Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized, reaction products with ethylene glycol

Endpoint summary

Description of key information

With regard to skin sensitisation, contradicting results are available: the two independent and valid LLNA tests gave a negative and a (weak) positive result, respectively. The tests were conducted in 2005 and 2007, when the issue of false positive results that might be obtained in LLNA studies with substances containing oleic acids was not yet known. Nowadays, a study protocol according to OECD 406 would be the choice. Due to animal welfare reasons, in-vitro tests (DPRA, MUSST, LuSens) were performed for clarification of the potentially false-positive results of the LLNA. In one test (MUSST), indications for a skin sensitizing potential were detected, but were in fact only weak positive. The other tests (DPRA and Lu-sens) were negative.Since the classification regarding skin sensitization potential is still unclear, a guinea pig maximization test (OECD 406) according to the Buehler protocol was ordered and is still ongoing.

For precautionary reasons, the substance is classified for skin sensitization 1B in the meantime, based on the results of the potentially false-positve LLNA.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007/07/05- 2007/07/25

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:

(2002)

Deviations:

yes

Remarks:

The relative humidity in the animal room was between appr. 30-84%. This deviation did not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Name of test material: Sovermol 1102
- Physical state: liquid at room temperature
- Analytical purity: > 99.5 %
- Lot/batch No.: CE62150013
- Expiration Date: August 2007

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands; B.V. Postbus 61 74; NL - 5960 AD Horst 1 The Netherlands
- Age at study initiation: 7 - 8 weeks
- Housing: individual caging
- Diet: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-331 78 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C
- Humidity: 30 - 84 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 a.m.

Vehicle:

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

Concentration:

Topical induction:
The test item was applied topically at concentrations of 25, 50 and 100 % (w/v) in acetone:olive oil (4+1). The application volume, 25 µL, was spread over the entire dorsal surface of 8 mm² of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

No. of animals per dose:

Preliminary test:
2 females for the pre- testing

Main trial:
4 females/ dose (3 x test item concentrations, 1 x vehicle control)

Details on study design:

MAIN STUDY
3 groups with each 4 females were treated with 25, 50 or 100 % of the test item by topical application onto the dorsum of both ear lobes for three consecutive days. A control group of 4 females was treated with the vehicle only. Five days after the first topical exposure the proliferative response of activated lymph node cells was assessed by the injection of 250 µL of 79.9 µCi/mL 3H- Methylthymidine (corresponding to 20.0 µCi/ mouse) by intravenous injection into the tail. 5 hours after injection the auricular lymph node was excised and pooled per group. The incorporated amount of 3H- Methylthymidine in proliferating lymph node cells was quantified by β- scintillation. Proliferation is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/ node) and as the ratio of the amount of 3H- Methylthymidine which is incorporated into lymph node cells of treated animals relative to the amount which is recorded for control lymph nodes (stimulation index S.I.). Before DPM/ node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EVALUATION CRITERIA
A test item is regarded as a sensitiser in the LLNA if the following criteria are fullfilled:
- Exposure to at least one concentration of the test item resulted in an incorporation of 3H- Methylthymidine past 3- fold or greater than that recorded in control mice, as indicated by the stimulation index.
- That the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

ADDITIONAL OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality/ viability: once daily (week day) from experimental start to necropsy.
- Body weights: prior to the first application and prior to treatment with 3H- Methylthymidine.
- Clinical signs (local / systemic): once daily (week day). Especially the treatment sites were observed carefully.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation: EC3 = (a-c) [(3-d)/(b-d)] + c ; where EC3 is the estimated concentration of the test item required to produce a 3- fold increase in draining lymph node cell proliferative activity.

Positive control results:

The positive control using alpha-Hexylcinnamaldehyde demonstrated an S.I. of 4.88 using a concentration of 25 % (w/v).

Parameter:

SI

Value:

2.3

Test group / Remarks:

25%

Parameter:

SI

Value:

3.19

Test group / Remarks:

50%

Parameter:

SI

Value:

3.5

Test group / Remarks:

100%

Key result

Parameter:

EC3

Value:

44.5

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS:
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS:
The body weight of the animals, recorded prior to the first application and prior to treatment with 3H-Methylthymidine, was within the range commonly recorded for animals of this strain and age.

MORTALITY/ VIABILITY:
No deaths occurred during the study period.

Concentration	Disintegrations per minute (DPM)
0%	698
25%	1619.5
50%	2228.1
100%	2441.5

Interpretation of results:

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

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

no guideline available

Principles of method if other than guideline:

Binding to Cys and Lys model peptides in chemico (Bauch C. et al 2011)

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct peptide binding assay

Justification for non-LLNA method:

With regard to skin sensitisation, contradicting results are available: the two independent and valid LLNA tests gave a negative and a (weak) positive result, respectively. The tests were conducted in 2005 and 2007, when the issue of false positive results that might be obtained in LLNA studies with substances containing oleic acids was not yet known. Nowadays, a study protocol according to OECD 406 would be the choice. Due to animal welfare reasons, in-vitro tests (DPRA, MUSST, LuSens) were performed for clarification of the potentially false-positive results of the LLNA.

Specific details on test material used for the study:

- Name of test substance: Sovermol 1102
- Batch identification: CE03250005
- Test substance No.: 11/0565-1

Details on the study design:

TEST ITEM PREPARATION
The test substance was prepared as a 100 mM stock solution in acetonitrile just prior to incubation with the synthetic peptides.

SYNTHETIC PEPTIDES:
- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- The peptides are custom material (Supplier: RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

CONTROLS
Negative control (NC): vehicle control = acetonitrile
Positive control (PC): p-Benzoquinone, puriss. (CAS-no. 106-51-4)

ANALYTICAL VERIFICATION OF TEST SUBSTANCE SOLUTION
Because the test-substance preparation was incubated with the peptide shortly after preparation, no analysis of the test substance in the vehicle was performed.

INCUBATION CONDITIONS
Three samples of the test substance were incubated with each peptide for 24h at room temperature. The incubation tubes were sealed. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The test substance was incubated with the C-containing peptide in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance, pH 7.5 phosphate buffer) and with the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance, pH 10.2 ammonium acetate buffer).

ANALYTICAL METHOD
The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition peptide standards of known concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analysis method. The mean peptide depletion of a test substance was calculated as the mean value of Cys containing peptide depletion and Lys-containing peptide depletion.

EVALUATION CRITERIA
According to the classification tree model described by Gerberick et al. for substances with known molecular weight highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitizer, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitizer, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitizer.

Positive control results:

All peptide was depleted by the positive control substance p-benzoquinone.

Run / experiment:

mean

Parameter:

other: mean Cysteine-peptide depletion (%)

Value:

11.2

Remarks on result:

other: unit: %

Run / experiment:

mean

Parameter:

other: mean Lysine-peptide depletion (%)

Value:

-0.4

Remarks on result:

other: Unit: %

Key result

Run / experiment:

mean

Parameter:

other: mean peptide depletion (%)

Value:

5.6

Remarks on result:

other: unit: %

Remarks:

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion

Other effects / acceptance of results:

OTHER EFFECTS:
- No co-elution of test substance and peptides was noticed.
- When mixed with the peptide stock solution the samples became opaque directly after preparation. After 24 hours precipitates were noticed in all samples. Thus all samples were centrifuged prior to HPLC analysis.

ACCEPTANCE OF RESULTS:
- Complete depletion was caused by the positive control substance.

Interpretation of results:

other: Non protein binding/peptide binding

Conclusions:

Based on the observed results and applying the prediction model proposed in Gerbreick et al. (2007), it was concluded that the test item shows a minimal chemical reactivity in the DPRA under the test conditions chosen. According to the classification tree model described by Gerberick et al., a test substance of minimal reactivity is predicted to be a non-sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vitro

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

Principles of method if other than guideline:

CD 86 expression in dentritic cell line as marker for stimulation (Myeloid U937 Skin Sensitization Test, MUSST)

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Dentritic cell activation assay

Specific details on test material used for the study:

- Name of test substance: Sovermol 1102
- Test substance No.: 11/0565-1
- Batch identification: CE03250005
- Date of production: Nov 21, 2010

Details on the study design:

MODEL
- U937 cell line (human dentritic-like cell line)
- Provider: German Resource Center for Biological Material (DSMZ, Germany, catalog no.: ACC 5).

CHOICE OF TEST CONCENTRATION
A pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (0.5 μg/mL up to 2000 μg/mL) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords at least 75% cell viability) was determined by linear regression from the concentration response curve to be 56 μg/mL. In the main test, test substance was used at six final concentrations determined with regard to the CV75 value: CV75 x 2, CV75 x 1.5, CV75 , CV75/2, CV75/4, CV75/8. After 48 hour exposure U937 cells were stained with FITC labeled anti-human-CD 86 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry.

ANTIBODIES:
Antibodies: FITC Mouse anti-human CD86 (BD Pharmingen) IgG1 FITC CD86 (BD Pharmingen)

EXPOSURE DURATION:
48h

CONCENTRATIONS:
7 - 112 μg/mL (affording at least 70% viability)

VEHICLE:
Culture medium

CONTROLS
True negative control: lactic acid 200 μg/Ml
Positive control: Ethylene diamine (EDA – 70 μg/mL)

EVALUATION CRITERIA
A test substance was predicted to have a dendritic cell activating potential when the marker expression exceeded the threshold of 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently non-cytotoxic (cell viability ≥ 70%) concentration in at least two independent experiments.

ACCEPTANCE CRITERIA
If the acceptance criteria mentioned below were not met, repetition of the test was considered. A tested concentration is not to be further evaluated when relative viability is less than 70%. A study is considered acceptable if the positive and negative and vehicle control data lie within the range of the historical data. The cell viability of untreated cells must yield at least 90%. The expression marker CD86 of the vehicle control cells should lie between 8% – 20%.

Positive control results:

Ethylene diamine gave the expected induction of CD86. For details, see 'any other information on results incl tables'

Remarks on result:

other: see 'any other information on results incl. tables'

Other effects / acceptance of results:

OTHER EFFECTS:
- A solubility experiment was performed. No precipitation in medium occurred, the test substance was a homogeneous suspension in culture medium. The dilutions of the test substance were solutions.
- The substance was active in the MUSST assay.

ACCEPTANCE OF RESULTS:
The level of CD86 expression was not induced after 48 hours treatment with lactic acid (negative control) and was induced after 48 hours exposure to EDA. The level of expression was within the range of the historical negative and positive control data.

Table 1: Viablility assays

Concentration(mg/L)	%PI negative cells, replicate 1	%PI negative cells, replicate 2	%PI negative cells, replicate 3	relative viability
control	98.9	98.8	98.8	100
0.5	99.0	98.6	98.8	99.9
1	99.0	98.7	98.8	100.0
5	98.9	98.7	98.8	99.9
10	98.4	98.5	98.5	99.6
50	80.4	74.8	77.6	78.5
100	53.5	43.8	48.7	49.2
500	39.1	43.1	41.1	41.6
1000	81.7	85.4	83.5	84.5
2000	51.4	39.5	45.5	46.0

PI = propidium iodide

Table 2: CD86 Induction

	experiment 1		experiment 2		experiment 3
concentration (mg/L)	CD86 induction	rel. Viability (%)	CD86 induction	rel. Viability (%)	CD86 induction	rel. Viability (%)
VC	1.0	100.0	1.0	100.0	1.0	100.0
7.0	0.8	99.9	1.7	100.2	0.9	99.9
14.0	0.8	99.2	1.7	99.6	0.9	99.6
28.0	0.8	97.2	1.3	97.4	0.8	98.4
56.0	1.9	79.7	1.4	92.2	1.3	91.0
112.0	1.4	34.0	2.9	46.1	1.4	56.6

Interpretation of results:

other: activation of dentritic cells in vitro

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes (LuSens, Bauch et al. 2012). lt employs the reporter gene for luciferase under the control of an antioxidant response element and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the up-regulation of the luciferase activity after 48 hour incubation with test substances. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010).

GLP compliance:

yes (incl. QA statement)

Type of study:

other: in-vitro keratinocyte activation assay (LuSens)

Specific details on test material used for the study:

- Name of test subtsance: Sovermol 1102
- Test substance No.: 11/0565-1
- Batch identification: CE03250005

Details on the study design:

MODEL
LuSens cells (derived from human keratinocyte cell line HaCaT). The reporter gene cell line LuSens was prepared in collaboration with the RWTH Aachen University. This keratinocyte cell line derived from HaCaT cells carries a reporter gene for luciferase under the control of an antioxidant-response-element (ARE) and hence monitors Nrf2 transcription factor activity. The ARE promoter belongs to the NADPH:quinone oxidoreductase1 gene from rats.

PREPARATION OF CELLS AND SUBSTANCE SOLUTIONS
LuSens cells were cultured in complete DMEM culture medium with high glucose supplemented with 10% fetal bovine serum (FBS),100 U/mL penicillin – 100 lg/mL streptomycin and 0.5 lg/mL puromycin in T75 culture flasks. Cells were grown for 24 h in 96-well plates prior to the substance treatment (10000 cells in 0.12 mL medium). Stock solutions of the substances were prepared by dissolving in DMSO (final concentration of 200 mM) and diluted in 2-fold dilutions. Test substance solutions were further diluted in medium containing 1% FBS w/o puromycin to obtain a DMSO concentration of 1%.

INCUBATION CONDITIONS AND ANALYTICAL METHOD
Substance incubation was performed under standard cell culture conditions for 48 h and luciferase activity was subsequently determined using SteadyGlo™ (Promega, Germany) according to manufacturer’s instructions.

DOSE RATIONALE
The highest tested concentration in the main experiment was 1.22 fold of the concentration affording a viability of 75% (CV75). The additional concentrations were obtained by a 1:1exp2 dilution series of the CV75.

CYTOTOXICITY ASSAY
Cytotoxicity was determined via the MTT assay.

NUMBER OF REPLICATES
Two independent experiments were performed. In each experiment, three duplicates of each treatment were tested.

CONTROLS
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA, 18 µg/ml)
Negative control (NC): OL-Lactic acid (LA, 450 µg/mL)
Vehicle control: 1% DMSO in culture medium

ACCEPTANCE CRITERIA
- A tested concentration was not further evaluated when relative viability is less than 70%.
- The cell viability of untreated cells must yield at least 90%. The positive control EGDMA should be ~3 fold induction and lactic acid <2 and viability ~70%. The average standard of the variability in the vehicle control wells for each plate should be below 20%.
- A study is considered acceptable if the positive and negative and vehicle control data lies within the range of the historical data.

EVALUATION CRITERIA
A test substance is concluded to exhibit an keratinocyte activating potential when the luciferase activity exceeds 2.0 fold induction with respect to the vehicle control and at concentrations that do not reduce viability below 70%.

Positive control results:

An upregulation of the luciferase activity after 48 hours treatment occurred with EGDMA.

Parameter:

other: Increase in luciferase activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: at concentrations that did not reduce cell viability below 70%

Other effects / acceptance of results:

CYTOTOXICITY (MTT):
The cytotoxicity of the test substance was evaluated by the MTT assay after 48 hours exposure, The CV75 value (estimated concentration that affords 75% cell viability) was derived from the concentration response curve. Under the chosen exposure conditions, the CV75 value for the test substance was determined to be 6.44 µg/mL.

ACCEPTANCE OF RESULTS:
Positive and negative controls: An upregulation of the luciferase activity after 48 hours treatment occurred with EGDMA but not with LA. The level was within the range of the historical negative and positive control data.

Interpretation of results:

other: No induction of antioxidant response genes in a keratinocyte cell line

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Deviations:

yes

Remarks:

age of the animals was 7-8 weeks (not 8-12).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Yellowish liquid, 100% active substance

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands; B.V. Postbus 61 74; NL - 5960 AD Horst 1 The Netherlands
- Age at beginning of acclimatization: 7 - 8 weeks
- Housing: individual caging
- Diet: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-331 78 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-3 °C
- Humidity: 30 - 70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 a.m.

Vehicle:

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

Concentration:

Topical induction:
The test item was applied topically at concentrations of 12.5, 25 and 50 % (w/v) in acetone:olive oil (4+1).

No. of animals per dose:

2 animals for the pre-test.
20 animals for the main study (3 dose groups of each 5, 1 control group of 5)

Details on study design:

MAIN STUDY
3 groups with each 5 females were treated with 12.5, 25 or 50 % of the test item by topical application onto the dorsum of both ear lobes for three consecutive days. The application volume, 25 µL, was spread over the entire dorsal surface of 8 mm² of each ear lobe once daily. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of the test item applied. A control group of 5 females was treated with the vehicle only. Five days after the first topical exposure the proliferative response of activated lymph node cells was assessed by the injection of 250 µL of 81.8 µCi/mL 3H- Methylthymidine (corresponding to 20.45 µCi/ mouse) by intravenous injection via a tail vain. 5 hours after injection the draining lymph nodes were excised and pooled per animal.

The incorporated amount of 3H- Methylthymidine in proliferating lymph node cells was quantified by β- scintillation. Proliferation is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/ node) and as the ratio of the amount of 3H- Methylthymidine which is incorporated into lymph node cells of treated animals relative to the amount which is recorded for control lymph nodes (stimulation index S.I.). Before DPM/ node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EVALUATION CRITERIA
A test item is regarded as a sensitiser in the LLNA if the following criteria are fullfilled:
- Exposure to at least one concentration of the test item resulted in an incorporation of 3H- Methylthymidine past 3- fold or greater than that recorded in control mice, as indicated by the stimulation index.
- That the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

ADDITIONAL EVALUATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality/ viability: once daily (week day) from experimental start to necropsy.
- Body weights: prior to the first application and prior to treatment with 3H- Methylthymidine.
- Clinical signs (local / systemic): oat 1 -2 hours after each application. Especially the treatment sites were observed carefully.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated.

Positive control results:

The positive control using alpha-Hexylcinnamaldehyde demonstrated an S.I. of 3.30 using a concentration of 25 % (w/v).

Parameter:

SI

Value:

1.18

Test group / Remarks:

12.5%

Parameter:

SI

Value:

1.35

Test group / Remarks:

25%

Parameter:

SI

Value:

1.49

Test group / Remarks:

50%

Parameter:

EC3

Value:

> 50

Cellular proliferation data / Observations:

EC3 CALCULATION
The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

CLINICAL OBSERVATIONS:
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3H-Methylthymidine, was within the range commonly recorded for animals of this strain and age.

MORTALITY
No deaths occurred during the study period.

Concentration	Disintegrations per minute (DPM)
0%	1109.8
12.5%	1311.2
25%	1495.0
50%	1658.1

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Two LLNA tests and an in vitro test battery for skin sensitisation are available for the substance itself. Summaries of the available data are given below.

 

Skin Sensitisation (LLNA)

The contact sensitizing potential of the test item was assessed in vivo using an LLNA test. The study was performed according to OECD 429, adopted in 2002. To investigate primary proliferation (induction phase) of sensitisation, 3 groups with each 4 females were treated with 25, 50 or 100 % of the test item by topical application onto the dorsum of both ear lobes for three consecutive days. A control group of 4 females was treated with the vehicle only. Five days after the first topical exposure the proliferative response of activated lymph node cells was assessed by the injection of 3H- Methylthymidine this is incorporating into the lymph node cells. Proliferation is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/ node) and as the ratio of the amount of 3H- Methylthymidine which is incorporated into lymph node cells of treated animals relative to the amount which is recorded for control lymph nodes (stimulation index S.I.). No mortality and no signs of systemic toxicity were observed. Also, no skin irritation was observed at the tested concentrations. A test item is regarded as a sensitizer if the exposure resulted in 3- fold or greater Stimulation Index (S.I.) compared to concurrent controls, namely the EC3. For the test item S.I. values of 2.32, 3.19, and 3.50 were determined with the test item at concentrations of 25, 50, and 100 % in acetone: olive oil (4+1), respectively. The calculated EC3 of the test item was 44.5 %. The test item is therefore considered to be a skin sensitizer (RCC 2007).

 

Skin Sensitisation (LLNA) 

The potential to induce skin sensitisation was evaluated in vivo in a LLNA study according to OECD 429. In the main study, 3 groups with each 5 females were treated with 12.5, 25 or 50 % of the test item by topical application onto the dorsum of both ear lobes for three consecutive days. A control group of 5 females was treated with the vehicle only. Five days after the first topical exposure, the proliferative response of activated lymph node cells was assessed by the injection of 3H- Methylthymidine, which is incorporated into lymph node cells of treated animals relative to the amount which is recorded for control lymph nodes (stimulation index S.I.). No mortality and no clinical signs were observed, the body weights of all animals were within the normal range. In this study, Stimulation Indices of 1.18, 1.35 and 1.49 were determined with the test item at concentrations of 12.5, 25 and 50% (w/v) in acetone:oliveoil (4+1). The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3 (RCC-CCR 2005).

 

Summary of the in vitro test battery for skin sensitisation

A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitisation as defined by OECD, has been conducted to assess the skin sensitisation potential.

-      Protein reactivity (DPRA)

-      Activation of keratinocytes (LuSens), and

-      Activation of dendritic cells (MUSST).

 

The results of the individual studies are summarized and evaluated to predict the presence of absence of skin sensitizing potential of the target substance. The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al. in 2012. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA, OECD 2010), the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans. A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing of this report.

In the DPRA (BASF 2012) and LuSens (BASF 2013) tests with the target substance, a negative result was obtained. The other study (MUSST) was positive (BASF 2012). Based on the results, the target substance is not peptide reactive, does not activate keratinocytes but activates dendritic cells.

 

With regard to skin sensitisation, contradicting results are available: the two independent and valid LLNA tests gave a negative and a (weak) positive result, respectively. The tests were conducted in 2005 and 2007, when the issue of false positive results that might be obtained in LLNA studies with substances containing oleic acids was not yet known. Nowadays, a study protocol according to OECD 406 would be the choice. Due to animal welfare reasons, in-vitro tests (DPRA, MUSST, LuSens) were performed for clarification of the potentially false-positive results of the LLNA. In one test (MUSST), indications for a skin sensitizing potential were detected, but were in fact only weak positive. The other tests (DPRA and Lu-sens) were negative.Since the classification regarding skin sensitization potential is still unclear, a guinea pig maximization test (OECD 406) according to the Buehler protocol was ordered and is still ongoing.

For precautionary reasons, the substance is classified for skin sensitization 1B in the meantime, based on the results of the potentially false-positve LLNA.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Though the available data was not consistent, the substance should be classified for precautionary reasons, as Skin Sens. 1B, H317: May cause an allergic skin reaction in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.