Registration Dossier

Administrative data

Description of key information

An in vitro test according to OECD 442 D showed no sensitising properties of the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1).

Due to the bad solubility of the test item no other OECD confirm in vitro test was possible to conduct. Therefore in vivo testing was required to determine the sensitising properties of the test item.

An in vivo test according to OECD 429 was considered not sufficient clear for concluding definitely about the skin sensitizing potential of the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid). Therefore additionally an in vivo test according to OECD 406 was conducted. The test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4tricarboxylic acid 1,2-anhydride (3:1) was not skin sensitizing.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-11-19 - 2018-11-30
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 05347/MA
- Expiration date of the lot/batch: retest date 2022-06-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature and darkness

Details on study design:
The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
Reference items:
Negative control: each culture plate contains 6 wells of solvent control (treatment medium with 1% DMSO final)
Treatment culture medium, 1% DMSO, 1% Non-heat inactivated foetal calf serum. The negative control is prepared just prior the test and used within the day.
Positive control: for each culture plate, the cinnamaldehyde is tested at 5 concentrations from 4 to 64 µM according to a geometric progression of ratio 2.
Blanks: 1 well by culture plate is left without cell and filled with negative control to assess background value.

The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

Test system:
Cells: KeratinoSens™ (Givaudan) maintained according to the current instruction of the test lab.
Cells are cultured in maintenance medium at 37°C, 5% CO2.
Cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current instruction of the test lab.
Cells were used at passage 17 in repetition 1 and passage 19 in repetition 2.

Media and reagents:
Stored at room temperature 20°C ± 5°C until opening
Dulbecco’s PBS Ca2+ and Mg2+ free*
DMSO CAS N° 67-68-5
Desorption solution: 10% SDS in water
EDTA CAS N°6381-92-6

Stored at 5°C ± 3°C
DMEM 1 g/l glucose
MTT powder CAS N°298-93-1
Geneticin 50 mg/ml
Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin
Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum
Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum
Dulbecco’s PBS Ca2+ and Mg2+ free complemented with 0.05% EDTA

Stored at - 20°C ± 5°C until opening
Non-heat inactivated foetal calf serum*
Trypsine (0.5 g/l) - EDTA (0.2 g/l)*

Others storage conditions
Luciferase substrate (Promega):
- Lyophilized Bright-Glo™ Substrate: - 20°C ± 5°C
- Bright-Glo™ Buffer: Room temperature 20°C ± 5°C
- Reconstituted reagent: - 80°C ± 10°C
Staining solution: 5 mg/ml MTT in solution in PBS - extemporaneously prepared and used within the day

* Stored at 5°C ± 3°C after opening.

The expiry after opening of the media and reagents used in the study is defined in the form FL REAC 05.

Equipment and consumables
Luminometer: GloMax™ (Promega)
MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -linearity range 0 - 2.200 units of Absorbance
White cell culture 96-well plates for luminescence reading
Transparent cell culture 96-well plates for absorbance reading
Plastic adhesive foils
Conventional cell culture laboratory equipment.

The equipment used is recorded in the study notebook.

Test protocol:
Cells seeding (first day)
The cells were harvested according to the current working instruction of the test lab. After removal the culture medium from the culture flask, the cell layer was rinsed with PBS 0.05% EDTA, Ca2 + and Mg² + free to remove all traces of serum. The solution of trypsin-EDTA was added and left for a few minutes at 37°C, 5% CO2 until the detachment of cells. The action of trypsin was stopped by addition of maintenance medium.
Cell concentration was determined on Malassez cell. Cells suspension was adjusted to a density of 8.104 cells/ml in seeding medium.
125 µl of the cell suspension at 8.104 cells/ml (i.e. 104 cells per well) were distributed in three white cell culture plates (96 wells) for the induction measurement and two transparent cell culture plates (96 wells) to assess the cytotoxicity. In order to ensure a good homogeneity of seeding, cells suspension was regularly mixed all along the seeding.
The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2.
Note: the H12 wells were left without cells and will enable the measurement of blanks.

Preparation of test item and positive control dilutions (second day)
Preparation of the test item stock solution
The stock solution was prepared at 200 mM in DMSO.

A volume of DMSO was calculated according to the following formula:

V = 5 x (p ÷100 ) x w /MW - w /1000

V is the volume of DMSO in ml to be added
p is the purity of the test item in %
MW is the molecular weight of the test item in g/mol
w is the exact weight of the test item in mg.

Preparation of the positive control stock solution
The positive control stock solution was prepared at 200 mM in DMSO according to the formula above then diluted to 6.4 mM in DMSO.

Preparation of the 100 X plate
A 100-fold concentrated dilutions series was prepared in 96-well plate.

Test item
The test item was placed in one of the rows B to F.
100 µl of DMSO were distributed from columns 1 to 11. 200 µl of the 200 mM stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl from column 12 to column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.

Positive control
100 µl of DMSO were distributed in row G from columns 7 to 10. 200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl from column 11 to column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.

Negative control
100 µl of DMSO were distributed row G from columns 1 to 6 and in the well H12.

Preparation of the 4 X dilution plate
The 100 X DMSO plate was diluted 25 fold in a new plate (4 X) with treatment medium.

Contact between the cells and the test and reference items (second day)
In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

Luciferase activity (day 4)
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferin + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis.
The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

Cell viability assessment with MTT method (day 4)
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5 mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37°C, 5% CO2).
After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.





Positive control results:
Test validation/historical data
• the gene induction must be statistically significant above the threshold of 1.5 in at least one of the tested concentration,
• the EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Follow-up of positive control: Cinnamaldehyde (CAS N°104-55-2):
Updated on 09/04/2018 - N = 233

Imax EC1.5 IC70
Mean value 4.94 12.38 µM > 64 µM
Standard deviation 2.91 4.71 -

Mean EC1.5 value ± 2 SD: 3 µM ≤ EC1.5 ≤ 22 µM

OECD validation dataset: between 7 µM and 30 µM.

Test results:
Reference item

Cinnamaldehyde 4 µM 8 µM 16 µM 32 µM 64 µM EC1.5 Imax
Rep 1 1.44 1.55 2.09 3.13 5.11 6.15 5.11
Rep 2 1.33 1.47 2.00 2.96 4.52 8.49 4.52
Mean 1.38 1.51 2.04 3.05 4.82 7.23* 4.82
*geometric mean



Key result
Parameter:
other: Imax
Remarks:
IC50 > 2000 µM; IC30 > 2000 µM
Run / experiment:
Rep 1
Value:
1.23
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Imax is lower than 1.5, no EC1.5 is determined
Key result
Parameter:
other: Imax
Remarks:
IC50 > 2000 µM: IC30 > 2000 µM
Run / experiment:
Rep 2
Value:
1.16
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Imax is lower than 1.5, no EC1.5 is determined
Key result
Parameter:
other: Imax
Run / experiment:
Mean
Value:
1.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Since all validity criteria are met, study is considered as valid.

No deviation or amendment to the Study Plan has been observed during this study.

Interpretation of the results:
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. Otherwise the KeratinosensTM prediction is considered as negative:

• the Imax is equal to or higher than 1.5 times and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s t-test on the raw RLU values),

• the EC1.5 value is strictly below 1000 µM,

• at the lowest concentration with a gene induction equal to or higher than 1.5, the cell viability must be strictly higher than 70%,

• there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.


Negative Control
The average coefficient of variation of the luminescence reading for the solvent controls (3 x 6 wells) should be below 20% in each repetition.

If for one repetition the validity criteria are not met, or in case of equivocal result additional repetitions should be considered.

The validation of the results is carried out by the Study Director according to the current working plan of the test lab.

Control solvent       CV %

control solvent

Rep 1                     10.5

Rep 2                     11.2

      

Interpretation of results:
GHS criteria not met
Conclusions:
Under the retained experimental conditions the test item REACTION PRODUCT OF GUERBET ALCOHOLS, C24-26, BRANCHED AND CYCLIC WITH 1,2,4-BENZENETRICARBOXYLIC ACID may be classified as not skin sensitizer.
Executive summary:

Under the retained experimental conditions the test item REACTION PRODUCT OF GUERBET ALCOHOLS, C24-26, BRANCHED AND CYCLIC WITH 1,2,4-BENZENETRICARBOXYLIC ACID may be classified as not skin sensitizer.

The test method KeratinoSensTM is considered scientifically valid to be used as part of an integrated approaches to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.

Endpoint:
skin sensitisation: in chemico
Type of information:
other: solubility test
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
pretest: Solubility test
Deviations:
not applicable
Remarks:
We can't obtain a 100 mM homogeneous limpid solution. The test item REACTION PRODUCT OF GUERBET ALCOHOLS, C24-26, BRANCHED AND CYCLIC WITH 1,2,4-BENZENETRICARBOXYLIC ACID cannot be tested according to the DPRA method.
GLP compliance:
no
Type of study:
other: pretest for the OECD 442C
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 05347/MA
- Expiration date of the lot/batch: 01/06/2022
Details on study design:
Expected test conditions according to the OECD guideline 442C
Solubility of the test chemical in an appropriate solvent should be assessed before performing the assay. An appropriate solvent will dissolve the test item completely. Visual inspection of the forming of a clear solution is considered sufficient to ascertain that the test item is dissolved. Acetonitrile is preferred, aiming a 100 mM concentration of the test item. Sonication (1 mn or less) may be used to help the solubilization. In the case where the test item is not soluble in acetonitrile, the following solvents must be successively tested (concentration of the test item 100 mM):

 Water (except for anhydrous product)
 Water/acetonitrile 1:1 (except for anhydrous product)
 Isopropanol
 Acetone

The test item has to be weighed in a glass vial and will be dissolved in the chosen solvent in order to prepare a 100 mM solution.
The weight is calculated according to the following formula:

Volume (ml) =0.01 × Vf × MW/Purity × Density

MW: molecular weight (g/mol) Vf: final volume (ml) Purity: purity of the test item (%). If unknown, it will be considered equal to 100%. Density of the test item (g/ml). If unknown, it will be assessed by weighing 1ml.

In case when a limpid stock solution is obtained, it is then diluted in peptide buffer for the HPLC analysis, ammonium buffer (lysine peptide / 1:50 ratio) and phosphate buffer (cysteine peptide / 1:10).

Solubilty test

Preliminary to the study, the solubility at 100 mM (i.e. stock solution) was assessed:

Step 1: - 412 µL into 3 mL of Acetonitrile (vial 1): Not soluble - Sonication: Not soluble

Step 2: - 412 µL into 3 mL of Water (vial 2): Not soluble - Sonication: Not soluble

Step 3: - Not tested because step 1 and 2 are not soluble

Step 4: - 274 µL into 2 mL of Isopropanol (vial 3): Not Soluble - Sonication: Not soluble

Step 5: - 274 µL into 2 mL of Acetone (vial 4) : Not soluble - Sonication: Not soluble

Interpretation of results:
other: test item cannot be tested in the DPRA-test because it is not soluble.
Conclusions:
We can't obtain a 100 mM homogeneous limpid solution. The test item REACTION PRODUCT OF GUERBET ALCOHOLS, C24-26, BRANCHED AND CYCLIC WITH 1,2,4-BENZENETRICARBOXYLIC ACID cannot be tested according to the DPRA method.
Executive summary:

We can't obtain a 100 mM homogeneous limpid solution. The test item REACTION PRODUCT OF GUERBET ALCOHOLS, C24-26, BRANCHED AND CYCLIC WITH 1,2,4-BENZENETRICARBOXYLIC ACID cannot be tested according to the DPRA method.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019-01-31 to 2019-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
05347/MA
- Expiration date of the lot/batch:
retest date 2022-06-01


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature and darkness

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941 Le Genest Saint Isle)
- Age at study initiation: 9 weeks
- Weight at study initiation: 19.9-23.4 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ENVIGO 2016 ad libitum
- Water: tap water from public distribution system ad libitum (Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE))
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2019-01-31 To: 2019-02-07
Vehicle:
methyl ethyl ketone
Concentration:
25; 50: 100 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse with the highest technically possible concentration. The mouse was treated by daily application of 25 µL of the test item undiluted (100%) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1 to day 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6. The concentration of 100% was chosen as the highest concentration for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: cell counting (The experimental protocol was established according to the O.E.C.D. Test Guideline No. 429 dated 22 July 2010 and to the Test method B.42 - Council regulation No 640/2012 of 06 July 2012 (E.U. Journal L193))
- Criteria used to consider a positive response: stimulation index (SI) >=1.4; together with consideration of dose-response, irritation level

TREATMENT PREPARATION AND ADMINISTRATION
Groups of four mice were treated with the test item undiluted (100%) and diluted at 50% and 25% in methyl ethyl ketone. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
Termination: On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes from the four mice were excised.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of DPBS (Ca2+ / Mg2+ - free) containing 0.5% BSA into a well of a multi-well 6. 10 µL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counted using a cell counter (Beckman Coulter Z2). For the run, the lower size selected was 5 µm and the upper size selected was 15 µm (the average size of a lymphocyte is 8 µm).

Stimulation index determination

The proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Results are expressed as the Stimulation Index (SI).

When using the pooled approach, the SI is calculated according to the following formula:
SI = cell count of treated group/cell count of control group

The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI > 1.4 will be classified as a "non-sensitiser".

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no statitistics performed
Positive control results:
The methods used on this study were periodically checked in the testing facility. Stimulation indices in recent positive control studies were 07 november to 12 November 2018 at concentrations of 5, 10 and 25% hexyl cinnamic aldehyde, thereby indicating that the test method employed is valid.
On the 12.11.2018 at concentration of 5, 10 and 25 % hexy cinnamic aldehyde the SI were 0.94, 1.14 and 1.82, therefore the EC1.4 = 15.74%.
Key result
Parameter:
SI
Value:
1.44
Test group / Remarks:
Test group 4 with a concentration of 100 %
Remarks on result:
other: unclear borderline result, no dose-response
Key result
Parameter:
SI
Value:
1.82
Test group / Remarks:
positive control at a concentration of 25 %
Remarks on result:
other: positive control

No mortality and no sign of systemic toxicity were noted in the treated and control animals during the test. No increase in ear thickness and in ear weight was recorded at the concentrations of 25%, 50% and 100%.

Body weight gain for animals receiving test substance was similar to controls.

Groups   test item Cell count / groups (x106 cells / mL)   Stimulation Index SI  Result  EC1.4 value
 1  MEK  23.60  n.a.  n.a.  n.a.

 25 %

 24.56

 1.04

 Negative

 

 3

 50 %

 24.58

 1.04

 Negative

 

 4

 100 %

 33.93

 1.44

 Positive

 95 %

Interpretation of results:
study cannot be used for classification
Conclusions:
The result obtained, may be considered as a borderline result (very close to the cut-off value 1.4 validated in the laboratory). Furthermore, a dose-response relationship was not noted during this study. As a pooled approach was performed; it is not possible to perform a statistical analysis of the data. The results of this study, under these experimental conditions, may be considered not sufficient clear for concluding definitely about the skin sensitizing potential of the test item Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid.
Executive summary:

The test was performed to assess the skin sensitization potential of the test item Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid in the CBA/J strain mouse following topical application to the dorsal surface of the ear.

Three groups of four animals were treated for the three consecutive days (D1, D2, D3) with 50 µL (25µL per ear) of the test item undiluted (100%) and diluted at concentrations of 50% and 25% in methyl ethyl ketone (MEK).

A further group of four animals was treated with MEK.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting.

The experimental protocol was established according to the O.E.C.D. Test Guideline No. 429 dated 22 July 2010 and to the Test method B.42 - Council regulation No 640/2012 of 06 July 2012 (E.U. Journal L193).

No mortality and no sign of systemic toxicity were noted in the treated and control animals during the test.

No increase in ear thickness and in ear weight was recorded at the concentrations of 25%, 50% and 100%.

Therefore, the test item has to be considered as not excessively irritant at these concentrations in accordance with the OECD criteria.

The stimulation increase (SI) calculated by pooled approach was 1.04, 1.04 and 1.44 for the treated groups at 25%, 50% and 100%, respectively.

The EC1.4 determined by linear regression was 95.00%.

The result obtained, may be considered as a borderline result (very close to the cut-off value 1.4 validated in the laboratory). Furthermore, a dose-response relationship was not noted during this study. As a pooled approach was performed; it is not possible to perform a statistical analysis of the data.

The results of this study, under these experimental conditions, may be considered not sufficient clear for concluding definitely about the skin sensitizing potential of the test item Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.06. - 18.07.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
the LLNA test gave no clear result
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (F-69210 Saint Germain-Nuelles)
- Age at study initiation: 5 or 6 weeks
- Weight at study initiation: 245 to 308 g
- Housing: in polycarbonate containers, florring covered with dust-free cuttings, stainless steel lid
- Diet (ENVIGO, 2040C): ad libitum
- Water (tap water from public distribution system): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
unchanged (no vehicle)
Remarks:
1st Induction
Concentration / amount:
three pairs of intradermal injections (ID) of 0.1 ml were performed:
2 ID Freund's Complete Adjuvant diluted at 50 % in corn oil
2 ID corn oil (control) or test item at 100 %
2 ID a mixture with equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and corn oil (control) or
- Freund’s Complete Adjuvant at 50% and test item at 100 %
Day(s)/duration:
6 d
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Remarks:
2nd Induction
Concentration / amount:
day 6: sodium lauryl sulfate at 10% in thick Vaseline
day 7: 0.5 ml corn oil (control) or test item at 100 % under occlusive dressing for 48 hours
day 9: occlusive dressing was removed
Day(s)/duration:
14 d
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: 1 sample with 50 % corn oil and one sample without vehicle
Concentration / amount:
Day 20: 1 sample cup saturated with the test item at 100% (MNIC) for 24 hours and
1 sample cup saturated with the test item at 50% in corn oil (1/2 MNIC) for 24 hours respectively
Day 21: removal of occlusive dressing
Day 22: 1st reading time - 24 hours after patch removal
Day 23: 2nd reading time - 48 hours after patch removal
Day(s)/duration:
4 d
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (5 control)
Details on study design:
Preparation of animals
Fifteen male albino guinea pigs of Dunkin-Hartley strain were supplied Charles River (F-69210 Saint Germain-Nuelles). At the beginning of the main test, the animals were 5 or 6 weeks old. The animals were nulliparous.
Prior to the test, the animals were kept for a minimum acclimatization period of 5 days, under stabling and nutritional conditions identical to those of the test.
Before the experimentation process, they were identified individually by marking with picric acid and by means of a numbered ring on the edge of one ear.
The animals were carefully shorn before each test item application: - On the inter-scapular zone for the induction phase, - On the dorso-lumbar zone for the challenge phase.
At least 3 hours before the first reading (challenge phase) they were shorn a second time in this dorsolumbar zone.
The animals were weighed at the beginning of the test, after the second induction and at the end of the test.

Animal welfare
The standard study plan related to this study has been approved by the registered Ethics Committee No. 76.
The study was performed in accordance with the guidelines regarding the care and use of animals for experimental procedures: - the European Communities Council Directive 2010/63/EU of 22 September 2010, - the French Decree No. 2013-118 of 01 February 2013.
The animals were provided with suitable environmental enrichment (Tunnel).
The study was designed and was conducted to cause the minimum of suffering or distress to the animals, according to the guidelines and to our internal animal welfare’s procedure.
At the end of the test, the animals were euthanized with sodium pentobarbital (Dolethal®).

Preliminary studies
-Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC)
This test was conducted for the purpose of defining a MNNC of the test item which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotizing concentration), should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation.
Two animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: undiluted (100%) and diluted at 75%, 50% and 25% in corn oil in view to determine the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after the injections.

-Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC)
This test, which allowed evaluating the irritancy potential of the test item, defined whether an application of sodium lauryl sulfate would be needed during topical induction phase.
The test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: undiluted (100%) and diluted at 75%, 50% and 25% in corn oil.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.

-Determination by topical application of the Maximal Non Irritant Concentration (MNIC)
This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase. Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (control) for the induction phase (i.e. corn oil and corn oil).
During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: undiluted (100%) and diluted at 75%, 50% and 25% in corn oil.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressing.

Main study
GROUP 1 (negative control) : 5 male guinea pigs identified n° C6928 to C6932 GROUP 2 (treated) : 10 male guinea pigs identified n° C6933 to C6942

- Induction phase
--1st Intradermal Induction:
Day 0 After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:

GROUP 1 (Control):
• 2 ID: Freund’s Complete Adjuvant diluted at 50 % in corn oil
• 2 ID: corn oil
• 2 ID: a mixture with equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and corn oil

GROUP 2 (Treated):
• 2 ID: Freund’s Complete Adjuvant diluted at 50 % in corn oil
• 2 ID: test item at 100%
• 2 ID: a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the test item at 100%

---2nd Topical Induction:
Day 6 The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick Vaseline, in order to create a local irritation.
Day 7 A topical application under occlusive dressing (25mm x 25mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 hours was performed on the injection sites of each animal.

GROUP 1 (control): 0.5 mL of corn oil.
GROUP 2 (treated): 0.5 mL of the test item at 100%

Day 9 The occlusive dressing was removed.

-Rest phase
The animals of both groups were left for 9 days.

- Challenge phase
Day 20 The experimental procedure of this phase was identical for both groups GROUP 1 (Negative control) and GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application, under occlusive dressing, was performed during 24 hours: - 1 sample cup saturated with the test item at 100% (MNIC) and 1 sample cup saturated with the test item at 50% in corn oil (1/2 MNIC).
Day 21 Removal of the occlusive dressing.
Day 22 1st reading time – 24 hours after the patch removal.
Day 23 2nd reading time – 48 hours after the patch removal.


Challenge controls:
identical treatment
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde
Positive control results:
The results of the last 11 positive groups (Reference substance: alpha-Hexylcinnamaldehyde) carried out as method sensitivity. The last test was carried out on the 12.3.2019 and 5.4.2019 with a concentration of 75 % (30 %) in corn oil. After 24 h 50 % (60 %) of animals sensitized, after 48 h 40 % and 50 %. The results were clearly positive.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 % and 100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 % and 100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 % and 100 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 % and 100 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
75 %
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
the no in the group not mentioned, calculated from 50 %
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
75 %
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
the no in the group not mentioned, calculated from 40 %
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
30 %
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
the no in the group not mentioned, calculated from 60 % response
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
30 %
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
the no in the group not mentioned, calculated from 50 %

Preliminary studies

- MNNC determination: 24 hours after the injections, no cutaneous reaction was noted at the tested concentrations of 100%, 75%, 50% and 25%.

The first induction of the Group 2 was carried out by intradermal injection at the maximal non necrosing concentration of 100%.

- Pre MNIC determination (Table 2): 24 hours after the removal of the occlusive dressings, no cutaneous reaction was noted at the tested concentrations of 100%, 75%, 50% and 25%.

In view of these results, the concentration selected was 100% for the 2nd induction of the Group 2 and the MNIC determination began at the concentration of 100%.

- MNIC determination (Table 3): 24 and 48 hours after the removal of the occlusive dressings, no cutaneous reaction was noted whatever the tested concentrations of 100%, 75%, 50% and 25%.

In view of this result, the concentrations selected were 100% (MNIC) and 50% (1/2 MNIC) for the challenge phase.

Main study Assessment of the sensitising potential

Challenge phase

Table 1: Macroscopic evaluation (readings at 24 and 48 hours) of cutaneous reactions

Groups  Reading time/h  Concentrations

Incidence

0/ 1/ 2/ 3

% of  positive responses >= 1

% of animal sensitized# 

Group 1

Control

24

48

100 %

100 %

5 /0/ 0/ 0

5 /0/ 0/ 0

0

0

 

24

48

50 %

50 %

5  /0/ 0/ 0

5 /0/  0/ 0

0

0

 

Group 2

Treated

24

48

100 %

100 %

10/ 0/ 0/ 0

10/ 0/ 0/ 0        

0

0

0

0

24

48

50 %

50 %

10/ 0/ 0/ 0

10/ 0/ 0/ 0    

0

0

0

Grading scale

0 No visible change

1 Discrete or patchy erythema

2 Moderate and confluent erythema

3 Intense erythema and swelling

A comparison of the intensities and persistence of reactions at the test item challenge sites in the test and control animals permits identification of sensitisation reactions. If the test item at the maximum non-irritant concentration produces reactions in test group animals at the 24 or 48-hour readings, these reactions were attributed to skin sensitisation. This pre-supposes that no similar reactions were observed in the test item challenge sites of any of the control group animals. If irritation was observed in the control group animals, only reactions in the test group animals that exceed the most severe reaction seen in the control group animals were attributed to skin sensitisation. The number of test group animals showing skin reactions greater than the most severe reaction observed in the control group animals, expressed as a percentage of test group animals.

Table 2: Sensitizing reponse indices

         Sensitizing response indices         
 Incidence of positive response    Severity    
 24 h 48 h 24 h 48 h
 Test Animals Treated at 100 %  0/10  0/10
 Control Animals at 100 % 0/5 0/5 
 Test Animals at 50 % 0/10   0/10
 Control Animals at 50 % 0/5 0/5  0

Table 3: weight evolution (In grams)

Negative Control

 Animals No Sex   Beginning of the test (D0 D9  End of the test (D23)  Body weight gain 
 1 267   307  417 150
 2 M 286  340  438  152 
M 272  323  451  179 
 4 259 337  439  180 
5 294  356  501  207 
 Mean     275.6 332.6  449.2  173.6 
 Standard-deviation      14.2  18.5  31.4  23.5 

Table 4: weight evolution (In grams)

Treated group

 Animal no Sex   Beginning of the test (D0)  D9  End of the test (D23)  Body weight gain
 1  M 265  288  366  101 
 2 275  316  418  143 
 3 273  334  483  210 
 4 275  343  460  185 
308  367  467  159 
 6 245  280  394  149 
 7 270  317  411  141 
 8  M 268  324  398  130 
 9  M 256  341  517  261 
 10 281  327  419  138 
 Mean     271.6 323.7  433.3  161.7 
 Standard-deviation    16.5  25.7  46.6 

45.9 

Interpretation of results:
GHS criteria not met
Conclusions:
In view of this results, Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) is not a skin sensitizer.
Executive summary:

The aim of the study was to evaluate the possible allergenic activity of the test item after intradermal and topical administration in guinea pigs.

According the results of the pretests, the induction phase (intradermic injection at 100% and topical application at 100%) was conducted with the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) to 10 Guinea pigs and a 9-day rest phase. The challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 100% and diluted at 50% in corn oil.  The experimental protocol was established according to the Magnusson and Kligman method (J. Invest. Dermatol. 1969. 52, 268-276) and in accordance with O.E.C.D. Test Guideline No.406 of July 17th, 1992.

In the treated group (treatment dose of 100%), no macroscopic cutaneous reactions attributable to allergy was recorded after the challenge phase. In the control group (associated with the treatment dose of 100%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

In the treated group (treatment dose of 50%), no macroscopic cutaneous reactions attributable to allergy was recorded after the challenge phase. In the control group (associated with the treatment dose of 50%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

In conclusion, in view of these results, under these experimental conditions, the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4tricarboxylic acid 1,2-anhydride (3:1) does not have to be classified in category 1 as a skin sensitizer, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures. No signal word or hazard statement is required.

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

The test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) was first tested in vitro according to OECD Guideline 442D. This test method, the KeratinoSensTM, is considered scientifically valid to be used as part of an integrated approaches to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.

Under the retained experimental conditions the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) may be classified as not skin sensitizer.

Due to the bad solubility of the test item no other OECD confirm in vitro test was possible to conduct. Therefore in vivo testing was required to determine the sensitising properties of the test item. It was not possible to conduct 3 in vitro tests according to OECD guidelines because of the properties of the test item.

An OECD 429 test was performed with the test item Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid (Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1)) in the CBA/J strain mouse following topical application to the dorsal surface of the ear. Three groups of four animals were treated for the three consecutive days (D1, D2, D3) with 50 µL (25µL per ear) of the test item undiluted (100%) and diluted at concentrations of 50% and 25% in methyl ethyl ketone (MEK).

A further group of four animals was treated with MEK. On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according to the O.E.C.D. Test Guideline No. 429 dated 22 July 2010 and to the Test method B.42 - Council regulation No 640/2012 of 06 July 2012 (E.U. Journal L193).

No mortality and no sign of systemic toxicity were noted in the treated and control animals during the test.

No increase in ear thickness and in ear weight was recorded at the concentrations of 25%, 50% and 100%.

Therefore, the test item has to be considered as not excessively irritant at these concentrations in accordance with the OECD criteria. The stimulation increase (SI) calculated by pooled approach was 1.04, 1.04 and 1.44 for the treated groups at 25%, 50% and 100%, respectively. The EC1.4 determined by linear regression was 95.00%. The result obtained, may be considered as a borderline result (very close to the cut-off value 1.4 validated in the laboratory). Furthermore, a dose-response relationship was not noted during this study. As a pooled approach was performed; it is not possible to perform a statistical analysis of the data. The results of this study, under these experimental conditions, may be considered not sufficient clear for concluding definitely about the skin sensitizing potential of the test item Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid.

Therefore it was required to conduct another in vivo test on the test item, an OECD 406 -test. According the results of the pretests, the induction phase (intradermic injection at 100% and topical application at 100%) was conducted with the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) to 10 Guinea pigs and a 9-day rest phase. The challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 100% and diluted at 50% in corn oil.  In the treated group (treatment dose of 100%), no macroscopic cutaneous reactions attributable to allergy was recorded after the challenge phase. In the control group (associated with the treatment dose of 100%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. In the treated group (treatment dose of 50%), no macroscopic cutaneous reactions attributable to allergy was recorded after the challenge phase. In the control group (associated with the treatment dose of 50%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

In conclusion, in view of these results, under these experimental conditions, the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) does not have to be classified in category 1 as a skin sensitizer, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures. No signal word or hazard statement is required.

In the supporting study according to OECD 406 to assess the sensitising properties of the similar substance 1,2,4 -benzenetricarboxylic acid, decyl octyl ester a maximisation test in guinea pigs has been conducted with test concentrations of 10% (induction) and 100% (challenge). No evidence of skin sensitisation was observed in any of the treated animals.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The results of the sensitisation tests show that the test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) does not need to be classified according to EU regulations (Directive 67/548/EEC and Regulation (EC) No 1272/2008.