(4E)-4-(3,3,4-trimethylcyclopentylidene)butanal

Endpoint summary

Description of key information

Skin irritation: irritant (QSAR predictions - ISafeRat & BfR, WoE, rel.2)

Eye irritation: not irritant (EpiOcular, OECD 492, GLP, K, rel.1)

Respiratory irritation: No data was available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion, other

Remarks:

in silico prediction

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

February 05th, 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source

Justification for type of information:

1. SOFTWARE
iSafeRat

2. MODEL (incl. version number)
iSafeRat Skin irritation/corrosion prediction model v1.0: iSafeRat SIC

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1CC(=CCCC=O)CC1(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
For details see attached QPRF

- Defined endpoint: Acute skin irritation/corrosion
- Unambiguous algorithm: yes
- Defined domain of applicability: yes
- Appropriate measures of goodness-of-fit: yes
- Mechanistic interpretation: yes

5. APPLICABILITY DOMAIN
- Descriptor domain: inside
- Structural domain: inside
- Mechanistic domain: inside
- Similarity with analogues in the training set: yes

6. ADEQUACY OF THE RESULT
iSafeRat skin irritation/corrosion model was trained with chemicals that had studies for skin irritation/corrosion which allowed to conclude on the GHS/CLP classification criteria. Therefore, the predictions from the iSafeRat SIC are considered to be equivalent to the GHS/CLP classification, as would be expected from an OECD TG 404 experimental study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

not applicable

Principles of method if other than guideline:

Model description: The iSafeRat skin irritation/corrosion prediction model determines whether the applied dose of a chemical substance causes cytotoxicity, thereby responsible for erythema and/or oedema as both lesions are driving the classification according to CLP and GHS. The dose applied and the physico-chemical properties of the test substance are the input of a series of algorithms to determine: the substance concentration in the viable epidermis and to establish if this concentration reaches a cytotoxic concentration for the keratinocytes. These concentrations are plotted against the logarithm of the subcooled water solubility of the substances (Log SCLS). The plot is subdivided into corrosive, irritant and non-irritant zones. The test item will fall into one of these zones, thereby allowing a classification.

The iSafeRat skin irritation/corrosion "aldehyde model" has an accuracy of 75% and a sensitivity for irritants (category 2) chemicals of 100% (9 irritant chemicals/9 total), based on the local model’s training set.

GLP compliance:

no

Remarks:

Not applicable (QSAR model)

Species:

rabbit

Irritation parameter:

erythema score

Time point:

24/48/72 h

Score:

> 2.3

Reversibility:

other: not applicable (QSAR prediction)

Remarks on result:

positive indication of irritation

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

QSAR prediction

Conclusions:

The test item was predicted as irritant to skin according to Regulation (EC) No. 1272/2008 (CLP) and to GHS using the iSafeRat skin irritation/corrosion v1.0. The test item falls within the applicability domain of the model’s training set and was hence reliably predicted for its skin irritation/corrosion potential. Therefore, this endpoint prediction can be considered reliable with restrictions. The prediction is accorded a Klimisch score K2, based on the results being “derived from a (Q)SAR model, with limited documentation/justification, but validity of model and reliability of prediction considered adequate.”

Executive summary:

The test item was predicted as irritating to skin using the iSafeRat skin irritation/corrosion prediction model. The prediction is considered reliable based on the evaluation of the applicability domain of the model, relative to the test item and the quality of the data used to generate the prediction.

Reference 2

Endpoint:

skin irritation / corrosion, other

Remarks:

in silico prediction

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

February 05th, 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source

Justification for type of information:

1. SOFTWARE
QSAR Toolbox v4.4

2. MODEL (incl. version number)
BfR skin irritation/corrosion (v.1.0)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1CC(=CCCC=O)CC1(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Acute skin irritation/corrosion
- Unambiguous algorithm: yes
- Defined domain of applicability: yes
- Appropriate measures of goodness-of-fit: yes
- Mechanistic interpretation: yes

See attached QPRF for details

5. APPLICABILITY DOMAIN
- Descriptor domain: inside
- Structural domain: inside
- Mechanistic domain: inside
- Similarity with analogues in the training set: yes

6. ADEQUACY OF THE RESULT
The BfR skin irritation/corrosion (v.1.0) prediction models outcome refers to skin corrosion EU risk phrases R34/R35 and to Skin irritation EU risk phrase R38 (Hulzebos et.al., 2005).

Principles of method if other than guideline:

Model description: The “Skin Exclusion rules by BFR” are based on the physico-chemical parameters of the test item (Gerner et. al., 2004) and “Skin Inclusion rules by BFR” are based on structural alerts (Hulzebos et. al., 2005). The SMILES code input is used by the QSAR toolbox software to generate/retrieve physico-chemical properties necessary to implement the “exclusion rules” and to detect structural fragment(s) within the test item’s structure to implement the “inclusion rules”.
The “Exclusion rules by BfR” of “log Kow < - 3.1” and “melting point > 200 °C”, where not met for the test item, as well as, for the “Group C” (Walker, et. al. 2005). Therefore, the test item is possibly irritant or corrosive to skin depending on “inclusion rules by BfR”. These exclusion rules were based on 56 out of 56 non-irritant chemicals (“log Kow < - 3.1”) and 291 out of 297 non-irritant chemicals for “melting point > 200 °C” (training set) based on an evaluation of 1833 chemicals total (Gerner et. al. 2004).

The “Inclusion rules by BfR” model structural alert “Aldehyde” triggered for the test item was based on 20 out of 20 aldehydes which can cause irritation or corrosion (training set, Hulzebos et.al. 2005).

GLP compliance:

no

Remarks:

Not applicable (QSAR model)

Irritation parameter:

erythema score

Time point:

24/48/72 h

Score:

> 2.3

Reversibility:

other: not applicable (QSAR prediction)

Remarks on result:

positive indication of irritation

Interpretation of results:

other: Irritant or Corrosive

Remarks:

QSAR prediction

Conclusions:

The test item was predicted using the BfR skin irritation/corrosion (v.1.0) model as Irritating or Corrosive to skin. The test item falls within the applicability domain of the model and was hence reliably predicted for its skin irritation/corrosion potential. Therefore, this endpoint prediction can be considered reliable with restrictions. The prediction is accorded a Klimisch score K2, based on the results being “derived from a (Q)SAR model, with limited documentation/justification, but validity of model and reliability of prediction considered adequate.”

Executive summary:

The test item was predicted as irritating or corrosive to skin using the BfR skin irritation/corrosion (v.1.0) prediction model. The prediction is considered reliable based on the evaluation of the applicability domain of the model, relative to the test item using the documentation available for the model.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

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:

2020-11-13 to 2020-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 492 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: Human Keratinocyte

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular™ EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcularTM Human Corneal Model (0.6 cm2 ) provided by MatTek In Vitro Life Science Laboratories, Bratislava – Slovakia. No biological contaminant detected.
- Cell line used, its source: Keratinocyte strain 4F1188.
- RhCE tissue used, including batch number: EpiOcular Tissue Lot Number: 30634.

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): 50 µL

Duration of treatment / exposure:

30 ±2 minutes at 37 °C, 5% CO2

Duration of post- treatment incubation (in vitro):

120 ±15 minutes at 37 °C, 5% CO2

Number of animals or in vitro replicates:

Duplicate cultures for test item, negative and positive controls

Details on study design:

- Details of the test procedure used: This method is based on the MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 02 October 2017.
- Volume of test chemical and control substances used: 50 µL
- Duration and temperature of exposure period: 30 ±2 minutes at 37 °C, 5% CO2
- Duration and temperature of post-exposure immersion period: 12 ±2 minutes at room temperature
- Duration and temperature of post-exposure incubation period: 120 ±15 minutes at 37 °C, 5% CO2
- Justification for the use of a different negative control than ultrapure H2O (if applicable): not applicable
- Justification for the use of a different positive control than neat methyl acetate (if applicable): not applicable
- Description of any modifications to the test procedure: none
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): not applicable
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): not applicable
- Description of the method used to quantify MTT formazan, if applicable: The optical density (OD) was measured at 570 nm (OD570) using a Labtech LT-4500 microplate reader. The upper limit of accuracy for measured absorbance of MTT Formazan at 570 nm (OD570), filter band pass 10 nm, was determined to be at an optical density of 2.6.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The relative mean tissue viabilities were compared to the mean of the negative control (n=2) treated tissues. The relative mean tissue viabilities were calculated as follows: Relative mean tissue viability = mean OD570 of test item mean / OD570 of negative control x 100. Mean Tissue Viability >60% : No Category / Mean Tissue Viability ≤60% : No prediction can be made according to the EU CLP and the GHS.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: In addition to the acceptability criteria a comparison of laboratory historical data on negative and positive controls was made to verify the functioning of the test system.
- Complete supporting information for the specific RhCE tissue construct used: yes, Certificate of Analysis from MatTek is attached to the report.
- Reference to historical data of the RhCE tissue construct: Acceptance criteria were met for Tissue Viability, Barrier function and Sterility.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not included in the report, but available upon request to the lab

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean of duplicate cultures

Value:

73.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

36.7%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Positive and negative control means and acceptance ranges based on historical data: All values for the control groups were within the ranges obtained by the testing laboratory.
- Acceptable variability between tissue replicates for positive and negative controls:
The relative mean tissue viability for the positive control treated tissues was 36.7% relative to the negative control treated tissues (below 50%). The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was 2.104 (> 0.8 and < 2.8). The negative control acceptance criterion was therefore satisfied.
- Acceptable variability between tissue replicates for the test chemical: The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (test items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Table 7.3.2/1: Individual Scores and Mean Scores for Corneal Effects – Test Item

 

Item	OD570 of tissues (tvt)	OD570 Tissues corrected for[tkt-ukt]	Correct Mean OD570 of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	2.209	-	2.104	105.0	100*	10.0
	1.999	-		95.0
Positive Control Item	0.737	-	0.771	35.0	36.7	3.3
	0.805	-		38.3
Test Item	1.540	1.498	1.539	71.2	73.2	3.9
	1.622	1.580		75.1

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

The purpose of this study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

Duplicate tissues were treated with the test item for an exposure period of 30 minutes. At the end of the exposure period each tissue was rinsed before incubating for 120minutes. The test item was found to directly reduce MTT and therefore additional non-viable, freeze-killed, 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. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and duplicate 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 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, corrected for direct MTT reduction, was 73.2%.

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

Under the test conditions, the test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

Since no key study was identified on the substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the skin corrosion/irritation potential of the substance:

 

	Element	Information	Conclusion	Comments
Existing data on physico - chemical properties	1a	Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?	NO
	1b	Is the substance an organic hydroperoxide or an organic peroxide?	NO
	1c	Is the pH of the substance ≤ 2.0 or ≥ 11.5?	NO
	1d	Are there other physical or chemical properties that indicate that the substance is corrosive/irritant?	NO
Existing human data	2	Are there adequate existing human data which provide evidence that the substance is a corrosive or irritant?	NO
Existing animal data from corrosion/irritation studies	3	Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?	NO	(at the initiation of the dossier, no test was available)
Existing data from general toxicity studies via the dermal route and from sensitisation studies	4a	Is the substance classified as acutely toxic by the dermal route (Category 1)?	NO
	4b	Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?	NO
	4c	Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated exposure?	NO	(at the initiation of the dossier, no test was available)
Existing/new (Q)SAR data and read -across	5a	Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion potential of the substance?	NO
	5b	Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?	YES	Predicted to be "Irritating to Skin" according to ISafeRat v1.0 and BfR v1.0
Existing in vitro data	6a	Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met	NO	(at the initiation of the dossier, no test was available)
	6b	Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO	(at the initiation of the dossier, no test was available)
	6c	Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?	NO
Weight-of- Evidence analysis	7	The  “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?	NO
New in vitro test for corrosivity	8	Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO
New in vitro test for irritation	9	Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO
New in vivo test for corrosion/irritation	10	To be used only as a last resort	NO

The test item was predicted as irritating to skin using the predictions of 2 in silico models (iSafeRat skin irritation/corrosion and BfR skin irritation/corrosion). The predictions are considered reliable based on the evaluation of the applicability domain of the models, relative to the test item and the quality of the data used to generate the prediction.

 

Eye irritation:

Since no key study was identified on the substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the eye corrosion/irritation potential of the substance:

	Element	Information	Conclusion	Comments
Conclusion of the information strategy on skin corrosion/irritation	0	Is the substance classified as a skin corrosive?	NO
Existing data on physico - chemical properties	1a	Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?	NO
	1b	Is the substance an organic hydroperoxide or an organic peroxide?	NO
	1c	Is the pH of the substance ≤ 2.0 or ≥ 11.5?	NO
	1d	Are there other physical or chemical properties that indicate that the substance causes serious eye damage or eye irritation?	NO
Existing human data	2	Are there adequate existing human data which provide evidence that the substance has the potential to cause serious eye damage or eye irritation?	NO
Existing animal data from corrosion/irritation studies	3	Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?	NO	(at the initiation of the dossier, no test was available)
Existing/new (Q)SAR data and read-across	4	Are there structurally related substances (suitable “read-across” or grouping), which are classified as causing serious eye damage/eye irritation, or indicating that the substance is non-irritant, or do valid (Q)SAR methods indicate serious eye damage/eye irritation or non-irritation of the substance?	NO
Existing in vitro data	5a	Has the substance demonstrated serious eye damage, eye irritation or non-irritating properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO	(at the initiation of the dossier, no test was available)
	5b	Are there acceptable data from (a) non-validated suitable in vitro test(s), which provide sound evidence that the substance causes serious eye damage/eye irritation?	NO
Weight-of- Evidence analysis	6	The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 0 – 5) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and – if so – how to classify and label?	NO
New in vitro tests for serious eye damage/eye irritation (Annex VII to the REACH Regulation)	7a	Does the substance demonstrate serious eye damage, eye irritation or non-irritant properties in (an) EU/OECD adopted in vitro test(s) for the eye hazard charaterisation? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions of Annex XI are met.	NO
	7b	Does the substance demonstrate serious eye damage or eye irritant properties in (a) non-validated suitable in vitro test(s) for serious eye damage/eye irritation?	YES	An OECD TG 492 (EpiOcular) study was performed. Mean tissue viability = 73,2% <=> Not Irritant or Corrosive to eyes.
New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)	8	Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?	NO

 

A new in vitro eye irritation study (EpiOcular, OCD 492, GLP, Covance, 2020, rel.1) was performed according to the OECD guideline No. 492 and in compliance with GLP, using the EpiOcular test model. The quality criteria required for acceptance of results in the tests were satisfied. The tissue viability of the test item was 73.2% after the 30 -minutes exposure period. Under the test conditions, with a tissue viability > 60%, the test item was considered to be non-irritant to eyes.

 

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on the available data, additional self-classification is proposed regarding both skin and eye irritation:

- skin irritant Category 2 according to the CLP and to the GHS

- not classified for eye irritation according to the CLP and to the GHS

No data was available regarding respiratory irritation.