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Description of key information

In a GLP study conducted according to OECD guideline 429, succinic anhydride dissolved in dimethylformamide was administered epicutaneously to the ears of mice once a day on three consecutive days at concentrations of 10%, 25%, or 31.3% w/w. On day 6, approximately 5 hours after intravenous administration of 3H-methyl thymidine, the draining auricular lymph nodes were excised and incorporation of radiolabeled 3HTdR was determined.
A stimulation index of 9.2, 11.6, and 11.0 was calculated for the low, mid and high dose groups, respectively, showing no clear dose response relationship. The potency for skin sensitisation according to ECHA guidance R8, appendix 8 -10, table R 8 -23, Succinic anhydride is assessed to be weak/moderate (EC3 (%) >2).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2009 -08 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-compliant, GLP proprietary study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
OECD 429 and Regulation (EC)No 440/2008 method B.42 standard LLNA method
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Mice were acclimated for 13 days and were approximately 8 weeks old at first administration and weighed 17.8 -22.2 g. Animals were housed singly in Makrolon cages type II with autoclaved aspen wood chips which were changed 1/week. Nesting material and a polycarbonate shelter was provided per cage. Average room temperature of 21.2 C and average relative humidity of 58.9%. Artificial light was provided from 6:00am to 6:00pm. Mice were given maintenance diet and tap water ad libitum.
Vehicle:
dimethylformamide
Concentration:
10, 25, and 31.3 % w/w succinic anhydride
No. of animals per dose:
5 animals/ group (including spare animals). The spare animal of the low dose group died and therefore, the spare animals of other groups were not used. The number of animals/group used for lymph node examination was 4.
Details on study design:
Solubility testing of the test substance showed the highest concentration suitable for application of the test substance can be achieved with DMF (31.3%, w/w). A range finding study was performed with 2 animals/concentration at 31.3 and 25% succinic anhydride. None of the animals showed overt systemic toxicity, excessive local skin irritation at the application sites or an important increase in ear thickness in the range finding study. Therefore, 31.3% was chosen as the highest test substance concentration and ear thickness measurement was not performed in the main study.Main study: Test solutions were prepared fresh on each day of administration and administered in 3 concentrations to the dorsal surfaces of the ears of mice. One negative control group and one positive control group were treated with DMF and HCA, respectively. Each animal was treated for 3 consecutive days. Five days after the first topical administration, each animal received 20 uCi 3HTdR by slow intravenous administration. Approximately 5 hours after 3HTdR injection, all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised. The proliferation of the lymphocytes of the draining lymph node was measured by the determination of the amounts of incorporated 3HTdR.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Stimulation Indices were calculated according to the standard method
Positive control results:
Application of 25% HCA in acetone:olive oil (4:1 v/v) resulted in an SI of 7.3.
Parameter:
SI
Remarks on result:
other: A stimulation index of 9.2, 11.6, and 11.0 was calculated for the low, mid and high dose groups, respectively. The negative and positive control groups had a stimulation index of 1 and 7.3, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 43520, 54949, and 52036 dpm for the low, mid and high dose groups, respectively. The negative and positive control groups had a dpm of 4733 and 34509, respectively.

One animal of the low dose group died on day 6 near application of radiolabeled H-methyl thymidine. This animal showed no adverse effects and no body mass loss during the study and gross necropsy was without observation. Therefore, this death was assumed to be random and not associated with test substance administration.

Table 1. Body mass index of animals on days 1 and 6

 Negative control           Low dose                    Mid dose        High dose              Positive control
  No.  b.m. day 1  b.m. day 6  b.m. gain   No.  b.m.day 1 b.m.day 6   b.m.gain No.   b.m.day 1 b.m.day 6   b.mgain   No.  b.m.day 1 b.m.day 6  b.m.gain    No.  b.m.day 1 b.m.day 6  b.m.gain 
 1  19.8 21.2  1.4   6  19.1 20.1  1.0   11 21.5  22.8  1.3   16 22.2  23.2  1.0   21 19.8  20.9  1.1 
 2  19.0 19.5  0.5  7 17.8   19.4 1.6   12 19.2  20.3  1.1   17  20.0 20.7  0.7   22  20.2 21.2  1.0 
 3  20.7 21.6  0.9   8  20.6 21.6  1.0   13  18.9 20.3  1.4   18  18.3 20.0  1.7   23  19.0 20.7  1.7 
 4  19.1 20.3  1.2   9  20.8 21.6  0.8   14 19.6  20.9  1.3   19  20.2 20.9  0.7   24  19.3 20.9  1.6 
 Mean  19.7 20.7  1.0  Mean   19.6 20.7  1.1  Mean  19.8  21.1  1.3  Mean  20.2  21.2  1.0  Mean  19.6  20.9  1.4 
 SD  0.8 0.9  0.4  SD  1.4  1.1  0.3  SD  1.2  1.2  0.1  SD  1.6  1.4  0.5  SD  0.5  0.2  0.4 
 n  4  4  4  n
    No. = Animal number; b.m.= body mass in g                                                     

Table 2. General observations and observations of the application sites on days 1 -6

 Finding  Group  No. of the affected animals  Observation time (day)
 General behavior normal  Negative control  1, 2, 3, 4  1 -6
   Low dose  6, 7, 8, 9  1 -6
   Mid dose  11, 12, 13, 14  1 -6
   High dose  16, 17, 18, 19  1 -6
   Positive control  21, 22, 23, 24  1 -6
 Application sites normal  Negative control  1, 2, 3, 4  1 -6
  Low dose   6, 7, 8, 9  1 -6
   Mid dose  11, 12, 13, 14  1 -6
   High dose  16, 17, 18, 19  1 -6
 Slight erythma  Positive control  21, 22, 23, 24  3 -4
Interpretation of results:
sensitising
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
A stimulation index of 9.2, 11.6, and 11.0 was calculated for the low, mid and high dose groups, respectively, showing no clear dose response relationship. The potency for skin sensitisation according to ECHA guidance R8, appendix 8 -10, table R 8 -23, Succinic anhydride is assessed to be weak/moderate (EC3 (%) >2).
Executive summary:

In a GLP study conducted according to OECD guideline 429, succinic anhydride was dissolved in dimethylformamide and administered to three groups of 4 female CBA/Ca mice. Succinic anhydride was administered epicutaneously to the dorsal surface of both ears, once a day on three consecutive days, at concentrations of 10%, 25%, or 31.3% w/w. The volume administered was 25 uL per ear. Positive and negative control groups were tested concurrently under identical conditions. Five days after topical administration, 3H-methyl thymidine was administered intravenously to all mice via a tail vein. Approximately 5 hours later, the draining auricular lymph nodes were excised, incorporation of radiolabeled 3HTdR was determined and compared with negative controls, and the SI index was calculated.

No adverse effects were noted in any animal. The SIs of the test substance concentrations were 9.2 (low dose), 11.6 (mid dose), and 11.0 (high dose). Although no clear dose response was observed, the SIs of all test substance concentrations were greater than 3 and succinic anhydride is regarded as a (weak/moderate) sensitizer in the LLNA.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Although no clear concentration related response was observed, succinic anhydride is regarded as a sensitizer in the LLNA according to OECD guideline 429 since the SIs of all examined test concentrations were greater than 3. This study does not provide information that can be used for quantitative risk assessment.

Calculation of an EC 3 value for succinic anhydride was not possible. The standard linear interpolation method requires a response on either side of the classification threshold of a 3.0 Stimulation Index.

In the study reported here the three SI values all greatly exceeded 3.0 but were not in a linear trend.

Succinic anhydride is an acylating agent which reacts with N-terminal amino acids and consequently falls within a chemical group with sensitising potential.

The sensitizing ability of succinic anhydride and other dicarboxylic acid anhydrides was investigated in guinea pigs. Animals were immunized intradermally by a single dose of 0.3 M succinic anhydride solution in olive oil. Specific IgG values were very low in the succinic anhydride group, which sensitized 7 out of 9 animals.

The most common site of reactivity of cyclic anhydrides in biological systems is the initial site of contact. Like other cyclic acid anhydrides, succinic anhydride is readily hydrolyzed to a dicarboxylic acid (WHO, 2009). Dicarboxylic acids are known irritants and the formation of the acid is the basis for skin and eye irritation seen with succinic and other anhydrides. Anhydrides, including succinic anhydride, aresensitizers. Experiments with sensitized animals have demonstrated the formation of anhydride-specific IgE and IgG (WHO, 2009).  Allergic reactions of the conjunctiva and respiratory tract have been reported in humans after exposure to the cyclic anhydrides.

Based on this information, succinic anhydride can be considered a sensitizing agent and is expected to cause respiratory sensitisation also.


Respiratory sensitisation

Link to relevant study records
Reference
Endpoint:
respiratory sensitisation
Remarks:
other: various tests assessed in the general overview
Type of information:
other: WHO CICAD 75 - the collective view of an international group of experts presented as a peer reviewed assessment of ciclic acid anhydrides effecs on human health
Adequacy of study:
key study
Study period:
Not stated
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: WHO CICAD 75 - the collective view of an international group of experts presented as a peer reviewed assessment of ciclic acid anhydrides effecs on human health
Principles of method if other than guideline:
Not applicable - assessment of human effects of the acid anhydride group via literature sources for a diversity of tests
GLP compliance:
not specified
Species:
human
Sex:
not specified
Details on test animals and environmental conditions:
No data
Route of induction exposure:
other: no data
Route of challenge exposure:
other: no data
Vehicle:
other: no data
Concentration:
refer to "any other information" and "results" fields
No. of animals per dose:
refer to "any other information" and "results" fields
Details on study design:
refer to "any other information" and "results" fields
Challenge controls:
refer to "any other information" and "results" fields

Effects of cyclic acid anhydride administration on humans

In humans, cyclic acid anhydrides can cause irritation and sensitization after direct contact with the skin and the mucous membranes or after exposure by inhalation.

The irritative symptoms (itching, lacrimation, sneezing, rhinorrhoea, cough, and dyspnoea) begin immediately following exposure to high concentrations of dusts or vapours.

The most common allergic diseases are rhinoconjunctivitis and asthma, both immediate-type IgE-mediated allergies. Also, late-type respiratory symptoms with specific IgG antibodies have been described. Less frequent consequences are the severe disease called pulmonary disease–anaemia syndrome, contact eczema, contact urticaria, allergic laryngitis, and allergic alveolitis.

Cases of respiratory irritation are common immediate responses to human exposure to acid anhydrides evident as a conjunctival, nasal and bronchial irritation. Contact with mucous membranes and sweating skin results in hydration of the anhydride to acid causing irritation, corneal damage, caustic dermatitis and burns. The human nasal threshold for irritation caused by phthalic anhydride is 30 mg/m3 and for maleic anhydride is 5.5 mg/m3. (Succinic anhyride can be assumed to be similar to maleic anhydride). Irritant haemorrhagic rhinitis is also reported following maleic anhydride exposure.

While allergic contact dermatitis to cyclic anhydrides is considered by the WHO review panel to be rare, they conclude that the proof of IgE mediation in immediate-type asthma or rhinitis due to acid anhydrides is convincing. Specific IgE to several acid anhydrides—phthalic anhydride, trimellitic anhydride, maleic anhydride, tetrachlorophthalic anhydride, hexahydrophthalic anhydride, tetrachlorophthalic anhydride, hexahydro-phthalic anhydride, himic anhydride, methyl hexahydrophthalic anhydride, methyl tetrahydrophthalic anhydride, and chlorendic anhydride—has been found.

Allergic reactions of the skin and conjunctiva and allergic respiratory manifestations are well known effects of occupational exposure to cyclic acid anhydrides. Respiratory diseases include occupational allergic rhinoconjunctivitis and occupational asthma. Urticaria and allergic rhinoconjunctivitis often precede asthma. Cases of haemorrhagic alveolitis, haemorrhagic anaemia, allergic alveolitis, and allergic laryngitis have also been reported in association with exposure to anhydrides. These manifestations of allergic respiratory sensitisation necessitate classification of succinic anhydride as R42 or H334 "may cause allergy or asthma symptoms or breathing difficulties if inhaled. Classification as H335 - May cause respiratory irritation is also indicated by the group read across to cyclic acid anhydrides.

A number of occupational cases of asthma or rhinoconjunctivitis due to exposure to different cyclic acid anhydrides have been reported. The symptoms are those of typical occupational asthma and rhinitis. After a symptom-free latency period, the worker experiences symptoms when exposed. The diagnosis has been based on the exposure, symptoms, and a cause–effect relationship proven with immunological tests or challenge tests. The induction time for positive specific IgE antibodies was 8.8 months when workers exposed to hexahydrophthalic anhydride, methyl hexahydrophthalic anhydride, and methyl tetrahydrophthalic anhydride were followed. Inhibition studies and passive transfer studies have supported the specificity of IgE antibodies, but cross-reactivity among some acid anhydrides has been reported. Specific IgG antibodies have been studied especially in connection with sensitization to trimellitic anhydride. Specific IgG antibodies against trimellitic anhydride–human serum albumin have been correlated with late-onset occupational asthma due to trimellitic anhydride. No cross-reactivity with phthalic anhydride, maleic anhydride, hexahydrophthalic anhydride, or tetrachlorophthalic anhydride was found when the specificity of IgG antibodies against trimellitic anhydride–human serum albumin conjugate was investigated. No results are presented for succinic anhydride cross reactivity.

WHO CICAD 75 contains results from occupational exposure scenarios for

phthalic anhydride - 118 workers - 28 workers (24%) had work-related rhinitis, 21 (18%) had asthma, and 13 (11%) had symptoms of chronic bronchitis.

trimellitic anhydride - five studies - 553 workers examined Frequent cases of respiratory sensitisation or irritation reported across various sites and exposures.

hexahydrophthalic anhydride - 56 workers in three cases - isolated cases of asthma or rhinitis observed.

other anhydrides - A worker in maleic anhydride production who previously had maleic anhydride–related asthmatic symptoms developed severe haemolytic anaemia. In another study of maleic anhydride–exposed workers, sensitization was found to be uncommon at the low exposure levels measured, 1.8–2.8 μg/m3.

Mode of action

Phthalic anhydride has been classified as a moderate sensitizer that causes type IV allergic contact dermatitis. Studies using cytokine stimulation concluded that phthalic anhydride, trimellitic anhydride, maleic anhydride, hexahydrophthalic anhydride, and methyl tetrahydrophthalic anhydride were not contact allergens. Case-reports in humans of allergic contact dermatitis are limited, suggesting that the potency of cyclic acid anhydrides is low by the dermal contact route.

Cyclic acid anhydrides have been observed to cause IgE-mediated contact urticaria in humans. There are some reports of contact urticaria via airborne exposure without skin contact. Allergic asthma is a well documented disease of cyclic acid anhydride exposure in workers. Allergic asthma is often preceded by rhinoconjunctivitis. IgE-mediated sensitization has been verified in exposed workers using skin prick tests with conjugates of the cyclic acid anhydrides and human serum albumin. Bronchial hyper-responsiveness has been correlated with specific sensitization.

The critical effects of cyclic acid anhydrides are considered to be sensitization and work-related symptoms. Sensitization and work-related respiratory symptoms have been reported at concentrations as low as 10–40 μg/m3 for trimellitic anhydride, 10–50 μg/m3 of mixed exposure to hexahydrophthalic anhydride and methyl hexahydrophthalic anhydride and 5–20 μg/m3 of exposure to methyl tetrahydrophthalic anhydride. For phthalic anhydride, the exposure level for sensitization and work-related respiratory symptoms was higher: 1500–17 400 μg/m3. For tetrachlorophthalic anhydride, the exposure level for sensitization and work-related respiratory symptoms was reported to be 140–590 μg/m3, but lower concentrations, between 4.1 and 66.7 μg/m3, have induced asthma reactions in challenge tests of sensitized workers with occupational asthma.

From all of the available information the cyclic acid anhydrides have been demonstrated to induce both respiratory irritation and respiratory sensitisation in humans and it can be assumed that the mode of action for succinic anhydride is similar to that of other anhydrides in the investigation group.

Interpretation of results:
other: cyclic acid anhydrides are irritating to mucous membranes of the respiratory system. Allergic effects are likely since anhydride specific IgE and IgG antibodies form and anhydride challenges to sensitised animals causes obstructive bronchial reactions
Conclusions:
Cyclic acid anhydrides are irritants to skin and mucous membranes of the eyes and respiratory system.In humans, cyclic acid anhydrides can cause irritation and sensitization after direct contact with the skin and the mucous membranes or after exposure by inhalation.The irritative symptoms (itching, lacrimation, sneezing, rhinorrhoea, cough, and dyspnoea) begin immediately following exposure to high concentrations of dusts or vapours.The most common allergic diseases are rhinoconjunctivitis and asthma, both immediate-type IgE-mediated allergies. Also, late-type respiratory symptoms with specific IgG antibodies have been described. Less frequent consequences are the severe disease called pulmonary disease–anaemia syndrome, contact eczema, contact urticaria, allergic laryngitis, and allergic alveolitis.Allergic reactions of the skin and conjunctiva and allergic respiratory manifestations are well known effects of occupational exposure to cyclic acid anhydrides. Respiratory diseases include occupational allergic rhinoconjunctivitis and occupational asthma. Urticaria and allergic rhinoconjunctivitis often precede asthma. Cases of haemorrhagic alveolitis, haemorrhagic anaemia, allergic alveolitis, and allergic laryngitis have also been reported in association with exposure to anhydrides. These manifestations of allergic respiratory sensitisation necessitate classification of succinic anhydride as R42 or H334 "may cause allergy or asthma symptoms or breathing difficulties if inhaled. Classification as H335 - May cause respiratory irritation is also indicated by the group read across to cyclic acid anhydrides
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

No value is presented for respiratory sensitisation - succinic anhydride is considered to be a respiratory sensitiser and is classified as such under Directive 67/548/EEC and in Annex I of CLP Regulation No 1272/2008.

Human exposure

Cyclic acid anhydrides are irritants to skin and mucous membranes of the eyes and respiratory system. In humans, cyclic acid anhydrides can cause irritation and sensitization after direct contact with the skin and the mucous membranes or after exposure by inhalation. The irritative symptoms (itching, lacrimation, sneezing, rhinorrhoea, cough, and dyspnoea) begin immediately following exposure to high concentrations of dusts or vapours. The most common allergic diseases are rhinoconjunctivitis and asthma, both immediate-type IgE-mediated allergies. Also, late-type respiratory symptoms with specific IgG antibodies have been described. Less frequent consequences are the severe disease called pulmonary disease–anaemia syndrome, contact eczema, contact urticaria, allergic laryngitis, and allergic alveolitis. Allergic reactions of the skin and conjunctiva and allergic respiratory manifestations are well known effects of occupational exposure to cyclic acid anhydrides. Respiratory diseases include occupational allergic rhinoconjunctivitis and occupational asthma. Urticaria and allergic rhinoconjunctivitis often precede asthma. Cases of haemorrhagic alveolitis, haemorrhagic anaemia, allergic alveolitis, and allergic laryngitis have also been reported in association with exposure to anhydrides. These manifestations of allergic respiratory sensitisation necessitate classification of succinic anhydride as R42 or H334 "may cause allergy or asthma symptoms or breathing difficulties if inhaled. Classification as H335 - May cause respiratory irritation is also indicated by the group read across to cyclic acid anhydrides


Migrated from Short description of key information:
Succinic anhydride is considered to be a respiratory irritant, requiring classification in accordance with the current level of classification indicated in CLP Regulation 1272/2008, i.e. succinic anhydride is considered to be a respiratory hazard. The information presented in WHO CICAD 75 indicates that as a group the cyclic acid anhydrides are responsible for cases of occupational respiratory irritation and respiratory sensitisation.

Justification for selection of respiratory sensitisation endpoint:
Review of the group cyclic anhydrides, of which succinic anhydride is a member.

Justification for classification or non-classification

Succinic anhydride is regarded as a skin sensitizer in the LLNA according to OECD guideline 429 and should be classified as "R43 May cause sensitization by skin contact" according to Directive 67/548/EEC. It is classified as H317 "May cause an allergic skin reaction" and H334 "May cause allergy or asthma symptoms or breathing difficulties if inhaled" with the hazard pictogram GHS08 and the signal word 'Danger'.

Manifestations of allergic respiratory sensitisation necessitate classification of succinic anhydride as R42 or H334 "may cause allergy or asthma symptoms or breathing difficulties if inhaled. Classification as R37 or H335 - May cause respiratory irritation is also indicated by the group read across to cyclic acid anhydrides and based on practical observation in human occupational exposure scenarios.