Registration Dossier

Administrative data

Description of key information

- skin corrosion: not corrosive based on GHS criteria; OECD TG 431 (29 July 2016); RL1; GLP; mean scores: relative mean tissue viability 59.7% for the 3 minute exposure period and 31.1% for the 60 minute exposure period.

- skin irritation: irritating to skin; OECD TG 439 ( 28 July 2015); RL1; GLP; mean scores: relative mean tissue viability 6.9% after the 15 minutes exposure period and 42 hours post exposure period.

- corrosive; OECD TG 402; RL1; GLP; full thickness necrosis and hemorrhage of the underlining tissues after 24 h exposure (semiocclusive) to teh neat substance

overall: corrosive

- eye irritation: Category 1 (irreversible effects on the eye) OECD TG 437: IVIS: 129.1

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:
key study
Study period:
2017-04-27 to 2017-05-06
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 29 July 2016)
Deviations:
yes
Remarks:
The coefficient of variation for the 10% v/v 60 minute exposure period was 30.6% with the acceptance criteria being 30%. This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a liquid: The test item was used as supplied and also prepared as 33% v/v and 10% v/v aqueous solutions.

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
recommended by guideline
Vehicle:
unchanged (no vehicle)
Remarks:
water for the 33% v/v and 10% v/v solutions
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 25810
- Production date:
- Shipping date:
- Delivery date: 03 May 2017
- Date of initiation of testing:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper and the tissue surface was swabbed. The rinsing procedure was then repeated once more to ensure the tissues were completely decontaminated.
- Observable damage in the tissue due to washing: no


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES: duplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze killed
- N. of replicates : duplicates
- Method of calculation used: True viability = mean OD tvt-(OD tkt-OD ukt)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): neat, 33% v/v and 10% v/v aqueous solutions

NEGATIVE CONTROL
- Sterile distilled water
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Potassium Hydroxide
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3-Minutes and 60-Minutes
Duration of post-treatment incubation (if applicable):
The plates stood overnight at room temperature, to allow MTT extraction to proceed
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
neat test item
Run / experiment:
3 minutes exposure period
Value:
59.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
4.0% tissue viability
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure period
Value:
31.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
3.9% tissue viability
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes; The test item was shown to directly reduce MTT and freeze-killed tissues were employed, the results of the MTT assay were therefore corrected as follows: True viability = mean OD tvt-(OD tkt-OD ukt)
- Colour interference with MTT: test item did not have the potential to cause color interference

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group were satisfied with the exception that the 10% v/v concentration of the test item after a 60 minute exposure period was borderline, this is reported as a deviation.
Interpretation of results:
other: not corrosive
Conclusions:
All three concentrations of the test item, 10%, 33% and neat, were considered to be non-corrosive to the skin.
Executive summary:

In a skin corrosion study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion) (adopted July 29, 2016), CGE-PMDA adduct (100%, 33% and 10% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 and 60 minutes respecitvely in triplicates.

Each approximately 50 µL of the test item were applied to the tissues. After 3 or 60 minutes exposure at room temperature (3 min) or 37°C (60 min exposure time), the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 3 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (8.0 N KOH) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after treatment with neat CGE-PMDA adduct compared to the negative control tissues was 59.7% for the 3 minute exposure period and 31.1% for the 60 minute exposure period. The relative mean tissue viability obtained after treatment with 33% CGE-PMDA adduct compared to the negative control tissues was 75.3% for the 3 minute exposure period and 15.8% for the 60 minute exposure period. The relative mean tissue viability obtained after treatment with 10% CGE-PMDA adduct compared to the negative control tissues was 94.4% for the 3 minute exposure period and 39.2% for the 60 minute exposure period.

Since the mean relative tissue viability for the neat test substance and both dilutions was ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure, the prediction to be considered according to EU CLP Regulation (EC) No 1272/2008 is non-corrosive.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2018 - 05 February 2018
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:
23 July 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
as recommended in OECD guideline 439
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: MTT interference control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm²)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of SDS 5% w/v
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
6.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
Mean OD570 of triplicate tissues: 0.859
Positive controls validity:
valid
Remarks:
3.3% tissue viability
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Item

OD570 of tissues

Mean OD570 of triplicate tissues

±SD of OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.959

0.859

0.088

111.6

100*

10.2

0.794

92.4

0.825

96.0

Positive Control Item

0.034

0.028

0.007

4.0

3.3

0.8

0.021

2.4

0.028

3.3

Test Item

0.067

0.059

0.008

7.8

6.9

0.9

0.058

6.8

0.052

6.1

 

Quality criteria

 

The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control treated tissues and the standard deviation value of the viability was 0.8%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.859 and the standard deviation value of the viability was 10.2%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 0.9%. The test item acceptance criterion was therefore satisfied.


OD= Optical Density.

SD=        Standard deviation.

*=         The mean viability of the negative control tissues is set at 100%.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The relative mean viability of the test item treated tissues was 6.9% after the 15 minute exposure period and 42 hours post exposure incubation period. Under the conditions of the study, the test item demonstrated the ability to cause a positive response.
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of CGE-PMDA adduct using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96-well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 6.9% after the 15-minute exposure period and 42-hours post-exposure incubation period.

The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of the study, the test item demonstrated the ability to cause a positive response.

Endpoint:
skin irritation / corrosion, other
Remarks:
acute dermal toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 May 2018 - 24 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Envigo RMS (UK) Limited, Oxon, UK
- Females nulliparous and non-pregnant: yes
- Weight at study initiation:
209 g
- Fasting period before study:
- Housing:
individually throughout the study in suspended solid floor polypropylene cages furnished with wood flakes
- Diet (e.g. ad libitum):
2014C Teklad Global Rodent diet , ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period:
least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light):
12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1000 mg/kg bw, corresponding to 0.96 mL/kg bw
- Concentration (if solution): neat substance

Duration of treatment / exposure:
24 h
Observation period:
1 day after dosing
Number of animals:
1
Details on study design:
The animal was observed for deaths or overt signs of toxicity 30 minutes, 1, 2, 4 and 6 hours after dosing and on Day 1. After removal of the dressings the test sites were examined for evidence of primary irritation.
The animal was humanely killed by cervical dislocation on Day 1. The animal was subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities and examination of the sub-cutaneous layer below the treatment site. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

OBSERVATION TIME POINTS
(indicate if minutes, hours or days)
Skin reactions were observed after 24 h

SCORING SYSTEM:
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4

Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and extending beyond the area of exposure) 4
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24 h
Score:
4
Max. score:
4
Reversibility:
not reversible
Remarks:
Full thickness necrosis and hemorrhage of the underlining tissues were noted at the visual inspection of the sub-cutaneous layer at the treatment site.
Remarks on result:
other: The animal was killed for humane reasons due to due to the corrosive nature of the test item after 1 d.
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24 h
Score:
3
Max. score:
4
Reversibility:
not reversible
Remarks:
Full thickness necrosis and hemorrhage of the underlining tissues were noted at the visual inspection of the sub-cutaneous layer at the treatment site.
Remarks on result:
other: The animal was killed for humane reasons due to due to the corrosive nature of the test item after 1 d.
Irritant / corrosive response data:
Dark green dermal necrosis over the majority of the treatment site was surrounded by a margin of blanching and moderate erythema. Full thickness necrosis and hemorrhage of the underlining tissues were noted at the visual inspection of the sub-cutaneous layer at the treatment site.
Other effects:
The animal was killed for humane reasons due to due to the corrosive nature of the test item in accordance with the severity limit set forth in the UK Home Office Project License.
Clinical signs noted one day after dosing were hunched posture, lethargy and tiptoe gait. It was considered that these signs may have been due to the dermal reactions causing uncomfortable movement as opposed to systemic toxicity.
The animal lost weight over the study period.
Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
In this study, the test item CGE-PMDA adduct was found to be corrosive.
Executive summary:

This study was performed to assess the acute dermal toxicity of CGE-PMDA adduct in the Wistar strain rat in accordance with OECD Guideline 402 (2017).

One female animal was given a single, 24-hour, semi‑occluded dermal application of the undiluted test item to intact skin at a dose level of 1000 mg/kg body weight. Based on the results of the initial animal, no further animals were treated. Clinical signs and body weight development were monitored during the study. The animal was subjected to gross necropsy.

The animal was killed for humane reasons due to due to the corrosive nature of the test item in accordance with the severity limit set forth in the UK Home Office Project License.

Clinical signs noted one day after dosing were hunched posture, lethargy and tiptoe gait.

Dark green dermal necrosis over the majority of the treatment site surrounded by a margin of blanching and moderate erythema was noted one day after dosing.

The animal lost weight over the study period.

Full thickness necrosis and hemorrhage of the underlining tissues were noted at the visual inspection of the sub-cutaneous layer at the treatment site. 

In this study, the test item CGE-PMDA adduct was found to be corrosive.

The acute dermal median lethal dose (LD50) of the test item could not be accurately evaluated.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 January 2018
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v

POSITIVE CONTROL USED
Ethanol

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others (e.g, pertinent visual observations, histopathology): No histopathology was required since a definitive result was achieved.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA:
IVIS UN GHS EU CLP
≤ 3 No Category Not classified for irritation
>3; ≤ 55 No prediction can be made No prediction can be made
> 55 Category 1 Category 1
H318: Causes serious eye damage
Irritation parameter:
in vitro irritation score
Value:
129.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
IVIS: 0.7
Positive controls validity:
valid
Remarks:
IVIS: 35.5
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In this study CGE-PMDA adduct was identified as Category 1 Causes serious eye damage.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short‑term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

 

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

129.1

Negative Control

0.7

Positive Control

35.5

 

CGE-PMDA adduct is therefore classified as Category 1 Causes serious eye damage.

 

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion/irritation

In a skin corrosion study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion) (adopted July 29, 2016), CGE-PMDA adduct (100%, 33% and 10% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 and 60 minutes, respectively, in triplicates.

Each approximately 50 µL of the test item were applied to the tissues. After 3 or 60 minutes exposure at room temperature (3 min) or 37°C (60 min exposure time), the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 3 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (8.0 N KOH) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after treatment with neat CGE-PMDA adduct compared to the negative control tissues was 59.7% for the 3 minute exposure period and 31.1% for the 60 minute exposure period. The relative mean tissue viability obtained after treatment with 33% CGE-PMDA adduct compared to the negative control tissues was 75.3% for the 3 minute exposure period and 15.8% for the 60 minute exposure period. The relative mean tissue viability obtained after treatment with 10% CGE-PMDA adduct compared to the negative control tissues was 94.4% for the 3 minute exposure period and 39.2% for the 60 minute exposure period.

Since the mean relative tissue viability for the neat test substance and both dilutions was ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure, the prediction to be considered according to EU CLP Regulation (EC) No 1272/2008 is non-corrosive.

 

A skin irritation study was performed in accordance with OECD Guideline 439. The purpose of this test was to evaluate the skin irritation potential of CGE-PMDA adduct using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a postexposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the postexposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to prelabeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTTloaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a prelabeled 96-well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 6.9% after the 15-minute exposure period and 42-hours post-exposure incubation period.

The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of the study, the test item demonstrated the ability to cause a positive response.

A study was performed to assess the acute dermal toxicity of CGE-PMDA adduct in the Wistar strain rat in accordance to OECD Guideline 402 (2017).

One female animal was given a single, 24-hour, semi‑occluded dermal application of the undiluted test item to intact skin at a dose level of 1000 mg/kg body weight. Based on the results of the initial animal, no further animals were treated. Clinical signs and body weight development were monitored during the study. The animal was subjected to gross necropsy.

The animal was killed for humane reasons due to due to the corrosive nature of the test item in accordance with the severity limit set forth in the UK Home Office Project License.

Clinical signs noted one day after dosing were hunched posture, lethargy and tiptoe gait.

Dark green dermal necrosis over the majority of the treatment site surrounded by a margin of blanching and moderate erythema was noted one day after dosing.

The animal lost weight over the study period.

Full thickness necrosis and hemorrhage of the underlining tissues were noted at the visual inspection of the sub-cutaneous layer at the treatment site. 

In this study, the test item CGE-PMDA adduct was found to be corrosive.

 

Overall, CGE-PMDA adduct is classified as Category 1 (corrosive) based on GHS criteria.

 

Eye irritation

An eye irritation study was performed according to OECD Guideline 437. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides shortterm maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

The IVIS of the test item was 129.1 in this test. Therefore, CGE-PMDA adduct is classified as Category 1 (Causes serious eye damage) based on GHS criteria.

 

Justification for classification or non-classification

Based on available and relevant data, the test substance is classified for skin irritation/corrosion according to regulation (EC) 1272/2008 as Category 1 and should be labeled with H314: Causes severe skin burns and eye damage.