Fragrance Skin Sensitization Testing: LLNA, DPRA, and KeratinoSens

Skin sensitization is the most consequential safety endpoint for fragrance materials. A positive result triggers GHS Category 1 labelling, IFRA use restrictions, EU CLP labelling, and in some cases outright prohibition. For B2B buyers, understanding which tests are run, what the numbers mean, and how they feed regulatory categorisation is essential to evaluating supplier safety files.

The Adverse Outcome Pathway

Modern sensitization assessment follows the OECD adverse outcome pathway (AOP), which describes four key events:

1. Molecular initiating event — covalent binding to skin proteins.
2. Keratinocyte activation — inflammatory signalling in the epidermis.
3. Dendritic cell activation — immune system priming.
4. T-cell proliferation — the in vivo manifestation measured in animal tests.

Each of the first three events has a validated in vitro or in chemico test, and the fourth has the historical animal test. Regulators now prefer defined approaches (DA) that combine multiple non-animal methods.

The Four Core Methods

1. LLNA — Local Lymph Node Assay (OECD TG 429)

The historical gold-standard in vivo test in mice. The test material is applied to the ear for three consecutive days, and lymph node cell proliferation is measured by radioactive thymidine incorporation on day six.

• Result: Stimulation Index (SI), the ratio of proliferation in treated versus control animals.
• Pass/Fail: SI greater than or equal to 3 = positive (sensitiser). SI below 3 = negative.
• Potency sub-categorisation: EC3 value (estimated concentration producing SI = 3) feeds GHS Category 1A (strong) versus 1B (weak).

LLNA is no longer required for EU cosmetics under the animal testing ban, but it remains accepted in many non-EU markets and for non-cosmetic uses.

2. DPRA — Direct Peptide Reactivity Assay (OECD TG 442C)

An in chemico test measuring covalent binding of the test material to synthetic cysteine and lysine peptides by HPLC.

• Result: Percent peptide depletion.
• Pass/Fail: Mean cysteine and lysine depletion above 6.38 percent = positive.
• Strength: Excellent for electrophilic materials such as aldehydes and Michael acceptors. Weak for pro-haptens that require metabolic activation.

3. KeratinoSens (OECD TG 442D)

An in vitro reporter gene assay using immortalised human keratinocytes carrying the ARE-Nrf2 luciferase construct. It measures activation of the antioxidant response element, a marker of keratinocyte stress.

• Result: Maximum luciferase induction (Imax) and the concentration producing 1.5-fold induction (EC1.5).
• Pass/Fail: Imax above 1.5, with a statistically significant dose response and viability above 70 percent at the EC1.5, = positive.

4. h-CLAT — Human Cell Line Activation Test (OECD TG 442E)

An in vitro test using the THP-1 monocytic cell line, measuring upregulation of CD86 and CD54 surface markers by flow cytometry.

• Pass/Fail: CD86 relative fluorescence intensity above 150 percent and CD54 above 200 percent, with viability above 90 percent, in at least two of three runs = positive.

Defined Approaches (DA)

No single non-animal method is sufficient on its own. OECD TG 497 defines Defined Approaches that combine at least two of the in chemico / in vitro methods with physico-chemical and metabolic information. A typical DA for a fragrance material uses:

• DPRA (key event 1)
• KeratinoSens (key event 2)
• h-CLAT (key event 3)

Pass/Fail Under a 2-out-of-3 DA

DPRA	KeratinoSens	h-CLAT	DA Conclusion
Positive	Positive	Positive	Sensitiser (Category 1)
Positive	Positive	Negative	Sensitiser
Positive	Negative	Positive	Sensitiser
Negative	Positive	Positive	Sensitiser
Negative	Negative	Negative	Non-sensitiser
Mixed single positive			Inconclusive; further testing required

From Test Result to GHS Category

• GHS Category 1A — strong sensitiser, supported by LLNA EC3 below 2 percent, human evidence, or DA potency data.
• GHS Category 1B — weak to moderate sensitiser.
• GHS Category 1, no sub-category — when potency cannot be resolved.

EU CLP additionally requires the EUH208 statement when a sensitiser is present above 0.1 percent in a mixture, naming the substance.

IFRA Implications

IFRA Standards use sensitization potency, alongside phototoxicity and systemic toxicity, to set maximum use levels by product category. A material classified as a strong sensitiser may be:

• Restricted to a low maximum use level (for example, 0.1 percent in leave-on products).
• Restricted to rinse-off categories only.
• Prohibited entirely, as with atranol and chloroatranol in oakmoss and treemoss extracts.

Documentation Buyers Should Require

• Full DA report citing OECD TG 497 with the underlying DPRA, KeratinoSens, and h-CLAT data.
• LLNA data where available, especially for older materials.
• GHS / CLP classification statement with category and sub-category.
• IFRA Certificate of Compliance with category-specific use levels.
• EUH208 trigger assessment for finished mixtures.

Closing Note

Skin sensitization data is the foundation of fragrance safety assessment. Buyers who require a complete defined approach, not just a single in vitro result, and who verify the GHS sub-categorisation and IFRA use levels, protect their products from late-stage reformulation and labelling surprises. Aromiso provides DA-based sensitization summaries, IFRA certificates, and CLP classification statements for every fragrance material in our library, and flags any material that would trigger EUH208 labelling at the proposed use level.