Registration Dossier

Administrative data

Description of key information

Based on the results of the read across studies, the test substance, 'iso and anteiso C10-40 AAP EDM-ES', is considered to be non-irritating to skin and irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 23, 2017 to September 04, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 29 August 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-035
Maintenance Medium lot number : 17-MAIN3-037
Assay Medium lot number : 17-ESSC-034
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26.3 μL/cm2)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
37ºC, 5% CO2 in air for 42 h
Number of replicates:
3 (Three)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test substance
Value:
ca. 113.6
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:
The relative mean tissue viability for the positive control treated tissues was 6.6% relative to the negative control treated tissues and the standard deviation value of the viability was 2.9%. The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.807 and the standard deviation value of the viability was 2.5%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test substance treated tissues was 1.4%. The test substance acceptance criterion was therefore satisfied.

Results

 

Direct MTT reduction

The MTT solution containing the test substance did not turn blue or purple which indicated that the test substance did not directly reduce MTT.

 

Assessment of color interference with the MTT endpoint

The solution containing the test substance was a very pale green color. This color was attributed to the intrinsic color of the test substance itself. It was therefore unnecessary to run color correction tissues.

 

Test substance, positive control substance and negative control substance

The individual and mean OD570values, standard deviations and tissue viabilities for the test substance, negative control substance and positive control substance are given in table 1. The mean viabilities and standard deviations of the test substance and positive control, relative to the negative control are also given in table 1.

 

Table 1: Mean OD570 values and viabilities for the negative control substance, positive control substance and test substance

Substance

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 Substance

0.794

0.807

0.020

98.4

100

2.5

0.830

102.9

0.797

98.8

Positive Control Substance

0.078

0.053

0.024

9.7

6.6

2.9

0.031

3.8

0.051

6.3

Test Substance

0.928

0.917

0.011

115.0

113.6

1.4

0.906

112.3

0.916

113.5

The relative mean viability of the test substance treated tissues was113.6% after a 15‑Minute exposure period and 42‑h post‑exposure incubation period. It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

Conclusion

The test substance was classified as non-irritant. The following classification criteria apply:

EU CLP not classified for irritation.

UN GHS not classified for irritation (category 3 cannot be determined).

Interpretation of results:
other: Not classified based on EU CLP Criteria
Conclusions:
Based on the results of the read across study, the test substance, iso and anteiso C10-40 AAP EDM-ES, can be considered to be non-irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), using EPISKIN TM reconstructed human epidermis model, according to the OECD Guideline 439 and EU Method B46, in compliance with GLP. Triplicate tissues were treated with the test substance (undiluted) for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. 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. Results were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). The relative mean viability of the test substance treated tissues was determined to be 113.6% after the 15 minute exposure period and 42 h post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-irritant to the skin (Envigo, 2017). Based on the results of the read across study, the test substance, 'iso and anteiso C10-40 AAP EDM-ES' can be considered to be non-irritating to the skin.

.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 14, 2017 to August 25, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 22 August 2017
EpiDermTM Tissues (0.63cm2) lot number: 25838
Assay Medium lot number: D81717TMC
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL undiluted test substance
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
37°C, 5% CO2 for 3 h
Number of replicates:
Two tissue replicates for each treatment group
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 3 minutes
Run / experiment:
Test substance
Value:
ca. 86.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 60 minutes
Run / experiment:
Test substance
Value:
ca. 38.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 3 and 60 minutes
Run / experiment:
Negative control
Value:
ca. 100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 3 minutes
Run / experiment:
Positive control
Value:
ca. 3.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control
Value:
ca. 3.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Corrosive

Results

Direct MTT Reduction

An assessment found the test substance was able to directly reduce MTT. Therefore, an additional procedure using freeze killed tissues was performed. The results of the freeze killed tissues were subtracted from the mean OD of the test substance treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

 

Assessment of Color Interference with the MTT endpoint

The solution containing the test substance did not become colored. This was taken to indicate the test substance did not have the potential to cause color interference.

Test Substance, Positive Control Substance and Negative Control Substance

 

Mean OD570values and viabilities for the negative control, positive control and test substance

Tissue

Exposure Period

Mean OD570of individual tissues (tvt)

Mean OD570of duplicate tissues

Corrected OD570(tvt- (tkt – ukt))

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.566

1.497

-

0.098

6.5

100*

1.428

60 Minutes

1.545

1.633

-

0.124

7.6

1.720

Positive Control

3 Minutes

0.062

0.059

-

0.004

na

3.9

0.056

60 Minutes

0.058

0.058

-

0.001

na

3.5

0.057

Test Substance

3 Minutes

1.268

1.319

1.297

0.071

5.4

86.6

1.369

60 Minutes

0.641

0.675

0.625

0.048

7.1

38.3

0.709

tvt = Treated viable tissue

tkt = Treated killed tissue

ukt = Untreated killed tissue

na = Not applicable

 

3 minute exposure corrected mean OD570=        0.139   (tkt) -  0.117   (ukt) = 0.022

60 minute exposure corrected mean OD570=      0.148   (tkt) -  0.098   (ukt) = 0.050

 

Relative mean viability (%) = mean OD570 of test substance / mean of OD570 of negative control × 100

Coefficient of variation = standard deviation / mean OD570 of duplicate tissues × 100

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Substance

3 minute

100*

3.9

86.6

60 minute

100*

3.5

38.3

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

 

Quality Criteria

The initial test was repeated due to a failure to meet the assay acceptance criteria. The mean OD570 for the negative control treated tissues was 1.497 for the 3‑Minute exposure period and 1.633 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.5% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

 

Conclusion

The test substance was considered to be non-corrosive to the skin.

Interpretation of results:
other: non corrosive based on EU CLP criteria
Conclusions:
Based on the results of the read across study, the test substance, iso and anteiso C10-40 AAP EDM-ES, can also be considered to be non-corrosive to skin.
Executive summary:

An in vitro study was conducted to determine the corrosivity potential of the read across substance, 'C18-unsatd. and C22-unsatd. AAP EDM-ES' (active: 104%), using the EpiDerm™ Human Skin Model, according to the OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. Duplicate tissues were treated with the 50 µL undiluted test substance and reference substances for exposure periods of 3 and 60 minutes. The test substance 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 exposure period the test substance was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570). Data were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). Percentage viability for the test substance at 3 and 60 minutes were 86.6% and 38.3% respectively. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-corrosive to the skin (Envigo, 2017). Based on the results of the read across study, the test substance, 'iso and anteiso C10-40 AAP EDM-ES', can also be considered to be non-corrosive to skin.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 14, 2017 to August 25, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 22 August 2017
EpiDermTM Tissues (0.63cm2) lot number: 25838
Assay Medium lot number: D81717TMC
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL undiluted test substance
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
37°C, 5% CO2 for 3 h
Number of replicates:
Two tissue replicates for each treatment group
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 3 minutes
Run / experiment:
Test substance
Value:
ca. 86.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 60 minutes
Run / experiment:
Test substance
Value:
ca. 38.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 3 and 60 minutes
Run / experiment:
Negative control
Value:
ca. 100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
Exposure: 3 minutes
Run / experiment:
Positive control
Value:
ca. 3.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control
Value:
ca. 3.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Corrosive

Results

Direct MTT Reduction

An assessment found the test substance was able to directly reduce MTT. Therefore, an additional procedure using freeze killed tissues was performed. The results of the freeze killed tissues were subtracted from the mean OD of the test substance treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

 

Assessment of Color Interference with the MTT endpoint

The solution containing the test substance did not become colored. This was taken to indicate the test substance did not have the potential to cause color interference.

Test Substance, Positive Control Substance and Negative Control Substance

 

Mean OD570values and viabilities for the negative control, positive control and test substance

Tissue

Exposure Period

Mean OD570of individual tissues (tvt)

Mean OD570of duplicate tissues

Corrected OD570(tvt- (tkt – ukt))

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.566

1.497

-

0.098

6.5

100*

1.428

60 Minutes

1.545

1.633

-

0.124

7.6

1.720

Positive Control

3 Minutes

0.062

0.059

-

0.004

na

3.9

0.056

60 Minutes

0.058

0.058

-

0.001

na

3.5

0.057

Test Substance

3 Minutes

1.268

1.319

1.297

0.071

5.4

86.6

1.369

60 Minutes

0.641

0.675

0.625

0.048

7.1

38.3

0.709

tvt = Treated viable tissue

tkt = Treated killed tissue

ukt = Untreated killed tissue

na = Not applicable

 

3 minute exposure corrected mean OD570=        0.139   (tkt) -  0.117   (ukt) = 0.022

60 minute exposure corrected mean OD570=      0.148   (tkt) -  0.098   (ukt) = 0.050

 

Relative mean viability (%) = mean OD570 of test substance / mean of OD570 of negative control × 100

Coefficient of variation = standard deviation / mean OD570 of duplicate tissues × 100

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Substance

3 minute

100*

3.9

86.6

60 minute

100*

3.5

38.3

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

 

Quality Criteria

The initial test was repeated due to a failure to meet the assay acceptance criteria. The mean OD570 for the negative control treated tissues was 1.497 for the 3‑Minute exposure period and 1.633 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.5% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

 

Conclusion

The test substance was considered to be non-corrosive to the skin.

Interpretation of results:
other: non corrosive based on EU CLP criteria
Conclusions:
Under study conditions, the test substance was considered to be non-corrosive to the skin.
Executive summary:

An in vitro study was conducted to determine the corrosivity potential of the test substance, 'C18-unsatd. and C22-unsatd. AAP EDM-ES' (active: 104%), using the EpiDerm™ Human Skin Model, according to the OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. Duplicate tissues were treated with the 50 µL undiluted test substance and reference substances for exposure periods of 3 and 60 minutes. The test substance 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 exposure period the test substance was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570). Data were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). Percentage viability for the test substance at 3 and 60 minutes were 86.6% and 38.3% respectively. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-corrosive to the skin (Envigo, 2017).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 23, 2017 to September 04, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 29 August 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-035
Maintenance Medium lot number : 17-MAIN3-037
Assay Medium lot number : 17-ESSC-034
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26.3 μL/cm2)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
37ºC, 5% CO2 in air for 42 h
Number of replicates:
3 (Three)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test substance
Value:
ca. 113.6
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:
The relative mean tissue viability for the positive control treated tissues was 6.6% relative to the negative control treated tissues and the standard deviation value of the viability was 2.9%. The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.807 and the standard deviation value of the viability was 2.5%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test substance treated tissues was 1.4%. The test substance acceptance criterion was therefore satisfied.

Results

 

Direct MTT reduction

The MTT solution containing the test substance did not turn blue or purple which indicated that the test substance did not directly reduce MTT.

 

Assessment of color interference with the MTT endpoint

The solution containing the test substance was a very pale green color. This color was attributed to the intrinsic color of the test substance itself. It was therefore unnecessary to run color correction tissues.

 

Test substance, positive control substance and negative control substance

The individual and mean OD570values, standard deviations and tissue viabilities for the test substance, negative control substance and positive control substance are given in table 1. The mean viabilities and standard deviations of the test substance and positive control, relative to the negative control are also given in table 1.

 

Table 1: Mean OD570 values and viabilities for the negative control substance, positive control substance and test substance

Substance

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 Substance

0.794

0.807

0.020

98.4

100

2.5

0.830

102.9

0.797

98.8

Positive Control Substance

0.078

0.053

0.024

9.7

6.6

2.9

0.031

3.8

0.051

6.3

Test Substance

0.928

0.917

0.011

115.0

113.6

1.4

0.906

112.3

0.916

113.5

The relative mean viability of the test substance treated tissues was113.6% after a 15‑Minute exposure period and 42‑h post‑exposure incubation period. It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

Conclusion

The test substance was classified as non-irritant. The following classification criteria apply:

EU CLP not classified for irritation.

UN GHS not classified for irritation (category 3 cannot be determined).

Interpretation of results:
other: Not classified based on EU CLP Criteria
Conclusions:
Under study conditions, the test substance was considered to be non-irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), using EPISKIN TM reconstructed human epidermis model, according to the OECD Guideline 439 and EU Method B46, in compliance with GLP. Triplicate tissues were treated with the test substance (undiluted) for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. 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. Results were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). The relative mean viability of the test substance treated tissues was determined to be 113.6% after the 15 minute exposure period and 42 h post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-irritating to the skin (Envigo, 2017).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
August 31, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
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:
not specified
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source of Bovine 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 prepared immediately on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
32 ± 1 ºC for 120 minutes
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
ca. 29.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction can be made
Other effects / acceptance of results:
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.

Results

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in below table:

Treatment

Cornea number

Opacity

Permeability (Optical density)

In Vitro Irritancy Score

Pre-treatment

Post-treatment

Post incubation

Post-incubation - Pre‑treatment

Corrected value

 

Corrected value

Negative control#

1

3

3

4

1

 

0.007

 

 

2

3

2

2

0

 

0.011

 

 

3

4

2

2

0

 

0.007

 

 

 

 

 

 

0.3*

 

0.008¨

 

0.5

Positive control#

4

6

40

37

31

30.7

0.794

0.786

 

5

3

31

33

30

29.7

1.960

1.952

 

6

2

24

26

24

23.7

1.885

1.877

 

 

 

 

 

 

28.0·

 

1.538·

51.1

Test substance

13

1

6

21

20

19.7

0.747

0.739

 

14

3

6

22

19

18.7

0.824

0.816

 

15

1

6

21

20

19.7

0.539

0.531

 

 

 

 

 

 

19.3·

 

0.695·

29.8

* = Mean of the post-incubation-pre‑treatment values                           

# = Control data shared with Envigo - Shardlow study number PS75FR

Corneal Epithelium Condition

The condition of each cornea is given in below table:

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control#

1

Clear

Clear

2

Clear

Clear

3

Clear

Clear

Positive Control#

4

Cloudy

Cloudy

5

Cloudy

Cloudy

6

Cloudy

Cloudy

Test Substance

13

Clear

Cloudy

14

Clear

Cloudy

15

Clear

Cloudy

# = Control data shared with Envigo - Shardlow study number PS75FR

The corneas treated with the test substance were clear post treatment and cloudy post incubation. The corneas treated with the negative control substance were clear post treatment and post incubation. The corneas treated with the positive control substance were cloudy post treatment and post incubation.

 

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Substance

29.8

Negative Control

0.5

Positive Control

51.1

 

Criteria for an Acceptable Test

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.

 

Conclusion

Based on the study results, the study author concluded that no prediction of eye irritation could be made.

Interpretation of results:
other: No prediction can be made based on EU CLP criteria
Conclusions:
Based on the results of the read across BCOP study, no prediction of eye irritation potential could be made for the test substance, 'iso and anteiso C10-40 AAP EDM-ES'.

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the the read across substance, C18-unsatd and C22-unsatd. AAP EDM-ES (active: 104%), using Bovine Corneal Opacity and Permeability (BCOP) method, according to the OECD Guideline 437 and EU B.47 Method, in compliance with GLP. The undiluted test substance and reference substances were applied to the test system for 10 minutes followed by an incubation period of 120 minutes. 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 substance was determined to be 29.8 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3; therefore no predictions could be made. Therefore based on EU CLP criteria, no prediction could be made about eye irrtation potential of the test substance. The IVIS of the positive control was within the range of 31.6 to 58.7, therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore the negative control acceptance criteria were satisfied. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, no prediction could be made about eye irritation potential of the test substance (Envigo, 2017). Based on the results of the read across BCOP study, no prediction of eye irritation potential could be made for the test substance, 'iso and anteiso C10-40 AAP EDM-ES'.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003
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:
other: HET-CAM test
Version / remarks:
modification of that described by Kemper and Luepke (1986).
GLP compliance:
yes
Species:
chicken
Details on test animals or tissues and environmental conditions:
- Chick embryo: chorioallantoic membrane (CAM)
- Fresh, fertile, White Leghor, eggs obtained from Avian Services in Frenchtown, New Jersey
- Acclimation: 7 days at 13°C before incubation
- Incubation: ca. 37°C with a relative humidity of 60 - 70% for 10 days (in a Kuhl incubator)
Vehicle:
water
Remarks:
10% test substance in water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
Test substance: 0.3 mL if liquid or 0.3 g if solid
Different concentrations: 0, 1, 5 and 10%
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
Up to 5 minutes
Duration of post- treatment incubation (in vitro):
-
Number of animals or in vitro replicates:
4
Details on study design:
After washing with physiological saline, membranes were observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects.
Irritation parameter:
other:
Run / experiment:
mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis
Value:
>= 0.25 - <= 2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: irritation potential: practically none
Other effects / acceptance of results:
CAM were exposed to the test substance at concentrations of 0, 1, 5 and 10% and the mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were 1.75, 2.00, 0.25 and 2.00, respectively. The test substance was considered to have almost no irritation potential to CAM.
Conclusions:
Under the study conditions, the test substance, 'iso and anteiso C10-40 AAP EDM-ES', was considered to have almost no irritation potential to eye (HET-CAM).

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'iso and anteiso C10-40 AAP EDM-ES', according to the HET-CAM test, in compliance with GLP. Chorioallantoic membrane (CAM) of the chick embryo were exposed for 20 seconds to 0.3 mL or 0.3 g test substance at concentrations of 0 (vehicle only: distilled water), 1, 5 and 10%. After washing with physiological saline, membranes were observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects. Mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were recorded and gave values of 1.75, 2.00, 0.25 and 2.00 for the concentrations 0, 1, 5 and 10%, respectively. Under the study conditions, the test substance, 'iso and anteiso C10-40 AAP EDM-ES' was considered to have almost no irritation potential to eye at 10% concentration (Nitka, 2003).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 31, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
not specified
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source of Bovine 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 prepared immediately on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
32 ± 1 ºC for 120 minutes
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
ca. 29.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction can be made
Other effects / acceptance of results:
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.

Results

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in below table:

Treatment

Cornea number

Opacity

Permeability (Optical density)

In Vitro Irritancy Score

Pre-treatment

Post-treatment

Post incubation

Post-incubation - Pre‑treatment

Corrected value

 

Corrected value

Negative control#

1

3

3

4

1

 

0.007

 

 

2

3

2

2

0

 

0.011

 

 

3

4

2

2

0

 

0.007

 

 

 

 

 

 

0.3*

 

0.008¨

 

0.5

Positive control#

4

6

40

37

31

30.7

0.794

0.786

 

5

3

31

33

30

29.7

1.960

1.952

 

6

2

24

26

24

23.7

1.885

1.877

 

 

 

 

 

 

28.0·

 

1.538·

51.1

Test substance

13

1

6

21

20

19.7

0.747

0.739

 

14

3

6

22

19

18.7

0.824

0.816

 

15

1

6

21

20

19.7

0.539

0.531

 

 

 

 

 

 

19.3·

 

0.695·

29.8

* = Mean of the post-incubation-pre‑treatment values                           

# = Control data shared with Envigo - Shardlow study number PS75FR

Corneal Epithelium Condition

The condition of each cornea is given in below table:

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control#

1

Clear

Clear

2

Clear

Clear

3

Clear

Clear

Positive Control#

4

Cloudy

Cloudy

5

Cloudy

Cloudy

6

Cloudy

Cloudy

Test Substance

13

Clear

Cloudy

14

Clear

Cloudy

15

Clear

Cloudy

# = Control data shared with Envigo - Shardlow study number PS75FR

The corneas treated with the test substance were clear post treatment and cloudy post incubation. The corneas treated with the negative control substance were clear post treatment and post incubation. The corneas treated with the positive control substance were cloudy post treatment and post incubation.

 

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Substance

29.8

Negative Control

0.5

Positive Control

51.1

 

Criteria for an Acceptable Test

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.

 

Conclusion

Based on the study results, the study author concluded that no prediction of eye irritation could be made.

Interpretation of results:
other: No prediction can be made based on EU CLP criteria
Conclusions:
Under study conditions, no prediction could be made about eye irritation potential of the test substance.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), using Bovine Corneal Opacity and Permeability (BCOP) method, according to the OECD Guideline 437 and EU B.47 Method, in compliance with GLP. The undiluted test substance and reference substances were applied to the test system for 10 minutes followed by an incubation period of 120 minutes. 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 substance was determined to be 29.8 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3; therefore no predictions could be made. Therefore based on EU CLP criteria, no prediction could be made about eye irrtation potential of the test substance. The IVIS of the positive control was within the range of 31.6 to 58.7, therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore the negative control acceptance criteria were satisfied. The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, no prediction could be made about eye irritation potential of the test substance (Envigo, 2017).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

Study 1: An in vitro study was conducted to determine the corrosivity potential of the read across substance, 'C18-unsatd. and C22-unsatd. AAP EDM-ES' (active: 104%), using the EpiDerm™ Human Skin Model, according to the OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. Duplicate tissues were treated with the 50 µL undiluted test substance and reference substances for exposure periods of 3 and 60 minutes. The test substance 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 exposure period the test substance was rinsed from each tissue before each tissue was taken for MTTloading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96well plate. The optical density (OD) was measured at 570 nm (OD570). Data were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). Percentage viability for the test substance at 3 and 60 minutes were 86.6% and 38.3% respectively. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-corrosive to the skin (Envigo, 2017). Based on the results of the read across study, the test substance, iso and antiso C10-40 AAP EDM-ES, can also be considered to be non-corrosive to skin.

Study 2: An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), using EPISKIN TM reconstructed human epidermis model, according to the OECD Guideline 439 and EU Method B46, in compliance with GLP. Triplicate tissues were treated with the test substance (undiluted) for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. 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. Results were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). The relative mean viability of the test substance treated tissues was determined to be 113.6% after the 15 minute exposure period and 42 h post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-irritant to the skin (Envigo, 2017). Based on the results of the read across study, the test substance, iso and anteiso C10-40 AAP EDM-ES can be considered to be non-irritating to the skin.

Eye:

Study 1: An in vitro study was conducted to determine the eye irritation potential of the the read across substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), using Bovine Corneal Opacity and Permeability (BCOP) method, according to the OECD Guideline 437 and EU B.47 Method, in compliance with GLP. The undiluted test substance and reference substances were applied to the test system for 10 minutes followed by an incubation period of 120 minutes. 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 substance was determined to be 29.8 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3; therefore no predictions could be made. Therefore based on EU CLP criteria, no prediction could be made about eye irrtation potential of the test substance. The IVIS of the positive control was within the range of 31.6 to 58.7, therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore the negative control acceptance criteria were satisfied. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, no prediction could be made about eye irritation potential of the test substance (Envigo, 2017). Based on the results of the read across BCOP study, no prediction of eye irritation potential could be made for the test substance, 'iso and anteiso C10-40 AAP EDM-ES'.

Study 2: A study was conducted to determine the in vitro eye irritation potential of the test substance, 'iso and anteiso C10-40 AAP EDM-ES', according to the HET-CAM test, in compliance with GLP. Chorioallantoic membrane (CAM) were exposed for 20 seconds to 0.3 mL or 0.3 g test substance at concentrations of 0 (vehicle only: distilled water), 1, 5 and 10%. After washing with physiological saline, membranes were observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects. Mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were recorded and gave values of 1.75, 2.00, 0.25 and 2.00 for the concentrations 0, 1, 5 and 10%, respectively. Under the study conditions, the test substance was considered to have almost no irritation potential to eye (Nitka, 2003).

Based on the in vitro BCOP study with the read across substance, no clear conclusion could be drawn as per the Guidelines. However, given the IVIS score (i.e., 29.8) from the read across study, which is in between the threshold for corrosive (i.e., >55) and non-corrosive limits (i.e., <=3), together with absence of skin irritation potential (which was assessed based on the in vitro skin irritation/corrosion read across studies), indicate that a corrosive potential can be ruled out and the test substance, ‘iso and anteiso C10-40 AAP EDM-ES' can be considered to be more likely to be irritating to the eyes (in a worst case).

Justification for classification or non-classification

Skin:

Based on the results of read across in vitro skin irritation studies, the test substance, 'iso and anteiso C10-40 AAP EDM-ES', does not warrant a classification for skin irritation according to the EU CLP criteria (Regulation 1272/2008/EC).

Eye:

Based on the results of read across in vitro skin and eye irritation studies, the test substance, 'iso and anteiso C10-40 AAP EDM-ES', is considered to warrant an 'Eye Irrit. 2-H319: Causes serious eye irritation' classification according to the EU CLP criteria (Regulation 1272/2008/EC).