Exposure of human skin to ultraviolet (UV) radiation can cause an array of phototoxic effects including induction of inflammatory responses that can lead to skin aging,¹ sunburns (erythema),² and skin cancer.^3,4 The UV light spectrum spans wavelengths from 100 to 400 nm and can be subdivided into three bands: UVA (315–400 nm), UVB (280–315 nm), and UVC (100–280 nm).⁵ Contained within the UVC band is a sub-band known as far-UVC that ranges from 200 to 235 nm. Each of these UV bands, characterized by their specific wavelengths and photon energy, are known to exert a variety of effects on the skin due to their penetrative capacity and interaction with different biophysical targets found within cells. The longest wavelengths, UVA, can penetrate deep into the dermis due to their lower photon energies that do not get absorbed by cellular biomolecules but can nevertheless induce the formation of damaging reactive oxygen species.⁶ In contrast, shorter wavelengths found within the UVB and UVC bands are mostly absorbed in the epidermis by interactions with major cellular biomolecules such as DNA and proteins.^2,7,8

The first line of defense of the skin against UV radiation is the outermost layer of the epidermis known as the stratum corneum (SC). Acting as a biomechanical barrier, the SC provides protection against various environmental factors and serves as an optical barrier against UV light.⁷ Research indicates that the SC absorbs UVC light most effectively, resulting in more significant mechanical and structural damage to the SC, than that induced by UVB and far less than that from UVA exposure.^7,9 The transmission and thickness of the stratum corneum determines the level of optical protection it provides to the dividing cells found in the underlying skin layers.^10–12 The high absorption of UVC light by the SC, particularly in the far-UVC region, is attributed to absorption by peptide bonds of proteins and lipids, while absorption in the UVB range is mainly due to keratin and melanin, and in the UVA range by melanin.^8,13–15

Due to the different harmful effects that each component of the UV spectrum has on the skin and eyes,⁵ guidance on maximal safety limits for UV exposure has been developed by national and international groups.^16,17 These guidelines were established to minimize both the risk of acute adverse effects (i.e., erythema and photokeratitis),¹⁸ as well as delayed effects (i.e., skin and ocular cancers).^19,20 These exposure limits depend on the wavelength and the photobiological responses induced, where thresholds for each biological effect vary strongly with wavelength. To this extent, action spectra have been developed that allows quantitative measure of the spectral effectiveness of each wavelength at eliciting specific biological effects.²¹

The earliest and most widely used guidelines for human exposure limits to radiation are issued by the American Conference of Governmental Industrial Hygienists (ACGIH) and are referred to as Threshold Limit Values (TLVs^®).²² TLVs^® have been defined as the dose to which nearly all healthy workers can be exposed during an 8-h period without experiencing acute adverse effects or an increased risk of developing delayed effects.^22,23 Similar guidelines on the limits of exposure have been implemented by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) in collaboration with the World Health Organization (WHO).¹⁷

In 2021, a review by Sliney and Stuck argued for raising the exposure limits for wavelengths below 300 nm.²⁴ They based their argument on numerous studies that found that the existing thresholds set for 222 and 207 nm to be overly conservative considering the minimal biological effects induced by these wavelengths.^24–31 Subsequently, in 2022, ACGIH increased the TLVs^® for wavelengths below 240 nm and established separate limits for eyes and skin at wavelengths below 300 nm. Notably, higher exposure limits were set for wavelengths below 300 nm for cases where human skin is exposed but the eyes are protected.³² The TLVs^® for the shorter wavelengths in the far-UVC region were increased for both eye and skin exposures. Radiation at 222 nm, which is of particular interest for its antimicrobial properties within occupied spaces using filtered KrCl excimer lamps,^33,34 had the TLVs^® raised from 23 to 160 mJ/cm² for eyes and to 480 mJ/cm² for the skin.^24,32,35

Even though considerable evidence suggests the new skin threshold limits for wavelengths below 240 nm pose minimal risk,^19,24 the standard erythema action spectra, which provide the quantitative basis for these limits, have not been extended below 240 nm.^16,36,37 Therefore, there is a need to expand the erythema action spectrum curve to include results down to 200 nm to better define the potential hazards of far-UVC exposures.^38–40 Previous research on murine models regarding far-UVC skin safety noted that erythema may not be the most accurate indicator of acute skin response at these wavelengths, as scale formation (flaking) has been observed to occur in the absence of erythema following 222 nm UV exposure.^27,38,41 Consequently, more appropriate evaluation methods have been adopted to determine the acute effects of far-UVC on the skin. These methods include a new threshold dose estimate termed the minimal perceptible response dose (MPRD), which considers the development of erythema, edema, scales, and fissures, rather than solely relying on the minimal erythema dose (MED). It has been observed that using MPRD evaluations to assess the effects of UVB radiation provides action spectra similar to the human erythema action spectra.³⁸ In the current study, we expand the erythema response spectrum down to 200 nm in hairless, albino, SKH-1 mice using MPRD evaluations. The SKH-1 experimental mouse model is often used in dermatological UVB exposure studies because this strain is particularly sensitive to UV effects and develops skin damage similar to that seen in humans.⁴⁰ Irradiations were performed with wavelengths between 200 and 270 nm in 5 nm increments using a UV monochromator with a narrow bandwidth emission.^24,42 The use of a narrow bandwidth provided more detailed information about the sharp increase in erythema response observed in the 230–240 nm range. This study reports minimal perceptible response threshold doses for mice skin for wavelengths across the UVC spectrum.

This study provides the most comprehensive analysis of mice acute skin response spectrum after UVC exposure (Figure 4). Previous reports of short-term skin responses to UV irradiation are limited for wavelengths below 250 nm primarily due to experimental inaccessibility to (1) a monochromator able to irradiate at spectral bandwidth less than 10 nm, and (2) limiting examinations to wavelengths from those principally emitted by gas lamps.^24,42 By taking proper consideration of the spectral bandwidth and utilizing narrow bandwidth exposures in 5 nm increments with minimal contributions of stray radiation (Figure 2), we are able to accurately report the effectiveness of UVC wavelengths at inducing short-term skin effects down to 200 nm. This approach helps reduce the uncertainty inherent in predicting the effect of each wavelength on specific photobiological outcomes.⁴²

The acute skin response spectrum reported in this study, derived from the predicted threshold doses (D₅₀), revealed a sharp increase in the doses required to trigger a response for UVC wavelengths below 240 nm (Figure 4 and Table 1). This increase in threshold doses needed to induce short-term skin effects have been attributed to the very short epidermal transmittance demonstrated by these short wavelengths in mammalian skin.^11,12 It has been shown that wavelengths within the far-UVC spectrum become highly absorbed by stratum corneum significantly reducing their penetration into the deeper layers of the epidermis.¹² The human stratum corneum has been determined to be typically 15–30 μm thick, while the epidermis has been found to range between 56 and 81 μm, varying depending on the body site.^44,45 During this study, we measured the average thickness of the stratum corneum in hairless mice dorsal skin to be 4.70 ± 0.88 μm, while the epidermis measured 20.86 ± 5.52 μm (Table S2).

This significant difference in stratum corneum and epidermis thickness between the SKH1 mouse model and human skin suggests that the skin responses observed in this study in hairless SKH1 mice, particularly for the far-UVC region,^7,12 may underestimate the human skin UV threshold.^40,46 Furthermore, to gain a better understanding of the overall impact of far-UVC radiation on the skin, it is crucial to investigate its effects on the stratum corneum, especially the extent of structural damage it causes. Methods that measure the level of mechanical and structural degradation inflicted by far-UVC, such as assessments of transepidermal water loss^47,48 or alteration in corneodesmosome junctions,⁷ should be pursued.

In an earlier far-UVC safety skin study that used hairless SKH1 mice models,⁴¹ it was stated that a more appropriate consideration of acute effects for UVC radiation involves the development of erythema, edema, scales, and fissures, rather than solely relying on erythema. This approach was established by Yamano et al.⁴¹ as a more accurate method to study the effects that far-UVC has on skin cutaneous responses,⁴⁷ as erythema was rarely observed, while edema and scale formation were more evident. While their study used excimer lamps emitting wavelengths at 207, 222, 235 nm to explore the effects of far-UVC light on 1-cm square irradiated skin regions, our study irradiated a smaller area of mice skin. Despite the significantly smaller irradiated area in our study compared to previous research, visualizing acute responses was not challenging, especially when compared to the surrounding unirradiated skin. However, this clarity did not extend to the photographic records of these acute reactions. Erythema was the most easily perceptible skin response in the photographs (Figure 1B,C). Recognizing the formation of scales and edema proved to be challenging, as we relied solely on visual inspections, whereas Yamano et al.⁴¹ utilized both visual and palpation inspections to make these observations. Additionally, the photographs revealed significant variability in the detectable skin reactions between mice (Figure 3), making it difficult to identify trends that could differentiate the effects triggered by the far-UVC region of the spectrum from other, longer wavelength, UVC radiation. If both visual and palpation inspections cannot be achieved in future skin acute studies, clearer recognition of the triggered acute reactions could be achieved by documenting descriptions in person during the experiments, as done by Yamano et al.

Based on the mouse skin acute response spectrum reported in this study, which covers UVC wavelengths from 200 to 270 nm (Figure 4), our data suggest that the revised skin TLVs^® established by ACGIH in 2022 for wavelengths below 230 nm is still overly conservative with respect to the risk of acute adverse skin effects. Specifically, we estimate that for wavelengths between 220 and 230 nm, the dose needed to induce a short-term effect on mouse skin ranges from 2310 to 177 mJ/cm² (Table 1), well above the current exposure skin limits set at 631–158.5 mJ/cm².³² While this mice skin acute threshold doses may not directly correspond to far-UVC safety in human skin, the findings of this study provides valuable data to guide development and planning of potential human acute skin exposure trials. In combination with studies to measure the effects of chronic far-UVC exposure, such as formation of UV-induced cyclobutane-pyrimidine dimers and 6–4 photoproducts, our data, and that of future studies, will inform the potential human health risks and acceptable threshold values for far-UVC radiation to enable its more widespread use as an antimicrobial technology.¹⁹