Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Endpoint summary

Description of key information

The registered substance is a multi-constituent substance.
It was proved not to be soluble in Milli-Q water dimethyl sulfoxide (DMSO) up to 10 mg/mL. In addition, a homogenous suspension could not be obtained.
The test item formed a usable homogenous suspension in Ethanol (EtOH) at 10 mg/mL.
The test item is also not soluble in complete medium (RPMI-1640) at 0.4 mg/mL. In addition, a homogenous suspension could not be obtained.
Therefore, it is concluded that the test item is not suitable to perform in vitro skin sensitisation testing, and that animal testing had to be conducted to investigate the skin sensitisation properties of the substance.
A LLNA was conducted on the test substance, which concluded that it was not sensitising to the skin.

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

Study period:

From 20 Oct 2020 to 10 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vitro skin sensitisation testing was not technically feasible. Therefore, it was required to conduct animal testing to comply with the requirements of REACH.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 21.3 to 23.7 g.
- Housing: up to 5 animals in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: Municipal tap-water was freely available to each animal via water bottles ad libitum
- Acclimation period: at least 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C
- Humidity: 40 to 70%
- Air changes: Ten or more air changes per hour
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

Vehicle:

methyl ethyl ketone

Concentration:

5, 10 or 20% w/w

No. of animals per dose:

Five animals/dose

Details on study design:

PRE-SCREEN TESTS:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 10% and 20% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed.
Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
- Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 µL/ear) with the test item, at approximately the same time on each day. The formulations were stirred with a magnetic stirrer until dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5%
trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.
- Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of ProSafe + as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to
Disintegrations Per Minute (DPM).

Positive control substance(s):

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

Statistics:

In case of borderline results, statistical analysis may be performed to determine the dose response relationship and pair wise comparisons between dose groups versus negative control.

Positive control results:

The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity

Parameter:

SI

Value:

1.2

Test group / Remarks:

20%

Parameter:

SI

Value:

1.4

Test group / Remarks:

10%

Parameter:

SI

Value:

1.2

Test group / Remarks:

5%

Cellular proliferation data / Observations:

Skin Reactions / Irritation
No irritation was observed in any of the animals.
White test item remnants were present on the dorsal surface of the ears of all test item treated animals between Days 1 and 3, which did not hamper scoring of the skin reactions.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The moderate body weight loss noted in one individual animal dosed at 10% was considered not toxicologically significant, since it concerns only one animal and no concentration-related incidence was apparent.

Macroscopic Examination of the Lymph Nodes and Surrounding Area All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 20% were 1462, 1653 and 1381 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1181 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.2, 1.4 and 1.2, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

A LLNA was conducted on the test substance, which concluded that it was not sensitising to the skin.

Executive summary:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 20%, Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.
The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Based on these results, Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Additional information:

In vitro skin sensitisation testing
The test item is not soluble in Milli-Q water dimethyl sulfoxide (DMSO) up to 10 mg/mL. In addition, a homogenous suspension could not be obtained.
The test item formed a usable homogenous suspension in Ethanol (EtOH) at 10 mg/mL. However, using this vehicle in the Keratinosens study the highest concentration in the assay will be 25 µg/mL. The recommended top concentration mentioned in the OECD guideline is 400 µg/mL.
The test item is also not soluble in complete medium (RPMI-1640) at 0.4 mg/mL. In addition, a homogenous suspension could not be obtained.
Therefore, it was concluded that the test item is not suitable to perform the Keratinosens (OECD 442D) and USENS (OECD 442E) tests. No conclusion can be made based on the obtained data.

skin sensitisation: in vivo (LLNA)
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 20%, Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.
The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Based on these results, Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Justification for classification or non-classification

Based on available testing on the registered substance, it does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).