Bisisopropyl peroxydicarbonate

Endpoint summary

Description of key information

Skin irritation

Based on the weight of evidence diisopropyl peroxydicarbonate is considered as irritant for the skin.

 

The skin irritation potential of Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) was evaluated using the EpiskinTM reconstructed human epidermis model (Grignard-Racinet, 2017). The study design was based upon OECD Guideline No. 439. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as a reference viability). In the preliminary tests, the test item was neither found to have direct MTT reducing properties, nor coloring potential.

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15-minute exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 9% with a Standard Deviation of 2% as assessed by the MTT assay. As the mean relative viability was < 50% after the MTT reduction, no determination of IL-1a concentration was performed and the culture medium samples were destroyed before finalization of the study report.

Moreover and since mean relative viability was < 50%, the results met the criteria for an irritant response. Under the experimental conditions of this study, Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) is considered to be irritant to skin. According to the results of this study, the classification of the test item should be Category 1 or Category 2 (UN GHS).

 

0.5 ml of 100 % diisopropyl peroxydicarbonate was applied to the intact and abraded skin of the back of 4 albino New Zealand rabbits, the substance was held in place occluded for 24 hours with a gauze bandage (Mastri, 1970). After 24 hours, the plastic wrapping and patches were removed. The skin was examined for any injury or irritation according to Draize scale at 24 and 72 hours. Individual mean erythema scores over 24 and 72h were 2.5 for the intact and abraded skin, corresponding edema scores were between 0.5 and 2. 100 % diisopropyl peroxydicarbonate was considered as a skin irritant but not corrosive.

 

The skin irritation potential of diisopropyl peroxydicarbonate was evaluated with the Toxtree module skin irritation. The substance was identified as irritant but not corrosive to skin.

 

Eye irritation

Based on the weight of evidence diisopropyl peroxydicarbonate is considered as severely irritant for the eyes.

 

The potential irritant and corrosive properties to the eye of Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test method which can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage (Grignard-Racinet, 2017). The design of this study was based on the OECD Guideline 437. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes and the open-chamber treatment method. Negative and positive controls were applied using the same treatment time but using the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Opacity and fluorescein fixation were observed on the three corneas treated with the test item. All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 16.

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation (UN GHS No Category).

 

A group of five New Zealand strain albino rabbits was used to evaluate the eye irritating properties of 100% diisopropyl peroxydicarbonate (IPP 100%) (Mastri, 1970). The test method employed was patterned after that of Draize et al. (1944). Exactly 0.1 ml of undiluted test material was instilled into the conjunctival sac of the right eye of each rabbit. The left eye of each animal served as a scoring control. One minute, one, 24, and 72 hours and 7, 14, and 21 days following instillation, the cornea, Iris, and palpebral conjunctiva were examined individually and graded for irritation and injury according to a standard scoring system. The maximum possible score at any examination and scoring period is 110 points which indicates maximal irritation and damage to all three ocular tissues. IPP 100% was rated extremely irritating. Sloughing of the corneal epithelium, opacity, and iridal and conjunctival irritation were noted within one hour after instillation. The irritation essentially peaked at this time and was still present in three rabbits alter 21 days.

Respiratory tract irritation

Exposure to vapors of bisisopropyl peroxydicarbonate and/or its degradation products is not irritating to the respiratory tract.

A 10 to 60-minute exposure to the fog and vapors of a 45% solution of bisisopropyl peroxydicarbonate in Soltrol 130 (ca 15 mg/L of bisisopropyl peroxydicarbonate) caused symptoms of respiratory disorders to hemorrhagic pulmonary edema (McNervey, 1961). No symptoms of abnormality and histopathological changes in lungs were noted during a 8-hour inhalation exposure to saturated vapor concentration of bisisopropyl peroxydicarbonate and/or its degradation products (Lalich, 1958).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 September 2017 - 25 September 2017

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiSkin Laboratories, Lyon, France

Justification for test system used:

The EpiskinTM model is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.
Based on the peer review of the results of an inter-laboratory study with the EpiskinTM model, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the EpiskinTM model can be used for distinguishing between skin irritant and non-irritant chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL evenly applied to the surface of each corresponding tissue, using a positive displacement pipette, taking care to spread it over the whole tissue area without damaging the tissue sample
- Concentration: neat (as supplied)

Duration of treatment / exposure:

exposure period of 15 minutes, at room temperature, followed by rinsing.

Duration of post-treatment incubation (if applicable):

42 hours recovery period at +37°C, 5% CO2 in a humidified incubator.

Number of replicates:

Each test or control item was applied on three tissues.
At the end of the viability assay, formazan level in tissues were assessed in duplicate for each tissue.
The mean blank OD values (mean ODblank) were then calculated from the six replicates on each plate.

Details on study design:

PRELIMINARY TESTS
Test for direct MTT reduction with the test item

As a test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test.
To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 10 µL of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 10 µL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 5 minutes).
Then the colour of the solutions obtained was evaluated.

Test for the detection of the colouring potential of the test item
The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 10 µL of test item to 90 µL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the colouration was evaluated.

MAIN TEST
One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.


PRE-INCUBATION OF THE TISSUES ON THEIR DAY OF ARRIVAL (Day 0)
A volume of 2 mL of pre-warmed (at +37°C) maintenance medium was added to 3 wells on a 12-well plate (one plate per item).
Each EpiskinTM tissue was then transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at +37°C, 5% CO2 in a humidified incubator for at least 24 hours.

TREATMENT OF TISSUES (Day 1)
A volume of 2 mL of pre-warmed maintenance medium was added into 3 wells on the 12-well plate for each item, respectively.
Each test or control item was then applied on three tissues for an exposure period of 15 minutes, at room temperature. The items were applied topically to the corresponding tissues and gently spread over the epidermal surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time intervals to ensure each tissue received an equal exposure period.
For the positive control only, the SDS solution was re-spread after 7 minutes (± 1 minute) contact time with a curved spatula to improve the distribution of the SDS for the remainder of the contact period.

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS (Day 1)
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

MTT VIABILITY ASSAY (Day 3)
Following the 42 hours incubation period, plates containing test item-treated tissues and negative control treated tissues were placed for 15 minutes (± 2 minutes) on a plate shaker to homogenise the released inflammatory mediators in the maintenance medium. Then, 1.6 mL of incubation medium from beneath each tissue were retained in pre-labelled micro tubes, and stored at -20°C until possible quantification for inflammatory mediators. Since no quantification was performed, these samples were then destroyed before the finalization of the study report.
A volume of 2 mL of a freshly prepared MTT solution (0.3 mg/mL) were added into 3 wells on each 12-well plate. The tissues were then transferred to the MTT filled wells and incubated for 3 hours (± 5 minutes) at +37°C, 5% CO2 in a humidified incubator.
At the end of the MTT incubation period, a total biopsy of the epidermis was made by using the Episkin™ biopsy punch. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. The epidermis was separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into micro tubes and incubated with 500 µL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at +5°C, protected from light until Day 6 of the experiment.

OPTICAL DENSITY MEASUREMENTS (Day 6)
At the end of the formazan extraction period, each tube was vortexed until the solution colour became homogenous.
Each tube was used to fill 2 wells of a 96-well plate with 200 µL of extract per well. One 96-well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item.
For each 96-well plate, the average Optical Density value (OD) of 6 wells containing 200 µL of acidified isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.

QUANTIFICATION OF IL-1a
The concentration of the IL-1a in the culture medium retained following the 42-hour recovery period was determined only for test items found with a mean relative viability > 50% after the MTT reduction. Since, the test item-treated tissues were found to have a mean relative viability < 50% (i.e. 9%), no determination of IL-1a concentration was performed and the retained culture medium samples were discarded before finalization of the study report.

Irritation / corrosion parameter:

% tissue viability

Remarks:

15 min

Run / experiment:

Mean

Value:

9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

5%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE CRITERIA FOR NEGATIVE AND POSITIVE CONTROLS
- the mean cOD of the three negative controls should be = 0.6 and the Standard Deviation (SD) value of the % viability should be = 18%,
- the relative mean viability of the positive control should be = 40% of the relative mean viability of the negative control and the SD value of the % viability should be = 18%.

EVALUATION OF THE COLOURATION OF TISSUES AT THE END OF THE MTT INCUBATION PERIOD
The qualitative evaluation of the MTT staining was performed with the naked eye.
All tissues treated with the negative control appeared blue which was considered indicative for viable tissues.
Conversely, all positive control and test item-treated tissues appeared white which was considered to be indicative of dead tissues.

EVALUATION OF THE MTT RESULTS
All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

Interpretation of results:

study cannot be used for classification

Remarks:

Category 1 or Category 2 according to UN GHS Classification

Conclusions:

Under the experimental conditions of this study, the test item is considered to be irritant to skin.
According to the results of this study, the classification of the test item should be Category 1 or Category 2 (UN GHS)

Executive summary:

The skin irritation potential of Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) was evaluated using the Episkin^TM reconstructed human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO₂in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as a reference viability). In the preliminary tests, the test item was neither found to have direct MTT reducing properties, nor colouring potential.

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15-minute exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 9% with a Standard Deviation of 2% as assessed by the MTT assay. As the mean relative viability < 50% after the MTT reduction,no determination of IL-1a concentration was performed and the culture medium samples were destroyed before finalization of the study report.

Moreover and since mean relative viability was < 50%, the results met the criteria for an irritant response.

Under the experimental conditions of this study, Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) is considered to be irritant to skin. According to the results of this study, the classification of the test item should be Category 1 or Category 2 (UN GHS).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Industrial Biotest Laboratory was found to use fraudulent practices in some of their studies and reports. Since these studies were performed before the implementation of Good Laboratory Practices, it is not possible to verify the scientific credibility of most of these studies.

Principles of method if other than guideline:

Draize method

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

24 hours

Observation period:

24 and 72 hours

Number of animals:

4

Irritation parameter:

erythema score

Basis:

animal: #1, 2 3 and 4

Time point:

other: 24/72h

Score:

2.5

Max. score:

4

Reversibility:

not fully reversible within: 72h

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 24/72h

Score:

0.5

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

edema score

Basis:

animal: #2 and 4

Time point:

other: 24/72h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 24/72h

Score:

1.5

Max. score:

4

Reversibility:

not fully reversible within: 72h

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Executive summary:

0.5 ml of 100 % diisopropyl peroxydicarbonate was applied to the intact and abraded skin of the back of 4 albino New Zealand rabbits, the substance was held in place occluded for 24 hours with a gauze bandage. After 24 hours, the plastic wrapping and patches were removed. The skin was examined for any injury or irritation according to Draize scale at 24 and 72 hours. Individual mean erythema scores over 24 and 72h were 2.5 for the intact and abraded skin, corresponding edema scores were between 0.5 and 2. 100 % diisopropyl peroxydicarbonate was considered as a skin irritant but not corrosive.

Reference 3

Endpoint:

skin irritation / corrosion, other

Remarks:

in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Toxtree (Estimation of Toxic Hazard - A Decision Tree Approach)

2. MODEL (incl. version number)
Version 2.6.13

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)OC(=O)OOC(=O)OC(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Skin irritation
- Unambiguous algorithm: see attached rules
- Defined domain of applicability: rules were developed for Identifying Chemical Substances with no Skin Irritation or Corrosion Potential whatever the chemical structure
- Appropriate measures of goodness-of-fit and robustness and predictivity: not relevant
- Mechanistic interpretation: according to the rules and decision tree of the specific end-point

5. APPLICABILITY DOMAIN
- Descriptor domain: Defined by the rules
- Structural and mechanistic domains: Defined by the rules
- Similarity with analogues in the training set: not relevant

6. ADEQUACY OF THE RESULT
The estimation is coherent with the available in vivo data

Qualifier:

according to guideline

Guideline:

other: REACH guidance. Chapter R.6: QSARs and grouping of chemicals

Version / remarks:

May 2008

Principles of method if other than guideline:

Toxtree (Estimation of Toxic Hazard - A Decision Tree Approach) (Version 2.6.13)

Remarks on result:

other: Irritating to skin

Irritant / corrosive response data:

 

 Q1.Melting Point[°C] > 200 No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q2.LogP-3.1 No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q3.Lipid Solubility[g/kg] 0.01 No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q4.Group C (C,H,O) Yes   CC(C)OC(=O)OOC(=O)OC(C)C
 Q5.Melting Point[°C] > 55 No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q6.Molecular Weight > 350.0 No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q7.Surface Tension[mN/m] > 62 No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q8.Vapour Pressure[Pa] 0.0001 No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q9.Group CN (C,H,O,N) No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q19.Group CNHal (C,H,O,N,F,Cl,Br or I ) No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q27.Group CNS (C,H,O,N,S) No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q33.Group CHal (C,H,O,F,Cl,Br or I ) No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q36.AlphaAlkynes No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q4.Group C (C,H,O) Yes   CC(C)OC(=O)OOC(=O)OC(C)C
 Q38.Acrylic Acids No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q39.O and P Quinones No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q40.AliphaticSaturatedAcidsAndHalogenatedAcids No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q41.Aldehydes No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q42.Phenols No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q43.CatecholsResorcinolsHydroquinones No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q43.CatecholsResorcinolsHydroquinones No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q45.AcidAnhydrides No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q46.Ketenes No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q47.BetaLactones No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q48.Lactone, fused to another ring, or 5- or 6-membered a,b-unsaturated lactone? No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q49.Epoxides No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q50.AcrylicAndMethacrylicEsters No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q51.Ketones No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q52.C10_C20AliphaticAlcohols No   CC(C)OC(=O)OOC(=O)OC(C)C
 Q53.EthyleneGlycolEthers Yes Class Irritating to skinCC(C)OC(=O)OOC(=O)OC(C)C

Interpretation of results:

other: Irritating

Executive summary:

The skin irritation potential of diisopropyl peroxydicarbonate was evaluated with the Toxtree module skin irritation. The substance was identified as irritant but not corrosive to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 September 2017 - 21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Species: bovine cattle (Bos taurus).
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France, immerged in containers filled with cooled buffered Hanks medium placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at CiToxLAB France. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].

Preparation of the corneas
Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL per cornea
- Concentration: undiluted

Duration of treatment / exposure:

Exposure period of 10 minutes (± 30 seconds) at +32°C, followed by rinsing.

Observation period (in vivo):

Not applicable.

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes) at +32°C

Number of animals or in vitro replicates:

Triplicate corneas for each item (test item, negative control, positive control)

Details on study design:

EYES SELECTION
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number.

TREATMENT
The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 10 minutes (± 30 secondes) in a water bath at +32°C (± 1°C). The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc.) was carried out in the same order for the three corneas of each series.

REMOVAL OF TEST SUBSTANCE
The purpose of rinsing was to eliminate as much item as possible, while taking care not to damage the cornea. On completion of the treatment period, test item, positive and negative controls were removed from the front opening of the anterior chamber (open-chamber method) and epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed three times with pre-warmed cMEM containing phenol red (i.e. until item had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

POST INCUBATION PERIOD
Following the 10-minute treatment and the rinsing step, the holders were incubated for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C). On completio of the 2-hour incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (+32°C (± 1°C)), the second opacity measurement (OPT2) was then performed.

SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
The change in opacity value of each individual cornea treated with test item, negative control or positive control was calculated by subtracting the initial base-line opacity measurement (OPT0) from the post treatment opacity reading (OPT2).
The average change in opacity for the corneas treated with the negative control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item or positive control to obtain a corrected opacity value (cOPT). When the average change in opacity for the corneas treated with the negative control was negative, it is considered equal to 0.
The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

- Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution at 4 mg/mL. The holders were incubated with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).
At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).
The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item or positive control was calculated by subtracting the average negative control cornea OD490 nm value from the original OD490 nm value of each cornea.
The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.

- Scoring:
In Vitro Irritancy Score (IVIS) = cOPT + (15 x cOD490 nm)

Irritation parameter:

in vitro irritation score

Remarks:

test item

Run / experiment:

mean

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

48.0 +/- 17.5

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

MACROSCOPIC EXAMINATION:
No notable opaque spots or irregularities were observed on negative control-treated corneas.
Opacity and fluorescein fixation were observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

ACCEPTANCE OF RESULTS:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
- the mean opacity and mean OD490 nm of the negative control corneas should be less than the established upper limit of historical mean.

Irritant / corrosive response data:

No prediction can be made.

Interpretation of results:

study cannot be used for classification

Remarks:

could not be identified as UN GHS Category 1 or as UN GHS No Category.

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation (UN GHS No Category).

Executive summary:

The potential irritant and corrosive properties to the eye of Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test method which can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the OECD Guideline 437. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes and the open-chamber treatment method. Negative and positive controls were applied using the same treatment time but using the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Opacity and fluorescein fixation were observed on the three corneas treated with the test item. All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 16.

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation (UN GHS No Category).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Industrial Biotest Laboratory was found to use fraudulent practices in some of their studies and reports. Since these studies were performed before the imple mentation of Good Laboratory Practices, it is not possible to verify the scientific credibility of most of these studies.

Principles of method if other than guideline:

Draize test

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.1 ml

Duration of treatment / exposure:

not rinsed

Observation period (in vivo):

1 min, 1, 24 and 72 hours, 7, 14 and 21 days

Number of animals or in vitro replicates:

5

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM: Draize scale

Irritation parameter:

other: Draize score

Basis:

mean

Time point:

24 h

Score:

50.4

Max. score:

110

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

other: Draize score

Basis:

mean

Time point:

72 h

Score:

66.6

Max. score:

110

Irritation parameter:

other: Draize score

Basis:

mean

Time point:

21 d

Score:

37

Max. score:

110

Irritant / corrosive response data:

IPP 100% was rated extremely irritating. Sloughing of the corneal epithelium, opacity, and iridal and conjunctival irritation were noted within one hour after instillation. The irrritation essentiallylly peaked at this time and was still present in three rabbits alter 21 days.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Executive summary:

A group of five New Zealand strain albino rabbits was used to evaluate the eye irritating properties of 100% diisopropyl peroxydicarbonate (IPP 100%). The test method employed was patterned after that of Draize et al. (1944). Exactly 0.1 ml of undiluted test material was instilled into the conjunctival sac of the right eye of each rabbit. The left eye of each animal served as a scoring control. One minute, one, 24, and 72 hours and 7, 14, and 21 days following instillation, the cornea, Iris, and palpebral conjunctiva were examined individually and graded for irritation and injury according to a standard scoring system. The maximum possible score at any examination and scoring period is 110 points which indicates maximal irritation and damage to all three ocular tissues. IPP 100% was rated extremely irritating. Sloughing of the corneal epithelium, opacity, and iridal and conjunctival irritation were noted within one hour after instillation. The irritation essentially peaked at this time and was still present in three rabbits alter 21 days.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

According to the available data and CLP/GHS criteria, diisopropyl peroxydicarbonate must be classified as skin irritant cat. 2 and eye irritant cat. 1.