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. 2025 Dec;57(1):2491664.
doi: 10.1080/07853890.2025.2491664. Epub 2025 Apr 17.

Methodological limitations and confounders in dermal toxicity evaluation of aqueous test substance by OECD technical guidelines 402, 410: our experience of testing ethanol based hand sanitizer

Rajendra Palkhade  1 , Deepali Sammal  2 , Jitendra Parmar  3 , Snehal Chavhan  4
Affiliations

Methodological limitations and confounders in dermal toxicity evaluation of aqueous test substance by OECD technical guidelines 402, 410: our experience of testing ethanol based hand sanitizer

Rajendra Palkhade et al. Ann Med. 2025 Dec.

Abstract

Background: In view of overzealous use of Alcohol based hand sanitizer (ABHS) during COVID-19 pandemic and associated alarming rise in the cases of hand eczema and dermatitis around the world. We conducted an in vivo dermal toxicity with objective of exploring the acute and subacute effects of ethanol based hand sanitizer (EBHS) on Sprague Dawley rats.

Aims: To evaluate acute and subacute dermal toxicity due to ethanol based hand sanitizer (EBHS) on Sprague Dawley rats.

Methods: In first phase, following Organisation for Economic Co-operation and Development (OECD) Technical Guidelines (TG) 402, we conducted acute dermal toxicity study with two rats and EBHS containing 72.34% ethanol. In second phase, sub-acute dermal toxicity study was conducted, following OECD TG 410 with five groups of rats (10 animals of either sexes in each group) at various doses.

Results: In both the studies, no erythema, oedema, and eschar was observed. Although no clinical signs of toxicity were observed in both the studies, one death was encountered in subacute study. Macroscopically skin was normal; however, microscopic changes such as hyperkeratosis, parakeratosis, erosion, and extracellular oedema in epidermis and diffuse inflammatory cell infiltration in dermis was observed, suggestive of spongiotic dermatitis and 'clinic-pathological discordance'. However, attributing this changes to ethanol is difficult due to methodological limitations and confounders.

Conclusion: In both the studies, ethanol based hand sanitizer (EBHS) was found to be non-irritant with LD50 of > 2000 mg/kg and classified as Class 5/Unclassified according to GHS classification. Although, spongiotic changes were observed, methodological limitation of absence of control group in TG 402 and confounding effect of water and occlusion in all the animals/groups in both studies prevented us to attribute it to ethanol.

Keywords: OECD TG 402; OECD TG 410; acute dermal toxicity; ethanol based hand sanitizer; subacute dermal toxicity.

Plain language summary

In COVID19 pandemic, an emergency measure of hand hygiene, i.e. Alcohol -based hand sanitizers (ABHS); Ethanol- based hand sanitizer (EBHS) in Indian context, has been used overzealously for prolonged period. This has led to rise in cases of skin changes in hands, which was reported by many dermatologists around the world. We tested a commonly used EBHS by applying it to rat skin on a single dose in acute dermal toxicity evaluation and repeated dose in subacute dermal toxicity evaluation. Although, we did not find gross skin changes in both the studies, microscopic changes in the epidermis and dermis suggestive of spongiotic dermatitis were observed. However, due to absence of control group in acute study, and presence of water in test substance along with occlusion of skin which independently can give rise to such microscopic picture, we could not implicate ethanol for microscopic changes in skin. Such situation demands modification in methodology in OECD TG 402 and 410 while testing an aqueous test substance. Also, simultaneous conduct of both acute and subacute studies is recommended for testing aqueous test substance because acute study conducted in isolation without control group may give erroneous results. In view of such inbuilt methodological limitations and confounder, use of newer in vitro, and molecular techniques is recommended for reliable toxicological evaluation of aqueous base substance.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
(a) Framework of acute dermal toxicity study. (b) Framework of Sub-acute dermal toxicity study.
Figure 2.
Figure 2.
Showing body weight changes observed during acute dermal toxicity evaluation.
Figure 3.
Figure 3.
Showing body weight changes observed during sub-acute dermal toxicity evaluation.
Figure 4.
Figure 4.
Showing histological findings of hyperkeratosis, parakeratosis, erosion/ulcer, extracellular oedema, and vesiculations in epidermis with diffuse inflammatory cell infiltration in dermis and normal Sub cutis observed in both the studies.
See this image and copyright information in PMC

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