Registration Dossier

Administrative data

Description of key information

Succinic anhydride was demonstrated to cause skin sensitising reactions in a GLP-compliant local lymph node assay (LLNA) conducted according to OECD TG 429 (Weber, 2010). At all concentrations tested (10%, 25%, 31.3% (w/w)), administration of succinic anhydride (in dimethylformamide) resulted in SI values of 9.2, 11.6, and 11.0. No clear dose-response relationship was observed. Potency categorisation for skin sensitisation according to ECHA Guidance R.8, Appendix R. 8 -10, Table R. 8 -23 was not possible.

No studies on the respiratory sensitisation potential of succinic anhydride itself were identified from literature. Therefore, the respiratory sensitising effects of succinic anhydride were evaluated in a read-across approach with maleic anhydride and other cyclic anhydrides.

In a respiratory sensitisation study with maleic anhydride (Hatoum, 1991), serum antibody titers specific against maleic anhydride were significantly elevated. Although prominent features of respiratory sensitization reactions in this rat model (such as increased numbers of external hemorrhagic lung foci, increased lung weight and volume, and extensive lung pathology) were not evident, these results indicate a respiratory sensitization potential of maleic anhydride.

In the review publication on the toxicology of cyclic anhydrides by the WHO CICAD 75 (2009), the results from studies investigating respiratory sensitisation indicate that cyclic acid anhydrides like (phthalic acid, hexahydrophthalic acid, trimellitic acid, methyl tetrahydrophthalic acid) are irritants to skin and mucous membranes of the eyes and respiratory system.

In animal studies described in the CICAD, it was demonstrated that antibody responses have been induced by cyclic anhydrides via bronchial, subcutaneous, intradermal and parenteral routes of exposure. Development of an allergic respiratory disease is dependent on the production of specific antibodies. In some of these studies, the development of allergic respiratory responses following sensitization with cyclic anhydrides was demonstrated.

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

Read- across is justified because a common feature of cyclic anhydride is that they readily hydrolyse to their corresponding dicarboxylic acids in aqueous solutions. The cyclic anhydrides used in the read-across approach generally demonstrate structural similarities with succinic anhydride and maleic anhydride in particular is a close structural analogue of succinic acid only differing in a double bond at position 2 of the molecule. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

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:
2010-01-26 to 2010-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
24 April 2002
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- White flakes with pungent odour.
- Solubility in water of 50 g/L (20° C, hydrolised)
- Density of 1.2340.
- Melting point of 119.6 C.
- Stable at room temperature.
- Name of test material (as cited in study report):succinic anhydride
- Substance type: dihydro-2,5-furandion cyclic acid anhydride
- Physical state: solid
- Analytical purity: 99.5%
- Purity test date: 22 October 2009
- Lot/batch No.: LEBA5A3070
- Expiration date of the lot/batch: 30 June 2010
- Stability under test conditions: stable
- Storage condition of test material: room temperature
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy, 1-33049 San Pietro all \latisone (UD).
- Females nulliparous and non-pregnant: yes
- Age at study initiation: About 8 weeks at the first administration.
- Weight at study initiation: 17.8 -22.2 g.
- Housing: Mice were individually housed in Makrolon cages type II, (22 cm x 16.S cm ground area, 15 cm high). Bedding material consisted of Aspen wood chips (Fa. ABEDD Dominik Mayr KEG, A-8580 Köflach). Germ reduction by autoclaving; Changed 1/week. Random samples of the bedding material are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are the same as with the feed.
- Diet: Maintenance diet for rats and mice R/M-H (item V1534-300), autoclavable, ad libitum.
Random samples of the feed are analysed for contaminants by the manufacturer: Ssniff Spezialdiaten GmbH, D-59494 Soest. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).
- Water (e.g. ad libitum): Tap water from Makrolon-bottles with stainless steel canules, ad libitum.
Random samples of the water are analysed for contaminants and germ content by the "AGES", A-1226 Vienna. The limits of tolerance are identical with that used for drinking water for humans in Austria.
- Acclimation period: Mice were acclimated for 13 days
- Indication of any skin lesions: not specifed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Average of 21.2°C (continuous monitoring and recording).
- Humidity (%): Average of 58.9 % (continuous monitoring and recording).
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): Only artificial light from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 2010-01-13 To: 2010-02-01

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 dimethylformamide (DMF) and hexyl cinnamic aldehyde (HCA), respectively. Each animal was treated for 3 consecutive days. 3 days after the last topical administration, each animal received 20 uCi 3HTdR by slow intravenous administration via the tail vein. 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 auricular 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 (see more details in section "Any other information on materials and methods incl. tables")
Positive control results:
Application of 25% HCA in acetone:olive oil (4:1 v/v) resulted in an SI of 7.3.
Parameter:
SI
Value:
1
Test group / Remarks:
negative control
Remarks on result:
other: dpm: 4733
Key result
Parameter:
SI
Value:
9.2
Test group / Remarks:
low dose (10% test substance)
Remarks on result:
other: dpm: 43520
Key result
Parameter:
SI
Value:
11.6
Test group / Remarks:
mid dose (25% test substance)
Remarks on result:
other: dpm: 54949
Key result
Parameter:
SI
Value:
11
Test group / Remarks:
high dose (31.3% test substance)
Remarks on result:
other: dpm: 52036
Parameter:
SI
Value:
7.3
Test group / Remarks:
positive control (25% HCA)
Remarks on result:
other: 34509
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION :
- The SIs of the test substance groups were between 9.2 and 11.6.

CLINICAL OBSERVATIONS:
- No abnormal behaviour or clinical signs were detected during the experiment in the animals.
- No local irritations were observed at the application sites of all animals of all test substance groups and the negative control group throughout the whole study. On Days 3 and 4 all animals of the positive control group had slight erythema on the application sites indicating slight irritative skin reactions.
- One animal (No. 10) of the low dosed group died on Day 6 near the application of 3H-methyl thymidine. Since this animal showed no adverse effects and no body mass loss during the study and the gross necropsy was without observation this death is assumed as random event which is not associated with the test substance application.

BODY WEIGHTS:
- Body masses and body mass gains of all animals were in the range to be expected from animals of the same strain, sex and age.

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:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The results from this local lymph node assay (OECD 429) indicate that succinic anhydride is a skin sensitizer based on stimulation indices of 9.2, 11.6, and 11.0, calculated for the low(10%), mid (25%) and high dose (31.3%) groups, respectively.
No clear dose response relationship was observed. Potency categorisation for skin sensitisation according to ECHA Guidance R.8, Appendix R. 8 -10, Table R. 8 -23 was not possible, since no EC3 value could be calculated for succinic anhydride from the data obtained in this study.
Executive summary:

In a dermal sensitization study conducted according to OECD TG 429 with succinic anhydride (99.5%) in dimethlformamide, three groups of 4 female young adult CBA/CaOlaHsd mice/dose group were tested in the local lymph node assay.

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 (25% hexyl cinnamic aldehyde in acetone:olive oil) and negative control (dimethylformamide) 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 radiolabelled 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 sensitizer in the LLNA.

Based on these results, classification of succinic anhydride as skin sensitizer Category 1 according to the CLP criteria set out in Regulation (EC) 1272/2008 is warranted.

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

In a dermal sensitization study conducted according to OECD TG 429 with succinic anhydride (99.5%) in dimethlformamide, three groups of 4 female young adult CBA/CaOlaHsd mice/dose group were tested in the local lymph node assay.

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 (25% hexyl cinnamic aldehyde in acetone:olive oil) and negative control (dimethylformamide) 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 radiolabelled 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 sensitizer in the LLNA.

Calculation of an EC 3 value for succinic anhydride was not possible. Therefore, this study does not provide information that can be used for quantitative risk assessment.

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

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, are sensitizers. 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 as well.


Respiratory sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
respiratory sensitisation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For details and justification of read-across please refer to the report attached in section 13 of IUCLID.
Reason / purpose for cross-reference:
read-across source
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:
other: guinea pig, rabbit, mouse, rat , humans
Sex:
not specified
Details on test animals or test system and environmental conditions:
No data
Route of induction exposure:
other: inhalation, subcutaneous, intradermal and parenteral
Route of challenge exposure:
other: inhalation, intratracheal, intradermal, subcutaneous
Details on study design:
Please refer to section "Any other information on results incl. tables".
Results:
For details on results, please refer to "Any other information on results incl. tables"

In animal studies described in the CICAD, it was demonstrated that antibody responses have been induced by cyclic anhydrides via bronchial, subcutaneous, intradermal and parenteral routes of exposure. Development of an allergic respiratory disease is dependent on the production of specific antibodies. In some of these studies, the development of allergic respiratory responses following sensitization with cyclic anhydrides was demonstrated.

Cyclic acid anhydrides are irritants of the skin and mucous membranes of the eyes and respiratory organs. This is due to rapid reactions with water, which form the corresponding acids responsible for the irritation. Experiments with sensitized animals have demonstrated the formation of anhydride-specific IgE and IgG antibodies. Phthalic anhydride, trimellitic anhydride, and hexahydrophthalic anhydride challenges to sensitized animals resulted in obstructive bronchial reactions and similar reactions may occur with the other cyclic acid anhydrides.

The mode of action for inducing bronchoconstriction has been extensively examined in animal studies (reported by WHO in CICAD 75), with histamine and thromboxane implicated in the response to anhydride challenge. Effects of respiratory irritation (bronchoconstriction, airway inflammation, eosinophilia) and respiratory sensitisation (development of anhydride -specific IgE and IgG, asthma-like symptoms, enhanced complement system response) were noted in the animal studies

Cyclic acid anhydrides can have similar effects on humans (Zeiss et al 1999 cf WHO CICAD 75). The results from human investigations are presented below.

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:
Category 1 (respiratory sensitising) based on GHS criteria
Conclusions:
Cyclic acid anhydrides are irritants to skin and mucous membranes of the eyes and respiratory system. These manifestations of allergic respiratory sensitisation necessitate classification of cyclic anhydrides for Respiratory sensitisation Category 1 and H334 ("May cause allergy or asthma symptoms or breathing difficulties if inhaled").
Executive summary:

In the review publication on the toxicology of cyclic anhydrides by the WHO CICAD 75 from 2009, the results from studies investigating respiratory sensitisation were cited.

Cyclic acid anhydrides are irritants to skin and mucous membranes of the eyes and respiratory system.

In animal studies described in the CICAD, it was demonstrated that antibody responses have been induced by cyclic anhydrides via bronchial, subcutaneous, intradermal and parenteral routes of exposure. Development of an allergic respiratory disease is dependent on the production of specific antibodies. In some of these studies, the developmentof allergic respiratory responses following sensitization with cyclic anhydrides was demonstrated.

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 cyclic anhydrides for Respiratory sensitisation Category 1 and H334 ("May cause allergy or asthma symptoms or breathing difficulties if inhaled").

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Endpoint:
respiratory sensitisation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For details and justification of read-across please refer to the report attached in section 13 of IUCLID.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Maleic anhydride
- Physical state: white briquettes

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
Route of induction exposure:
inhalation
Route of challenge exposure:
inhalation
Vehicle:
not specified
Concentration:
The analytical time-weights average concentrations of maleci anhydride for the five part 1 and part 2 exposures and one challenge were 500 µg/m³ and 317 µg/m³, respectively. The time-weightes average challenge concentration for TMA was 448 µg/m³.
No. of animals per dose:
Part 1) 10 males and 10 females/dose
Part 2) 12 males/dose
Details on study design:
MAIN STUDY: Part 1
1A. INDUCTION EXPOSURE: Part 1
- No. of exposures: 5
- Exposure period: 6 hrs
- Test groups: one test group
- Control group: two, non-exposed
- Frequency of applications: daily
- Duration: 5 days
- Concentrations: 500 µg/m³

1B. CHALLENGE EXPOSURE: Part 1
- No. of exposures: 1
- Day(s) of challenge: 26
- Exposure period: 6 hrs
- Test groups: two test groups
- Control group: one, non-exposed
- Concentrations: 317 µg/m³
- Evaluation (hr after challenge): 18 hrs

MAIN STUDY: Part 2
2A. INDUCTION EXPOSURE: Part 2
- No. of exposures: 5
- Exposure period: 6 hrs
- Test groups: two test group
- Control group: none
- Frequency of applications: daily
- Duration: 5 days
- Concentrations: 500 µg/m³

2B. CHALLENGE EXPOSURE: Part 2
- No. of exposures: 1
- Day(s) of challenge: 26
- Exposure period: 6 hrs
- Test groups: one test group exposed to trimellitic anhydride
- Control group: one, non-exposed
- Concentrations: 448 µg/m³ trimellitic anhydride
- Evaluation (hr after challenge): 18 hrs

Challenge controls:
unexposed control groups
Negative control substance(s):
none
Results:
Part 1: The maleic anhydride-exposed/maleic anhydride-challenged animals had small, but statistically significant (p < 0.05), increases in maleic anhydride-specific serum IgG antibody compared to the controls (challenged and nonchallenged; females higher than males). A few external hemorrhagic lung foci were noted in several of the rats in all three groups; no rats were considered to have responded positively (i.e. having > 10 foci/lung). It has to be noted, that two rats of the MA-exposed/nonchallenged group in part 2 of the experiment had more than 10 lung foci (i.e., positive response); however, mean values for lung foci, weight, and volume were not significantly different from control values. Accumulation of alveolar macrophages was seen histologically in one control rat and no maleic anhydride-exposed rats. Microscopic lung lesions were minimal and provided no evidence of pulmonary sensitization.

Part 2: A mean of 3 foci/lung was seen in both the maleic anhydride-exposed/non-challenged and the maleic anhydride-exposed/trimellitic anhydride-challenged rats. Two rats in each group were considered to have responded positively (i.e. having > 10 foci/lung). Serum IgG antibody levels were similar in the two groups and the serum IgG levels of both were significantly increased over those of the challenged and non-challenged control males of part 1. Antibody levels of both the non-challenged and trimellitic anhydride-challenged rats were significantly increased over those of the maleic anhydride-exposed and challenged animals.
The microscopic lesions observed (i.e. accumulation of alveolar macrophages, alveolar haemorrhage and perivascular acute and chronic inflammation) were minimal in severity, focal in distribution and were each seen in only one rat in the maleic anhydride-exposed/non-challenged group. No microscopic lung lesions were seen in the selected maleic anhydride-exposed/trimellitic anhydride-challenged rats.
Negative control results:
Accumulation of alveolar macrophages was seen histologically in one control rat and no maleic anhydride-exposed rats.

Part 1: The analytical time-weighted averaged concentration of maleic anhydride was 500 and 317 µg/m³, for the induction and challenge phases, respectively.

Part 2: The time-weighted average concentration of maleic anhydride in the induction phase was 500 µg/m³, time-weighted average concentrations for trimellitic anhydride was 448 µg/m³.

Interpretation of results:
Category 1 (respiratory sensitising) based on GHS criteria
Conclusions:
Based on the results of this study, maleic anhydride is considered to be a respiratory sensitiser. In accordance with CLP regulation 1272/2008 classification as Resp. Sens. 1, H334 is warranted.
Executive summary:

In a respiratory sensitisation study, the sensitisationof maleic anhydride was tested in two parts:

In the first part, the respiratory sensitisation potential of maleic anhydride was investigated in three groups of 10 Sprague-Dawley rats/sex. One group was exposed to 500 µg/m³ maleic anhydride for 6 hours/day for 5 days by inhalation. The other two groups were not exposed. After a 3 -week rest period, the maleic anhydride-exposed rats were challenged together with one unexposed group with 317 µg/m³ for 6 hours while the third group remained unexposed and all groups euthanized 18 hours later.

There were statistically significant increases in maleic acid-specific serum IgG antibody levels in the maleic acid-treated male and female rats in the absence of other major signs of respiratory sensitisation reactions like increased numbers of hemorrhagic foci, increased lung weight and volume and severe lung pathology in a specific group.

Based on these results, there is evidence for a potential respiratory sensitising mechanism of maleic anhydride.

In the second part, cross-sensitisation reactions between maleic anhydride and trimellitic anhydride were investigated. Two groups of 12 Sprague-Dawley rats were exposed to 500 µg/m³ maleic anhydride for 6 hours/day for 5 days. After a 3 -week rest period, one group was challenged with 448 µg/m³ trimellitic acid, while the other group was not challenged.

One rat died in the challenged group during the first week of the rest period. Two rats in each group were considered to have responded positively (i.e. having > 10 foci/lung), while mean foci/lung were not different between the two groups. There were no differences in serum IgG levels in both groups, but IgG levels of both were significantly increased compared to the challenged and non-challenged control males of part 1 and increased over the maleic anhydride-exposed and challenged animals. Microscopic lesions were minimal in severity, focal in distribution and were seen in only one rat in the maleic anhydride-exposed/non-challenged group. No microscopic lung lesions were seen in the challenged rats.

Based on these results, no cross-reactivity was evident between maleic anhydride and trimellitic anhydride.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

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

In a respiratory sensitisation study, the sensitisationof maleic anhydride was tested in two parts:

In the first part, the respiratory sensitisation potential of maleic anhydride was investigated in three groups of 10 Sprague-Dawley rats/sex. One group was exposed to 500 µg/m³ maleic anhydride for 6 hours/day for 5 days by inhalation. The other two groups were not exposed. After a 3 -week rest period, the maleic anhydride-exposed rats were challenged together with one unexposed group with 317 µg/m³ for 6 hours while the third group remained unexposed and all groups euthanized 18 hours later.

There were statistically significant increases in maleic acid-specific serum IgG antibody levels in the maleic acid-treated male and female rats in the absence of other major signs of respiratory sensitisation reactions like increased numbers of hemorrhagic foci, increased lung weight and volume and severe lung pathology in a specific group.

Based on these results, there is evidence for a potential respiratory sensitising mechanism of maleic anhydride.

In the second part, cross-sensitisation reactions between maleic anhydride and trimellitic anhydride were investigated. Two groups of 12 Sprague-Dawley rats were exposed to 500 µg/m³ maleic anhydride for 6 hours/day for 5 days. After a 3 -week rest period, one group was challenged with 448 µg/m³ trimellitic acid, while the other group was not challenged.

One rat died in the challenged group during the first week of the rest period. Two rats in each group were considered to have responded positively (i.e. having > 10 foci/lung), while mean foci/lung were not different between the two groups. There were no differences in serum IgG levels in both groups, but IgG levels of both were significantly increased compared to the challenged and non-challenged control males of part 1 and increased over the maleic anhydride-exposed and challenged animals. Microscopic lesions were minimal in severity, focal in distribution and were seen in only one rat in the maleic anhydride-exposed/non-challenged group. No microscopic lung lesions were seen in the challenged rats.

Based on these results, no cross-reactivity was evident between maleic anhydride and trimellitic anhydride.

In the review publication on the toxicology of cyclic anhydrides by the WHO CICAD 75 from 2009, the results from studies investigating respiratory sensitisation were cited.

Cyclic acid anhydrides are irritants to skin and mucous membranes of the eyes and respiratory system.

In animal studies described in the CICAD, it was demonstrated that antibody responses have been induced by cyclic anhydrides via bronchial, subcutaneous, intradermal and parenteral routes of exposure. Development of an allergic respiratory disease is dependent on the production of specific antibodies. In some of these studies, the development of allergic respiratory responses following sensitization with cyclic anhydrides was demonstrated.

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 cyclic anhydrides for Respiratory sensitisation Category 1 and H334 ("May cause allergy or asthma symptoms or breathing difficulties if inhaled").


The information presented in WHO CICAD 75 indicates that cyclic acid anhydrides as a group are responsible for cases of occupational respiratory irritation and respiratory sensitisation.

Justification for classification or non-classification

Based on the harmonised classification according to Annex VI of Regulation (EC) 1272/2008, classification of Succinic anhydride Skin Sens. 1, H317 ("May cause an allergic skin reaction") and Resp. Sens. 1, H334 "(May cause allergy or asthma symptoms or breathing difficulties if inhaled") is warranted.