[No public or meaningful name is available]

Endpoint summary

Description of key information

MDP is a multi-constituent substance that contains four main components, each with methacrylate functional groups and three with phosphate functional groups.  Since the DPRA, h-CLAT and KeratinoSens assays (OECD 442 series) have not been validated for use with multi-constituent substances, alternative data sources were compiled to determine the skin sensitization potential of MDP while avoiding unnecessary animal testing.  Methacrylates are generally well-documented to be either weak skin sensitizers or nonsensitizers (Kimber et al., 2019). Phosphate compounds are generally accepted to be nonsensitizers (Johnson et al., 2021) so the presence of the phosphate functionality in several of the minor components of MDP is not expected to add to the sensitization potential of the overall multi-constituent substance.

 

The skin sensitization of this multi-constituent substance (EC 944-391-4) was evaluated using a Weight of Evidence (WoE) approach to avoid unnecessary animal testing.  An LLNA was conducted with the major constituent of this substance (CASRN 6701-13-9 is 60-70% of the multi-constituent) and the results indicate that it is a GHS Category 1B sensitizer.  Three additional constituents are present at lower levels in the multi-constituent (see section 1.2 of the dossier for full details).  The skin sensitization potential of the three minor constituents were addressed via the OECD QSAR Toolbox DASS Protocol, computational profiling and literature data (see dossier section 7.4 endpoint summary for a summary of the WoE argument).

The four main constituents of MDP are:

 

1: 1,10-Decanediol-dimethacrylate: An LLNA was conducted with this component (GHS Category 1B sensitizer)

2: 10-Methacryloyl-oxy-decylphosphate (OECD QSAR Toolbox DASS Protocol - not expected to be a skin sensitizer based on profiling)

3: Bis-(10-methacryloyl-oxy-decyl)-phosphate (OECD QSAR Toolbox Profilers - same sensitization alerts returned as the main constituent)

4: Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate (OECD QSAR Toolbox Profilers - same sensitization alerts returned as the main constituent)

The skin sensitization potential of 10-(phosphonooxy)decyl methacrylate was predicted in the OECD

QSAR Toolbox ‘EC3 from LLNA or Skin sensitization from GPMT assay for defined approaches (SS AW for

DASS)’ automated workflow that is utilized in OECD 497. Based on the result of the prediction, 10-(phosphonooxy)decyl methacrylate is not expected to be a skin sensitizer.

The skin sensitization potential of Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate were evaluated using the skin sensitization and protein binding profiling schemes available in OECD QSAR Toolbox v. 4.5.  Both substances returned the same profiling alerts for skin sensitization and protein binding as the main constituent (1,10-Decanediol-dimethacrylate) for which there is experimental data indicating that it is a weak skin sensitizer (GHS Category 1B).  

1,10-Decanediol-dimethacrylate (main constituent), Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate all returned the following skin sensitization alerts when profiled using OECD QSAR Toolbox v 4.5.

General Mechanistic:

Protein binding by OASIS and OECD: Michael addition

Protein binding potency Cys: DPRA above 21%

Endpoint Specific:

Protein binding potency GHS: Slightly reactive (GSH)

Protein binding alerts for skin sensitization according to GHS: Skin Sensitization Category 1A

Protein binding potency h-CLAT: alpha, beta-Unsaturated esters

 

These alerts were common to 1,10-Decanediol-dimethacrylate, Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate with 1,10-Decanediol-dimethacrylate having experimental data indicating that it is a GHS Category 1B sensitizer.

 

Additionally, toxicological literature summarizing the skin sensitization potential of methacrylate and phosphate functional group-containing structures was reviewed to determine if the two lowest level components of the multi-constituent MDP (Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate) would be expected to add to the overall skin sensitization potential of the substance. Generally, methacrylates are well-documented to be weak skin sensitizers (as demonstrated by1,10-Decanediol-dimethacrylate) or nonsensitizers (Kimber et al., 2019). Some higher molecular weight methacrylates (i.e., isodecyl methacrylate and dodecyl methacrylate) are not skin sensitizers in the local lymph node assay, indicating a trend that skin sensitization potency of methacrylates may decrease as molecular weight increases. Both Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate have much higher molecular weights (546 and 627 g/mol, respectively) when compared to isodecyl methacrylate and dodecyl methacrylate (226 and 254 g/mol, respectively). Furthermore, both are higher in molecular weight than the main component of MDP, 1,10-Decanediol-dimethacrylate, which has a molecular weight of 310 g/mol. Based on Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate returning the same structural alerts for sensitization as 1,10-Decanediol-dimethacrylate, the documented skin sensitization profiles of the functional moieties present in the structures (methacrylate and phosphate groups), and the inverse relationship between molecular weight and skin sensitization potency, Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate are not expected to increase the overall skin sensitization potency of MDP relative to the known potency of the major constituent. 

Based on this information, the overall weight of evidence suggests that a GHS Category 1B skin sensitization classification is most appropriate for MDP.

 

References:

Johnson W, Boyer I, Bergfeld WF, et al. Safety Assessment of Phosphoric Acid and Its Salts as Used in Cosmetics. International Journal of Toxicology. 2021; v. 40(1 supp):34S-85S

Kimber, I., The activity of methacrylate esters in skin sensitization test methods: a review. Regulatory Toxicology and Pharmacology. 2019; v. 104; pp. 14-20.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2023

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source

Justification for type of information:

1. SOFTWARE : OECD QSAR Toolbox Version 4.5

2. MODEL: Automated workflow for EC3 from LLNA or Skin sensitization from GPMT assays for defined approaches (SS AW for DASS)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(=C)C(=O)OCCCCCCCCCCOP(O)(O)=O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL

- Defined endpoint: Skin sensitization
- Unambiguous algorithm: See attached prediction report and DASS protocol documentation attachments.
- Defined domain of applicability: See attached prediction report and DASS protocol documentation attachments. The profiling method deemed the structure to be inside the applicability domain of the profiling scheme.
- Appropriate measures of goodness-of-fit and robustness and predictivity: See attached prediction report and DASS protocol documentation attachments.
- Mechanistic interpretation: See attached prediction report and DASS protocol documentation attachments.

5. APPLICABILITY DOMAIN
See attached prediction report and DASS protocol documentation attachments.

6. ADEQUACY OF THE RESULT
This prediction was utilized as part of a larger Weight of Evidence to determine the skin sensitization potency of MDP. See the endpoint summary (7.2) for more detail.

Qualifier:

no guideline available

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR Toolbox Version 4.5.
- Model(s) used: Automated workflow for EC3 from LLNA or Skin sensitization from GPMT assays for defined approaches (SS AW for DASS)
- Model description: see field 'Justification for non-standard information'
- Justification of QSAR prediction: see field 'Justification for type of information'

GLP compliance:

no

Specific details on test material used for the study:

NA: Computational profiling prediction.

Key result

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the result of the prediction, 10-(phosphonooxy)decyl methacrylate is not expected to be a skin sensitizer.

Executive summary:

The skin sensitization potential of 10-(phosphonooxy)decyl methacrylate was predicted in the OECD QSAR Toolbox EC3 from LLNA or Skin sensitization from GPMT assay for defined approaches (SS AW for DASS) automated workflow. Based on the result of the prediction, 10-(phosphonooxy)decyl methacrylate is not expected to be a skin sensitizer.

Reference 2

Endpoint:

skin sensitisation, other

Type of information:

other: See 'Remarks'

Remarks:

Computational profiling results and literature data were utilized as part of a Weight of Evidence approach to address skin sensitization.

Adequacy of study:

weight of evidence

Study period:

2023

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: See 'Remarks'

Remarks:

Standard profiling alerts from OECD Toolbox and literature data were utilized to address skin sensitization potential.

Justification for type of information:

MDP is a multi-constituent substance that contains four main components, each with methacrylate functional groups and three with phosphate functional groups. Since the DPRA, h-CLAT and KeratinoSens assays (OECD 442 series) have not been validated for use with multi-constituent substances, alternative data sources were compiled to determine the skin sensitization potential of MDP while avoiding unnecessary animal testing. Methacrylates are generally well-documented to be either weak skin sensitizers or nonsensitizers (Kimber et al., 2019). Phosphate compounds are generally accepted to be nonsensitizers (Johnson et al., 2021) so the presence of the phosphate functionality in several of the minor components of MDP is not expected to add to the sensitization potential of the overall multi-constituent substance.

The skin sensitization of this multi-constituent substance (EC 944-391-4) was evaluated using a Weight of Evidence (WoE) approach to avoid unnecessary animal testing. An LLNA was conducted with the major constituent of this substance (CASRN 6701-13-9 is 60-70% of the multi-constituent) and the results indicate that it is a GHS Category 1B sensitizer. Three additional constituents are present at lower levels in the multi-constituent (see section 1.2 of the dossier for full details). The skin sensitization potential of the three minor constituents were addressed via the OECD QSAR Toolbox DASS Protocol, computational profiling and literature data (see dossier section 7.4 endpoint summary for a summary of the WoE argument).

The four main constituents of MDP are:

1: 1,10-Decanediol-dimethacrylate: An LLNA was conducted with this component (GHS Category 1B sensitizer)
2: 10-Methacryloyl-oxy-decylphosphate (OECD QSAR Toolbox DASS Protocol - not expected to be a skin sensitizer based on profiling)
3: Bis-(10-methacryloyl-oxy-decyl)-phosphate (OECD QSAR Toolbox Profilers - same sensitization alerts returned as the main constituent)
4: Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate (OECD QSAR Toolbox Profilers - same sensitization alerts returned as the main constituent)

The skin sensitization potential of Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate were evaluated using the skin sensitization and protein binding profiling schemes available in OECD QSAR Toolbox v. 4.5. Both substances returned the same profiling alerts for skin sensitization and protein binding as the main constituent (1,10-Decanediol-dimethacrylate) for which there is experimental data indicating that it is a weak skin sensitizer (GHS Category 1B). Additionally, literature reviews summarizing the skin sensitization potential of methacrylates and phosphate containing structures was reviewed to determine if the two lowest level components of the multi-constituent MDP (Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate) would be expected to add to the overall skin sensitization potential of the substance. Based on Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate returning the same structural alerts for sensitization as 1,10-Decanediol-dimethacrylate, the documented skin sensitization profiles of the functional moieties present in the structures (methacrylate and phosphate groups), and the inverse relationship between molecular weight and skin sensitization potency, Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate are not expected to increase the overall skin sensitization potency of MDP relative to the known potency of the major constituent.

References:

Johnson W, Boyer I, Bergfeld WF, et al. Safety Assessment of Phosphoric Acid and Its Salts as Used in Cosmetics. International Journal of Toxicology. 2021;v. 40(1 supp):34S-85S

Kimber, I., The activity of methacrylate esters in skin sensitization test methods: a review. Regulatory Toxicology and Pharmacology. 2019; v. 104; pp. 14-20.

Qualifier:

no guideline required

Principles of method if other than guideline:

The skin sensitization potential of Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate were evaluated using the skin sensitization and protein binding profiling schemes available in OECD QSAR Toolbox v. 4.5. Both substances returned the same profiling alerts for skin sensitization and protein binding as the main constituent (1,10-Decanediol-dimethacrylate) for which there is experimental data indicating that it is a weak skin sensitizer (GHS Category 1B). Additionally, literature reviews summarizing the skin sensitization potential of methacrylates and phosphate containing structures was reviewed to determine if the two lowest level components of the multi-constituent MDP (Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate) would be expected to add to the overall skin sensitization potential of the substance.

GLP compliance:

no

Type of study:

other: Computational profiling and literature data from review articles used as part of a Weight of Evidence.

Key result

Remarks on result:

other: See 'Remarks'

Remarks:

Positive indication of weak dermal sensitization potential for high molecular weight methacrylates.

Interpretation of results:

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

Conclusions:

The skin sensitization potential of Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate were evaluated using the skin sensitization and protein binding profiling schemes available in OECD QSAR Toolbox v. 4.5. Both substances returned the same profiling alerts for skin sensitization and protein binding as the main constituent (1,10-Decanediol-dimethacrylate) for which there is experimental data indicating that it is a weak skin sensitizer (GHS Category 1B). Additionally, literature reviews summarizing the skin sensitization potential of methacrylates and phosphate containing structures was reviewed to determine if the two lowest level components of the multi-constituent MDP (Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate) would be expected to add to the overall skin sensitization potential of the substance. Based on Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate returning the same structural alerts for sensitization as 1,10-Decanediol-dimethacrylate, the documented skin sensitization profiles of the functional moieties present in the structures (methacrylate and phosphate groups), and the inverse relationship between molecular weight and skin sensitization potency, Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate are not expected to increase the overall skin sensitization potency of MDP relative to the known potency of the major constituent.

Executive summary:

MDP is a multi-constituent substance that contains four main components, each with methacrylate functional groups and three with phosphate functional groups.  Since the DPRA, h-CLAT and KeratinoSens assays (OECD 442 series) have not been validated for use with multi-constituent substances, alternative data sources were compiled to determine the skin sensitization potential of MDP while avoiding unnecessary animal testing.  Methacrylates are generally well-documented to be either weak skin sensitizers or nonsensitizers (Kimber et al., 2019). Phosphate compounds are generally accepted to be nonsensitizers (Johnson et al., 2021) so the presence of the phosphate functionality in several of the minor components of MDP is not expected to add to the sensitization potential of the overall multi-constituent substance.

 

The skin sensitization of this multi-constituent substance (EC 944-391-4) was evaluated using a Weight of Evidence (WoE) approach to avoid unnecessary animal testing.  An LLNA was conducted with the major constituent of this substance (CASRN 6701-13-9 is 60-70% of the multi-constituent) and the results indicate that it is a GHS Category 1B sensitizer.  Three additional constituents are present at lower levels in the multi-constituent (see section 1.2 of the dossier for full details).  The skin sensitization potential of the three minor constituents were addressed via the OECD QSAR Toolbox DASS Protocol, computational profiling and literature data (see dossier section 7.4 endpoint summary for a summary of the WoE argument).

The four main constituents of MDP are:

1: 1,10-Decanediol-dimethacrylate: An LLNA was conducted with this component (GHS Category 1B sensitizer)

2: 10-Methacryloyl-oxy-decylphosphate (OECD QSAR Toolbox DASS Protocol - not expected to be a skin sensitizer based on profiling)

3: Bis-(10-methacryloyl-oxy-decyl)-phosphate (OECD QSAR Toolbox Profilers - same sensitization alerts returned as the main constituent)

4: Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate (OECD QSAR Toolbox Profilers - same sensitization alerts returned as the main constituent)

The skin sensitization potential of Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate were evaluated using the skin sensitization and protein binding profiling schemes available in OECD QSAR Toolbox v. 4.5.  Both substances returned the same profiling alerts for skin sensitization and protein binding as the main constituent (1,10-Decanediol-dimethacrylate) for which there is experimental data indicating that it is a weak skin sensitizer (GHS Category 1B).  

1,10-Decanediol-dimethacrylate (main constituent), Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate all returned the following skin sensitization alerts when profiled using OECD QSAR Toolbox v 4.5.

General Mechanistic:

Protein binding by OASIS and OECD: Michael addition

Protein binding potency Cys: DPRA above 21%

Endpoint Specific:

Protein binding potency GHS: Slightly reactive (GSH)

Protein binding alerts for skin sensitization according to GHS: Skin Sensitization Category 1A

Protein binding potency h-CLAT: alpha, beta-Unsaturated esters

These alerts were common to 1,10-Decanediol-dimethacrylate, Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate with 1,10-Decanediol-dimethacrylate having experimental data indicating that it is a GHS Category 1B sensitizer.

Additionally, toxicological literature summarizing the skin sensitization potential of methacrylate and phosphate functional group-containing structures was reviewed to determine if the two lowest level components of the multi-constituent MDP (Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate) would be expected to add to the overall skin sensitization potential of the substance. Generally, methacrylates are well-documented to be weak skin sensitizers (as demonstrated by1,10-Decanediol-dimethacrylate) or nonsensitizers (Kimber et al., 2019). Some higher molecular weight methacrylates (i.e., isodecyl methacrylate and dodecyl methacrylate) are not skin sensitizers in the local lymph node assay, indicating a trend that skin sensitization potency of methacrylates may decrease as molecular weight increases. Both Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate have much higher molecular weights (546 and 627 g/mol, respectively) when compared to isodecyl methacrylate and dodecyl methacrylate (226 and 254 g/mol, respectively). Furthermore, both are higher in molecular weight than the main component of MDP, 1,10-Decanediol-dimethacrylate, which has a molecular weight of 310 g/mol. Based on Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate returning the same structural alerts for sensitization as 1,10-Decanediol-dimethacrylate, the documented skin sensitization profiles of the functional moieties present in the structures (methacrylate and phosphate groups), and the inverse relationship between molecular weight and skin sensitization potency, Bis-(10-methacryloyl-oxy-decyl)-phosphate and Bis-(10-methacryloyl-oxy-decyl)-P,P’-diphosphate are not expected to increase the overall skin sensitization potency of MDP relative to the known potency of the major constituent.

References:

Johnson W, Boyer I, Bergfeld WF, et al. Safety Assessment of Phosphoric Acid and Its Salts as Used in Cosmetics. International Journal of Toxicology. 2021;v. 40(1 supp):34S-85S

Kimber, I., The activity of methacrylate esters in skin sensitization test methods: a review. Regulatory Toxicology and Pharmacology. 2019; v. 104; pp. 14-20.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

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:

22 July, 2010

Deviations:

no

Remarks:

No deviations that adversely impacted the study results ocurred.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source not reported, Batch E1026992VS
- Purity, including information on contaminants, isomers, etc.: Reported as "Considered 100%" in the study report.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70% RH)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicle was assessed: AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted). The formulation at 50% (w/v) using AOO as vehicle was suitable for the test. The 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) formulations appeared to be solutions by visual examination. The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd. Analytical determination of the test item concentration, stability and homogeneity was not performed
because of the character and the short period of study

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
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 / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 18.3 – 21.2 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 7 days
Note: In the Preliminary Experiment, mice of 12 weeks of age (22.9-25.0 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.6 – 25.6°C
Relative humidity: 30 - 80 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Food and feeding: Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” produced by ssniff Spezialdiäten GmbH, 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 supply: Animals received tap water from the municipal supply from 500 mL bottles, ad libitum.
Bedding: Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).
Identification and randomisation: A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups.
The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Vehicle:

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

Concentration:

The formulation at 50% (w/v) using AOO as vehicle was suitable for the test.

No. of animals per dose:

Preliminary Irritation/Toxicity Test: 2 animals/dose
Main Test: 4 animals/dose

Details on study design:

Formulation
The solubility of the test item was examined in a short Preliminary Compatibility Test.
The following standard OECD vehicle was assessed: AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted). The formulation at 50% (w/v) using AOO as vehicle was suitable for the test.
The 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) formulations appeared to be solutions by visual examination.
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100% (undiluted) and 50% (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 and the radioactive proliferation assay was not performed.
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. Both ears of each mouse were observed for erythema and scored (see “Any other information” for Erythema Scoring table). 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.
During the Preliminary Irritation / Toxicity Test, no mortality was observed. No test item precipitate was observed on the ears of the animals.
Marked body weight loss (>5% decrease) was detected for both animals in the 100% (undiluted) and 50 % (w/v) groups. The mean body weight loss of these groups was more than 5 %. There were no indications of any irritancy at the site of application.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The ear thickness values and ear punch weights were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were slightly enlarged than normal for both animals of the 100% (undiluted) and for one animal of the 50% (w/v) dose groups and they were normal for the other animal of the 50% (w/v) dose group (subjective judgement by analogy with observations of former experiments).
Taking into account these results, the body weight difference was close to the 5% weight loss in 2 animals of the 100% (undiluted) and in 2 animals of the 50% (w/v) dose group. With the small group sizes in the preliminary test, it is difficult to be certain where the threshold for systemic toxicity exists; hence, to try to be sure of a valid main study and according to the Study Plan, five dose groups will be used in the main experiment so there should be three acceptable dose groups for evaluation. Therefore, 100% (undiluted), 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) doses will be examined in the main test.

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 euthanized 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 each mouse were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of 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). 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 resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended 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 ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The ß-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
The proliferative response of lymph node cells from the lymph nodes of each individual animal is expressed as radioactive disintegrations per minute (DPM) per animal. 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 disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal following the industry standard for data presentation.
The stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
The use of the individual approach to calculate the SI made the use of a statistical analysis available.

Interpretation of Results
The test item is regarded as a sensitizer 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:

The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values.
Note: In the report, the mean DPM values are shown, but all the data of the individual measurements are kept and archived in the raw data binder.
Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of intergroup differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorow-Smirnow test. In the case of no normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using the Mann-Whitney U-test.

Positive control results:

The result of the positive control substance a-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/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 12.0) 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 group included 4 animals.

Key result

Parameter:

EC3

Value:

17.5

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the main study. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the mean body weight changes in the main study.

PROLIFERATION ASSAY
The appearance of the lymph nodes was larger than normal for all animals of the 100% (undiluted) dose group and for three animals of the 50% (w/v) dose group and in the positive control group. They were slightly enlarged than normal for one animal of the 50% (w/v) dose group and for all animals of the 25% (w/v) dose group, and they were normal for all animals of the 10% (w/v) and 5% (w/v) dose groups and in the negative control group.
The stimulation index values were 7.0; 6.7; 4.4; 1.6 and 2.2 at concentrations of 100% (undiluted), 50% (w/v) 25 % (w/v), 10% (w/v) and 5% (w/v) respectively.

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
2292 2314 2306 2312	1 2 3 4	Negative (vehicle) control AOO     Mean	21.2 19.6 19.2 18.9 19.7	22.5 20.4 20.2 20.6 20.9	6.1 4.1 5.2 9.0 6.1
2377 2295 2305 2311	5 6 7 8	1,10-decanediyl bis methacrylate 100% (undiluted)     Mean	21.2 19.6 19.2 18.6 19.7	21.0 20.1 20.3 19.9 20.3	-0.9 2.6 5.7 7.0 3.6
2293 2378 2304 2379	9 10 11 12	1,10-decanediyl bis methacrylate 50 (w/v)% in AOO     Mean	21.2 19.2 19.8 18.8 19.8	22.0 21.3 21.4 19.8 21.1	3.8 10.9 8.1 5.3 7.0
2299 2307 2316 2300	13 14 15 16	1,10-decanediyl bis methacrylate 25 (w/v)% in AOO     Mean	21.1 20.6 19.8 18.3 20.0	22.0 20.9 21.2 19.2 20.8	4.3 1.5 7.1 4.9 4.4
2308 2302 2369 2376	17 18 19 20	1,10-decanediyl bis methacrylate 10 (w/v)% in AOO     Mean	20.8 20.0 19.4 19.6 20.0	21.1 20.8 21.0 21.2 21.0	1.4 4.0 8.2 8.2 5.5
2320 2353 2317 2351	21 22 23 24	1,10-decanediyl bis methacrylate 5 (w/v)% in AOO     Mean	20.4 20.3 20.2 18.9 20.0	21.6 20.2 21.8 19.1 20.7	5.9 -0.5 7.9 1.1 3.6
2355 2318 2366 2354	25 26 27 28	Positive control 25 (w/v)% HCA in AOO     Mean	20.5 20.4 19.0 19.4 19.8	21.9 20.7 20.5 19.1 20.6	6.8 1.5 7.9 -1.5 3.7

Notes:

^*: Terminal body weights were measured on Day 6

^#: =(Terminal Body Weights – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Identity Number	Mean DPM^Note	Number of lymph nodes	DPN	Mean DPN	Stimulation Index
Background (5% (w/v) TCA)	-	-	-	-	-	-
Negative control (AOO)	1 2 3 4	724.0 705.5 1552.0 476.5	2 2 2 2	362.0 352.8 776.0 238.3	432.3	1.0
1,10-decanediyl bis methacrylate 100% (undiluted)	5 6 7 8	4443.5 5096.5 5158.5 9531.5	2 2 2 2	2221.8 2548.3 2579.3 4765.8	3028.8	7.0^*
1,10-decanediyl bis methacrylate 50 (w/v)% in AOO	9 10 11 12	1388.0 5409.5 9606.0 6653.0	2 2 2 2	694.0 2704.8 4803.0 3326.5	2882.1	6.7^*
1,10-decanediyl bis methacrylate 25 (w/v)% in AOO	13 14 15 16	3351.5 3426.5 5367.5 3091.5	2 2 2 2	1675.8 1713.3 2683.8 1545.8	1904.6	4.4^*
1,10-decanediyl bis methacrylate 10 (w/v)% in AOO	17 18 19 20	795.0 2279.0 1771.5 797.5	2 2 2 2	397.5 1139.5 885.8 398.8	705.4	1.6^NS
1,10-decanediyl bis methacrylate 5 (w/v)% in AOO	21 22 23 24	2148.5 1953.5 793.0 2637.5	2 2 2 2	1074.3 976.8 396.5 1318.8	941.6	2.2^*
Positive control (25% HCA) in AOO	25 26 27 28	7507.5 9976.5 14103.0 9979.5	2 2 2 2	3753.8 4988.3 7051.5 4989.8	5195.8	12.0^*

Notes:

1. NS = Not Significant (p<0.05, Mann-Whitney U-Test versus negative control)

2. * = Significant (p<0.05, Mann-Whitney U-Test versus negative control)

3. ** = Significant (p<0.01, Mann-Whitney U-Test versus negative control)

4. Note: The background values were in the 32 – 35 range.

 

Summarized Clinical Observations

Group	Animal No.	Identity No.	CLINICAL OBSERVATIONS
			DAY 1	DAY 2	DAY 3	DAY 4	DAY 5	DAY 6
Negative control (AOO)	2292   2314   2306   2312	1   2   3   4	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 100% (undiluted)	2377   2295   2305   2311	5   6   7   8	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 50 (w/v)% in AOO	2293   2378   2304   2379	9   10   11   12	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 25 (w/v)% in AOO	2299   2307   2316   2300	13   14   15   16	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 10 (w/v)% in AOO	2308   2302   2369   2376	17   18   19   20	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 5 (w/v)% in AOO	2320   2353   2317   2351	21   22   23   24	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
Positive control (25% (w/v) HCA in AOO)	2355   2318   2366   2354	25   26   27   28	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free

Note:

1. BT: before treatment; AT: after treatment

 

Results of the Preliminary Irritation / Toxicity Test

 

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
2075 2084	1 2	100% (undiluted) 100% (undiluted) Mean	24.5 24.0 24.3	23.2 22.8 23.0	-5.3 -5.0 -5.2
2067 2074	3 4	50% (w/v) 50% (w/v) Mean	25.0 22.9 24.0	23.7 21.2 22.5	-5.2 -7.4 -6.3

Notes:

1.^*: Terminal body weights were measured on Day 6.

2.^#= (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals

Animal Number	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
			R	L	R	L	R	L
2075 2084 2067 2074	1 2 3 4	100% (undiluted) 100% (undiluted) 50% (w/v) 50% (w/v)	0.21 0.21 0.21 0.21	0.21 0.22 0.21 0.21	0.23 0.23 0.22 0.22	0.23 0.23 0.21 0.22	0.21 0.21 0.22 0.22	0.22 0.22 0.21 0.22	17.34 16.78 15.67 16.20

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (=25%)

2. R: Right; L: Left

 

Summarized Clinical Observations

Period	Group	Identity No.	Animal No.	Clinical observations
DAY 1	100% (undiluted)	1	2075	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	2084	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	50% (w/v)	3	2067	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	2074	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 2	100% (undiluted)	1	2075	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	2084	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	50% (w/v)	3	2067	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	2074	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 3	100% (undiluted)	1	2075	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	2084	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	50% (w/v)	3	2067	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	2074	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 4	100% (undiluted)	1	2075	Symptom-free, ES: 0
		2	2084	Symptom-free, ES: 0
	50% (w/v)	3	2067	Symptom-free, ES: 0
		4	2074	Symptom-free, ES: 0
DAY 5	100% (undiluted)	1	2075	Symptom-free, ES: 0
		2	2084	Symptom-free, ES: 0
	50% (w/v)	3	2067	Symptom-free, ES: 0
		4	2074	Symptom-free, ES: 0
DAY 6	100% (undiluted)	1	2075	Symptom-free, ES: 0
		2	2084	Symptom-free, ES: 0
	50% (w/v)	3	2067	Symptom-free, ES: 0
		4	2074	Symptom-free, ES: 0

Notes:

1. The clinical observations of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score.

 

 Historical Control Data

 

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2015)

 

CBA/CaOlaHsd mice
	Vehicles
	Acetone : Olive oil 4:1 (AOO)			1% Pluronic PE9200 in water (1% Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	415.2	2922.6	7.5	197.7	1825.3	10.0
Range:  min                max	111.3 847.8	890.3 7674.5	3.3 15.5	23.0 680.8	154.0 6755.8	3.0 33.1
Number of cases	32	32	30	134	134	128

 

	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	244.6	2522.6	10.8	488.7	3212.1	7.8
Range:  min                max	140.8 505.8	1201.3 4804.6	6.3 21.3	238.5 934.6	2017.2 4877.5	3.1 14.5
Number of cases	21	21	21	13	13	12

 

	Vehicles
	Propylene glycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	235.4	2371.8	10.0	260.2	4888.8	19.5
Range:  min                max	63.3 506.0	817.3 4978.0	6.5 14.4	183.5 383.3	2456.3 8682.5	8.9 36.3
Number of cases	14	14	14	9	10	10

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Interpretation of results:

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

Conclusions:

When tested according to OECD 429, 1,10-decanediyl bis methacrylate was shown to have sensitization potential. The calculated EC3 value of 1,10-decanediyl bis methacrylate is 17.5%(w/v).

Executive summary:

The skin sensitization potential of 1,10-decanediyl bis methacrylate was evaluated in mice in a local lymph node assay. The study was conducted according to OECD 429 in compliance with OECD GLP.

 Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100% (undiluted). The formulation at 50% (w/v) using AOO as vehicle was suitable for the test. 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100% (undiluted) and 50% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 100% (undiluted) was selected as top dose for the main test. In the main assay, twenty eight female CBA/CaOlaHsd mice were allocated to seven groups of four animals each:

- five groups received 1,10-decanediyl bis methacrylate at 100% (undiluted); or diluted in AOO at 50% (w/v), 25% (w/v), 10% (w/v) and 5%(w/v) concentrations,

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

- the positive control group received 25 % (w/v) HCA (dissolved in 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. 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 signs of systemic toxicity were observed during the study. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals. No treatment related effects were observed on the mean body weight changes in the main study.

The stimulation index values were 7.0; 6.7; 4.4; 1.6 and 2.2 at concentrations of 100% (undiluted), 50% (w/v) 25 % (w/v), 10% (w/v) and 5% (w/v) respectively.

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present 1,10-decanediyl bis methacrylate was shown to have sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of present 1,10-decanediyl bis methacrylate is 17.5%(w/v).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

The overall weight of evidence suggests that a GHS Category 1B skin sensitization classification is most appropriate for MDP.