Didodecyl fumarate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

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 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: mean: 19.6 g
- Housing: single housing in Makrolon cage, type II
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

methyl ethyl ketone

Concentration:

10, 25, 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was a 50% (w/w) dilution in MEK.
- Irritation: No signs of local skin irritation were observed. One animal, treated with 25 %, showed white test item residues prior the 2nd application only.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU incoroporation measured in a photometer
- Criteria used to consider a positive response: The proliferative response of the lymph node cells is expressed as the mean S.I.. The S.I. is derived by dividing the mean BrdU labeling index/mouse within each test substance group as well as for the positive control group by the mean BrdU labeling index for the vehicle group.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of BrdU at least 1.6-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response (Ehling et al. (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay 2nd round. Toxicology, 212, 69–79.). Furthermore, according to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation.

TREATMENT PREPARATION AND ADMINISTRATION: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 10, 25 and 50% (w/w) in MEK. The application volume 25 µL/ear/day was spread over the entire dorsal surface (diameter ca. 8 mm) of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25 % α-hexyl cinnamaldehyde dissolved in MEK. For injection BrdU was dissolved in DPBS (10 mg/mL) before administration and stored in a refrigerator until use. Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5mg/per mouse/injection) was intraperitoneally injected. Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a 70 µm nylon mesh. The lymph node cells were re-suspended in 6 mL DPBS. After cell count with a cell counter (Casy Cell Counter, Innovatis), cell suspensions of 100 000 cells/mL were adjusted.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A Mann-Whitney-U test for non-parametric comparison was applied as statistical test. Statistical significance was set at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Positive control results:

25 % α-hexyl cinnamaldehyde: S.I. = 5.0

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Vehicle control: mean: S.I. = 1.0 10% test item: mean: S.I. = 1.2 25% test item: mean: S.I. = 0.9 50% test item: mean: S.I. = 0.9 A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentration results in a 1.6-fold or greater increase in incorporation of BrdU compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 1.6 is referred to as the EC1.6 value. An EC1.6 value could not be determined as all S.I.s obtained were below the threshold of 1.6

Observations:

No deaths occurred during the study period. No symptoms of systemic toxicity were observed during the study period.No local findings (ears) were observed in the test item dose groups. Scaling and incrustations were noted in the positive control. The body weight of the animals recorded prior to the first application and prior to sacrifice were within the range commonly recorded for animals of this strain and age.

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. In the positive control a statistically significant and biological relevant increase was observed.

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biological relevant increase in ear weights was not observed in any test item treated group in comparison to the vehicle control group.

According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group. In the positive control a statistically significant and biological relevant increase was observed.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not sensitising
DSD: not sensitising

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

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 Sensitisation
1	2402-58-6	Experimental result: not sensitising
2	10341-03-4	RA: CAS 2402-58-6
3	68610-90-2	RA: CAS 2402-58-6
4	68921-51-7	RA: CAS 2402-58-6
5	68921-52-8	RA: CAS 2402-58-6
6	68921-53-9	RA: CAS 2402-58-6

Skin sensitisation

There is a reliable GLP guideline study (OECD 442B, Höger, 2013) in order to assess the skin sensitising potential of Didodecyl fumarate (CAS 2402-58-6). In this local lymph node assay five female CBA mice per group were tested with the test item at concentrations of 10, 25 and 50% (w/w) in the vehicle methyl ethyl ketone (MEK). The animals did not show any signs of systemic toxicity or local findings during the course of the study and no cases of mortality were observed. In the positive control (alpha-hexylcinnamaldehyde) scaling and incrustations were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off-value for a positive response regarding the ear weight index of 1.25 was not exceeded in any dose group.

A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentration results in a 1.6-fold or greater increase in incorporation of BrdU compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 1.6 is referred to as the EC1.6 value.

In this study Stimulation Indices (S.I.) of 1.2, 0.9 and 0.9 were determined with the test item at concentrations of 10, 25 and 50 % (w/w) in MEK. An unusual dose response was observed. An EC1.6 value could not be determined as all S.I. obtained were below the threshold of 1.6.

A statistically significant or biologically relevant increase in BrdU incorporation and also in lymph node weight, cell count and ear weight measurement was not observed in any of the test item dose groups in comparison to the vehicle control group. Furthermore, the cut off-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded in any of the tested dose groups (Ehling et al., 2005).

As expected, a statistical and biological relevant increase in BrdU labelling, lymph node weight, lymph node cell count and ear weight measurement was determined in the positive control.

The test item was thus not a skin sensitiser under the test conditions of this study.

 

Conclusion for sensitisation

An in vivo skin sensitisation study was performed using Didodecyl fumarate (CAS 2402-58-6), showing no potential for skin sensitisation. As dermal absorption is 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 and thus lowest log Pow, represents a worst-case within the category. 

In conclusion, the available data indicate that no hazard for skin sensitisation for any category member has to be expected.

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

Migrated from Short description of key information:
The overall assessment of the available information gave no indication for sensitising properties of the category members.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

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 sensitisation 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.