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Administrative data

Description of key information

In vivo (LLNA):

The skin sensitizing potential of C9-11 Methacrylate was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/CaOlaHsd mice each were treated with 2.5%, 10% and 25% (w/w) preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. The high concentration was selected based on the presence of ear irritation in a pretest by using a 50% preparation. The study used 3 test groups and 1 control group. Each test animal was treated with 25μL per ear of the appropriate test-substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25μL per ear of the vehicle alone. Three days after the last application, 20μCi 3H-thymidine in 250μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed in all animals during general observation. When applied as 10% and 25% preparations in MEK, the test substance induced a biologically relevant (increase above the cutoff Stimulation Index of 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, the 10% and 25% test-substance preparations in MEK induced a biologically relevant and statistically significant response (SI ≥ 1.5) in the auricular lymph node cell counts. In addition, statistically significant increases in lymph node weights were noted at the 10% and 25% concentration. Also, the 25% test-substance preparation caused biologically revelvant (SI1.25) and statistically significant increase in ear weights demonstrating ear skin irritation. A statistically significant, but not biologically relevant increase in ear weights was noted at the 10% concentration. Slight scaling at the ear root was observed on four animals at the 10% concentration on study day 5.

A very slight erythema was observed on all animals at the 25% concentration on study days 1 and 2. Slight scaling and a slight incrustation were observed on all animals at the 25% concentration on study day 2. On study day 5, the scaling and incrustation on all animals were moderate.

Thus, it is concluded that C9-11 Methacrylate exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >2.5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.1% and 8.6%, respectively (BASF, 2018).

In vitro (Skin Sensitisation Test Battery):

The objective was to assess the skin sensitizing potential of C9-11 Methacrylate. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy: protein reactivity (DPRA), activation of keratinocytes (LuSens), and activation of dendritic cells (h-CLAT).

DPRA: The reactivity of C9-11 Methacrylate towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were emulsions at the time of preparation and at visual observation after the 24-hour incubation time. No co-elution of test substance and peptides was present. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that C9-11 Methacrylate shows low chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the values for peptide depletion could therefore be under-predictive.

LuSens: The keratinocyte activating potential of test substance C9-11 Methacrylate was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In summary, after 48 hours of exposure to test substance C9-11 Methacrylate luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance C9-11 Methacrylate does not have a keratinocyte activating potential. However, information is available that the log KOW of the test substance is >5 and keratinocyte activation is known to be under-predictive in this case (OECD TG 442D). A “negative” result should be considered “inconclusive” in this case.

h-CLAT: The potential of test substance C9-11 Methacrylate to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry. In summary, after 24 hours of exposure to test substance C9-11 Methacrylate CD86 and CD54 expression was not induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance C9-11 Methacrylate does not induce dendritic cell activation. However, information is available that the log KOW of the test substance >3.5 and the h-CLAT is known to be under-predictive in this case (OECD TG 442E). A “negative” result should be considered “inconclusive” in this case.

Test Method

Test Result

Test Evaluation

Direct Peptide Reactivity

Assay (DPRA)

13.20% mean peptide depletion (26.39% cysteine peptide depletion; -2.81% lysine peptide depletion).a

Positive

Keratinocyte Activation

Assay - LuSens

In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.b

Negative / Inconclusivec

Dendritic Cell Line

Activation Assay Human

Cell Line Activation Test

(h-CLAT)

In at least two independent experiments no biologically relevant induction of the expression of CD86 and CD54 was observed.

Negative / Inconclusived

aNegative depletions were considered to be “zero” for calculation of mean peptide depletion. Further, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the values for peptide depletion could therefore be under-predictive.

bFor details of evaluation criteria see section 3.10.2.

cInformation is available that the log KOWof the test substance is >5 and keratinocyte activation is known to be under-predictive in this case (OECD TG 442D). A “negative” result should be considered “inconclusive” in this case.

dInformation is available that the log KOWof the test substance is >3.5 and the h-CLAT is known to be underpredictive in this case (OECD TG 442E). A “negative” result should be considered “inconclusive” in this case.

Based on the results and applying the evaluation criteria, C9-11 Methacrylate is peptide reactive. The potential of the test substance to activate keratinocytes and to activate dendritic cells could not be conclusively evaluated. Applying the evaluation criteria, the skin sensitization potential of C9-11 Methacrylate can not be conclusively evaluated (BASF, 2018).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
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)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS GmbH, C/O Postfach 553 NL-5800 AN Venray
- Age at study initiation: 8 weeks (main test); 9 weeks (pretest)
- Weight at study initiation: 17.2 g- 20.1 g (main test), 20.1 g - 22.9 g (pretest)
- Housing: single (Polycarbonate cages type MII)
- Diet (e.g. ad libitum):Kliba mouse/rat maintenance diet "GLP" supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days before the first application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
methyl ethyl ketone
Concentration:
2.5, 10 and 25% (w/w)
No. of animals per dose:
5
Details on study design:
The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice. Prior to first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions. Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Obvious signs of systemic toxicity and/or local inflammation atthe application sites were noted for each animal in the raw data. A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays
and on public holidays.
Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions. Application volume of 25 µL per ear. Site of application dorsal part of both ears. Frequency of application 3 consecutive applications (day 0 – day 2) to the same application site.
The animals of control group 1 and test groups 2-4 were treated with the vehicle or a test-substance preparation as given in section 2.2.
On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice.
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 6 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter.
The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.
Positive control substance(s):
other: A positive control with a substance that has been proven to cause skin sensitization in the animal strain used will not be included in this study. A separate study with a positive control is carried out in the laboratory twice a year.
Statistics:
Cell count, 3H-thymidine incorporation, lymph node weight and ear weight, WILCOXON - Test
Parameter:
EC3
Remarks:
%
Value:
9.1
Parameter:
other: EC1,5
Remarks:
%
Value:
8.6

Test item concentration

Group Calculation

Mean DPM per animal (2 lymph nodes)

SD

S.I.

Vehicle Control Group (vehicle: MEK)

304.9

125.3

1.00

2.5% C9-11 Methacrylat

 

350.4

108.2

1.15

10%C9-11 Methacrylat

 

995.7

207.6

3.27

20% C9-11 Methacrylat

 

2144.8

611.8

7.03

Stimulation indices:

Test Group

Treatment

3H-thymidine incorporation Stimulation Index1

Cell Count Stimulation Index1

Lymph Node Weight

Stimulation Index1

Ear Weight Stimulation Index1

1

Vehicle MEK

1.00

1.00

1.00

1.00

2

2.5% in MEK

1.15

0.92

0.92

1.01

3

10% in MEK

      3.27 ##

    1.63 ##

    1.56 ##

   1.16 #

4

25% in MEK

     7.03 ##

    2.28 ##

    2.17 ##

     1.70 ##

1SI-calculation versus vehicle control

The statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.005, ## for p ≤ 0.01)

The threshold concentration for sensitization induction was >2.5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.1% and 8.6%, respectively.

No relevant influence on the body weights was observed during the study.

No signs of systemic toxicity were noticed in all animals during general observation.

Slight scaling at the ear root was observed on four animals at the 10% concentration on study day 5. A very slight erythema was observed on all animals at the 25% concentration on study days 1 and 2. Slight scaling and a slight incrustation were observed on all animals at the 25% concentration on study day 2. On study day 5, the scaling and incrustation on all animals were moderate.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Thus, it is concluded that C9-11 Methacrylate exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In vitro Skin Sensitisation Test Battery:

In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone in-house validation using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

Based on the results of the in house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90% and an accuracy of 90%.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauchet al. 2012; Table 1). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA

LuSens/

KeratinoSensTM

MUSST/

h-CLAT

Test battery evaluation

positive

positive

positive

sensitizer

positive

positive

negative

sensitizer

positive

negative

positive

sensitizer

positive

negative

negative

non-sensitizer

negative

positive

positive

sensitizer

negative

positive

negative

non-sensitizer

negative

negative

positive

non-sensitizer

negative

negative

negative

non-sensitizer

Each individual assay was performed under GLP and the cell based assays LuSens and h-CLAT consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays. The DPRA and the keratinocyte activation assay follow the procedures of the respective OECD testing guidelines (OECD 442C and D). An OECD testing guideline for the dentritic cell activation assay is under preparation.

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

Sensitization involves a number of key steps in order to take place, and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The studies carried out for this substance address these three key steps. The results are then used in a predefined evaluation scheme to determine hazard classification as a sensitizer by a weight-of-evidence approach.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item is classified as skin sensitization cat. 1B (H317) according to Regulation (EC) No 1272/2008 (CLP).