Registration Dossier

Administrative data

Description of key information

The in vitro skin irritating/corrosion potential of the target substance was tested in three in vitro skin irritation tests conducted in accordance with current OECD guidelines (OECD 431, OECD 435 and OECD 439). In the in vitro test for corrosion (OECD 431), the test item was considered to be corrosive. In the other both in vitro tests, contradictory results were obtained. In both experiments, the test item showed no irritating effects.

Succinic anhydride caused severe injury to rabbit eyes when applied undiluted and as a 15 % solution for an 18–24 hour exposure.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14-Apr-2014 to 18-Apr-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: EU method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline, no. 431: In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 25 mg of the test substance was applied undiluted ( with 25 µl Milli-Q water to ensure close contactto the tissue)NEGATIVE CONTOL:- Amount(s) applied (volume or weight with unit): 50 µl Milli-Q waterPOSITIVE CONTROL- Amount(s) applied (volume or weight with unit): 50 µl KOH- Concentration (if solution): 8 N
Duration of treatment / exposure:
Exposure:3 minutes and 1 hourPost incubation period: 42 hours
Details on study design:
TEST SITE- Area of exposure: human skin model- % coverage: 0.6 cm2REMOVAL OF TEST SUBSTANCE- Washing (if done): phosphate buffered saline- Time after start of exposure: 3 minutes and 1 hourPOST INCUBATION PERIOD- 42 hoursSCORING SYSTEM:- After treatment the medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm
Irritation / corrosion parameter:
other:
Value:
96
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 3 minutes. (migrated information)
Irritation / corrosion parameter:
other:
Value:
12
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 1 hour. (migrated information)
Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
Succinic anhydride is corrosive in the in vitro skin corrosion test
Executive summary:

The test substance was tested for skin corrosion potential in an in vitro skin corrosion study (OECD 431).The positive control had a mean relative tissue viability of 9% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 23% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Succinic anhydride compared to the negative control tissues was 96% and 12%, respectively. Because the mean relative tissue viability for Succinic anhydride was below 15% after the 1-hour treatment, it is considered to be corrosive.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Details on test system:
Test systemCorrositex® is a test system that is composed of two components, a hydrated collagen matrix (bio-barrier) on a supporting filter membrane and CDS, an underlying aqueous solution of two pH indicator dyes.
The Corrositex® kit contained:
- Bio-barrier matrix.
- Bio-barrier diluent
- Vials with Chemical Detection System (= CDS).
- Membrane discs.
- Compatibility test tubes.
- Timescale categorize test tubes with buffer A and B.
- Confirm reagent. The batch number of the kit used for the experiment was CT051214.

Rationale: Recommended test system in international guideline (OECD 435).
Source InVitro International Inc., Irvin CA, USA

Succinic acid anhydride was classified into one of the two timescale categories (see Table 1). This category determined how the penetration response time (if one occurs) was interpreted. The two different penetration response timescales were based on the acid or alkali reserve of the chemical. Succinic acid anhydride (100 mg) was added to both buffers A and B, shaken vigorously for 10 seconds and after 1 minute the colour change was read on the chart.In case the test substance was immiscible, the colour change was read at the interface after another minute.Buffer A detected weak or strong acids and buffer B detected strong or weak bases. In case no colour change was observed in both buffers a conformation test was performed. Two drops of confirm reagent were added to buffer B. The tube was shaken vigorously for 5 seconds and the colour of the solution was read on the chart confirming that the substance is a timescale category 2 substance.

The test was performed on a total of 4 membrane discs with bio-barrier matrix together with a negative and positive control. A disc was placed on a vial with CDS fluid. Within two minutes after the disc was placed on the CDS fluid, the test substance (503.9 to 509.3 mg) was applied on top of the matrix. One disc was exposed to 500 μL citric acid (10%, negative control) and one disc was exposed to 507.1 mg of the positive control sodium hydroxide. The test substance and controls were evenly distributed.Each vial was at least monitored for the first 5 minutes and 5 minutes before and after each packing cut-off time. The time of a change in the CDS fluid was recorded. Changes in the CDS may be colour changes (red, orange or lightening) and flaking or precipitation. The elapsed time between application and penetration of the membrane was determined.

The in vitro membrane barrier test is considered acceptable if it meets the following criteria:a) The negative control should be non-corrosive (category obtained from OECD 435) and have a penetration response time of> 60 minutes.b) The breakthrough time of the positive control sodium hydroxide should be between the 3 and 30 minutes (GHS corrosive subcategory 1B; category obtained from OECD 435).
Irritation / corrosion parameter:
penetration time (in minutes)
Run / experiment:
mean
Value:
> 60
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
For detailed results please refer to box "Any other information on results incl. tables".

 Chemical  Timescale category  Mean penetration time (minutes) UN Packing group (GHS subcategory) determined with Corrositex® 
 Citric acid (10%)

 2*

 > 60

 Non-corrosive
 Succinic acid anhydride

2

>60

 Non-corrosive
 Sodium hydroxide (as it is)

 1*

 16  II (GSH 1B)

*Not determined in the present study; category obtained from OECD 435

Interpretation of results:
GHS criteria not met
Conclusions:
Succinic anhydride is classified as non-corrosive in the Corrositex® assay.
Executive summary:

In vitro membrane barrier test for skin corrosion (Corrositex®) with Succinic acid anhydride.

This report describes the ability of Succinic acid anhydride to pass through a bio-barrier and to elicit a colour change in the underlying liquid chemical detection system.

The study procedures described in this report were based on the most recent OECD guideline.

Batch LEBA5A9071 of Succinic acid anhydride were white flakes with a purity of 99.5%. The test substance compatibility test showed that Succinic acid anhydride was compatible with the chemical detection system. Based on the acid or reserve, the test substance was classified as a timescale 2 compound. Succinic acid anhydride was crushed and approximately 500 mg of Succinic acid anhydride was applied on top of the bio-barrier matrix. The elapsed time between application and penetration was determined by monitoring changes in the chemical detection system.

The penetration time of Succinic acid anhydride was >60 minutes. Since Succinic acid anhydride was a timescale 2 test substance and showed a mean penetration time of >60 minutes, Succinic acid anhydride is classified as non-corrosive.

The negative control citric acid (10%) showed a penetration time of >60 minutes and was therefore classified as non-corrosive. The positive control sodium hydroxide (as it is) showed a penetration time of 16 minutes and was therefore classified as UN packing group II (=GSH 1B). It was therefore concluded that the test system functioned properly.

Finally, it is concluded that this test is valid and that Succinic acid anhydride is classified as non-corrosive in the Corrositex® assay under the experimental conditions described in this report.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28-Apr-2014 to 05-May-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 14-EKIN-015). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 25 hours at 37 °C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.5 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 34.5 - 36.0°C) and humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test for reduction of MTT by the test substance
Succinic anhydride was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 10.6 mg of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (10.6 to 11.8 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µl PBS (negative control) and 3 tissues with 25 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

Cell viability measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck,
Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Succinic anhydride consisted of white flakes with a purity of 99.7%. Skin tissue was moistened with 5 µl of Milli-Q water and 10.6 to 11.8 mg of Succinic anhydride was applied directly on top of the skin tissue for 15 minutes.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
1 042
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
Succinic anhydride is non-irritant in the in vitro skin irritation test.
Executive summary:

The test substance was tested for skin irritating potential in an in vitro skin irritation study (OECD 439). Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Succinic anhydride compared to the negative control tissues was 102%. Since the mean relative tissue viability for Succinic anhydride was above 50% after 15 minutes treatment it is considered to be non-irritant. The positive control had a mean cell viability of 8% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1938-1946
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline-similar study reported in sufficient detail to enable confident assessment of the method and interpretation of the results and published in a peer-reviewed journal
Qualifier:
no guideline available
Principles of method if other than guideline:
Grade the severity of eye burns from a large number of chemicals and translate each injury into a numerical score.
GLP compliance:
not specified
Species:
rabbit
Strain:
other: normal, albino
Vehicle:
other: propylene glycol or water
Controls:
not specified
Amount / concentration applied:
0.005 mL undiluted, 5% solution and 15% solution
Duration of treatment / exposure:
18-24 hours
Number of animals or in vitro replicates:
Usually 5
Details on study design:
Rabbit eyes were selected on the basis of absence of grossly visible staining by the 5% aqueous solution of fluorescein sodium, flushed with distilled water for 20 seconds after application. After a 2-hr interval to allow the eye to return to normal, 0.005 mL of the undiluted material is applied to the center of the cornea while the lids are retracted. About one minute later, the lids are released. Eighteen to 24 hours later, the eye is examined in strong diffuse daylight, then stained with fluorescein, and the injury scored. Additional applications of the test material are made until the chemical can be assigned to a specific grade (see Table 2) . If large volumes are applied, lids are held closed for one minute before the animal is released.The individual numerical scores of each eye (see Table 1) were added together and then divided by the number of eyes (usually 5) to obtain the score of the injury caused by treatment. A total of 180 chemicals were arranged according to injury grades.
Irritation parameter:
other: grade 8
Basis:
mean
Remarks:
15% solution scores over 5.0; 5% solution not over 5.0
Remarks on result:
other: A score of 5.0 corresponds to necrosis, visible only after staining and covering about three-fourths of the surface of the cornea; or a more severe necrosis covering a smaller area.
Irritant / corrosive response data:
Succinic anhydride was assigned grade 8 on the basis that 0.005 mL and 15% solutions yielded scores of over 5.0 and the 5% solution was not over 5.0.

Table 3. Injury grades of some chemicals when applied to the rabbit eye

 Grade 8 (0.005 mL and 15% solution yield scores of over 5.0, 5% solution not over 5.0)   
 Caproic acid  Phenyl methyl ketone
 *Di-2 -ethylhexyl amine Soap (granulated white) 
 Di-n-hexyl amine Succinic acid 
 *Dimethyl sulfate Succinic anhydride 
 *Ethylene diamine  Tetramethyl ethylene diamine
 *Formaldehyde  Trimethyl adipic acid
 Lactic acid  
 Grade 10 (1% solution yields score of over 5.0)   
 *Maleic anhydride  *Sodium hydroxide
    * Compounds known to have caused severe human eye injury
Interpretation of results:
corrosive
Remarks:
Migrated informationCriteria used for interpretation of results: expert judgment
Conclusions:
Succinic anhydride caused severe injury to rabbit eyes when applied undiluted and as a 15% solution for an 18- 24 hour exposure; a 5% solution did not cause this eye damage grade.Succinic acid was graded in the same effect group.Maleic anhydride caused severe injury to rabbit eyes when applied as a 1% solution for an 18- 24 hour exposure.
Executive summary:

Undiluted succinic anhydride (0.005 mL) was applied to the center of the cornea of approximately 5 albino rabbits while the lids were retracted. Lids were released after one minute. Larger volumes of 5% and 15% succinic anhydride solutions were also applied and lids were held closed for one minute before the animal was released. Eighteen to 24 hours later, the eye was examined, stained and scored such that each injury was translated into a numerical score developed by the authors. The individual numerical scores of each eye were added together and then divided by the number of eyes to obtain the score of the injury caused by treatment. These scores were then arranged according to injury grades developed by the authors. Undiluted and 15% succinic anhydride yielded an injury score of over 5.0 and the 5% solution yielded an injury score not over 5.0. The authors described succinic anhydride as grade 8, which causes severe injury to rabbit eyes.

Another cyclic anhydride also tested in this scheme for eye injury. Maleic anhydride was scored as grade 10 for injury to the rabbit eye and a solution of 1% yielded an injury score of over 5.0.

Sufficient information is available on succinic anhydride and a separate eye irritation study is not proposed and cannot be justified based upon animal welfare considerations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance was tested for skin irritating potential in an in vitro skin irritation study (OECD 439). Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Succinic anhydride compared to the negative control tissues was 102%. Since the mean relative tissue viability for Succinic anhydride was above 50% after 15 minutes treatment it is considered to be non-irritant. The positive control had a mean cell viability of 8% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

In a second in vitro test, the study procedures described in this report were based on the OECD test guideline 435. The test substance compatibility test showed that Succinic acid anhydride was compatible with the chemical detection system. Based on the acid or reserve, the test substance was classified as a timescale 2 compound. Succinic acid anhydride was crushed and approximately 500 mg of Succinic acid anhydride was applied on top of the bio-barrier matrix. The elapsed time between application and penetration was determined by monitoring changes in the chemical detection system. The penetration time of Succinic acid anhydride was >60 minutes. Since Succinic acid anhydride was a timescale 2 test substance and showed a mean penetration time of >60 minutes, Succinic acid anhydride is classified as non-corrosive.

In a third in vitro test, the test substance was tested for skin corrosion potential in an in vitro skin corrosion study (OECD 431). The positive control had a mean relative tissue viability of 9% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 23% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Succinic anhydride compared to the negative control tissues was 96% and 12%, respectively. Because the mean relative tissue viability for Succinic anhydride was below 15% after the 1-hour treatment, it is considered to be corrosive.

Based on the available data, it is justified to classify the substance as Skin Irrit 2, H315 in a weight-of evidence approach. The irritating potential is also supported by the findings of an acute dermal toxicity study. In this study, 2000 mg/kg bw were applied dermally to skin of rats. 3/5 males and all females exhibited erythema and eschar formation at the application site. These local effects were observed 1 day after administration lasting until a maximum of 7 days post administration.

Eye Irritation:

The key study is a non-GLP and non-TG conform study (Carpenter and Smyth, 1946). The grade of severity of eye burns from a large number of chemicals have been examined and the injury has been translated into a numerical score. Albino rabbit eyes have been treated with 0.005 ml of undiluted material to the center of the cornea while the lids are retracted. Depending on the outcome, additional applications were made. Succinic anhydride and succinic acid have the same grading (injury grade 8 out of 10), which indicates a corrosive potential of the test substance. Grade 8 is defined that 5% solution gives injury of up to 5.0 points, and 15% solution scores over 5.0. A score of 5.0 corresponds to necrosis, visible only after staining and covering about three-fourths of the surface of the cornea; or a more severe necrosis covering a smaller area. The 5% solution corresponds to 0.25 µl test solution. The supporting - read across - study carried out with succinic acid has been conducted under GLP conditions and is a TG conform study (OECD guideline 405). Approximate equivalent of 0.1 ml succinic acid has been applied to one eye of one rabbit. No additional animals were tested, since severe eye lesions have been observed. Severe irreversible corneal alterations were observed (score 4) until 21 days post application with the majority of cornea affected. The Iris could not be examined due to corneal alterations. The palpebral and bulbar conjunctivae were diffuse beefy red up to the 6 d post application. The degree of redness of the conjunctivae decreased continuously with time. The conjunctivae chemosis index decreased with time and there was no swelling observed on day 21. In the first 72h p.a. the swelling lead to closed lids (more or about of the half). The study outcome demonstrates that succinic acid meets the criteria laid down in the Regulation (EC) No. 1272/2008 to be classified as eye damage 1 (H318: Causes serious eye damage). The third study has been carried out with maleic anhydride (Winter, 1950). The outcome of the study substantiates the corrosive potential of the monocylic acid anhydrides.

In conclusion, based on the evidence coming from a comparative study that succinic anhydride and succinic acid possess adverse effects on the eye in the same order of magnitude (grade 8 out of 10) (Carpenter and Smyth, 1946) and due to the fact that the anhydride form is rapidly hydrolysed to the succinic acid form in aqueous solution, the study carried out with the acid form is valid for evaluation. The study carried out with succinic acid is a GLP and guideline conform study and unambiguously demonstrates that succinic acid has to be classified for its severe damage to eyes. Furthermore, also the structural similar compound maleic anhydride warrants a classification regarding its adverse effects on the eye (eye damage 1).

 

Justification for classification or non-classification

Based on the available results and assessing them in a weight-of-evidence approach, the target substance warrants classification as Eye Dam 1, H318 and Skin Irrit 2, H315.