Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Calculated SI values < 3 at the 3 test item concentrations tested in a LLNA study (OECD guideline 429, GLP).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
other justification
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint:
skin sensitisation, other
Remarks:
Solubility Assessment for the Non-animal Skin Sensitization Studies KeratinoSens and U-SENS
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)
Version / remarks:
June 2018
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)
Version / remarks:
June 2018
GLP compliance:
yes (incl. QA statement)
Type of study:
other: The objective of this study was to evaluate the solubility of the test substance in various solvents. These data will be used to help assessing the applicability of the test item in the KeratinoSens assay and U-SENS assay.
Remarks on result:
other: See the section "Any other information on results incl. tables" below

- Solubility assessement KeratinoSensTM


The following solvents were evaluated: DMSO, Milli-Q water and ethanol. 


The test item formed a homogenous suspension in DMSO and Milli-Q water at 200 mM. In contrast, non-homogenous suspensions were obtained with ethanol at 800 mM (Tabe 1). Therefore, the next steps were not performed with ethanol.


The solubility of the test item in DMSO and Milli-Q water was evaluated in KeratinoSensTM test conditions at a concentration range of 16 – 2000 µM (2-fold dilution series). The results are presented in Table 2. Using DMSO, microscopic or slight precipitation was observed upon preparation at 125 µM and upwards and, after incubation (i.e. 48 hours at 37+/- 1°C in the presence of 5 % CO2), at 16 µM and upwards. Using Milli-Q water, microscopic or slight precipitation was observed upon preparation at 63 µM and upwards and, after incubation, at 16 µM and upwards.


Based on the solubility assessment the preferred solvent for the KeratinoSensTM is DMSO.


Table 1: Solubility of the Test Item in an Appropriate Solvent (KeratinosensTM)

























Test item stock solution
concentration (mM)



Solvent tested



Result of the visual inspection



200



Milli-Q



White homogenous suspension



DMSO



White homogenous suspension



800



Ethanol



Non-homogenous suspension



 


Table 2: Solubility in 96-well plate test conditions (KeratinosensTM)






























































































Solvent of the test item stock solution



Concentration in assay (µM)



Result of the visual inspection upon preparation



Result of the visual inspection after incubation



DMSO



2000



Slight precipitation



Slight precipitation



1000



Slight precipitation



Slight precipitation



500



Microscopic precipitation



Slight precipitation



250



Microscopic precipitation



Slight precipitation



125



Microscopic precipitation



Microscopic precipitation



63



No precipitation



Microscopic precipitation



31



No precipitation



Microscopic precipitation



16



No precipitation



Microscopic precipitation



Milli-Q water



2000



Slight precipitation



Slight precipitation



1000



Slight precipitation



Slight precipitation



500



Microscopic precipitation



Microscopic precipitation



250



Microscopic precipitation



Microscopic precipitation



125



Microscopic precipitation



Microscopic precipitation



63



Microscopic precipitation



Microscopic precipitation



31



No precipitation



Microscopic precipitation



16



No precipitation



Microscopic precipitation



 


- Solubility assessement U-SENSTM


The following solvents were evaluated: RPMI medium and DMSO. The test item formed a homogenous suspension in RPMI medium at 0.4 and 50 mg/mL and in DMSO at 50 mg/mL (see table 3 below).


The solubility of the test item in RPMI medium and DMSO was evaluated in U-SENS(TM) test conditions at a concentration of 200 µg/mL. At this concentration, the test item showed precipitation upon preparation and after incubation (i.e. 48 hours at 37+/- 1°C in the presence of 5 % CO2) with either RPMI medium and DMSO as solvents (Table 4).


Based on the solubility assessment the preferred solvent for the U-SENS(TM) is RPMI medium. But due to the precipitation observed in the test conditions, the outcome of the assay runs will likely be predermined as positive and therefore a negative result is unlikely to be obtained.


Table 3: Solubility of the Test Item in an Appropriate Solvent (U-SENSTM)

























Test item stock solution
concentration (mg/ml)



Solvent tested



Result of the visual inspection



50



RPMI



Cloudy pink suspension



DMSO



Pink suspension



0.4



RPMI



Cloudy pink suspension



 


Table 4: Solubility in 96-well plate test conditions (U-SENSTM)
























Solvent of the test item stock solution



Concentration in assay (µg/ml)



Result of the visual inspection upon preparation



Result of the visual inspection after incubation



RPMI



200



Precipitation



Precipitation



DMSO



200



Precipitation



Precipitation



 


 


- Discussion:


The substance is an inorganic UVCB under powder form with no Log Kow and, with a melting point > 300°C, no vapor pressure was measured. The substance is expected to have a low water solubility based on solubility data of its constituents (a transformation/dissolution study on the substance is currently under progress, see Section 4.8 for further information). Therefore, the skin sensitisation properties can not be evaluated using QSARs models as well as in the in chemico skin sensitisation assays (OECD TG 442C) since metals and/or inorganic compounds are out of the applicability domain.


According to the solubility assessment for the in vitro skin sensitization studies, both Keratinosens (OECD TG 442-D) and U-SENS (OECD TG 442-E) studies are in principle applicable, as a suitable vehicle can be found. However, due to the precipitation observed in the culture medium used for the U-SENS study (OECD TG 442-E) upon preparation and after incubation for 48 hours at 37°C with the tested solvents, the outcome of the assay runs will likely predetermined as positive according to the OECD TG 442E. But, whatever the results of the Keratinosens (OECD TG 442-D) if it had been run, further data/information will have been needed according to the ECHA Guidance on Information Requirements and Chemical Safety Assessment - R.7a, (version 6.0, 2017) and the OECD Guidance Document n° 497 (June 2021) to predict with confidence the skin sensitisation hazard of the substance and/or potency for sub-categorisation.


Indeed, in case of negative/inconclusive results in the Keratinosens, considering the predetermined positive results in the U-SENS assay, skin sensitization hazard of the substance could not have been predicted due to non-concordant results in both assays. In case of positive results in the Keratinosens, considering the predetermined positive results in the U-SENS assay and the non-applicability of in chemico assays and QSARs methods, prediction for the potency sub-categorisation could not have been done with confidence, and thus, triggering further testings.


Therefore, from the above mentioned reasons, an in vivo study was launched in accordance with Annex VII, section 8.3.2, and the LLNA study was chosen based on solubility pre-tests.


 

Interpretation of results:
study cannot be used for classification
Remarks:
The objective of this study was to evaluate the solubility of the test subsatnce in various solvents. These data will be used to help assessing the applicability of the test item in the KeratinoSensTM assay and U-SENSTM assay. According to the solubility assessment for the in vitro skin sensitization studies, both Keratinosens (OECD TG 442-D) and U-SENS (OECD TG 442-E) studies are in principle applicable, as a suitable vehicle can be found. Due to the precipitation observed in the culture medium used for the U-SENS study (OECD TG 442-E) upon preparation and after incubation for 48 hours at 37°C with the tested solvents, the outcome of the assay runs will likely predetermined as positive according to the OECD TG 442E. However, considering that the in chemico skin sensitisation assays (OECD TG 442C) is not applicable to metals/ inorganic compounds, whatever the results of the Keratinosens (OECD TG 442-D) if it had been run (Positive or negative/unconclusive, further data/information will have been needed according to the ECHA Guidance on Information Requirements and Chemical Safety Assessment - R.7a, (version 6.0, 2017) and the OECD Guidance Document n° 497 (June 2021) to predict with confidence the skin sensitisation hazard of the substance and/or potency for sub-categorisation.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 11/08/2021 yo 14 /09/2021
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:
July 2010
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 20.7 to 25.6 g.
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Animal Enrichement: For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures was 18 to 24°C
- Humidity (%): Target humidity was 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained.
- IN-LIFE DATES: From: To: 25/08/2021 -14/09/2021
Vehicle:
dimethylformamide
Remarks:
The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.
Concentration:
2, 5 and 10 %
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: yes
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; 10% and 25% w/w in DMF. The highest concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

- Results of the pre-screen test:
At a concentration of 25% w/w test substance in DMF, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values in one animal and therefore this concentration did not meet the selection criteria.
At 10% w/w in DMF, no signs of systemic toxicity and no irritation and no effects on ear thickness were observed. Therefore, this concentration was selected as highest concentration for the main study.
White test item remnants were present on the dorsal surface of the ears of all animals on Days 1-4, which did not hamper scoring of the skin reactions.

MAIN STUDY:
ANIMAL ASSIGNMENT
- Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study. Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

TREATMENT
- Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle (DMF).
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The formulations were stirred with a magnetic stirrer until dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After 5 hours, all animals were euthanized. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue processing for radioactivity - Day 6: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactive measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

- OBSERVATIONS and measurements:
* Mortality/moribundity checks: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
* Clinial observation: Post-dose observations were performed once daily on Days 1-6 (on Days 1-3 at least 3 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.
* Body weight: Animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy).
* Irritation: Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing), according to the following numerical scoring system:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4
Furthermore, a description of all other (local) effects was recorded.
* Terminal procedures: No necropsy was performed, since all animals survived until the end of the observation period.


- Criteria used to consider a positive response:
A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3):
SI < 3 => Not a skin sesnitiser
SI ≥ 3 => Cat 1 Skin sensitizer; EC3 value ≤ 2%: sub-category 1A; EC3 value ≤ 2%: sub-category 1A

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by the laboratory. In this study, performed in May 2021, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKCD3159 (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO).
The results are the following :
Group* % HCA Mean DPM ± SEM SI ± SEM
1 0% (AcOO) 236 ± 45 1.0 ± 0.2
2 5% 272 ± 56 1.2 ± 0.2
3 10% 518 ± 59 2.2 ± 0.2
4 25% 1317 ± 130 5.6 ± 0.5
* Five females per group

The SI values calculated for the item concentrations 5, 10 and 25% were 1.2, 2.2 and 5.6, respectively. An EC3 value of 13.5% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.3, 12.8, 9.0, 10.9 and 8.0%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed in the Laboratory is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
ca. 1.5
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
ca. 1.4
Test group / Remarks:
5 %
Key result
Parameter:
SI
Value:
ca. 1.3
Test group / Remarks:
2 %
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% w/w in DMF were 278, 280 and 318 DPM, respectively. The mean DPM/animal value for the vehicle control group was 207 DPM (see Table 2 in "Any other information on results incl. tables").

DETAILS ON STIMULATION INDEX CALCULATION: The mean SI values calculated for the test item concentrations 2, 5 and 10% w/w in DMF were 1.3, 1.4 and 1.5, respectively (see Table 2).

EC3 CALCULATION: Since there was no indication that the test subsatnce could elicit a SI ≥ 3 when tested up to 10% w/w in DMF, it was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the experimental or control animals.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (See Table 1 in "Any other information on results incl. tables).

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): No skin irritation was observed in any of the animals. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals (See Tables 1 and 2 in "Any other information on results incl. tables).

Table 1: Main Study - Body Weights and Skin Reactions


































































































































































































































































































































































































IT1 (%)



Animal n°



Day 1



Day 2



Day 3



Day 4



Day 5



Day 6



 



Erythema3



Erythema



Erythema



Erythema



Erythema



Erythema



 



Bw (g)²



Left



Right



Left



Right



Left



Right



Left



Right



Left



Right



Left



Right



Bw (g)



0



1



24.8



0



0



0



0



0



0



0



0



0



0



0



0



27.1



2



25.6



0



0



0



0



0



0



0



0



0



0



0



0



27.2



3



23.6



0



0



0



0



0



0



0



0



0



0



0



0



25.1



4



21.4



0



0



0



0



0



0



0



0



0



0



0



0



23.6



5



23.4



0



0



0



0



0



0



0



0



0



0



0



0



24.3



2



6



20.7



0



0



0



0



0



0



0



0



0



0



0



0



20.4



7



23.3



0



0



0



0



0



0



0



0



0



0



0



0



23.2



8



20.9



0



0



0



0



0



0



0



0



0



0



0



0



22.7



9



23.1



0



0



0



0



0



0



0



0



0



0



0



0



23.6



10



22.7



0



0



0



0



0



0



0



0



0



0



0



0



23.7



5



11



23.8



0



0



0



0



0



0



0



0



0



0



0



0



25.4



12



22.1



0



0



0



0



0



0



0



0



0



0



0



0



24.6



13



24.8



0



0



0



0



0



0



0



0



0



0



0



0



24.7



14



24.0



0



0



0



0



0



0



0



0



0



0



0



0



24.9



15



24.4



0



0



0



0



0



0



0



0



0



0



0



0



27.3



10



16



23.5



0



0



0



0



0



0



0



0



0



0



0



0



25.4



17



22.3



0



0



0



0



0



0



0



0



0



0



0



0



25.3



18



23.7



0



0



0



0



0



0



0



0



0



0



0



0



22.7



19



23.8



0



0



0



0



0



0



0



0



0



0



0



0



25.1



20



21.9



0



0



0



0



0



0



0



0



0



0



0



0



22.2



1   TI = test item (% w/w).


2   Body weight (grams).


3   Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear):


    0 = No erythema


 


Table 2: Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)






















































































































































IT1 (%)



Animal n°



Size Nodes 2



DPM 3 / Animal



Mean DPM


± SEM 4



Mean SI


± SEM



left



right



0



1



n



n



249



207 ± 29



1.0 ± 0.1



2



n



n



234



3



n



n



242



4



n



n



217



5



n



n



95



2



6



n



n



219



278 ± 72



1.3 ± 0.3



7



n



n



324



8



n



n



168



9



n



n



139



10



n



n



538



5



11



n



n



255



280 ± 44



1.4 ± 0.2



12



n



n



340



13



n



n



167



14



n



n



226



15



n



n



414



10



16



n



n



152



318 ± 74



1.5 ± 0.4



17



n



n



183



18



n



n



529



19



n



n



446



20



n



n



278



1   TI = test item (% w/w).


2   Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).


3     DPM = Disintegrations per minute.


4     SEM = Standard Error of the Mean.

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that test substance could elicit a SI ≥ 3 when tested up to 10% w/w in DMF, it was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%. Therefore, the test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the GHS and CLP criteria.
Executive summary:

The potential of the test item to induce skin sensitization was tested in mice a LLNA study performed according to OECD guideline 429 and in compliance with GLP.


Three groups of 5 female CBA/J mice were treated with test item concentrations of 2, 5 or 10% w/w in DMF on 3 consecutive days, by open application on the ears. A group of 5 mice were similarly treated with the vehicle alone. Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after 5h the draining (auricular) lymph nodes were excised and pooled per animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group using the individual animal approach.


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


Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% w/w in DMF were 278, 280 and 318 DPM, respectively. The mean DPM/animal value for the vehicle control group was 207 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% w/w in DMF were 1.3, 1.4 and 1.5, respectively.


Since there was no indication that the time item could elicit a SI ≥ 3 when tested up to 10% w/w in DMF, it was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%..


Based on these results, the test time would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. Therefore, the test item does not have to be classified for sensitization by skin contact according to the GHS and CLP criteria.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

- Skin sensitisation:


The SI values calculated for the test substance at the 3 concentrations tested were all found to be < 3 in a study performed according to OECD guideline 429 and in accordance with GLP. Thereore, according to this result, the test substance does not have to be classified according to the GHS and CLP criteria.


- Respiratory sensitisation : No data available.