Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-13 to 2010-11-10
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
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
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate
EC Number:
700-321-2
Molecular formula:
C30H26O6Zn, C46H42O6Zn, C62H58O6Zn
IUPAC Name:
hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Experimental Animals
Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals/treatment group
Sex: Female, nulliparous, non pregnant
Age of animals: Young adult, 11-12 weeks old, age-matched within one week
Body weight range at starting: 20.0 – 23.5 g
Acclimatisation time: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchange/hr
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
From: 2010-10-07 to 2010-11-10

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test:
Concentration test substance: 100 and 50 (w/v) %

Main Test:
Concentration test substance: 100, 50, 25 and 10 (w/v) %
No. of animals per dose:
Preliminary test:
2 females/dose

Main Test:
4 females/dose
Details on study design:
Preliminary Test

The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429 (2010). The maximum concentration based on the solubility of the test item was 100 (w/v) %, the highest recommended concentration.
A Preliminary Irritation / Toxicity Test was performed in groups of 2 mice (CBA/J Rj mice were used) at test item concentrations of 100 and 50 (w/v) % in AOO. This preliminary experiment was conducted under similar experimental conditions as the main study but was ended on Day 6 with a body weight measurement. A radioactive proliferation assay was not performed.
During the Preliminary Irritation / Toxicity Test no mortality, systemic toxicity or local irritation were observed. No treatment related effect on body weights was observed in the 100 (w/v) % treated group. In the 50 (w/v) % treated group slight body weight loss was observed. Ear thickness was measured by using a thickness gauge on Day 1. However, measurement on Day 3 and Day 6 could not be performed due to the chemical nature of the test item (glue-like behaviour of the test item was observed; i.e. after treatment the test item formed a dried layer on the surface of the ears, which made them very rigid, sticky and unevenly surfaced).
Additional quantification of the ear thickness was performed by ear punch weight determination after euthanasia of the experimental animals. The weights of the ear punches (2 per animal) were recorded, revealing weights above the normal range. In general, this could be an indication of irritation. However, as these results are quite variable, a correlation between weight of the ear punches and residual test item upon can not be excluded. Furthermore, no visual signs of irritation were observed in any dose group. Therefore, the highest dose (100 %) was considered to be acceptable for the main test.
In order to make sure that there will be three acceptable dose groups, an additional dose group was used in the main experiments. Additionally, ears of the animals used in the main test were taken at termination and preserved in neutral buffered 10% formalin for potential examination of irritation, if it is needed. The preserved ears will be discarded after the finalisation of the report if no further examination will be performed.


Main Study

Administration of the Test Item
During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 µL of the appropriate formulation applied using a pipette. 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 20 µCi (approximately) of 3HTdR using a gauge 25Gx1" 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 euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision). 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. Before discarding the carcasses, death was confirmed by cervical dislocation or by cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group were pooled and collected in a Petri dish containing 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 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were resuspended and 10 mL of PBS were added to the tubes. The washing step was repeated twice. This procedure was performed for each group of pooled lymph nodes.

Determination of incorporated 3HTdR:
After the final washing step, supernatants were removed. Pellets were gently resuspended, then 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 (approx. 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 suitable sized scintillation vials 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 expressed the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). The background level was 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) all animals were observed at least once daily (twice - before / after treatment - on treatment days) for any clinical signs, including local irritation and systemic toxicity. 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 (“Group DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solution was used as the background DPM value. The results were expressed as “DPN” (Group 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.

In general, a stimulation index of 3 or greater is an indication of a positive result. However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.
Interpretation of results
The test item is regarded as a sensitiser if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- 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)
Statistics:
NA

Results and discussion

Positive control results:
The positive control group animals were treated with 25 (w/v) % HCA solution in a relevant vehicle (AOO) concurrent to the test item groups. The positive control substance was chosen according to the OECD guideline.

No mortality, cutaneous reactions or signs of toxicity were observed in the animals of the positive control group. A significant lymphoproliferative response (stimulation index value of 21.5) was noted in this experiment for a-Hexylcinnamaldehyde. The results of the positive control group demonstrated the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
- Test substance: Stimulation index values of the test item were 6.4, 89.7, 56.8 and 17.9 at treatment concentrations of 100, 50, 25 and 10 (w/v) %, respectively. These stimulation index values were compatible and in line with the biological dose-related response. The low value obtained for the 100% dose is not unusual and can sometimes be observed in connection with strong sensitisers as cytotoxicity to the immunocytes cannot be excluded. - Positive Control: α-Hexylcinnamaldehyde (25 (w/v) % dissolved in AOO). A stimulation index value of 21.5 was noted for the positive control chemical, indicating a significant lymphoproliferative response. This result confirmed the validity of the assay.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“Group DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solution was used as the background DPM value. The results were expressed as “DPN” (Group 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. In general, a stimulation index of 3 or greater is an indication of a positive result. However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive. For details on DPM results see table in "Any other information on results incl. tables" below.

Any other information on results incl. tables

DPM (Disintegrations per minute)

 Test Group Name  Measured DPM/group  Group DPM  No. of Nodes  DPN  Stimulation Index Values
 Background (5 (w/v) % TCA ) 33  -  -  -  -
 Negative control (vehicle) AOO 432  399  8 49.9 1.0
 Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc (100 % in AOO) 2570 2537  8 317.1 6.4
  Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc (50 % in AOO) 35821 35788  8 4473.5 89.7
   Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc (25 % in AOO) 22698  22665  8 2833.1 56.8
   Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc (10 % in AOO) 7179  7146  8 893.3 17.9 
 Positive control 25 % HCA in AOO 8603  8570  8 1071.3 21.5

Clinical Signs and Mortality

No mortality or signs of systemic toxicity were observed during the study. No significant effects were observed on animal body weights. Individual and mean body weights were recorded. Dried test item and fixed or slightly fixed ears were observed in the animals of the 100 and 50 (w/v) % treated groups on Days 1-6. Alopecia was observed in the animals of the 100 and 50 (w/v) % treated groups on Days 2-6 around the treated site. Peeling was observed on the ears of the animals of the 50 (w/v) % treated groups on Days 5 and 6.

Proliferation Assay

Significant lymphoproliferative response (SI >= 3) of Reaction product of

2-hydroxybenzoic acid, styrene and oxozinc was observed at all the concentrations applied compared to the negative control. Stimulation index values of the test item were 6.4, 89.7, 56.8 and 17.9 at treatment concentrations of 100, 50, 25 and 10 (w/v) %, respectively.

Reliability of the Assay

The positive control group animals were treated with 25 (w/v) % HCA solution in a relevant vehicle (AOO) concurrent to the test item groups. The positive control substance was chosen according to the OECD guideline. No mortality, cutaneous reactions or signs of toxicity were observed in the animals of the positive control group. A significant lymphoproliferative response (stimulation index value of 21.5) was noted in this experiment for alpha-Hexylcinnamaldehyde. The results of the positive control group demonstrated 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.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay under the test conditions used in this study.
Executive summary:

A Local Lymph Node Assay according to OECD 429 (2010) and Commission Regulation (EC) No 440/2008, 30 May 2008, B.42 was performed with CBA/J Rj mice in order to determine the skin sensitisation potential of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc.

The test item was an amber coloured solid; vehicle compatibility was verified in a Preliminary Compatibility Test with Acetone: Olive oil 4:1 (v:v) mixture (AOO). The test item was soluble in this vehicle. The maximum available concentration was 100% using this vehicle.

A Preliminary Irritation / Toxicity Test was performed with the test item at concentrations of 100 % and 50 % in the selected vehicle. The 100 % concentration was considered acceptable as the maximum concentration in the main assay.

In the main assay twenty-four female CBA/J Rj mice were allocated to six groups of four animals each:

- four groups received the appropriate formulation of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc at concentrations of 100, 50, 25 and 10 (w/v) %,

- the negative control group received the vehicle (AOO),

- the positive control group received 25 (w/v) % HCA in the relevant vehicle (AOO).

The test item 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 treatment related effect on the body weights was observed. Dried test item and fixed or slightly fixed ears were observed in the animals of the 100 and 50 (w/v) % treated groups on Days 1-6. Alopecia was observed in the animals of the 100 and 50 (w/v) % treated groups on Days 2-6. Peeling was observed on the ears of the animals of the 50 (w/v) % treated groups on Days 5 and 6.

Significant lymphoproliferative response (SI >= 3) of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was observed at the concentrations applied compared to the negative control. Stimulation index values of the test item were 6.4, 89.7, 56.8 and 17.9 at treatment concentrations of 100, 50, 25 and 10 (w/v) %, respectively. These stimulation index values were compatible and in line with the biological dose-related response. The low value obtained for the 100% dose is not unusual and can sometimes be observed in connection with strong sensitisers as cytotoxicity to the immunocytes cannot be excluded.

α-Hexylcinnamaldehyde (25 (w/v) % dissolved in AOO) was used as the positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 21.5) was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay under the test conditions used in this study.