if one or more constituents of the UVCB substance Soybean meal, protein extn. Residue (CAS No. 91081-84-4), are pre and/or pro-haptens, and in the absence of other information/data to support a negative conclusion, an OECD 429 LLNA would be required.

Hence, regardless of the outcome of the in vitro tests to address key events 2 and 3, a conclusion on classification and risk assessment would not be possible. To achieve this , an OECD 429 LLNA would be required.

Conclusion

Existing information/data are not available to allow conclusion on classification and risk assessment as required by point 8.3.1 of Annex VII of the REACH Regulation for skin sensitisation of Soybean meal, protein extn. Residue (CAS No. 91081-84-4). Furthermore, the OECD 442C in chemico DPRA to address key event 1 of the AOP, is not applicable to assessment of the UVCB substance and the conduct of available in vitro tests to address key events 2 and 3 will not generate adequate information for classification and risk assessment. Conduct of an OECD 429 LLNA will therefore be necessary and omission of the in vitro tests is justified.

References:

1. Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

2. Commission Regulation (EU) 2017/706 of 19 April 2017 amending Annex VII to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards skin sensitisation and repealing Commission Regulation (EU) 2016/1688.

3. OECD (2010). The Local Lymph Node Assay. OECD Guideline for the Testing of Chemicals No. 429, Organisation for Economic Cooperation and Development, Paris.

4. OECD (2015). In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA). OECD Guideline for the Testing of Chemicals, No. 442C. Organisation for Economic Cooperation and Development, Paris.

5. OECD (2018). Key Event Based Test Guideline 442D In Vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation. Organisation for Economic Cooperation and Development, Paris.

6. OECD (2018). Key Event Based Test Guideline 442E In Vitro Skin Sensitisation Assays Addressing the Key Event on Activation of Dendritic Cells on the Adverse Outcome Pathway for Skin Sensitisation. Organisation for Economic Cooperation and Development, Paris.

7. Patlewicz G, Casati S, Basketter DA, Asturiol D, Roberts DW, Lepoittevin J-P, Worth A and Aschberger K (2016). Can currently available non-animal methods detect pre and pro haptens relevant for skin sensitisation? Regul Toxicol Pharmacol, 82, pp 147-155.

Silvia Casati, Karin Aschberger, David Asturiol, David Basketter, Sabcho Dimitrov, Coralie Dumont, Ann-Therese Karlberg, Jean-Pierre Lepoittevin, Grace Patlewicz, David W. Roberts and Andrew Worth; Ability of non-animal methods for skin sensitisation to detect pre- and pro-haptens: Report and recommendations of an EURL ECVAM expert meeting; EUR 27752 EN; doi:10.2788/01803

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to sensitisation.