Reaction mass of 29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper and Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, (dimethylamino)methyl derivs.

Endpoint summary

Description of key information

Skin Corrosion:

The mean viability of 2 discs exposed to the test item is 110.6%.

Skin Irritation:

The mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is 95.8% of the mean viability of the negative controls.

Eye Irritation in vitro:

Mean maximum cornea opacity score: 0.67

Mean fluorescein retention score: 1.0

Mean maximum of corneal swelling percentage: 0.5 after 75 min and 1.1 after 240 min.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 July 2016 - 15 July 2016

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:

The purity reported is different to the Study Plan due to a new Certificate of Analysis being provided by the sponsor.

Deviations:

yes

Remarks:

this fact has no impact on the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Version / remarks:

The purity reported is different to the Study Plan due to a new Certificate of Analysis being provided by the sponsor.

Deviations:

yes

Remarks:

this fact has no impact on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: 150205
Description: Blue powder
Manufacture date: 05 February 2015
Expiry date: 05 February 2019
Purity: As a UVCB, purity is considered as 100%
Storage conditions: Controlled room temperature (15-25°C, below 70% RH%).

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: main basal, supra basal, spinous and granular layers and a functional stratum corneum

Source strain:

not specified

Details on animal used as source of test system:

Manufactured tissues, reconstructed epidermis

Justification for test system used:

The model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

physiological saline

Details on test system:

SKIN DISC PREPARATION
- Procedure used: supplied by manufacturer
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25.2-26.4°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: no
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: yes
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if both disks have mean viability of <35% and the variability is less than 50%
- The test substance is considered to be non-corrosive to skin if both disks have mean viability of ≥35% and the variability is less than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

- 20 mg of test item was applied evenly to the epidermal surface of each of two test units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

4 hours (±10 min)

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

108.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

112.5

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:

After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions in each case.
The mean OD value of the two negative control tissues was in the recommended range (0.834).
The positive control treated tissues showed 1.9% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 3.3%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with the test item, the mean cell viability was 110.6% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro model test with the test item, the results indicate that the test item is non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 October 2016 - 07 October 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: 150205
Description: Blue powder
Expiry date: 05 February 2019
Purity: As a UVCB, purity is considered as 100%
Storage conditions: Controlled room temperature (15-25°C, below 70% RH%)

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Cell source:

other: main basal, supra basal, spinous and granular layers and a functional stratum corneum

Source strain:

not specified

Justification for test system used:

The model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: provided by manufacturer
- Quality control for skin discs: The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C, the kits were found to be in good order at reception.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: (23.3-24.8°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: none
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

test item: 10 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three replicates were used for the test item.
Three negative controls and three positive control.
Two additional test item-treated killed epidermis.
Two negative control treated killed epidermis.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

95.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean OD value of the three negative control tissues was in the recommended range (0.764). Standard deviation of the viability results for negative control samples was 6.8.
The positive control treated tissues showed 5.3% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.2.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.6.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with the tesi item, the mean cell viability was 95.8% (after adjustment for non-specific MTT reduction) compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.
The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKINTM(SM) model test with the test substance (Batch number: 150205), the results indicate that the test item is non-irritant to skin (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

11 April 2016 - 11 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

Due to unscheduled delay of reporting (The Study was on hold for Sponsor’s request),
the Draft Report was issued later than indicated in the Study Plan. The purity reported is different to the Study Plan due to a new Certificate of Analysis being provided by the
sponsor.

Deviations:

yes

Remarks:

has no impact on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: 150205
Description: Blue powder
Expiry date: 05 February 2019
Purity: As a UVCB, purity is considered as 100%
Storage conditions: Controlled room temperature (15-25°C, below 70% RH%).

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: commercial abattoir
- Age at study initiation: approximately 7 weeks old
- Acclimatisation: approximately 45 to 60 minutes.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

30, 75, 120, 180 and 240 minutes after the post-treatment rinse

Duration of post- treatment incubation (in vitro):

Observation: 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
If the cornea was in good condition, the eyeball was carefully removed from the orbit.

EQUILIBRATION AND BASELINE RECORDINGS
The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time.

NEGATIVE CONTROL USED: Physiological saline solution (0.9% (w/v) NaCl)

POSITIVE CONTROL USED: Imidazole

OBSERVATION PERIOD: 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL in physiological saline at ambient temperature. Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes
- Damage to epithelium based on fluorescein retention:yes
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: yes

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: based on the OECD guideline quantitative assessments

Irritation parameter:

cornea opacity score

Remarks:

mean maximum

Run / experiment:

1

Value:

0.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Remarks:

mean retention

Run / experiment:

1

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Remarks:

mean maximum up to 75 min

Run / experiment:

1

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum up to 240 min

Run / experiment:

1

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Test item was stuck on all cornea surfaces during the full the post-treatment period and could not be rinsed off. All three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. The particles stuck to the cornea could mask corneal effects underneath the particles and could potentially result in mechanical damage in vivo.
The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test, the test item is not classified as a severe irritant and not classified as non-irritant.
It is concluded that an in vivo study is required for proper classification.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor: 150205
- Expiration date of the lot/batch: 05 February 2019
- Purity: As a UVCB, purity is considered as 100%
- Description: Blue powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 °C, below 70% RH)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S&K-LAP Kft. , 2173 Kartal, Császár út 135, Hungary
- Age at study initiation: 13-14 weeks old
- Weight at study initiation: 3431 g – 3898 g
- Housing: individually housed in AAALAC approved metal wire rabbit cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 23.5 °C
- Humidity (%): 37 – 80%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g of the test substance

Duration of treatment / exposure:

As the solid test item remained in the eye sac in all animals at the one hour observation time point, the treated eye of test animals was rinsed with physiological saline solution.

Observation period (in vivo):

1, 24, 48 and 72 hours after the treatment

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes one hour after the treatment with Physiological saline (0.9% NaCl)

SCORING SYSTEM: Draize

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

Animal 1 (No: 4453) clinical observation:
At one hour after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.
At 24 and 48 hours after the application, conjunctival redness (score 1) and discharge (score 1) were noted in the rabbit.
At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 2 (No: 4467) clinical observation:
At one hour after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.
At 24 and 48 hours after the application, conjunctival redness (score 1) and discharge (score 1) were noted in the rabbit.
At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 3 (No: 4455) clinical observation
At one hour and 24 hours after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 2) were noted in the rabbit. Test item remnant was seen in the
eye sac at one hour. At 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

The general state and behaviour of animals were normal throughout the study period.

No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

The test item B440, applied to rabbit eye mucosa, caused conjunctival effects at 1, 24 and 48 hours after application which were fully reversible within 72 hours.

The body weight of the animals was considered to be within the normal range of variability.

There was no mortality observed during the study.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item B440, applied to rabbit eye mucosa, caused conjunctival effects at 1, 24 and 48 hours after application which were fully reversible within 72 hours.
According to Regulation (EC) No 1272/2008, B440 does not require classification as an eye irritant.

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, B440 does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Skin Corrosion:

If both disks have mean viability of ≥35% and the variability is less than 50%, the test item is considered as Non Corrosive.

Skin Irritation:

The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), as the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.

Eye Irritation in vitro:

Based on the endpoints not classifiable for No Category and for Category 1.

Particles of test item were stuck to the cornea and could not be washed off during the study.

Eye Irritation in vivo:

The test item B440, applied to rabbit eye mucosa, caused conjunctival effects at 1, 24 and 48 hours after application which were fully reversible within 72 hours. The test item therefore did not meet the classification criteria.