Reaction product of salicylaldehyde and formaldehyde and phenol

Endpoint summary

Description of key information

Skin Sensitisation, in vivo (LLNA). Hargitai (2013)

Under the conditions of this study, the test material was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay.

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:

28 November 2012 to 04 December 2012

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)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old (age-matched, within one week). In the preliminary experiment, mice of 12 weeks of age were used.
- Weight at study initiation: 20.9 - 22.6 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.) In the preliminary experiment, mice weighing 25.1 - 26.2 grams were used.
- Housing: Group caging / mice were provided with glass tunneltubes. Cage type: Type II. polypropylene / polycarbonate. Bedding was available to animals during the study.
- Diet: ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water: Animals received tap water from the municipal supply from 500 mL bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly.
- Acclimation period: 13 days
- Indication of any skin lesions: Only healthy animals were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.4 - 25.0 °C
- Humidity: 30 - 70 %
- Air changes: 15 - 20 air exchange/hour
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

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

Concentration:

5, 10, 25 and 50 (w/v) %

No. of animals per dose:

4 females per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The applicable vehicle of the test material was selected based on the results of a short Preliminary Compatibility Test. Due to the physical characteristics of the test material, 100 (w/v) % concentration was not achievable. However, the resulted formulation at 50 (w/v) % using AOO (acetone:olive oil 4:1 (v:v) mixture) as vehicle was suitable for the test. As AOO is one of the vehicles recommended by the relevant OECD guideline, it was selected for vehicle of the study.
- Irritation and Systemic toxicity: A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using two doses (2 animals/dose), at test material concentrations of 50 and 25 (w/v) % in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. The maximum concentration of test material in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 50 (w/v) %. In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site.
- Ear thickness measurements: Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
- Erythema scores: Both ears of each mouse were observed for erythema and scored.

MAIN STUDY
- Dose Selection: In the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. Marked body weight loss (>5 %) was observed in one experimental animal in the 50 (w/v) % dose group. Alopecia and rigid ears were observed for all animals in the 50 and 25 (w/v) % groups on Days 3-6. Test material residue was observed on the ears of all animals on Days 2-6. The revealing ear punch weights and ear thickness data were out of the historical control range. However it could not be excluded that test material remaining on the ears may have contributed to the thickness and weight. The draining auricular lymph nodes of the animals were visually examined: they were considered to be larger than normal in both dose groups (subjective judgement by analogy with observations of former experiments). Based on these results, 50, 25, 10 and 5 (w/v) % doses were selected for the main test. Ear thickness of the experimental animals in the main test was measured by ear punch weight determinations, which were performed on Day 6 after the animals were humanely killed. The ear punches were retained in formalin.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test material was weighed and formulations prepared daily on a weight: volume basis (as (w/v) %) in the Central Dispensary Unit of CiToxLAB Hungary Ltd. Analytical determination of the test material concentration, stability and homogeneity was not performed because of the character and the short period of study.
- Topical application: During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. 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
- Injection of Tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the 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 euthanised by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes 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 pooled lymph node cells (LNCs) 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). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently re-suspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
- Determination of Incorporated 3HTdR: After the final washing step, supernatants were removed. Pellets were gently agitated re-suspended and 3 mL of 5 (w/v) % TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 (w/v) % TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 (w/v) % TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS
- Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Measurement of Body Weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

EVALUATION OF THE RESULTS
- DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
- Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
- Interpretation of Results: The test material is regarded as a sensitiser if both of the following criteria are fulfilled: That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index and, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

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

Positive control results:

- The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/J Rj mice.
- No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 9.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
- Furthermore, the DPN values observed for the positive control substance in this experiment were within the historical control range.

Key result

Parameter:

SI

Value:

86.1

Test group / Remarks:

50 (w/v) %

Key result

Parameter:

SI

Value:

67.5

Test group / Remarks:

25 (w/v) %

Key result

Parameter:

SI

Value:

66.4

Test group / Remarks:

10 (w/v) %

Key result

Parameter:

SI

Value:

34

Test group / Remarks:

5 (w/v) %

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
- No mortality or signs of systemic toxicity were observed during the study. Test material precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 1-6 and in the 25 % (w/v) group on Day 2-6. Alopecia was observed in the 50 % (w/v) dose group on Days 2-6, in the 25 % (w/v) group on Days 3-6 and for one animal in this group on Days 2-6. Rigid ears were observed in the 50 and 25 % (w/v) dose groups on Days 4-6 and in the 10 % (w/v) dose group on Day 4.

BODY WEIGHT MEASUREMENT
- No marked body weight loss (< 5 %) was observed on the experimental animals.

EAR THICKNESS MEASUREMENTS
- Increased biopsy weight values were observed in the 50, 25 and 10 % dose groups. Test material remaining on the ears may have contributed to the weight.

PROLIFERATION ASSAY
- The appearance of the lymph nodes was normal in the negative (vehicle) control group. Larger than normal lymph nodes were observed in all test material treated and positive control groups.
- The stimulation index values were 86.1, 67.5, 66.4, and 34.0 at concentrations of 50, 25, 10 and 5 (w/v) %, respectively.

INTERPRETATION OF OBSERVATIONS
- The test material was a light red solid, which was formulated in AOO. Since, there was a clear positive effect with a dose response, which even at the lowest dose was over 11 times the threshold for a positive response; the test material was clearly a positive sensitiser. There were no confounding effects of irritation or systemic toxicity at the lower concentration range. Any possible irritant effects at high dose levels do not influence this interpretation. The preserved ears were discarded as no further examination is required.

RELIABILITY OF THE TEST
- The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/J Rj mice.
- No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 9.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
- Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control groups included 4 animals.

Table 1: DPM, DPN and Stimulation Index Values for all Groups

Test Group	Measured DPM/Group	DPM	Number of Lymph Nodes	DPN	Stimulation Index
Background (5 (w/v) % TCA)	36	-	-	-	-
Negative (vehicle) control (AOO)	1573	1537.0	8	192.1	1.0
Test Material 50 (w/v) % in AOO	132446	132410.0	8	16551.3	86.1
Test Material 25 (w/v) % in AOO	103755	103719.0	8	12964.9	67.5
Test Material 10 (w/v) % in AOO	102024	101988.0	8	12748.5	66.4
Test Material 5 (w/v) % in AOO	52269	52233.0	8	6529.1	34.0
Positive control (25 (w/v) % HCA in AOO)	15163	15127.0	8	1890.9	9.8

Interpretation of results:

other: EU Criteria: Skin sensitiser (Category 1)

Conclusions:

Under the conditions of this study, the test material was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions.

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test material was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration was 50 (w/v) %.

The Preliminary Irritation / Toxicity Tests were performed in CBA/J Rj mice using two doses: 50 and 25 (w/v) % in the selected vehicle. Based on the observations recorded in the preliminary test, the 50 (w/v) % was selected as top dose for the main test.

In the main assay, twenty four female CBA/J Rj mice were allocated to six groups of four animals each: four groups received the test material (formulated in AOO) at 50, 25, 10 and 5 (w/v) % concentrations, the negative control group received the vehicle (AOO) and the positive control group received 25 (w/v) % HCA (dissolved in AOO). The test material solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No marked body weight loss was detected for test material treated animals. Test material precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 1-6 and in the 25 % (w/v) group on Day 2-6. Alopecia was observed in the 50 % (w/v) dose group on Days 2-6, in the 25 % (w/v) group on Days 3-6 and for one animal in this group on Days 2-6. Rigid ears were observed in the 50 and 25 % (w/v) dose groups on Days 4-6 and in the 10 % (w/v) dose group on Day 4.

The observed stimulation index values were 86.1, 67.5, 66.4, and 34.0 at concentrations of 50, 25, 10 and 5 (w/v) %, respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 9.8) was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of this study, the test material was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin Sensitisation, in vivo (LLNA). Hargitai (2013)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test material was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration was 50 (w/v) %.

The Preliminary Irritation / Toxicity Tests were performed in CBA/J Rj mice using two doses: 50 and 25 (w/v) % in the selected vehicle. Based on the observations recorded in the preliminary test, the 50 (w/v) % was selected as top dose for the main test.

In the main assay, twenty four female CBA/J Rj mice were allocated to six groups of four animals each: four groups received the test material (formulated in AOO) at 50, 25, 10 and 5 (w/v) % concentrations, the negative control group received the vehicle (AOO) and the positive control group received 25 (w/v) % HCA (dissolved in AOO). The test material solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No marked body weight loss was detected for test material treated animals. Test material precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 1-6 and in the 25 % (w/v) group on Day 2-6. Alopecia was observed in the 50 % (w/v) dose group on Days 2-6, in the 25 % (w/v) group on Days 3-6 and for one animal in this group on Days 2-6. Rigid ears were observed in the 50 and 25 % (w/v) dose groups on Days 4-6 and in the 10 % (w/v) dose group on Day 4.

The observed stimulation index values were 86.1, 67.5, 66.4, and 34.0 at concentrations of 50, 25, 10 and 5 (w/v) %, respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 9.8) was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of this study, the test material was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified as a skin sensitiser, H317: may cause an allergic skin reaction.