Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

Endpoint summary

Description of key information

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine was tested  in the local lymph node assay: BrdU-ELISA at concentrations of 2.5%, 5% or 10% (w/w) in acetone/olive oil (3+1 v/v), based on preliminary testing. Stimulation indices of 0.972, 0.974 and 1.394 were found for the various concentrations, respectively. The results did not reveal any sensitising properties.

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC method B.51. Skin Sensitisation: Local Lymph Node Assay: BrdU-ELISA, Commission Regulation (EC) No. 640/2012 of 6 July 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier S.A.S, Route des Chênes Secs, C.S. 4105, Le Genest-St-Isle, 53941 St Berthevin Cedex, France
- Age on test day 1: 10 to 11 weeks
- Weight on test day 1: 18 - 21 g
- Fasting period before study: Not provided
- Housing: Before application the animals were housed in groups in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. After application the animals were housed singly in order to prevent their licking off the test item from the ears of other animals. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
- Diet (e.g. ad libitum): ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed.
- Water (e.g. ad libitum): Drinking water was offered ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): 12-18 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 21, 2013 To: April 17, 2013

Vehicle:

other: acetone/olive oil (3+1 v/v)

Concentration:

Three concentrations of the test item (2.5%, 5% and 10%, w/w) diluted with acetone/olive oil (3+1 v/v) were tested. The test item preparation was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.

No. of animals per dose:

Preliminary Test: 2
Main Test: 5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: No data
- Irritation: Excessive local irritation is indicated by an erythema score ≥3 and/or ear thickness of ≥25% on any day of treatment. The highest dose selected for the main LLNA: BrdU-ELISA was the next lower dose in the pre-screen concentration series that did not induce systemic toxicity and/or excessive local skin irritation.
In a pre-screen test, concentrations of 0.5, 1, 2.5, 5, 10, 25, 50 and 100% of test item, employing 2 animals per concentration, were examined. At a concentration of 25% very slight erythema was noted on the left and right ear of both animals. A moderate to severe erythema or a severe erythema to eschar formation were noted on the left and right ear of one animal each. At the highest concentration a severe erythema to eschar formation was noted on the left and right ear of both animals. On day 6 the ear thickness was increased by more than 25% starting at a test item concentration of 25%.
- Lymph node proliferation response: The pre-screen test was conducted under conditions identical to the main LLNA:BrdU-ELISA study, except there was no assessment of lymph node proliferation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA: BrdU-ELISA study
- Criteria used to consider a positive response:
Results for each treatment group were expressed as the mean SI. The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control group by the mean BrdU labelling index for the solvent/vehicle control group. The average SI for the vehicle controls was then one.
The BrdU labelling index was defined as:
BrdU labelling index = (ABS em – ABS blank em) – (ABS ref – ABS blank ref)
Where; em = emission wavelength; and ref = reference wavelength.
The decision process regarded a results a result as positive when SI ≥ 1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses have also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) was declared positive.
For a borderline positive response between an SI of 1.6 and 1.9, it may be necessary to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positives. Consideration will be also given to various properties of the test item, including whether it has a structural relationship to known skin sensitizers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observation.
In addition, the average ear weights per group and the average ear thickness per group are compared to the vehicle control group as an indication for possible irritating properties.
Statistical analyses include linear regression or Williams’ test to assess dose-response trends, and Dunnett’s test for pair-wise comparisons.

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal was identified and recorded. Any clinical observation was recorded. 25 µL of the appropriate dilution of the test item, the vehicle alone, or the positive control was applied to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Day 4:
No treatment.
Day 5:
0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected inter-peritoneally.
Day 6:
The weight of each animal and any clinical observation was recorded. Approximately 24 hours after BrdU injection the animals were humanely killed. The draining auricular lymph nodes from each mouse ear was excised and processed separately in phosphate buffered saline (PBS) for each animal. To further monitor the local skin response in the main study, additional parameters such as scoring of ear erythema and ear thickness measurements (obtained by using a thickness gauge and ear punch weight determinations at necropsy) were carried out.

Positive control substance(s):

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

Statistics:

Statistical analyses include linear regression or Williams’ test to assess dose-response trends, and Dunnett’s test for pair-wise comparisons.

Positive control results:

The positive control group caused the expected increases in the BrdU labelling index. Therefore, the study can be regarded as valid.

Parameter:

SI

Remarks on result:

other: In the main study, treatment with the test item at concentrations of 2.5%, 5% or 10% did not exceed the stimulation index for the BrdU labelling index of 1.6. Hence, the test item is classified as not sensitising.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not applicable

Table 1. Main study results: stimulation indices (SI)

Parameter	Group 1, negative control	Group 2, 2.5%	Group 3,  5%	Group 4, 10%	Group 5, positive control
BrdU labelling index	1.000	0.972	0.974	1.394	1.611
Ear weight	1.000	0.977	1.081	1.208**	1.191**
Difference of ear thickness (TD 3)	1.000	1.019	1.009**	1.066**	1.160**
Difference of ear thickness (TD 6)	1.000	1.028	1.085**	1.170**	1.217**

                                             

**significantly increased compared to control at p ≤ 0.01

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions, the test item at concentrations of 2.5%, 5% or 10% (w/w) in acetone/olive oil (3+1 v/v) did not reveal any sensitising properties in the local lymph node assay: BrdU-ELISA and, therefore, the test item should not be classified and labelled according to (EC) No.: 1272/2008 and its subsequent regulations.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions, Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine requires no labelling.

Executive summary:

The purpose of this study was to determine the sensitising potential of Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine in the local lymph node assay (LLNA): BrdU-ELISA in mice. The study was performed according to OECD guideline 442B. Stimulation indices (SI) were calculated for the BrdU labelling index, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle control treated ones. SI-values above 1.6 (BrdU labelling index) are considered positive. Three concentrations of the test item (2.5%, 5% and 10%, w/w) diluted with acetone/olive oil (3+1 v/v) were tested in five female CBA/JN mice per group and compared to a vehicle control group. In a pre-screen test, concentrations of 0.5%, 1%, 2.5%, 5%, 10%, 25%, 50% and 100% of test item, employing 2 animals per concentration, were examined. At a concentration of 25% very slight erythema was noted on the left and right ear of both animals. A moderate to severe erythema or a severe erythema to eschar formation were noted on the left and right ear of one animal each. At the highest concentration a severe erythema to eschar formation was noted on the left and right ear of both animals. On day 6 the ear thickness was increased by more than 25% starting at a test item concentration of 25%. In addition, a positive control group (25% solution v/v of α-hexyl cinnamic aldehyde in acetone/olive oil (3+1 v/v)) was employed.

In the main study treatment with the test item at concentrations of 2.5%, 5% or 10% did not exceed the stimulation index for the BrdU labelling index of 1.6. Stimulation indices of 0.972, 0.974 and 1.394 were found for the various concentrations, respectively. Hence, the test item is classified as not sensitising. The ear weight (punch biopsies) in the high dose group and the difference of ear thickness in the mid and high dose groups were slightly increased (statistically significant at p ≤ 0.01). This is considered to be an indication for irritating properties of the test item. The positive control group caused the expected increases in the BrdU labelling index. Therefore, the study can be regarded as valid. No signs of systemic intolerance were recorded. No erythema was observed. The animal body weight was not affected by the treatment..

In conclusion, under the present test conditions, the test item at concentrations of 2.5%, 5% or 10% (w/w) in acetone/olive oil (3+1 v/v) did not reveal any sensitising properties in the local lymph node assay: BrdU-ELISA.

.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a key study performed according to OECD guideline 442B, the sensitising potential of the test item was determined in the local lymph node assay (LLNA): BrdU-ELISA in mice (Haferkorn, 2013c). Stimulation indices (SI) were calculated for the BrdU labelling index, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle control treated ones. SI-values above 1.6 (BrdU labelling index) are considered positive. Three concentrations of the test item (2.5%, 5% and 10%, w/w) diluted with acetone/olive oil (3+1 v/v) were tested in five female CBA/JN mice per group and compared to a vehicle control group. In a pre-screen test, concentrations of 0.5%, 1%, 2.5%, 5%, 10%, 25%, 50% and 100% of test item, employing 2 animals per concentration, were examined. At a concentration of 25% very slight erythema was noted on the left and right ear of both animals. A moderate to severe erythema or a severe erythema to eschar formation were noted on the left and right ear of one animal each. At the highest concentration a severe erythema to eschar formation was noted on the left and right ear of both animals. On day 6 the ear thickness was increased by more than 25% starting at a test item concentration of 25%. In addition, a positive control group (25% solution v/v of α-hexyl cinnamic aldehyde in acetone/olive oil (3+1 v/v)) was employed.

In the main study treatment with the test item at concentrations of 2.5%, 5% or 10% did not exceed the stimulation index for the BrdU labelling index of 1.6. Stimulation indices of 0.972, 0.974 and 1.394 were found for the various concentrations, respectively. Hence, the test item is classified as not sensitising. The ear weight (punch biopsies) in the high dose group and the difference of ear thickness in the mid and high dose groups were slightly increased (statistically significant at p ≤ 0.01). This is considered to be an indication for irritating properties of the test item. The positive control group caused the expected increases in the BrdU labelling index. Therefore, the study can be regarded as valid. No signs of systemic intolerance were recorded. No erythema was observed. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, the test item at concentrations of 2.5%, 5% or 10% (w/w) in acetone/olive oil (3+1 v/v) did not reveal any sensitising properties in the local lymph node assay: BrdU-ELISA.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance was negative in the LLNA test and should not be classified and labelled according to (EC) No.: 1272/2008 and EC-Commission directive 67/548/EEC relating to the classification, packaging and labelling of dangerous substances.