Acetic acid, C11-14-isoalkyl esters, C13-rich

Endpoint summary

Description of key information

In the key skin sensitisation study, conducted according to OECD TG 429 and in compliance with GLP, the test item, Acetic acid, C11-14-isoalkyl esters, C13-rich, was reported to be not sensitising (Envigo, 2016).

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:

30 June 2016 to 31 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was conducted to determine homogeneity, concentration or stability of the test formulation.

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 27 May 2017
- Purity test date: 30 May 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: It was assumed that the formulation was stable for 2 hours prior to application.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was freshly prepared as a solution in acetone/ olive oil 4:1. The vehicle was chosen as it produced the most suitable formulation at the required concentration.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no information

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated within 2 hours of being applied to the test system. No analysis was conducted to determine homogeneity, concentration or stability of the test formulation.
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: The test item was used undiluted and freshly prepared as a solution in acetone/ olive oil 4:1.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: no data
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: food, ad libitum
- Water: mains tap water, ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15/ hour
- Photoperiod (hrs dark / hrs light): 12/ 12

Vehicle:

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

Concentration:

Preliminary study: undiluted test material and 50% v/v in acetone/ olive oil 4:1
Main study: 50%, 25% or 10% v/v in acetone/ olive oil 4:1

No. of animals per dose:

Preliminary study: 2 mice
Main study: 5 mice

Details on study design:

PRE-SCREEN TESTS:
The mice were treated by daily application 25 µL of the undiluted test item or the test item at a concentration of 50% v/v in acetone/ olive oil 4:1, on the dorsal surface of each ear for three consecutive days. The mice were observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Local skin irritation was scored daily according. Any clinical signs of toxicity were also recorded. Body weights were recorded prior to dosing on day 1 and on da 6. Mitutoyo 547-300S gauge was used to measure the thickness of each ear treated with undiluted test material, pre-dose on day 1, post-dose on day 3 and on day 6. Similarly, the thickness of each ear treated with 50% v/v was measured pre-dose and 1 hour post-dose on day 1, 1 hour post-dose on days 2 and 3 and 4 to 6. On day 6 the animals were killed by carbon dioxide asphyxiation followed by cervical separation.


MAIN STUDY
The mice were treated by daily application of 25 µL of 50%, 25% or 10% v/v in acetone/ olive oil 4:1 to the dorsal surface of each ear for three consecutive days. The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of 5 mice received the vehicle alone in the same manner. Local skin irritation was scored daily. The thickness of each ear was measured and recorded by Mitutoyo 547-300S gauge pre-dose and 1 hour post-dose on day 1, 1 hour post-dose on days 2 and 3 and 4 to 6. Five days following the first topical application, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing ³H-methyl thymidine giving a total of 20 µCi to each mouse. All animals were observed on days 4, 5 and 6. Any signs of toxicity during the test were recorded. Body weights were recorded prior to dosing on day 1 and prior to termination on day 6. Five days following ³H-methyl thymidine administration all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes were excised and processed for each animal. A single cell suspension of the lymph node cells for each animal was prepared. The lymph node cells were rinsed and the suspension was transferred into a centrufuge tube. The lymph node cells were pelleted at 1400 rpm for 10 minutes. To precipitate the radioactive material the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA). After the cells were incubated for 18 hours at 4°C, the precipitates were recovered by centrifugation at 2100 rpm for 10 minutes, re-suspended and transferred to 10 mL of scintillation fluid. β-scintillation counting was used to measure ³H-methyl thymidine. The samples and scintillation fluid were stored in dark for 20 minutes. Then, the number of radioactive disintegrations per minute was measured.


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: The test item was considered to be a sensitiser if at least one concentration of the test item results in a threefold or greater increase in ³H-methyl thymidine incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25 µL of 50%, 25% or 10% v/v in acetone/ olive oil 4:1 to the dorsal surface of each ear for three consecutive days. The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of 5 mice received the vehicle alone in the same manner.

Positive control substance(s):

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

Statistics:

The parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Positive control results:

α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

1.13

Test group / Remarks:

10% v/v in acetone/ olive oil 4:1

Remarks on result:

other: negative

Key result

Parameter:

SI

Value:

2.14

Test group / Remarks:

25% v/v in acetone/ olive oil 4:1

Remarks on result:

other: negative

Key result

Parameter:

SI

Value:

1.68

Test group / Remarks:

50% v/v in acetone/ olive oil 4:1

Remarks on result:

other: negative

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Not specified

DETAILS ON STIMULATION INDEX CALCULATION: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of ³H-methyl thymidine into lymph node cells of test nodes relative to that recorded for the control.

EC3 CALCULATION: No data

CLINICAL OBSERVATIONS: No deaths occurred throughout the study period. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals was comparable to that of control group.

Interpretation of results:

GHS criteria not met

Conclusions:

In the skin sensitisation study, conducted according to OECD TG 429 and in compliance with GLP, the test item, Acetic acid, C11-14-isoalkyl esters, C13-rich, was reported to be not sensitising.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

In the key skin sensitisation study, conducted according to OECD TG 429 and in compliance with GLP, the test item, Acetic acid, C11-14-isoalkyl esters, C13-rich, was reported to be not sensitising (Envigo, 2016).

Based on results from a preliminary study, the chosen concentrations for the main study were 10%, 25%, and 50% of test substance in v/v in acetone/ olive oil 4:1. In each phase of the study the mice were treated by daily application of 25 µl of each concentration, or of vehicle control, to the dorsal surface of both ears for three consecutive days. No deaths occurred during the study period. No clinical signs of toxicity were noted in any of the animals. Five days following the first topical application of the test substance (day 6) all mice were injected via the tail vein with 250µl of phosphate buffered saline containing ³H-methyl Thymidine giving a total 20 µCi to each mouse. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application by measurement of the incorporation of ³H-methyl Thymidine by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node and as the ratio of ³H-methyl Thymidine incorporation into LNC of test nodes relative to that recorded for control ratios.

An insult patch test/ photosensitisation study in human subjects was also available, in which the irritating, fatiguing and sensitising properties of Acetic acid, C11-14-isoalkyl esters, C13-rich were investigated (Biosearch, 1979). Based on the results obtained, the test substance was not a primary skin irritant or a fatiguing agent. No sensitisation reactions were noted. Exposure to UV-A light does not appear to initiate or increase the incidence or severity of irritation, fatigue or sensitisation.

Justification for classification or non-classification

Based on the available data for Acetic acid, C11-14-isoalkyl esters, C13-rich, no classification is required for skin sensitisation according to Regulation (EC) No. 1272/2008.