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/corrosion tests conducted in accordance with current OECD guidelines (OECD 431, OECD 435 and OECD 439). In the EpiDerm in vitro test for skin corrosion (OECD 431), the test item was considered to be corrosive. In contrast, the EpiSkin (OECD 439) and the membrane barrier test (OECD 435) showed contradictory results. In both experiments, the test item did not show irritating/corrosive effects.

Based on the available harmonised classification according to Annex VI of Regulation (EC) 1272/2008, it is justified to classify the substance as Skin Corr. 1, H314.

Succinic anhydride caused severe injury to rabbit eyes when applied undiluted and as a 15 % solution for an 18–24 hour exposure. In addition, these observations are supported by studies identified in a read-across approach to the substances succinic acid and maleic anhydride. Application of these substances to the rabbit eye likewise caused severe damage to the eye. Read-across is feasible because succinic anhydride readily hydrolyses to succinic acid in aqueous solutions. Maleic anhydride is a close structural analogue of succinic anhydride only differing in a double bond at position 2 of the molecule. These results are in line with the harmonised classification of succinic anhydride as Eye Dam. 1, H318 according to Annex VI of Regulation (EC) 1272/2008.

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 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:
2014-04-18 to 2014-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 13 April 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of the test material used in the report: Succinic anhydride
- Batch no.: LEBA3A7021
- Purity: 99.7%
- Storage temperature: In refrigerator (2 to 8 °C)
- Expiry date: 31 December 2014

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No correction was made for the purity/composition of the test substance. The test substance was crushed and ground in a mortar with pestle. Twenty-five mg of Succinic anhydride was applied directly on top of the skin tissue. Succinic anhydride was spread to match the size of the tissue.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test substance by assessment of its effect on a three-dimensional human epidermis model.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200). MatTek Corporation, Ashland MA, USA
- Tissue batch number(s): Lot no.: 19989 kit D

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 83%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 - 36.8 °C). Temperature and humidity were continuously monitored throughout the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL, MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: The DMEM 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.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 4 tissues per test substance (two tissues were used for 3 min and two for a 1-hour exposure)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test substance is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance considered non-corrosive (viability  50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test substance is decreased below 15%.
A test substance is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: Succinic anhydride was crushed and ground in a mortar with pestle. Twenty-five mg of Succinic anhydride was applied directly on top of the skin tissue which was moistened with 25 μl of Milli-Q water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl 8N KOH
Duration of treatment / exposure:
3 min or 1 hour
Number of replicates:
The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Succinic anhydride and two for a 1-hour exposure
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure, mean of replicates
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: in combination with the 1-hour exposure: corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure, mean of replicates
Value:
12
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: in combination with the 3-min exposure: corrosive
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes , The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: The mean relative tissue viability following
3-minute exposure to the positive control was 9%.
- Acceptance criteria met for variability between replicate measurements: 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%.

Table 1: Mean absorption in the in vitro skin corrosion test with Succinic anhydride

3-minute application 1-hour application
A (OD540) B (OD540) Mean (OD540) SD A (OD540) B (OD540) Mean (OD540) SD
Negative control 1.493 1.347 1.42 0.103 1.408 1.361 1.385 0.033
Succinic anhydride 1.279 1.454 1.366 0.124 0.189 0.147 0.168 0.03
Positive control 0.124 0.118 0.121 0.004 0.097 0.085 0.091 0.008

Table 2: Mean tissue viability in the in vitro skin corrosion test with Succinic anhydride

3-minute application
viability (percentage of control)
1-hour application
viability (percentage of control)
Negative control 100 100
Succinic anhydride 96 12
Positive control 9 7
Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
In this study under the given conditions the test item showed corrosive effects. The mean relative tissue viability (% negative control) was below 15% after 60 min treatment and above 50% after 3 min treatment. Therefore, the test item is classified as corrosive.
Executive summary:

In a primary skin corrosion study conducted according to OECD testing guideline 431, two EpiDerm tissues per dose group were exposed to 25 mg of Succinic anhydride (99.7% purity) for 60 min and 3 min. Cytotoxicity was measured in comparison to the concurrent negative controls via the MTT reduction assay and irritation was scored by the method of mean relative tissue viability. The test item showed corrosive effects. The mean relative tissue viability (% negative control) was below 15% (12%) after 60 min treatment and greater than 50% (96%) after a 3 min treatment. The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on the results from this study, the test item can be classified as corrosive.

The study is acceptable and satisfies the guideline requirements for an in vitro skin corrosion study (OECD 431).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-12-22 to 2015-02-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Version / remarks:
adopted 19 July 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of the test material used in the report: Succinic anhydride
- Batch no.: LEBA5A9071
- Purity: 99.5%
- Storage temperature: In refrigerator (2 to 8 °C)
- Expiry date: 28 February 2015

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Succinic acid anhydride was applied (approximately 500 mg), directly on top of the bio-barrier. The test substance was weighed in the dark (yellow light).
Test system:
artificial membrane barrier model
Justification for test system used:
The test is based on the ability of corrosive chemicals or chemical mixtures to pass through, by diffusion and/or destruction/erosion, a bio-barrier and to elicit a colour change in the underlying liquid chemical detection system (CDS). The bio-barrier is composed of a hydrated collagen matrix in a supporting filter membrane and the CDS is composed of water and pH indicator dyes.The time it takes for a test compound to penetrate the bio-barrier and to produce a colour change in the CDS is used to determine corrosivity/noncorrosivity and to identify the appropriate UN GHS subcategory and the UN transport packing group.
Vehicle:
unchanged (no vehicle)
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).SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
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

WAS THE COMPATIBILITY TEST PERFORMED:
Prior to performing the membrane barrier penetration study, Succinic acid anhydride was evaluated for the compatibility with the CDS. Succinic acid anhydride (100 mg) was added to the compatibility test tubes filled with CDS fluid. The tube was shaken to dissolve solids. In case the test substance was immiscible, the vial was shaken and the colour change was read at the interface after 1 minute. If the CDS solution detected a colour or consistency change within 5 minutes, the test substance was tested for corrosivity using the Corrositex® kit. In case the CDS solution detected no colour or consistency change the membrane barrier penetration study was not performed with Corrositex®.

WAS THE TIMESCALE CATEGORY TEST PERFORMED:
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.

TEMPERATURE USED DURING TREATMENT:
- All tests were performed at room temperature. The samples were at room temperature when applied.

METHOD OF DETECTION
- liquid chemical detection system (CDS), aqueous solution of two pH indicator dyes.

METHOD OF APPLICATION:
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.

NUMBER OF REPLICATES:
The test was performed on a total of 4 membrane discs with bio-barrier matrix together with a negative and positive control.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if <60 minutes elapsed between application of the test substance to the membrane barrier and barrier penetration (0 to 3 min for Cat. 1A, >3 to 30 min for Cat. 1B and >30 to 60 min for Cat. 1B).
- The test substance is considered to be non-corrosive to skin if a cut-off time value >60 minutes was exceeded
- Justification for the selection of the cut-off point(s): Timescale Category 2 substance (determined from timescale category test)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: Succinic acid anhydride was crushed and approximately 500 mg of Succinic acid anhydride was applied on top of the bio-barrier matrix

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): The negative control was 10% citric acid (Merck, Nottingham, United kingdom; casnumber 5949-29-1; batchnumber k45326644408 (see protocol deviation 1); purity ≥ 99%). Approximately 500 μL was applied, directly on top of the biobarrier.

POSITIVE CONTROL
- Amount(s) applied: The positive control was sodium hydroxide (GHS corrosive subcategory 1B; category obtained from OECD 435) as it is (Boom, Meppel, The Netherlands; batchnumber 201311PROD13840 (see protocol deviation 1); purity ‘pure’, ≥ 95%). Approximately 500 mg was applied, directly on top of the biobarrier.
Duration of treatment / exposure:
The elapsed time between application and penetration of the membrane was determined.
Number of replicates:
The test was performed on a total of 4 membrane discs with bio-barrier matrix together with a negative and positive control.
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
Remarks on result:
other: non-corrosive
Other effects / acceptance of results:
For detailed results please refer to box "Any other information on results incl. tables".

Table 1: Results and Determination of the UN Packing Group or UN GHS sub-category

 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 (OECD 435).
Executive summary:

Skin corrosivity of Succinic anhydride (99.5% purity) was determined in an in vitro membrane barrier test using the Corrositex® system. The test was performed according to OECD TG 435. The corrosivity of the test substance was determined by 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 test substance compatibility test showed that Succinic anhydride was compatible with the chemical detection system. Due to the lack of a color change in the timescale category test, the confirmation test with buffer B revealed that the test substance was classified as a timescale 2 compound.

In the actual membrane barrier penetration test, Succinic anhydride was crushed and approximately 500 mg (503.9 to 509.3 mg) were applied on top of the four Corrositex® membrane discs with 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 anhydride was >60 minutes. Since Succinic anhydride was a timescale 2 test substance and showed a mean penetration time of >60 minutes, Succinic anhydride is classified as non-corrosive.

All acceptability criteria were fulfilled. 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 showed a penetration time of 16 minutes and was therefore classified as UN packing group II (=GHS 1B). It was therefore concluded that the test system functioned properly. It is concluded that this test is valid and that Succinic 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:
2014-05-05 to 2014-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of the test material used in the report: Succinic anhydride
- Batch no.: LEBA3A7021
- Purity: 99.7%
- Storage temperature: In refrigerator (2 to 8 °C)
- Expiry date: 31 December 2014

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
No correction was made for the purity/composition of the test compound. The solid test substance (10.6 to 11.8 mg) was crushed and ground in a mortar with pestle to improve the consistency and was applied directly on top of the skin tissue. Succinic anhydride was spread to match the size of the tissue.
Test system:
human skin model
Source species:
human
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:
Test system:
EPISKIN Small Model^TM (EPISKIN-SMTM, 0.38 cm², Batch no.: 14-EKIN-015, source SkinEthic Laboratories, Lyon, France). 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.

Preparation and preincubation:
Tissues:
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.

Study design:
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:
for 15 minutes.
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
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 of triplicates
Value:
102
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: not specified
- Colour interference with MTT: no colour change

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: yes, the positive control had a mean cell viability after 15 minutes exposure of 8%. The standard deviation of the positive control was 0.047.
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviation value of the percentage viability of three tissues treated identically was less than 9%.

For detailed results please refer to Table 1 and 2 in box "Any other information on results incl. tables).

Table 1: Mean absorption in the in vitro skin irritation test with Succinic anhydride

A (OD570) B (OD570) C (OD570) Mean (OD570) SD
Negative control 1.107 0.969 0.961 1.012 0.082
Succinic anhydride 1.063 1.043 1.000 1.035 0.033
Positive control 0.044 0.131 0.056 0.077 0.047

Table 2: Mean tissue viability in the in vitro skin irritation test with Succinic anhydride

Mean tissue viability (percentage of control)
Negative control 100
Succinic anhydride 102
Positive control 8
Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro skin irritation study (OECD 439), the test item is considered to be non-irritant to the skin.
Executive summary:

In an in vitro skin irritation study conducted according to OECD guideline 439, the EpiSkin-SM™-Model was topically exposed to Succinic anhydride (99.7% purity) for 15 min followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with PBS. The mean relative tissue viability (% negative control) was >50% (102%). The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on this result, the test item is considered to be non-irritating to the skin.

The study is acceptable and satisfies the guideline requirements for an in vitro skin irritation study (OECD 439).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
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
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
4
Max. score:
4
Reversibility:
not reversible
Remarks:
reactions persisted to termination on day 21
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
other: Extensive corneal opacity persisted to termination on day 21 precluding assessment or ophthalmological examination of the iris.
Remarks on result:
not determinable
Remarks:
It was not possible to provide a score for iridial change recorded due to extensive corneal opacity to termination on day 21 precluding assessment or ophthalmological examination of the iris. A maximum score of 2 was assumed.
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Although reactions showed some amelioration over the three week observation period, some conjunctivitis remained at termination
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.7
Max. score:
4
Reversibility:
fully reversible within: 15 days
Remarks on result:
other: marked chemosis persisted to 72 h after instillation but reactions lessened over the first week and the conjunctival swelling had overtly resolved by day 15
Irritant / corrosive response data:
As severe eye lesions were observed, only one rabbit was treated with the test substance. Corneal opacification affected the entire corneal surface from Day 1 to Day 21 with no resolution or improvement. The severity of the corneal obfuscation precluded any assessment of iris response or iridial changes. Conjunctival reactions include a diffuse beefy red discoloration that persisted for 6 days and gradually reduced in severity but did not fully resolve before termination on day 21. The degree of conjunctival swelling, initially severe, improved slowly and had resolved fully by day 15
Other effects:
At 48 and 72 hours the region located dorsally to the treated eye was also swollen
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In an acute eye irritation/corrosion study, instillation of succinic acid to one rabbit eye resulted in severe and persistent irritant reactions that require classification of the test material as Cat. 1 (serious damage to eyes) according to CLP criteria as set out in Regulation 1272/2008.
Executive summary:

In a primary eye irritation/corrosion study performed according to OECD TG 405, 100 mg of succinic acid (purity: min. 99.5%) was instilled into the conjunctival sac of the right eye of one female New Zealand White rabbit for 24 hours. After the instillation of the test substance, the conjunctival sacs of both eyes were rinsed with warm tap water to remove residual test substance. Anmals were then observed for 21 days. Irritation was scored by the method of Draize.

Severe and irreversible corneal alterations were observed until day 21. The iris was not discernible because of severe corneal opacity. Pronounced conjunctival redness was observed which gradually decreased in severity but did not fully resolve. Severe conjunctival swelling was noted which was reversible until day 15. In addition, a dermal swelling dorsal of the test eye was noted.

In this study, succinic acid caused severe damage to the eye, based on irreversible ocular lesions that failed to fully resolve within 21 days after instillation.

Based on these results, classification of succinic anhydride as Cat. 1 (serious damage to eyes) is warranted according to CLP criteria as set out in Regulation 1272/2008.

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 (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation/Corrosion:

The test substance Succinic anhydride (99.7% purity) was tested for skin irritating potential in an in vitro skin irritation study (OECD 439) using the EPISKIN-SMTM human skin model. Skin irritation is expressed as the remaining cell viability after exposure to the test substance as measured by the reduction of MTT at the end of the treatment. The relative mean tissue viability obtained after 15 minutes treatment with 10.6 to 11.8 mg 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.

All acceptability criteria were fulfilled. 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 performed according to OECD TG 435, corrosivity was determined in the membrane barrier test using the Corrositex® system.

The test substance compatibility test showed that Succinic anhydride (purity: 99.5%) was compatible with the chemical detection system. Due to the lack of a color change in the timescale category test, the confirmation test with buffer B revealed that the test substance was classified as a timescale 2 compound.

In the actual membrane barrier penetration test, Succinic anhydride was crushed and approximately 500 mg (503.9 to 509.3 mg) were applied on top of the four Corrositex® membrane discs with 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 anhydride was >60 minutes. Since Succinic anhydride was a timescale 2 test substance and showed a mean penetration time of >60 minutes, Succinic acid anhydride is classified as non-corrosive.

All acceptability criteria were fulfilled.

In a third in vitro test, the test substance Succinic anhydride (purity: 99.7%) was tested for skin corrosion potential in an in vitro skin corrosion study (OECD 431) using the EpiDerm (EPI-200) human skin model.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance as measured by the reduction of MTT at the end of the treatment. The relative mean tissue viability obtained after 3-minute and 1-hour topical treatments with 25 mg Succinic anhydride compared to the negative control tissues was 96% and 12%, respectively. Because the mean relative tissue viability for Succinic anhydride was above 50% after 3 minute exposure and below 15% after the 1-hour treatment, the test item is considered to be corrosive. All acceptability criteria were fulfilled.

Based on the available harmonised classification according to Annex VI of Regulation (EC) 1272/2008, it is justified to classify the substance as Skin Corr. 1, H314.

Eye Irritation:

The key study carried out with the source substance, succinic acid has been conducted under GLP conditions and is a TG conform study (OECD guideline 405). Approximate equivalents of 0.1 mL succinic acid have 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 72 h p.a. the swelling lead to closed lids (more or about of the half).

An other eye irritation study has been carried out with the source substance, maleic anhydride (Winter, 1950). The outcome of the study substantiates the corrosive potential of the monocyclic acid anhydrides.

Futhermore, in a non-GLP and non-TG conform study (Carpenter and Smyth, 1946) with the target substance, succinic anhydride, 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.

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.

The study outcomes are therefore in line with the harmonised classification according to Annex VI of Regulation (EC) 12721/2008 as Eye Dam. 1 (H318: Causes serious eye damage).

Furthermore, also the structurally similar compound maleic anhydride warrants harmonised classification according to Annex VI of Regulation (EC) 12721/2008 regarding its adverse effects on the eye (Eye Dam. 1, H318).

 

Justification for classification or non-classification

Based on the available harmonised classification according to Annex VI of Regulation (EC) 1272/2008, classification of succinic acid as Eye Dam. Cat 1, H318 and Skin Corr. 1, H314 is warranted.