Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-16 to 2016-11-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

EC Number:

279-510-2

EC Name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Cas Number:

80584-99-2

Molecular formula:

n.a.

IUPAC Name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Test material form:

liquid

Details on test material:

- Batch No: 3300081
- Physical state: liquid
- Colour: colourless to yellow
- Purity: 100 %
- Expiry date: 2018-05-16
- Storage conditions: Room temperature (20 ± 5 °C), keep away from light

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Based on the preliminary test results the test item was examined in the main test at concentrations of 25%, 10%, 5% and 2.5% (w/v). Formulations (apparently solutions) were prepared with N,N-Dimethylformamide (DMF). The test item was weighed and formulations prepared daily on a weight: volume basis in a final volume of 1 mL using volumetric vials and mechanical agitation. Formulations were freshly prepared just before the treatments.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca Ola Hsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT, H-1103, Budapest, Cserkesz u. 90.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 11-12 weeks old
- Weight at study initiation: 19.0 - 21.7 g, the weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight
- Housing during acclimatization period: Grouped caging in small groups
- Housing during the test: Grouped caging (4 animals/cage)
- Diet (e.g. ad libitum): ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water (e.g. ad libitum): ad libitum, tap water from watering bottles
- Acclimation period: 14 days
- Indication of any skin lesions: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30–70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 2.5, 5, 10 and 25%

No. of animals per dose:

4 animals per dose

Details on study design:

Dose Range Finding Study:
The pre-experiments on formulation evaluation of the test item and the Dose Range Finding Tests (DRFs) were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report, but the raw data of these tests are archived under the study code of present study. The maximum dose selection was performed according to the relevant guidelines. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used. Two consecutive DRFs have been performed. In both tests groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test. Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Both ears of each mouse were observed for erythema and scored according to criteria depicted in Table 1. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
In the first DRF the test item was evaluated at concentrations of 100% (as the undiluted test item) and as 50% and 25% (w/v) formulations in AOO. Both the undiluted test item and the formulations (apparently solutions) were adequately applicable on the ears of animals. No mortality was observed during the test. No significant signs of systemic toxicity were observed in any dose group. Although body weights decreased by ≥ 5 % were observed in the 50% (w/v) dose group (1/2 animals, 5 % decrease) and in the 25% (w/v) dose group (1/2 animals, 12% decrease) the effect was not dose related. No erythema was observed in any dose group. As a sign of an irritation significantly increased ear thickness values (an increase of >= 25 % compared to the initial value) were observed on Day 6 in all dose groups (the maximum increase was 38.1% in the 50% (w/v) dose group; the minimum increase was 13.6% in the 25% (w/v) dose group). The results obtained from this test were considered equivocal. It was also considered that the vehicle (AOO) may adversely interfere with the results (especially at 50% (w/v) test concentration), hence the DRF was repeated with test item formulations prepared with another suitable vehicle (DMF). In the second DRF the 100% (undiluted), 50% and 25% (w/v) concentrations were tested. Formulations, prepared with DMF, were adequately applicable on the ears of animals. No mortality was observed during the test. No significant effect on the body weights or other symptom of systemic toxicity was observed in any dose group. As an adverse effect (systemic or local) loss of hair (on the head) was observed in the 100% (w/v) dose group for both animals on the non-treatment days (Days 4-6). No similar effect was observed in the 50% or 25% (w/v) dose groups. No erythema was observed in any dose group. Significantly increased ear thickness values were observed in the 50% (w/v) dose group on Day 6 (2/2 animals, the maximum and minimum increase was 28.6% or 23.8%, respectively). Although an increase of 25% was observed also in the 25% (w/v) dose group for one animal it was considered not obvious sign of a significant irritation. Based on the overall results the undiluted test item (100% (w/v) test concentration) was considered not suitable for further testing. DMF was considered to be more suitable vehicle than AOO and to have no adverse impact on the test results. According to this the test item was formulated in the main test with DMF and the 25% (w/v) was used as the maximum attainable (non-toxic, non-irritant) concentration. The test item was tested also at three additional, lower concentrations (10%, 5% and 2.5%, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines. The effect on the ear thickness observed in the 25% (w/v) dose group in the second DRF was considered not significant but could not be negligible hence ear thickness was monitored in the test item treated groups during the main test.

Main Test Design:
Animals in the treatment groups were treated with the relevant vehicles (DMF or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test. For detailed data see the table 2 in box "Any other information on materials and methods incl. tables".

In vivo Treatment:
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance or the vehicles (see table 3 in box "Any other information on materials and methods incl. tables".) using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation Assay:
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test: all animals treated were processed. Therefore no treatment group was excluded from the evaluation.

Injection of 3^HTdR:
On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 µCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes:
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node cells:
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3^HTDR:
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5% (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5% TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the ß-scintillation counter. 3^HTdR incorporation was measured for up to 10 minutes per sample. The ß -counter expressed the 3^HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3^HTdR levels were measured in two 1 mL aliquots of 5% TCA. Instrument used for the measurement: Name: Tri-Carb 3100TR, Liquid Scintillation Analyzer, Serial Number: 072971

Observations in Main Test:
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test (Day 1 to Day 6). Additionally, ear thickness was measured in the test item treated groups. Ear thickness measurement was taken by using a digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Individual records were maintained for all observations.

Measurement of Body Weight:
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3^HTdR injection) with a precision of ± 0.1 g.

Evaluation of the Results:
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5% (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) of the test item was calculated according to published method. All calculations were made by Microsoft Excel Software.

Interpretation of the Results:
The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3^HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

Positive control substance(s):

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

Statistics:

n.a.

Results and discussion

Positive control results:

- The positive control item (25% (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 8.0). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

mean of four animals

Value:

15.3

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Remarks:

mean of four animals

Value:

6.8

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Remarks:

mean of four animals

Value:

4

Test group / Remarks:

5%

Results 4

Key result

Parameter:

SI

Remarks:

mean of four animals

Value:

2.7

Test group / Remarks:

2.5%

Cellular proliferation data / Observations:

Proliferation Assay:
Since no failed treatment, obvious sign of systemic toxicity or excessive irritation was observed during the test no treatment group was excluded from the evaluation.Visually larger lymph nodes compared to the relevant vehicle control (AOO or DMF) were observed in the positive control group and in the 25% (w/v) dose group. Appearance of the lymph nodes was normal in both vehicle control groups (AOO or DMF) and in the other test item treated groups (10%, 5% or 2.5%, w/v). Significant lymphoproliferative response (SI >= 3) compared to the relevant vehicle control (DMF) was noted for VOELOFA Monomer at 25%, 10% and 5% (w/v) test concentrations. No significantly increased lymphoproliferation was observed in the 2.5% (w/v) dose group. The observed stimulation index values were 15.3, 6.8, 4.0 and 2.7 at test item concentrations of 25%, 10%, 5% and 2.5% (w/v), respectively.
Significance of the dose-response was evaluated by linear regression using the SI values. Significant dose-response relationship was observed (p= 0.000045, r = 0.99995). For the results of the proliferation assay please refer to table 4 in box "Any other information on results incl. tables".
According to evaluation criteria of the relevant guidelines the significantly increased lymphoproliferation (indicated by an SI >= 3) observed at the maximum feasible (non-toxic, non-irritant) concentration of 25% (w/v) and also at lower concentrations (10% and 5%, w/v) and the strong dose-response relationship are considered evidence that VOELOFA Monomer is a skin sensitizer. Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve. The calculated EC3 value for the test item was 3.1 % (w/v) in this LLNA. Using this value VOELOFA Monomer can be ranked among moderate skin sensitizers according to the published data for classification of contact allergens.

Body Weight Measurement:
No significant, treatment related effect on body weights was observed during the test. Body weights, decreased by > 5 % were observed in the AOO group (2/4 animals, 7 % decrease both) but the effect was considered neither significant nor treatment related.

Clinical Observations and Other Observations:
No mortality or symptoms of systemic toxicity were observed in any treatment group. Ear thickness was measured in the test item treated groups. Ear thicknesses increased by ≥ 25 % (compared to the initial values) were observed on Day 6 in the 25 % (w/v) dose group in case of 3 animals: the maximum increase was 45 %. No significantly increased ear thickness values were observed for the fourth animal in this dose group. Similarly, no significantly increased ear thickness values were observed in the other dose groups (10 %, 5 % or 2.5 %, w/v). No erythema or any other local effect was observed in any treatment group.

Any other information on results incl. tables

Table 4: DPM and Stimulation Index Values for all Groups in the Main Test

Dose Group	Measured	Group^*	DPM/Mouse^#	Stimulation
	DPM/group	DPM		Index Values
Vehicle control for the positive control:	1	5019.5	1254.9	1.0
AOO
Positive control:	2	40191.5	10047.9	8.0
25 % HCA in AOO
Vehicle control for the test item:	3	1176.5	294.1	1.0
DMF
VOELOFA Monomer	4	18018.5	4504.6	15.3
25 % in DMF
VOELOFA Monomer	5	7979.5	1994.9	6.8
10 % in DMF
VOELOFA Monomer	6	4658.5	1164.6	4.0
5 % in DMF
VOELOFA Monomer	7	3200.5	800.1	2.7
2.5 % in DMF

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results from a mouse local lymph node assay (LLNA, EC3 value of 3.1%), the test item is considered to be a moderate skin sensitizer.

Executive summary:

In a dermal sensitization study conducted according to the guideline OECD 429, VOELOFA Monomer (100% purity) dissolved in DMF, young adult female CBA/CaOlaHsd mice (4 per dose group) were tested at concentrations of 2.5%, 5%, 10% and 25% in a local lymph node assay (LLNA). No mortality and no other adverse signs of toxicity were observed. Significant lymphoproliferative response (stimulation index >= 3) compared to the relevant vehicle control (DMF) was noted for VOELOFA Monomer at test item concentrations of 25% (15.3), 10% (6.8) and 5% (4.0) (w/v). At 2.5% test item concentration the stimulation index was 2.7. Based on the results, the calculated EC3 value for the test item was 3.1%. Based on this result, a sub-categorisation is possible and in accordance with CLP classification as Skin Sens 1B, H317 is warranted.