Didodecyl fumarate

Endpoint summary

Description of key information

None of the category members of the PFAE fumarate group is considered to be skin or eye irritating.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200)

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: MatTek Corporation, Ashland MA, USA

TEST METHOD
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 6-well plates containing 900 µL assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1 °C, 5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: 90-95%

Type of coverage:

other: open - in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

other: minimally moistened with PBS

Controls:

other: concurrent control tissues treated with the vehicle served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume (about 15 mg)
- Concentration (if solution): 50% (v/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): 50%

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: SDS, 5% (v/v)

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable. Post-treatment incubation period: 42 ± 4 h

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 60 min
- Post-treatment incubation period: 42 ± 4 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed about 42 h after the incubation period. Therefore, tissues were incubated in 300 µL MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissue in isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of the solvent controls (PBS). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

4

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (5% SDS). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

96

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Results:

test substance		tissue 1	tissue 2	tissue 3	mean	SD
NC	mean OD570	2.404	2.415	2.401	2.407
	viability [% of NC]	99.9	100.3	99.8	100	0.29
test item	mean OD570	2.281	2.049	2.589	2.306
	viability [% of NC]	94.8	85.1	107.6	96	11.27
PC	mean OD570	0.096	0.09	0.105	0.097
	viability [% of NC]	4	3.7	4.4	4	0.32

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - non official national or international study, well documented

Qualifier:

no guideline followed

Principles of method if other than guideline:

The potential of didodecyl fumarate to cause ocular irrritation was assessed by a single topical application of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcularTM). The study was performed according to the methods described in the following publications: MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010 and Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: EpiOcularTM; reconstructed three-dimensional human cornea model (OCL-200)

Details on test animals or tissues and environmental conditions:

TEST SKIN MODEL
- Source: MatTek Corporation, Ashland MA, USA

TEST METHOD
The model represents a reconstructed three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratiozytes used to model the human corneal epithelium. Irritant materials are identified by their ability to induce cytotoxicity (= loss of viability) at the surface of the EpiOcularTM tissue. The decrease in cell viability is determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 6-well plates containing 1 mL assay medium per well and preincubated in a humidified incubator for 16 to 24 hours (37 ± 1 °C, 5% CO2) before use. After the pre-incubation the tissue were pre-treated with 20 µL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: 90-95%

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with the water served as negative controls, positive controls were exposed to 50 µL methyl acetate

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL bulk volume (about 22 mg)

NEGATIVE CONTROL:
- 50 µL of sterile de-ionized water

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: 50 µL methyl acetate

Duration of treatment / exposure:

90 minutes

Observation period (in vivo):

Not applicable. Post-treatment period: 18 hours

Number of animals or in vitro replicates:

Not applicable. The test was performed in duplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the tissue three times with phosphate buffered saline (PBS). In order to remove residual test substance, washed tissue were immediatly immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion). After 12 minutes of post-soak immersion, each tissue was dried and transferred to fresh 6-well plates filled with pre-warmed medium.
- Time after start of exposure: 90 min
- Post-treatment incubation period: 18 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 18 h after the incubation period. Therefore, tissues were incubated in 300 µL MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissue in isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: mean value of the negative control

Time point:

other: 90 min

Score:

100

Reversibility:

other: not applicable

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: mean value of positive control

Time point:

other: 90 min

Score:

4

Reversibility:

other: not applicable

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: mean value of the test item

Time point:

other: 90 min

Score:

96

Reversibility:

other: not applicable

Results:

test substance		tissue 1	tissue 2	mean	SD
NC	mean OD570	1.646	1.393	1.52
	viability [% of NC]	108.3	91.7	100	16.6
test item	mean OD570	1.133	1.349	1.241
	viability [% of NC]	74.6	88.7	82	14.2
PC	mean OD570	0.43	0.344	0.387
	viability [% of NC]	28.3	22.6	25	5.7

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not irritant
DSD: not irritant

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for grouping of substances and read-across

The PFAE fumarates (Polyfunctional Aliphatic Ester) category consists of six members, which are either well-defined mono-constituent substances or related UVCB substances, with varying fatty alcohol chain lengths. The distinguishing feature of this category of chemicals is that its members are diester derivatives of fumaric acid (CAS 110-17-8). The alcohol moiety of the dicarboxylic esters generally falls in the C8-C22 carbon number range, including linear, even numbered alcohols. 

In order to avoid the need to test every substance for every endpoint, the category concept is applied for the assessment of environmental fate, environmental toxicity and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by inter- or extrapolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. Structural similarities and similarities in properties and/or activities of the source and target substances in the category are the basis of read-across.

The available studies providing information on the human health hazard assessment within the PFAE fumarates category were conducted with the category member Didodecyl fumarate (CAS 2402-58-6). This substance was selected for testing, because it represents the category member with the shortest fatty alcohol side chain, and consequently with the lowest molecular weight, which is regarded as worst-case approach in terms of hazard assessment of the PFAE fumarates for the local as well as for systemic effects.

Furthermore, the category is supported by another polyfunctional aliphatic ester, namely Bis(2-ethylhexyl) adipate (CAS 103-23-1). This supporting chemical is used to cover toxicological endpoints, exclusively. The read across of Bis(2-ethylhexyl) adipate (CAS 103-23-1) to the PFAE fumarate category is justified due to the similar structural and physico-chemical properties, as well as their toxicological, and ecotoxicological profiles.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Sections 7.1 and 13) and within Chapter 5.1 of the CSR.

Endpoint specific data matrix

ID #	CAS	Skin irritation/corrosion	Eye irritation
1	2402-58-6	Not irritating	Not irritating
2	10341-03-4	RA: CAS 2402-58-6	RA: CAS 2402-58-6
3	68610-90-2	RA: CAS 2402-58-6	RA: CAS 2402-58-6
4	68921-51-7	RA: CAS 2402-58-6	RA: CAS 2402-58-6
5	68921-52-8	RA: CAS 2402-58-6	RA: CAS 2402-58-6
6	68921-53-9	RA: CAS 2402-58-6	RA: CAS 2402-58-6

Irritation/corrosion:

CAS 2402-58-6

Skin irritation:

The potential of Didodecyl fumarate (CAS 2402-58-6) to cause dermal irritation was assessed by a single topical application of 25 µL bulk volume (about 15 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model according to OECD 439 (Remmele, 2013). Three EpiDerm TM tissue samples were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. Tissue destruction was determined by measuring the reduction of mitochondrial dehydrogenase activity, indicated by reduced formazan production after incubation with a tetrazolium salt (MTT).The formazan production after incubation with a substance treated epidermal tissues is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability. The EpiDerm TM skin irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test substance treated tissues determined after an exposure period of 1 hour and after 42 hours post-exposure was 96%.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show skin irritation potential in the EpiDerm TM skin irritation test under the test conditions chosen.

 

Eye irritation:

The potential of Didodecyl fumarate (CAS 2402-58-6) to cause ocular irritation was assessed by a single topical application of 50 µL bulk volume (about 22 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular TM) (Remmele, 2013). Two EpiOcular TM tissue samples were incubated with the test substance for 90 minutes followed by an 18-hour post-incubation period. Tissue destruction was determined by measuring the reduction of mitochondrial dehydrogenase activity, indicated by reduced formazan production after incubation with a tetrazolium salt (MTT). The formazan production after incubation with a substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiOcular TM eye irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test substance treated tissues was 82%.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular TM eye irritation test under the test conditions chosen.

 

Conclusion for irritation

In conclusion, the skin irritating properties of the PFAE fumarate category members have been investigated in a validated in vitro study indicating no or very low skin irritating properties. Moreover, skin irritation is expected to decrease with increasing fatty alcohol chain length. Thus skin irritating properties of the PFAE fumarate category members have been investigated using the mono-constituent category member Didodecyl fumarate (CAS 2402-58-6) with the shortest fatty alcohol side chain, which is regarded as worst-case approach. Therefore, based on the study results with Didodecyl fumarate (CAS 2402-58-6) none of the category members of the PFAE fumarate group is considered to be skin irritating.

In conclusion, the eye irritating properties of the PFAE fumarate category members have been investigated in a reliable in vitro study indicating no eye irritating properties. As eye irritating properties are expected to decrease with increasing fatty alcohol chain length, Didodecyl fumarate (CAS 2402-58-6) the category member with the shortest fatty alcohol side chain, therefore representing a worst case, was used for testing. Therefore, based on the study results with Didodecyl fumarate (CAS 2402-58-6) none of the category members of the PFAE fumarate group is considered to be eye irritating.

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the group concept is applied to the members of the PFAE fumarate category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labeled on this basis.

Therefore, based on the group concept, all available data on skin and eye irritation / corrosion do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.