Reaction product of Fatty acids, C18 alkyl with amines, polyethylenepoly-tetraethylenepentamine fraction

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 to 28 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to relevant testing guidelines, with no deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 2

Reference substance name:

Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction

IUPAC Name:

Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction

Constituent 3

Reference substance name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

EC Number:

292-587-7

EC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Cas Number:

90640-66-7

IUPAC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Constituent 4

Reference substance name:

lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction

Molecular formula:

for example : C26H53N5O, C26H55N5O...

IUPAC Name:

lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report):TOFA DimerFA TEPA PAA
- Physical state:Yellow-red, transparent, viscous liquid
- Analytical purity: 9% free amine
- Storage condition of test material:Room temperature (15°C to 30°C)
-Sponsor batch: BB000649V1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

The test animals were nulliparous, non-pregnant female CBA/CaCrl mice, obtained from Charles River (UK) Ltd., Margate. The animals were 8 to 10 weeks old on Day 1 of the study, and weighed 17 to 21 g on the day prior to dosing. Individual body weights were within ± 20% of the mean body weight. The animals were group housed during acclimatisation and individually housed from Day –1. Mice were fed SQC(E) Rat and Mouse Maintenance Diet No 1, from Special Diets Services Ltd, Witham, UK ad libitum. Mains water was provided ad libitum via cage-mounted bottles. The temperature was maintained at 20 to 24°C, and the humidity was 45 to 65%. Lighting was provided on a 12 hour cycle, and there were 15 to 20 air changes per hour.
The mice were acclimatised for 8 to 14 days.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0%, 10% w/v, 25% w/v, 50% w/v

No. of animals per dose:

4

Details on study design:

A preliminary study was conducted in one mouse to determine the systemic toxicity/irritancy potential of the test material. The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in acetone in olive oil) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3), and observed daily for five days from the initiation of treatment. Death or signs of systemic toxicity/excessive irritation were not observed, therefore concentrations of 10%, 25% and 50% were selected for the main study.

The test material or control (0.025 mL/pinna) was applied directly to the outer surface of both pinnae. Applications were made once daily on Days 1, 2 and 3. On Day 6 the mice were placed in a warming cabinet in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of tritiated ³H-methyl thymidine (³HTdR) into a tail vein of each mouse by slow bolus injection. After this treatment the mice were returned to their cages. Approximately five hours after intravenous injection of the ³HTdR, all mice were killed by exposure to a rising concentration of carbon dioxide. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes. Once death was confirmed, a mid-line incision from the lower jaw to the sternum was made and the cervical structures were exposed by reflecting the skin. The auricular lymph nodes were located and removed, any connective tissue was removed from the capsule of the nodes. The auricular lymph nodes of all mice from each dose group were placed into petri dishes containing 5 mL phosphate buffered saline.

The test result is not valid for those groups producing an SI value of 3.0 or more when the sites of application have shown excessive irritation and for those groups that have shown indications of systemic toxicosis. The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above. The test article is classified as a non sensitiser when the maximum value of the SI is less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

Formulations were prepared fresh in 80% v/v acetone in olive oil, on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing. They were mixed by multiple inversion of the containers prior to administration to ensure homogeneity. Concentrations of test article were expressed gravimetrically and in terms of test article received (without regard to purity or active content). An aliquot of 5.75 mL phosphate buffered saline was mixed with 0.5 mL ³HTdR shortly before intravenous dosing commenced on Day 6. The final product contained ³HTdR at 80 μCi/mL.

Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation. Routine health checks were made twice daily during the acclimatisation and study periods. Mice were weighed the day prior to dosing, and on Day 6 prior to ³HTdR administration.

Positive control substance(s):

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

Statistics:

Not required.

Results and discussion

Positive control results:

Positive control tests are conducted every 6 months at the test facility. The results from the most recent test (October 2012) gave stimulations indices of 1.6, 2.6 and 12.1 for α-Hexylcinnamaldehyde concentrations of 5%, 10% and 25%, respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

13.1

Test group / Remarks:

10% concentration

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM were 2329, 30421, 30706 and 35131 for the vehicle control, and 10%, 25% and 50% test material concentrations, respectively.

Results 3

Parameter:

SI

Value:

13.2

Test group / Remarks:

25% concentration

Results 4

Parameter:

SI

Value:

15.1

Test group / Remarks:

50% concentration

Any other information on results incl. tables

There were no deaths during the study, and no clinical signs of toxicity were observed. No signs of irritation were noted at the test sites. All animals had greasy fur to the back of the head and neck on Day 1, with greasy and sticky fur on the back of the head and neck noted on Day 2. Greasy fur to the back of the head and neck was noted in the control group from Day 3 to 6 with sticky and thinning fur to the back of the head and neck noted in the treated groups from Day 3 to 6. There were no effects of treatment on body weight.

Table 1. Scintillation counts and stimulation indices

Sample identity	No. sites yielding lymph nodes	Disintegrations per minute* (DPM)	Disintegrations per minute per node (DLM)	Stimulation Index (SI)
Scintillation fluid with 5% w/v trichloroacetic acid	-	45	-	-
Vehicle control	8	2329	291	-
Test article, 10% w/v	8	30421	3803	13.1
Test article, 25% w/v	8	30706	3838	13.2
Test article, 50% w/v	8	35131	4391	15.1

* All scintillation counts corrected for the blank

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results of the study, the test material is considered to be a skin sensitiser.

Executive summary:

The skin sensitisation potential of TOFA_DimerFA_TEPA_PAA was evaluated in the Local Lymph Node Assay (LLNA), according to OECD Test Guideline 429 and GLP. A preliminary study was conducted in one female CBA/Ca mouse to determine concentrations to be used for the main study. The main study animals were female CBA/Ca mice (4 per group). The test material was prepared for administration at 0, 10, 25 and 50% w/v in 80% v/v acetone in olive oil. The test material or vehicle control was applied to the outer aspect of the auditory pinnae of the mice once daily on Days 1, 2 and 3. On Day 6 a 20 µCi dose of tritiated ³H-methyl thymidine was injected intraveneously into each mouse. Approximately 5 hours later the auricular lymph nodes were recovered from each animal. Nodes from the same treatment group were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter. The DPM value (disintegrations per minute in a ten minute period) was used to calculate the DLM value (disintegrations per minute per lymph node). The DLM value of each test group was divided by the DLM value for the vehicle control group to provide the Stimulation Index for each group. Stimulation indices of 13.1, 13.2 and 15.1 were obtained for test concentrations 10%, 25% and 50%, respectively. Therefore, TOFA_DimerFA_TEPA_PAA is considered to be a skin sensitiser in the LLNA. On the basis of these results, TOFA_DimerFA_TEPA_PAA requires classification as a Category 1 Skin Sensitiser according to Regulation (EC) No 1272/2008.