Registration Dossier

Administrative data

Description of key information

Assessment of contact hypersensitivity in the Mouse, LLNA test: Not sensitising

Assessment of contact hypersensitivity in the albino Guinea Pig, Maximisation test: Not sensitising

Skin sensitization in Guinea pigs, Buehler test:  Not sensitizing.

Skin sensitization in human, case reports: Some evidence of transient skin sensitization but not sufficient for assessment on the sensitization potential of Azodicarbonamide.

Respiratory effects in animal: After repeated inhalations in guinea pigs, no sign of respiratory sensitization has been highlighted.

Respiratory effects in human: respiratory health effects in case reports and health surveillance data after occupational exposure.

Key value for chemical safety assessment

Skin sensitisation

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

Animal data

A local lymph node assay in mice was conducted to determine the potential for contact hypersensitivity of ADCA (WIL Research Europe B.V. 2014, 506582).

The study was conducted in accordance with OECD test guidelines, and in compliance with GLP.

Fifteen female mice (three groups of five animals) were treated with one test substance concentration per group, one group of five animals was treated with vehicle alone. The dorsal surface of both ears was topically treated with application of a 0, 10, 25 or 50% (w/w) solution of the test substance in propylene glycol. This process was performed at the same time each day for three consecutive days, this is the induction phase of the study. On day 6 each animal was injected with phosphate buffered saline (pbs) containing 20 μCi of 3H-methyl thymidine, 5 hours later the animals were killed and the draining (auricular) lymph node of each ear excised. The lymph node cells (LNC) were extracted with PBS and centrifugation and the DNA precipitated with 5% trichloroacetic acid (TCA). The DNA was resuspended in TCA , scintillation fluid added and the incorporation of radioactivity into the LNC measured by liquid scintillation counting. The Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI≥3 the test substance may be regarded as a skin sensitiser.

No irritation of the ears were seen in any of the animals examined. No mortalities occurred and no clinical signs of systemic toxicity were observed in test animals in the main study.

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.2, 1.2 and 1.1, respectively.

Since there was no indication that the test substance elicited a SI ≥ 3 when tested up to 50%, ADCA was not considered to be a skin sensitizer.

A maximisation test in the Albino Guinea Pig was conducted to determine whether ADCA induces contact hypersensitivity after intradermal and epidermal exposure (WIL Research Europe B.V. 2014, 506583). The Maximisation test was selected in order to assess the risk of the test substance to act as a respiratory sensitiser in man.

The study was conducted in accordance with OECD test guidelines, and in compliance with GLP.

Induction

Day 1 - was performed in ten female albino guinea pigs by intradermal injections of three pairs of injections into the clipped scapular region with

a) a 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection;

b) Intradermal injection of the first animal with a 20% test substance concentration was technically difficult and the injected volume could not be established reliably. Therefore the remaining animals were injected at a 10% concentratio;.

c) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant..

Day 3 - the dermal reactions caused by intradermal injections were assessed for irritation.

Day 7 - The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate in vaseline using a spatula. This concentration of SDS is known to provoke a mild inflammatory reaction.

Day 8 - The 10% SDS treated area between the injection sites was treated with 0.5 mL of a 50% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with tape and subsequently an elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

Challenge

Day 22 - One flank of all animals was clipped and treated by epidermal application of a 50% test substance concentration and the vehicle (0.1 mL each), using Patch Test Plasters. The patches were held in place with tape and subsequently an elastic bandage.

The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

No skin reactions were evident after the challenge exposure in the experimental and control animals.

There was no evidence that ADCA had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 50% test substance concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

A skin sensitization test was conducted to determine the potential for skin sensitization of ADCA (tested under the name of Unifoam AZ SO- NL)(HLS 1988, OCI68/88708D).

The study was conducted in accordance with OECD test guidelines, and in compliance with GLP.

Ten female albino guinea pigs were treated with topical application of a 50% (w/w) solution of the test substance in water for approximately 6 hours. This process was performed at weekly intervals over three occasions. A concurrent control group of ten female guinea pigs was exposed in the same fashion, however no test material was used. In the challenge procedure, all control and test animals were exposed to a topical application of the 50% (w/w) test substance solution for six hours. The challenge application was made two weeks after the third induction application. The challenge site was evaluated for signs of oedema and erythema 24, 48, and 72 hours after the removal of the patch (topical application).

No dermal reactions were seen in any of the test or control animals.

In this screening test, performed in ten albino guinea- pigs, Unifoam AZ SO- NL did not produce any evidence of delayed contact hypersensitivity.

Human data

Three case reports on skin sensitization have been published and are discussed in IUCLID section 7.10.

Respiratory sensitisation

Endpoint conclusion
Additional information:

Animal data

A study was conducted by Gerlach et al., to investigate reports by industry workers exposed repeatedly by inhalation to azodicarbonamide (ADCA) or respiratory problems, possibly indicating pulmonary sensitisation. A non-invasive method was used for measuring specific airway conductance to evaluate the potential for repeated exposure to ADCA to cause respiratory sensitisation to the Guinea pig.

Two groups of 30 male guinea pigs were exposed to aerosolised ADCA for 6 hours per day, 5 days per week, for four weeks. Within each group, dose groups of ten animals were exposed to ADCA at 51 mg/m3, 200 mg/m3, or to filtered air (control group). One of the groups was assessed for specific airway sensitisation by a challenge procedure with inhaled ADCA exposure at the same concentration as the exposure concentration (or 200 mg/m3in the case of the controls). The other group was assessed for non-specific sensitization by a challenge procedure with increasing concentrations of histamine. Challenge procedures were performed one week before the start and three days after the end of the exposure period. Skin tests for immunological responses to ADCA were also performed.

Repeated ADA exposure did not increase the sensitivity of Guinea Pigs to either Histamine or ADA (non-specific or specific sensitisation, respectively). There were no significant differences between pre- and post- exposure histamine response concentrations, either within or amongst the treatment groups. Few Guinea pigs responded to the ADA challenge with a 50% or greater reduction in sGAW (Specific airway conductance) either before or after the repeated ADA exposure. Increases in the number of individual animals displaying a difference between pre- and post- exposure challenge-induced change in sGAW were similar for all dose groups (control, 51, and 200 mg/m3). The only response to intradermal injections (skin tests) was a concentration-related erythematous response. There were no statistically relevant differences between reaction sizes of ADA-exposed and control guinea pigs in the ADA challenge group at any observation time. Among the histamine-challenged groups, the mean reaction size of the 200 mg/m3group was significantly larger than that of the controls at 72 h.

The test substance ADCA did not induce sensitization to either the skin or respiratory tract in guinea pigs, following repeated inhalation exposure 5 days per week for four weeks.

Human data

Case reports and epidemiological data showing evidence of a link between exposure to azodicarbonamide and respiratory symptoms in workers are available in IUCLID Section 7.10.

An expert review was performed looking at the case for considering whether ADCA was a respiratory allergen or not. The reviewer considered the case for low molecular weight (LMW) molecules being able to cause respiratory sensitisation. The definition of an asthmagen and the etiology of asthma and the evidence for ADCA being considered to be a respiratory allergen from available human data were also discussed. As only limited human data is available the experimental animal data was also considered especially in the light of new study designs and understanding of the applicability of skin sensitising data to respiratory sensitisation. ADCA did not show any skin sensitisation potential in any of the studies performed.

Based on all the information the reviewer concluded that ADCA would be the first low molecular weight chemical classified as respiratory sensitiser not testing positive in any dermal skin sensitisation test which is highly unlikely. It was noted that ADCA manufacture has evolved over recent decades, becoming purer, cleaner, and free of historic secondary components (such as chromium compounds). The recently tested ADCA in the LLNA and GPMT (as reported in the skin sensitisation section of this summary) had a purity of >99%. It is therefore assumed that it has not been ADCA itself but an impurity that may have caused the respiratory symptoms. Additionally, or alternatively, ADCA may have caused non-immunological respiratory symptoms upon exposure at high concentrations such as Reactive Airways Dysfunction Syndrome (RADS) or Irritant-Induced Asthma for which effects still may have been present when bronchial challenge tests were done. The reviewer concluded that overall, as such, ADCA should not be considered a respiratory sensitiser.

A second expert review was performed looking at the question of whether ADCA has the ability to cause allergic sensitisation of the respiratory tract. The reviewer looked at the reason why ADCA was considered to be a respiratory sensitiser (epidemiological data on the use of ADCA in the workplace) and then assessed whether ADCA has the ability to cause allergic sensitisation of the respiratory tract. The history of chemical respiratory allergy and the process of the development of occupational asthma was discussed with relevant examples, as well as the mechanism for the developement of non-immunological occupational asthma. The available in vitro and in vivo testing methods together with the experimental results from tests performed with ADCA were examined and the results explained in context of the classification. An ambiguity in the immunological classification of ADCA is also discussed.

The reviewer concluded that, based upon the failure of ADCA to elicit positive responses in either the LLNA or guinea pig maximisation test, the conclusion drawn was that ADCA is not a respiratory allergen and lacks the potential to cause allergic sensitisation of the respiratory tract. All other known chemical respiratory allergens that have been tested in the LLNA yield positive responses and there is no reason to believe that ADCA should be an exception in that respect.

Although ADCA is not a chemical respiratory allergen, and should not be classified as such, it does appear to have some potential to cause irritation of the upper respiratory tract and this could induce in some exposed workers occupational asthma, or related health effects such as RADS (Reactive Airways Dysfunction Syndrome).

Justification for classification or non-classification

A local lymph node assay (LLNA) contact hypersensitivity study was conducted in mice, ADCA was not considered to be a skin sensitiser.

A Guinea Pig Maximisation test was conducted to assess contact hypersensitivity in Albino Guinea Pigs, ADCA was shown to have a sensitisation rate of 0 per cent and there was no evidence that ADCA caused skin hypersensitivity.

A skin sensitization test was conducted in guinea pigs, and no evidence of skin sensitization was seen.

A number of cases of transient skin sensitisation have been reported by industrial workers, however these were not sufficient to allow an assessment on the skin sensitisation potential of ADCA.

On the basis of the available data it is concluded that ADCA is not a skin sensitiser and should therefore not be classified as such.

ADCA is listed in Annex VI to the CLP Regulation (Index Number 611-028-00-3) which specifies a classification of respiratory sensitiser category 1 (Hazard phrase H334 "May cause respiratory sensitisation"). This classification is based on historical accounts of sensitisation amongst industrial users although the effect cannot be replicated in laboratory tests. It is noted that in recent years no new cases of sensitisation related to industrial use of ADCA have been reported; in addition testing for skin sensitisation has indicated that ADCA does not appear to present any sensitisation potential. Two recent expert reviews (please refer above) suggest that in light of the absence of skin sensitisation potential observed for ADCA it is unlikely that the substance will possess respiratory sensitisation properties. It is furthermore suggested that the historical incidence of occupational asthma which has been attributed to ADCA exposure may in fact have been due to impurities (which are no longer present in the substance as it is manufactured now) or due to Reactive Airways Dysfunction Syndrome (RADS) which is caused by physical irritation rather than an immunological effect.

It should be noted that although the available data tends to suggest that ADCA is not a true sensitiser, the classification defined in Annex VI of the CLP Regulation is legally binding, and must therefore be applied.