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Description of key information

A DEREK assessment, a DPRA assay and in vitro KeratinoSensTM assay were performed. Based on these data no conclusion could be drawn on the skin sensitisation of 1,3-bis (4-hydroxy benzoyl) benzene. Therefore, an in vivo study (LLNA) was performed to determine the skin sensitising potential and potency. Based on the results of the LLNA 1,3-bis (4-hydroxy benzoyl) benzene was concluded not to be a sensitiser.

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:
09 January 2018 - To be added
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The results obtained from the available in silico/in chemico/in vitro studies are not adequate for classification and risk assessment.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No 640/2012, Part B: Skin sensitization: "Local Lymph Node Assay"
Version / remarks:
July 2012
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Specific details on test material used for the study:
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 19.2 - 22.9 g
- Housing: Animals were group housed (up to 5 animals together) in labeled Makrolon cages containing sterilized sawdust as bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (daily mean)
- Temperature (°C): 22 - 23
- Humidity (%): 41 - 43
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 10 January 2018 - 5 February 2018
Vehicle:
dimethylformamide
Concentration:
0, 10, 25, 60% (w/w) (main study)
25, 60% (w/w) (pre-test)
No. of animals per dose:
5 (main study)
2 (pre-test)
Details on study design:
RANGE FINDING TESTS:
Two test item concentrations were tested: a 25% and 60% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (prior to dosing on days 1 and 3, and on day 6.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item and were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.

Rationale for vehicle: The vehicle was chosen from the vehicles specified in the test guideline. The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.


Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.


Observations:
Mortality/Viability: Twice daily.
Body weights: On day 1 (pre-dose) and day 6 (prior to necropsy).
Clinical signs: Once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on days 1-6 (on days 1 - 3 within 1 hour after dosing) according to a numerical scoring system (according to guideline). Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was performed, since all animals survived until the end of the observation period.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
0.8
Variability:
0.0 (standard error of the mean)
Test group / Remarks:
10%
Remarks on result:
other: Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, the substance was not considered to be a skin sensitizer
Key result
Parameter:
SI
Value:
0.8
Variability:
0.1 (standard error of the mean)
Test group / Remarks:
25%
Remarks on result:
other: Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, the substance was not considered to be a skin sensitizer
Key result
Parameter:
SI
Value:
0.8
Variability:
0.1 (standard error of the mean)
Test group / Remarks:
60%
Remarks on result:
other: Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, the substance was not considered to be a skin sensitizer
Cellular proliferation data / Observations:
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 836, 830 and 828 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1055 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 0.8, 0.8 and 0.8, respectively.

Results Pre-screen test:

At a 25% and 60% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore the 60% concentration was selected as highest concentration for the main study.

Other results - main study:

Skin Reactions / Irritation

No irritation was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all test item treated animals throughout the observation period, which did not hamper scoring of the skin reactions

Macroscopic  Examination of the Lymph Nodes and Surrounding Area

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Systemic Toxicity and bodyweight

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:
GHS criteria not met
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines and in compliance with GLP principles, 1,3-Bis (4-hydroxy benzoyl) benzene was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 60% (w/w).
Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 60% w/w. No irritation was observed in any of the animals. All auricular lymph nodes were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 836, 830 and 828 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1055 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 0.8, 0.8 and 0.8, respectively. As the SI appeared not to be ≥ 3 when tested up to 60% w/w, 1,3 -Bis (4-hydroxy benzoyl) benzene was not considered to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A Weight-of-Evidence approach has been used to fulfill information requirements about skin sensitisation potential of the test substance. The weight of evidence approach was used according to Annex XI, sections 1.2-1.5, to the REACH Regulation.

The following studies were used:

- Using DEREK NEXUS software (version 5.0.2), the structure of the test item yielded:

*an alert (equivocal) for 1,3 -Bis (4 -hydroxy benzoyl) benzene for skin sensitization based on the presence of a substituted phenol group. Equivocal alert means there is an equal weight of evidence for and against the proposition.

*a predicted EC3 of 0.022% (extreme sensitizer) based on data on closest structurally-related substances. The potential mechanism is by pre-/pro-hapten producing electrophilic Michael acceptor or by pre-/pro-hapten producing free radical generator.

- A valid DPRA test was performed according to OECD 442C guidance and following GLP principles. For the DPRA assay, 1,3-bis (4-hydroxy benzoyl) benzenewas dissolved in Dimethylsulphoxide (DMSO):acetonitrile (ACN) 1:9 (v/v) at 100 mM and formed a clear solution by visual inspection. Precipitation was observed upon the addition of the test item to the synthetic peptides containing cysteine (SPCC) solution.No co-elution of the test item with SPCC or synthetic peptides containing lysine (SPCL) was observed.In the cysteine reactivity assay the test item showed 2.0% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.0% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC peptide solution, it is unknown how much test item remained in the solution to react with the SPCC peptide. Consequently, this negative result is uncertain and should be interpreted with caution.

- A valid KeratinoSensTM assay was performed according to OECD 442D guidance and according to GLP principles. For the KeratinoSensTMassay 1,3-bis (4-hydroxy benzoyl) benzene was dissolved in DMSO to a final concentration of 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was considered to be the maximum testable concentration based on solubility in DMSO and is the highest dose required in the current guideline. The test item precipitated at the dose level of 500, 1000 and 2000 μM in experiment 1 only. Two independent experiments were performed.The test item showed toxicity (IC30values of 100 μM and 100 μM and IC50values of 140 μM and 119 μM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5values of 9.7 μM and 50 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.51-fold and 1.54-fold in experiment 1 and 2 respectively. The test item is considered as positive in the KeratinoSensTMassay since positive results (>1.5-fold induction) were observed at test concentrations <1000 μM with a cell viability of >70% compared to the vehicle control.

 

- An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 60% w/w. No irritation was observed in any of the animals. All auricular lymph nodes were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 836, 830 and 828 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1055 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 0.8, 0.8 and 0.8, respectively. As the SI appeared not to be ≥ 3 when tested up to 60% w/w, 1,3 -Bis (4 -hydroxy benzoyl) benzene was not considered to be a skin sensitiser.

Interpretation and conclusion of the Weight-of-evidence:

Based on a positive DEREK NEXUS assessment, an inconclusive DPRA assay and a positive KeratinoSensTMassay, no conclusion could be drawn on skin sensitization properties of 1,3-bis(4 -hydroxy benzoyl) benzene. Performance of an additional in vitro assay, for example the U-SENSTMassay, would not be sufficient for potency assessment in case the test were positive and in case of negative test, the results would not be sufficient to conclude. Therefore, a third in vitro study was omitted and an in vivo study (LLNA) was been performed to determine the skin sensitizing properties of 1,3-bis (4-hydroxy benzoyl) benzene. According to the result of the in vivo study, 1,3-Bis (4-hydroxy benzoyl) benzene was considered not to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance does not have an harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

According to EU CLP regulation (Consolidated version 01 -01 -2017), skin sensitisers shall be classified in category 1 where data (human or animal data) are not sufficient for sub-categorisation. Current available data are in vitro/in chemico tests (DEREK Nexus assessment, in vitro DPRA test and in vitro KeratinoSens test) and one in vivo test (LLNA according to OECD 429 guidelines).

Based on the results of these studies, 1,3-Bis (4-hydroxy benzoyl) benzene is not classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).