Registration Dossier

Administrative data

Description of key information

GMPT: negative (BASF 1994)

LLNA: positive, EC 3 = 29% (Evonik, 2002)

in vitro: positive (BASF, 2017)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(adopted 24 April 2002)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 17 - 21 g
- Housing: individually
- Diet: Free access to mains tap water and food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthom, Bicester, Oxon, UK)
- Water: free access to tap water
- Acclimation period: at least five days

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

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50, and 100% (v/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% (v/v), this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). A further group of four mice received the vehicle alone in the same manner. Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

15% (v/v) in acetone/olive oil 4:1 = 10.91

Alpha-Hexy1cinnamaldehyde (purity: 85%) was considered to be a sensitiser under the conditions of the test.
Parameter:
EC3
Value:
ca. 29

Concentration (%) dpm/Node*  SI
Vehicle 748.88  na
25 1802.35  2.41
50 4819.02  6.43
100 3557.64  4.75

* Disintegration per minute/node obtained by dividing the disintegration per minute value by 8 (total number of lymph nodes)

There were no deaths. No signs of systemic toxicity were noted in the test or contral animals during the test. Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Endpoint:
skin sensitisation: in vivo (non-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:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Remarks:
Department of Toxicology, BASF SE, Germany
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test already available
Species:
guinea pig
Strain:
other: Pirbright White, Dunkin Hartley Crl: (HA)BR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH - Wiga, Sulzfeld
- Weight at study initiation: 275-335 g
- Housing: 5 per cage, Makrolon type IV
- Diet: Kliba laboratory diet, 341 ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
Induction - Intradermal: 5% in olive oil DAB 10 or in Freund's adjuvant/0,9% aqueous NaClsolution (1 : 1)
Induction - Epicutaneous, occlusive: 100% (unchanged)
Challenge - Epicutaneous, occlusive: 100% (unchanged)
Route:
epicutaneous, occlusive
Vehicle:
olive oil
No. of animals per dose:
test group: 20 animals;
control groups: 10 animals each
Details on study design:
RANGE FINDING TESTS:
Amount applied:
2 x 2 cm filter paper strips were applied to the skin of the flanks under an occlusive dressing (the bandage consists of rubberized linen patches 4 x 4 cm from Russka, test patch 5 x 5 cm of "Idealbinde" from Pfälzische Verbandstoff-Fabrik, and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG) . The test filter paper strip was soaked in the respective test substance formulation; thus, the animals were exposed to about 0.15 g of the test substance.

Exposure period:
The test substance was applied 2 times for 24 hours (within a period of 96 hours) in order to detect non-specific phenomena that are not caused by a sensitization reaction but could possibly be attributed to a shift in the irritation threshold.

Site of application:
- flank, respective on the same area

Number of test animals:
- 4 per test concentration

Readings:
- about 24 and 48 h after application
Assessment of skin findings (according to Draize, J .H. (1959): Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States Austin, Texas):

1. Erythema and eschar formation
0) No erythema
1) Very slight erythema (barely perceptible)
2) Well-defined erythema
3) Moderate to severe erythema
4) Severe erythema (beet redness) to slight eschar formation (injuries in depth)

2. Edema formation
0) No edema
1) Very slight edema (barely perceptible)
2) Slight edema (edges of area well defined by definite raising)
3) Moderate edema (raised approximately 1 mm)
4) Severe edema (raised more than 1 mm and extending beyond the area of exposure)


MAIN STUDY
A. INDUCTION EXPOSURE
A1. Intradermal induction:
- No. of exposures: 6 intradermal injections in groups of two per animal
- Test groups: intradermal injections: front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1:1; middle row: 2 injections each of 0.1 ml of the test substance formulation; back row : 2 injections each of 0.1 ml Freund's adjuvant / 0.9% aqueous NaCI-solution (1 : 1) with test substance
- Control group: The animals were given the same injections but without the test substance, only with the formulating agent
- Site: shoulder
- Frequency of applications: day 0 intradermal induction;
- Duration: 24 hours
- Concentrations: undiluted

A2: Percutaneous induction:
- No. of exposures: 1 percutaneous induction (one week after intradermal induction)
- Test groups: 2x4 cm filter paper stripes were applied to the skin of the shoulder under an occlusive dressing. The filter paper was soaked in the test substance. Thus the animals were exposed to about 0.3 g of the test substance.
- Control group: left untreated since the test substance was applied unchanged and thus no solvent was used
- Site: shoulder
- Frequency of applications: day 7 percutaneous induction
- Duration: 48 hours
- Concentrations: 2x4 cm filter paper stripes soaked with the test substance (about 0.3 g of the test substance)
- Readings: 48 hours after application
B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 21 days and 28 days after intradermal induction
- Exposure period:
- Test groups: receiving the test substance at a non-irritant concentration
- Control group: 1st challenge: 1st control group received test substance, 2nd remained untreated; 2nd challenge both control groups treated with test substance
- Site: shaved intact flank skin
- Concentrations: non-irritant concentration (2x2 cm filter paper soaked in the test material; animals received about 0.15 g of the test substance)
- Evaluation (hr after challenge): 24h and 48h after removal of the patch

Challenge controls:
1st challenge:previously untreated animals receiving either the same treatment as the test group (1st control group) or left untreated (2nd control group);
2nd challenge: both control groups received the same treatment as the test group
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene
Positive control results:
The positive control with 1-chloro-2,4-dinitrobenzene showed that the test system was able to detect sensitizing compounds under the laboratory conditions chosen.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
scaling in 1 animal
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: scaling in 1 animal.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Pretest:

In the preliminary test after two 24-hour percutaneous occlusive applications within 96 hours the unchanged test substance was found to be non-irritant.

Intradermal injection of 5% of the test substance caused topical reactions as described below.

Intradermal induction:

Control group animals (Freund's adjuvant : 0.9% aqueous NaCI-solution 1:1): well-defined erythema and slight edema was observed at the injection sites.

Control group animals (olive oil DAB 10): well-defined erythema.

Test group animals (Freund's adjuvant : 0.9% aqueous NaCI-solution 1:1): well-defined erythema and slight edema was observed at the injection sites

Test group animals (test substance preparation in olive oil DAB 10): well-defined erythema (20/20) animals and very slight edema (7/20)

Test group animals (test substance preparation in Freund's adjuvant : 0.9% aqueous NaCI-solution 1:1): well defined erythema and slight edema.

Percutaneous induction:

Test group: incrustation, partially open (caused by the intradermal induction) in addition to well-defined erythema and slight edema.

1st challenge:

Test group: 4/20 animals exhibited very slight erythema.

Control group 1: no skin reactions

Control group 2: no application of the test substance

2nd challenge:

Test group: no skin reaction

Control group 1: no skin reaction

Control group 2: no skin reaction

Tables:

Readings

Animal

1st Challenge – Test group

Erythema

Edema

Additional findings

24 h

1

0

0

2

0

0

3

1

0

4

0

0

5

0

0

6

1

0

7

0

0

8

1

0

9

1

0

10

0

0

48 h

1

0

0

2

0

0

3

0

0

4

0

0

5

0

0

6

1

0

7

0

0

8

0

0

scaling

9

1

0

10

0

0

Mean

24, 48 h

1

0

0

2

0

0

3

0.5

0

4

0

0

5

0

0

6

1

0

7

0

0

8

0.5

0

9

1

0

10

0

0

Mean 24, 48 h

1-10

0.3

0

Readings

Animal

2nd Challenge – Test group

Erythema

Edema

Additional findings

Mean

24, 48 h

1

0

0

2

0

0

3

0

0

4

0

0

5

0

0

6

0

0

7

0

0

8

0

0

9

0

0

10

0

0

Mean 24, 48 h

1-10

0

0

Positive control: (data derived from project no. 30H0267/922123, 20.09.1993 )

1st challenge

2nd challenge

1-chloro-2,4-dinitrobenzene 0.5% in EtOH

EtOH

1-chloro-2,4-dinitrobenzene 0.5% in EtOH

EtOH

Control group 1

1/10

0/10

5/10

0/10

Control group 2

no application of 1-chloro-2,4-dinitrobenzene

0/10

0/10

0/10

Test group

20/20

0/20

20/20

0/20

After application of the test substance no abnormalities in the body weight gain were observed in the treated animals. No additional findings were reported.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the study under the test conditions chosen and applying the evaluation criteria it was concluded that Oxoöl 9 N does not have a sensitizing effect on the skin of the guinea pig in the Maximization Test .
Endpoint:
skin sensitisation: in vitro
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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted February 2015
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted February 2015
Qualifier:
according to
Guideline:
other: OECD 442E (h-CLAT)
Version / remarks:
adopted July 2016
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) 2017/735, B.59 (DPRA)
Version / remarks:
adopted February 2017
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) 2017/735, B.60 (LuSens)
Version / remarks:
adopted February 2017
GLP compliance:
yes (incl. certificate)
Type of study:
other: Combined evaluation of direct peptide binding and activation of keratinocytes and dendritic cells
Details on study design:
All: no analysis of the test substance in the vehicle was performed, since it was applied shortly after preparation.

DPRA:
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)
concentrations: 5% (50.3mg/ml) and undiluted (99.3% pure) - a gravimetric method was used, since no molecular weight could be assigned to the complex mixture of hydrocarbons (UVCB)
ratio for incubation:
5% dilution: 1:7 and 1:32 (cysteine and lysine containing peptides), based on absolute mass - tested as emulsion
undiluted: 1:10 and 1:50 (cysteine and lysine containing peptides) - tested as emulsion
vehicle controls (propanol) and all concentrations performed as triplicate
positive control: EGDMA (CAS 97-90-5)
co-elution control performed to detect possible interferences of the test substance with the peptides


LuSens (Keratinocyte activation)
cell line: Human transgenic keratinocyte cell line derived from HaCaT cells
cell densitiy: 0.83 x10e5 /ml
incubation time: 48h at 37°C
concentrations based on MTT assay:
first experiment
4.5, 5.4, 6.5, 7.8µg/mL
fourth experiment (experiments 2 + 3 did not meet acceptance criteria)
1.1, 1.3, 1.5, 1.8, 2.2, 2.6, 3.1, 3.8, 4.5, 5.4, 6.5µg/mL
next higher concentration caused a reduction in viability of more than 30%
dilutions were prepared by stirring
positive control: EGDMA, 18µg/ml (CAS 97-90-5)
negative control: DL-lactiv acid, 450µg/ml in 1% DMSO (CAS 50-21-5)
vehicle control 1% DMSO in culture medium
Replicates: triplicates


h-CLAT (activation of dendritic cells)
induction of CD86 and CD54
cell line: human monocytic leukemia cells THP-1
cell densitiy: 2x10e6/ml
incubation time: 24h at 37°C
cytotoxicity: determined by PI - DNA intercalation (goal: viability > 50%, due to a calcuation error for viability, too low concentrations were used in the first experiments)
concentrations:
first experiment: 40, 48, 58, 69, 83, 100, 120, 144µg/mL
second experiment: 83, 100, 120, 144, 173, 208, 249, 299µg/ml
third through fifith experiment: 120, 144, 173, 208, 249, 299, 359, 431µg/mL
Dilutions were prepared by stirring and ultra-sonication
positive control: DNCB, 4µg/ml (CAS 97-00-7)
negative control: DL-lactiv acid, 1000µg/ml (CAS 50-21-5)
Duplicates
vehicle control 0.2% DMSO in culture medium
Parameter:
other: Peptide depletion
Remarks:
(mean of both peptides)
Run / experiment:
5%
Value:
2.99
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Remarks:
substance tested as emulsion, so the test may be underpredictive
Parameter:
other: peptide depletion
Remarks:
mean of both peptides
Run / experiment:
undiluted
Value:
0.54
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Remarks:
potentially underpredictive, since substance tested as emulsion
Parameter:
other: EC 1.5 (µg/ml)
Remarks:
(LuSens)
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: EC200 (µg/ml)
Remarks:
CD54 (h-CLAT)
Run / experiment:
1
Value:
132
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
(no increase for CD 86)
Parameter:
other: EC200 (µg/ml)
Remarks:
CD54 (h-CLAT)
Run / experiment:
5
Value:
50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
(no increase for CD86)
Other effects / acceptance of results:
DPRA
no Co-elution of the substance with the peptide occurred

LuSens
no precipitation occurred

h-CLAT
no precipitation occurred
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
DPRA: negative, but potentially underpredictive, since substance tested as emulsion. Result thus rendered inconclusive
LuSens: positive (EC1.5 = 1.5µg/mL9
h-CLAT: positive (EC 200 >=50 <=132µg/mL) based on CD54 induction. No induction of CD86
Overalls interpretation: positive
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Maximization Test:

The test substance was tested for its sensitizing effect on the skin of the guinea pig in the Maximization test based on the method of Magnusson and Kligman according to OECD Guideline 406 (BASF AG, 30H0116/932042, 1994). The intradermal induction with the 5% test substance preparations caused very slight to well-defined signs of irritation in the test group animals. After percutaneous induction with the unchanged test substance incrustation, partially open (caused by the intradermal induction) in addition to well-defined erythema and slight edema could be observed in the test group animals. The first challenge with the unchanged test substance caused very slight erythema in 4 out of 20 test group animals 24 hours after removal of the patches. After the second challenge no skin reactions could be observed. Based on the results of the study under the test conditions chosen it was concluded that the test substance does not have a sensitizing effect on the skin of the guinea pig in the Maximization Test .

LLNA

In an LLNA according to OECD 429, CBA/CA mice were treated with 25, 50, and 100% of the registered substance in aceton/olive oil. Based on a pre-test, all concentrations should not cause excessive irritation. Lymph nodes were pooled per group. Concentrations of 50% and 100% caused a relevant increase in radioactivity. The EC3 value was calculated as 29%.

In vitro assays according to OECD 442C, D, E

The three tests used in the “2 out of 3” testing strategy assess protein binding in chemico (DPRA), triggering an antioxidant response in keratinocytes (LuSens) and activation of dendritic cells (h-CLAT), which cover different key events of the skin sensitization Adverse Outcome Pathway. In the DPRA, no or minimal peptide binding was observed. 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 result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case. Luciferase activity was enhanced more than 1.5 fold in at least two consecutive concentrations in tow independent experiments at a cell viability above 70%. The EC1.5 value was 1.5µg/ml. No activation of CD86 was observed in dendritic cells, but expression of CD54 was enhanced more than 2 fold in at least two independent experiments. Consequently, two positive results were obtained in the LuSens and h-CLAT and one inconclusive result in the DPRA. Following the overall evaluation strategy, the substance is considered a sensitizer in this test system.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

In a weight of evidence approach, the substance is considered a moderate skin sensitizer. Based on the overall positive result in the in vitro assays and the EC3 value obtained in the LLNA assay of 29%, classification according to GHS, EU as skin sensitizer cat. 1B is required.