EN 455-5:2025 – chemical residues in gloves

Some chemical residues in single-use medical gloves can cause allergic contact dermatitis.

The requirements for single-use medical gloves relevant to the EU market are contained within the EN 455 series of standards. In August 2025, this series was expanded with the introduction of part 5 – ‘Extractable chemical residues’. This is a significant new addition for a very important parameter that was previously not included in the series.

Part 5 standardises the testing conditions and identifies 27 relevant chemicals, so manufacturers’ claims regarding extractable allergenic substances can be homogenised. This will help to provide new information to healthcare professionals when selecting suitable single-use gloves. In this article, we will explain the background to the standard, what is assessed and how testing is performed.

Before EN 455-5 was developed, the biological evaluation of medical gloves was considered through the requirements in both EN 455-3:2023 (for endotoxins, powder-free glove claims and leachable proteins), and the requirements in the EN ISO 10993 series of standards for medical devices.

Some of the parts of EN ISO 10993 utilised animal testing to determine the irritation and sensitisation potential of medical gloves. In addition to ethical considerations, such techniques are seen as a crude screening test which did not provide specific information about potential allergens.

Prior to the existence of EN 455-5, there was no standardised procedure to test for extractable residues which could cause allergic reactions or contact dermatitis. This gave the potential for manufacturers’ claims that were unable to be independently verified.

Allergic reactions from chemical additives extracted from single-use gloves will only appear on the area which has been in contact with the allergen. Therefore, the reactions usually only result in hand dermatitis. This is in contrast to the potentially life-threatening reactions from a Type I latex protein allergy. Natural rubber latex used in many medical gloves contains proteins from its botanic source (Hevea brasiliensis). Several of these proteins are strong allergens which could potentially cause an immediate Type I allergic reaction, resulting in symptoms ranging from mild itching to life-threatening anaphylaxis. Methods to mitigate the risk of Type I reaction from medical gloves allergy are considered in EN 455-3:2023, whereas the assessment of chemical residues that could cause Type IV allergies are considered in EN 455-5.

Type I and Type IV allergic reactions

‘Type I’ reaction: An immediate allergic response that happens when the body overreacts to something that many people consider to be relatively harmless (like food or insect venom), causing symptoms such as itching, swelling, wheezing or, in severe cases, anaphylaxis.

‘Type IV’ reaction: A delayed allergic reaction where symptoms appear hours or days after contact with a substance, because immune cells (rather than antibodies) slowly cause inflammation in the affected area.

Type IV allergies are characterised as a delayed hypersensitivity reaction that generally occurs more than 12 hours after exposure to the allergen. Type IV allergies could be career-altering, as the dermatitis will not heal until the exposure is avoided or significantly reduced. However, because of the requirement for healthcare workers to wear gloves in most situations involving physical patient contact, sufferers can become sensitised to specific allergens. This means that very small concentrations of a chemical residue could initiate a reaction. So, if medical gloves that initiate a reaction in the wearer cannot be avoided, sufferers may need to review their career options.

Why might chemical additives be present?

The manufacturing process for disposable gloves involves dipping hand formers into liquid elastomers (see the photograph at the top of this page). The dipped former coated with the liquid elastomer requires curing to turn the liquid into a solid film or membrane, and to provide the desired chemical and physical properties of the polymer. Curing requires chemical cross-linking of the rubber polymer chains using sulphur, zinc oxide and heat – a process called ‘vulcanisation’.

This generally uses chemical accelerators to speed up the rate and the efficiency of the reaction. Residual chemicals such as ‘accelerators’ can be contained in the glove. These may migrate to the surface over time and come into contact with the wearer’s skin.

However, knowing the hazards from chemicals added to the manufacturing process does not necessarily give a true picture of the allergens to which the wearer will be exposed. The elastomer’s composition changes during manufacturing due to the chemical reactions between the ingredients forming new chemical bonds, but there could be unreacted residual accelerators in the manufactured gloves which could cause contact dermatitis in the wearer.

An example of accelerators that were widely used are ‘thiruams’. However, because these are strong contact allergens, they have generally been replaced by similar but less allergenic compounds such as zinc dithiocarbamates, 2-Mercaptabenzothiazole (see figure 1) and guanidines. Alternative methods of manufacturing are theoretically possible which avoid the use of accelerators. These include the use of ultra-violet (UV) light, peroxides in place of sulphur-dependent accelerators, or thermoplastic elastomers which do not require curing or vulcanisation.

Chemicals known as ‘activators’ (which enhance the effectiveness of accelerators), antioxidants (which protect the rubber from degradation caused by environmental factors such as heat, UV light and ozone) or donning aids (added to increase friction to make putting on and taking off the gloves easier) are also potential sources of Type IV allergic reactions.

Testing procedure and analysis

The 27 chemicals included in Annex E of EN 455-5 are listed in table 1. This is a non-exhaustive list of contact allergens relevant to medical gloves. In order to draw out any residues of these 27 chemicals from medical gloves, an extraction procedure must be utilised. The CEN committee working group responsible for the development of EN 455-5 decided upon a ‘simulated use’ extraction, which replicates the level of chemicals likely to be leached out of the glove while it is being worn.

Alternative ‘exaggerated extraction conditions’ would provide a total amount of the chemicals present, which results in a greater amount of the chemicals being extracted compared to the amount released during simulated use extraction. The simulated use extraction method selected in EN 455-5 determines the amount of leachable chemicals present on the inner surface of two whole gloves. This uses a 75 per cent ethanol in water extraction solution in an attempt to mimic the in-use extraction of chemicals onto the wearer’s hand.

The principle of the extraction is that residual chemicals are extracted from the inner surfaces of two whole gloves following a pre-conditioning step. The solubility of residual chemicals in the glove material can vary significantly with temperature, and this has a direct effect on the amount that can be extracted. Storage at lower temperatures can result in ‘blooming’ of the chemical to the glove surface, making it easier to extract from the glove.

Table 1: The 27 chemicals listed in Annex E of EN 455-5:2025
Chemical name	Abbreviation	CAS number
2-Mercaptobenzothiazole	MBT	149-30-4
Zinc 2-mercaptobenzothiazole	ZMBT	155-04-4
2-(Morpholinothio)benzothiazole	MOR, MBS	102-77-2
2,2’Dithiobis(benzothiazole)	MBTS	120-78-5
N-Cyclohexyl-2-benzothiazolesufenamide	CBS	95-33-0
N,N-Dicyclohexylbenzothiazole-2-sulphenamide	DCBS	4979-32-2
Zinc diethyldithiocarbamate	ZDEC	14324-55-1
Zinc dibutyldithiocarbamate	ZDBC	136-23-2
Ziram, zinc dimethyldithiocarbamate	ZDMC	137-30-4
Zinc bis(N-ethyl-N-phenyldithiocarbamate)	ZEPC	14634-93-6
Zinc pentamethylenedithiocarbamate	ZPD	13878-54-1
Zinc dibenzyldithiocarbamate	ZBEC	14726-36-4
Sodium dibutyldithiocarbamate	SDBC	136-30-1
Sodium cyclohexyl ethyldithiocarbamate	SHEC	7346-67-0
Tetramethylthiuram monosulfide	TMTM	97-74-5
Tetraethylthiuram disulfide	TETD	97-77-8
Tetramethylthiuram disulfide	TMTD	137-26-8
Dicyclopentamethylenethiuram disulfide	DPTD	94-37-1
N,N’-Dibutylthiourea	DBTU	109-46-6
N,N’-Diethylthiourea	DETU	105-55-5
N,N’-Diphenylthiourea	DPTU	102-08-9
1,3-Diphenylguanidine	DPG	102-06-7
1,2 Benzisothiazolinone	BIT	2634-33-5
2-Mercaptobenzimidazole	MBI	583-39-1
4,4’-thiobis(6-tert-butyl-meta-cresol) (Lowinox 44S36)	–	96-69-5
Butylated hydroxyanisole	BHA	9009-68-1
Cyclohexylthiophthalimide	CTP	17796-82-6

Pre-conditioning

The pre-conditioning step before the extraction uses two conditions for the storage of gloves before they are extracted. These are i) seven days at 4°C and ii) seven days at 25°C. If extractable residues will be higher in one of the temperatures, that temperature is to be used for the pre-conditioning. In reality, there are many different glove polymer formulations that may contain some of the 27 chemicals listed in Annex E, so the conditions likely to provide the highest extractable values are unlikely to be known. The approach taken by SATRA will therefore be to pre-condition gloves at both temperatures, with the result of the test coming from the conditions which give the highest extractable values.

After pre-conditioning, testing is carried out on eight gloves of the same size and from the same production lot. Gloves are separated into four pairs, and the mean chemical content of the four determinations is the overall result, which is expressed in the unit ‘µg/g’ (that is, µg of chemical per gram of glove material). The reporting requirements in EN 455 specify that the test report must state the limit of detection and limit of quantification. These limits are not defined in the test procedure. Therefore, laboratories must determine these for each of the 27 chemicals as part of their method validation process. It is acknowledged in the introduction to this standard that laboratories will need several months to develop their testing and become accredited before testing to EN 455-5 can be offered.

It was mentioned earlier that the inner surfaces of two whole gloves are extracted with the simulant. This is achieved by turning one glove inside out (so the surface that would be in contact with the wearer’s skin is on the outside) and inserting that glove inside the second glove. The extraction solution is introduced between the inner and outer gloves, and the gloves are clamped at a distance of 200 mm from the middle finger.

These are then shaken at a speed of approximately 3 hertz for 15 minutes. The extract is transferred to a flask, filtered and analysed by High Performance Liquid Chromatography (HPLC). Quantification is made by comparing separated integrated peak areas in the chromatograms with those from standard solutions. Unfortunately, there is no single analysis method to analyse all 27 residual chemicals simultaneously. Three simple HPLC analytical methods are provided in informative annexes. However, due to solubility, stability and peak separation challenges, these analysis methods are not comprehensive and require full validation by laboratories before testing services can be made available to the industry.

Requirements

EN 455-3:2023 – ‘Medical gloves for single use – Requirements and testing for biological evaluation’ includes the requirements for labelling and the disclosure of information relevant to the applicable test methods. When EN 455-5 was published, the intention was that EN 455-3 would be revised with an amendment to include additional requirements related to chemical residues. At the time of this article being written, these requirements are at a draft stage, and are expected to mandate the inclusion of extractable values for all 27 chemicals in Annex E. However, that will be confirmed once the revision is published.

At SATRA we are validating our internal standard operating procedures (SOPs) based on the three methods in the annexes and hope to offer testing shortly.

In conclusion

EN 455-5 is a new part to the single-use medical gloves series of standards which establishes a test protocol to determine the amount of 27 chemicals after extracting the inner surfaces of two gloves with 75 per cent ethanol in water. Those 27 chemicals are relevant contact allergens in medical gloves and have been associated with contact dermatitis. Requirements for the labelling of medical gloves will be included in an amendment to EN 455-3, which is likely to be published before the end of 2026.

---