Fatty acids, C18-unsatd., dimers, reaction products with fatty acids, C14-18 and C16-18-unsatd. and propylidynetrimethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see “remarks”

Remarks:

GLP-Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

147256-33-5

EC Number:

604-580-1

Cas Number:

147256-33-5

IUPAC Name:

147256-33-5

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: individually in suspended soil-floor polypropylene cages furnnished with softwood woodflakes
- Diet: 2014C Teklad Global Rodent diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: mains tap water, ad libitum
- Acclimation period: at least 5 days

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

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

100; 50; and 25% (v/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Irritation: Using available information regarding the systemic toxicity and irritancy of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of the undiluted test item to the dorsal surface of each ear for three consecutive days.(Days 1, 2, and 3). The mouse was observed twice daily on days 1, 2, and 3, and once daily on Days 4, 5, and 6. Local irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 prior to dosing and on Day 6. The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post-dose on day 3, and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine (3HTdR) incorporation determined by ß-scintillation
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index, SI). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdr incorporation will be classified as a non-sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of 5 mice were treated with the undiluted test item or the test item at concentrations of 50 or 25% (v/v) in acetone/olive oil 4:1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of each ear using the tip of the pipette. A further group of 5 mice received the vehicle alone in the same manner. The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item at a concentration of 25% (v/v)in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
5 days following the first application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR:80µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.
5 h following 3HTdR administration all mice were sacrificed and for each animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL PBS was added to the lymph nodes.
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish. The lymph node cell suspension was then transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove the remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at approximately 190 x g for 10 min. The pellet was resuspended in 10 mL of PBS and re-pelleted. To prcipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).
After approxiately 18 h incubation at approximately 4 °C, the precipitates were recovered by centrifugation at approximately 450 x g for 10 min, resuspended in 1 mL TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measureed by ß-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer for approximately 20 min. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 min, the vials were shaken vigorously. The number of radioactive disintegrations per min was then measured.

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per min and standard deviations where appropriate. Individual and group mean disintegrations per min values were assessed for dose response relationships by ANOVA. In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Metod was used.
Probability values (p) are presented as follows:
p<0.001 ***; p<0.01 **; p<0.05 *; p≥0.05 (not significant)

Results and discussion

Positive control results:

- the measured mean disintegrations per min was as follows: 16427.98±2134.26 dpm***
- the calculated Stimulation Indicex was as follows: 9.91***
In conclusion, the positive control substance produced a Stimulation Index greater than 3, and thus can be considered to be sensitising. Based on this data the positive control was considered to be valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The calculated Stimulation Indices were as follows: - vehicle control: 1 - test item 25% (v/v): 1.68 - test item 50% (v/v): 1.54 - test item 1000% (v/v): 1.82 In conclusion, no significant or dose-dependent increase in the Stimulation Indeces was observed in the test groups, and none of the groups produced a Stimulation Index equal to or greater than 3. Thus, no EC3 value could be determined and the test item can be considered to be not sensitising.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The measured mean disintegrations per min were as follows: - vehicle control: 1657.10±637.26 dpm - test item 25% (v/v): 2790.56±812.83 dpm - test item 50% (v/v): 2557.70±950.03 dpm - test item 1000% (v/v): 3011.09±2544.30 dpm

Any other information on results incl. tables

Preliminary Screening Test:

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the undiluted test item and the test item at concentrations of 50 and 25% (v/v) in acetone/olive oil 4:1 were selected for the main test.

Main Test:

Table 1: The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment group	Concentration	Stimulation Index	Result
Vehicle control	acetone/olive oil 4:1	1	-
Test item	25% (v/v) in acetone/olive oil 4:1	1.68	Negative
	50% (v/v) in acetone/olive oil 4:1	1.54	Negative
	100% (undiluted)	1.82	Negative
Positive control	25% (v/v) in acetone/olive oil 4:1	9.91***	Positive

*** p<0.001

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified