Registration Dossier

Administrative data

Description of key information

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:
13 to 27 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to relevant testing guidelines, with no deviations.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
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 9 to 10 weeks old on Day 1 of the study, and weighed 17 to 19 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 7 to 14 days.
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.
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.
Parameter:
SI
Remarks on result:
other: The stimulation indices were 8.0, 9.5 and 9.9 for concentrations of 10%, 25% and 50%, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM were 2461, 196660, 23297 and 24426 for the vehicle control, and 10%, 25% and 50% test material concentrations, respectively.

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 or sticky fur to the back of the head from Day 1. Animals treated with the test material also had hard and thinning fur to the back of the head from Day 3. 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

-

46

-

-

Vehicle control

8

2461

308

-

Test article, 10% w/v

8

19660

2458

8.0

Test article, 25% w/v

8

23297

2912

9.5

Test article, 50% w/v

8

24426

3053

9.9

* All scintillation counts corrected for the blank

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_TETA_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. Female CBA/Ca mice (4 per group) were used in the main study. 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 8.0, 9.5 and 9.9 were obtained for test concentrations 10%, 25% and 50%, respectively. Therefore, TOFA_DimerFA_TETA_PAA is considered to be a skin sensitiser in the LLNA. On the basis of these results, TOFA_DimerFA_TETA_PAA requires classification as a Category 1A Skin Sensitiser according to Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitisation potential of Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine was evaluated in the Local Lymph Node Assay (LLNA), in a study conducted according to OECD Test Guideline 429 and GLP (Dreher, 2013d). A preliminary study was conducted in one female CBA/Ca mouse to determine concentrations to be used for the main study. Female CBA/Ca mice (4 per group) where used in the main study. 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 8.0, 9.5 and 9.9 were obtained for test concentrations 10%, 25% and 50%, respectively. On the basis of these findings, Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine is considered to be a skin sensitiser.


Migrated from Short description of key information:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine was considered to be a skin sensitiser in the Local Lymph Node Assay (LLNA) conducted according to OECD Test Guideline 429.

Justification for selection of skin sensitisation endpoint:
Sole study providing data from a guideline compliant study. Under the conditions of the study, the test substance was found to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine was considered to be a skin sensitiser in the local lymph node assay. On this basis, the substance meets the criteria for classification for skin sensitisation as: Category 1, H317 “May cause an allergic skin reaction” according to Regulation (EC) No 1272/2008 and as Xi, R43 “May cause sensitisation by skin contact” according to Directive 67/548/EEC.