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Diss Factsheets

Administrative data

Description of key information

The skin sensitization potential of the substance was evaluated according to the OECD Guideline No. 429 "Skin Sensitization: Local Lymph Node Assay". Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the undiluted substance or the substance as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of four animals were treated with acetone/olive oil 4:1 alone. The Stimulation Indices are as follows: 25% - 4.33; 50% - 10.96; 100% - 17.12. The concentration of the substance expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 21.8% (extrapolated). Therefore the substance would be considered to be a sensitizer based on the results of this study. However the results of this study are thought to be questionable so an additional in vivo study (Guinea Pig Maximisation test, GPMT) was commissioned in order to confirm or refute the LLNA result.

 

The substance was tested in a GPMT according to OECD Guideline No. 406 in which male guinea pigs were administered intradermally (together with Freund's adjuvant) and epicutaneously (lowest irritating concentration) with the substance. Twenty-one days after first administration, the substance was applied epicutaneously at the highest non-irritating concentration (100% and 50%). The local reactions observed after the first challenge were considered to be of irritant nature since irritation was noted in the control animals. So a second challenge phase was performed using the substance diluted to 25% and 10%. No macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. Based on these findings the substance is not considered to be a skin sensitizer.

 

The skin sensitizing potential of an analogue substance was investigated in a GPMT according to OECD 406. Male guinea pigs were treated intradermally (together with Freund's adjuvant) and epicutaneously (lowest irritating concentration) with the test item. Twenty-one days after first administration, the substance was applied epicutaneously at the highest non-irritating concentration. The local reactions recorded in the eight test animals (40 %) after the challenge were considered to be of irritant nature. This interpretation is supported by the fact that the reactions faded at the 48-hour reading and that 60 % of the control animals also showed a local erythema at the 24 -hour reading. Based on these findings the analogue substance is not classified as a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to K1 study therefore K2 is the maximum Klimisch value.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was conducted prior to LLNA being accepted as a TG.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS Albino Dunkin Hartley Guinea Pig, CRL:(HA)BR, SPF (Specific Pathogen Free)
- Source: Charles River, Kisslegg, Germany
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 538-704 g
- Housing:Individually in Makrolon type-4 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard guinea pig breeding / maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland) ad lib.
-Water (e.g. ad libitum):Community tap water ad lib.
- Acclimation period: under laboratory conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 30-70
- Air changes (per hr): at least 15/h
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 2008-11-12 to 2008-12-09
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
5% for intradermal induction, 100% for epidermal induction;
75% for epidermal challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5% for intradermal induction, 100% for epidermal induction;
75% for epidermal challenge
No. of animals per dose:
10 control animals and 20 test animals
Details on study design:
RANGE FINDING TESTS:
Intradermal pretest (together with Freund's) in two animals, 75-50-25-15-10 and 5 %. Dermal reactions were assessed 24 hours later based on the results, the test item concentration of 5 % (w/w) was selected for intradermal induction in the main study.
Epidermal pretest (after Freund's) in two animals, 100-75-50 and 25 %. Based on the results, the test item concentration of 75 % (w/w) was selected for epidermal challenge and 100% as lowest irritating concentration for epidermal induction.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1x intradermal, 1 x epicutaneous
- Exposure period: intradermal-continuous, epicutaneous: 48 h
Test group:
intradermal induction:
in parallel intradermal injection of Freund's complete adjuvant, test item (5% ) in purified water, Freund's/test item (5%) 1:1
epidermal induction:
100% test item, occlusive conditions, 48 h
- Control group:
intradermal induction:
in parallel intradermal injection of Freund's complete adjuvant, purified water, Freund's/purified 1:1
epidermal induction:
purified water, occlusive conditions, 48 h
- Site: dorsal skin from the scapular region (intradermal and epicutaneous)
- Frequency of applications: once



B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: day 22
- Exposure period: 24 h
- Test groups: test item (75%) / water
- Control group: test item (75%) / water
- Site: left flank
- Evaluation (hr after challenge): 24 h, after depilation
Challenge controls:
No re-challenge
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamaldehyde
Positive control results:
90% of positive responders in test group, 0% in control group
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 8.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE at 1 % in PEG 300
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE at 1 % in PEG 300
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
ALPHAHEXYLCINNAMALDEHYDE at 0.1 % in PEG 300
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
discrete/patchy erythema
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
No skin sensitizing potential was identified in the guinea pig Maximization-test with the substance.
Executive summary:

The skin sensitizing potential of the substance was investigated in a guinea pig maximisation test according to OECD 406. Male guinea pigs were treated intradermally (together with Freund's adjuvant) and epicutaneously (lowest irritating concentration) with the test item. Twenty one days after first administration, the substance was applied epicutaneously at the highest non-irritating concentration. The local reactions recorded in the eight test animals (40 %) after the challenge were considered to be of irritant nature. This interpretation is supported by the fact that the reactions faded at the 48-hour reading and that 60 % of the control animals also showed a local erythema at the 24 -hour reading. Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 2001/59/EC, the substance does not have to be classified and labelled as a skin sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2018 - 20 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 17th, 1992
Deviations:
yes
Remarks:
A number of environmental conditions were breached during the study, but as no effect was noted on the health of the animals, this deviation is considered as without impact on the conclusion of the study.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The substance was tested for skin sensitization potential in a LLNA study and the results revealed Stimulation Indices higher than three at all dose concentrations and increasing in a dose-dependent manner. Based on these results, this substance would normally be classified as a skin sensitizer. However, there are several reasons why the validity of the LLNA result is open to question:
 
i) The chemical structure(s) do not contain any obvious features which are normally associated with skin sensitization i.e. the presence or metabolic/non-metabolic formation of protein reactive functional groups.
ii) The analogue substance Triethanolamine, propoxylated gave a positive result in a LLNA, but a negative response in a reliable GPMT.
iii) There is significant discussion in the toxicology literature regarding the validity of positive results obtained in the LLNA on surfactant substances such as Palm oil, propoxylated.

Based on the uncertainties listed above, the validity of the positive LLNA result is questionable. Therefore, this in vivo skin sensitization testing (GPMT) was commissioned to confirm or refute the validity of the LLNA result. 
Specific details on test material used for the study:
Batch No.: 1023R26015
Storage: room temperature, darkness
Form: pasty liquid
Colour: yellow
Expiry date: 16 September 2018
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
Prior to the test, the animals were kept for a minimum acclimatization period of 5 days.
The drinking water (tap water from public distribution system) and food were supplied ad libitum.
The animals were housed in groups of 5 at the maximum in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted with a stainless steel lid with a feeding device and drinking device of 500 mL.
The temperature and relative humidity of the main test were controlled to remain within target ranges of 19°C to 25°C and 30% to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
100% and 50%.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% and 100%
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
25% and 10%
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
GROUP 1 (negative control): 10 male guinea pigs
GROUP 2 (treated): 20 male guinea pigs
Details on study design:
RANGE FINDING TESTS:
Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC)
Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC)
Determination by topical application of the Maximal Non Irritant Concentration (MNIC)

MAIN STUDY
A. INDUCTION EXPOSURE
GROUP 1 (control):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in corn oil
· 2 ID: corn oil
· 2 ID: a mixture with equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and corn oil
GROUP 2 (Treated):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in corn oil
· 2 ID: substance at 50% in corn oil
· 2 ID: a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the substance at 100%
2nd Topical Induction:
- Exposure duration: 48 hours
GROUP 1 (control): 0.5 mL of corn oil.
GROUP 2 (treated): 0.5 mL of the substance at 100%

B. CHALLENGE EXPOSURE
First challenge:
1 Sample cup saturated with the substance at 100% (MNIC) and 1 sample cup saturated with the test item at 50% in corn oil (1/2 MNIC) under occlusive dressing for 24 hours.
Reading were taken at 24, 48 and 72 hours after patch removal.
Second challenge:
1 Sample cup containing the substance diluted at 25% and 1 sample cup containing the substance diluted at 10% in corn oil under occlusive dressing for 24 hours.
Reading were taken at 24 and 48 hours after patch removal.
Positive control substance(s):
yes
Remarks:
The results of the last 11 positive groups (Reference substance: alpha-Hexylcinnamaldehyde) were included.
Key result
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
A discrete to moderate erythema was noted in 30% (3/10) of the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
A moderate erythema was noted in 10% (1/10) of the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reaction was noted.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reaction was noted.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reaction was noted.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reaction was noted.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
Discrete to moderate erythema in 20% (4/20).
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
A moderate to intense erythema was noted in 35% (7/20) of animals.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
Discrete to moderate erythema in 15% (3/20).
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
A moderate to intense erythema was noted in 10% (2/20) of animals.
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
test chemical
Dose level:
100%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Discrete to moderate erythema in 10% (2/20).
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
test chemical
Dose level:
50%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
A moderate to intense erythema was noted in 5% (1/20) of animals.
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Under these experimental conditions, the substance did not produce positive skin sensitisation results and therefore does not have to be classified as a skin sensitizer.
Executive summary:

A Guinea Pig Maximisation Test was undertaken to evaluate the possible allergenic activity of the substance after intradermal and topical administration in guinea pigs. The study was conducted accxordign to OECD Test Guideline No.406 of July 17th, 1992, and the test method B.6 of council regulation No. 440/2008 of May 30th 2008. The induction phase (intradermic injection at 50% and topical application at 100) was conducted with the substance to 20 Guinea pigs followed by a 10-day rest phase. The challenge phase conducted under occlusive dressing for 24 hours,

consisted of a single topical application of the substance at 100% and diluted at 50% in corn oil. In the treated group (treatment dose of 100%), a discrete to moderate erythema was noted in 20% (4/20), 15% (3/20) and 10% (2/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively. In the control group (associated with the treatment dose of 100%), a discrete to moderate erythema was noted in 30% (3/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted at 48 and 72 hours. In the treated group (treatment dose of 50%), a moderate to intense erythema was noted in 35% (7/20), 10% (2/20) and 5% (1/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively. In the control group (associated with the treatment dose of 50%, a moderate erythema was noted in 10% (1/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted at 48

and 72 hours. As irritation was noted in the control group animals, only reactions in the test group animals that exceed the most severe reactions seen in the control group animals were attributed to skin sensitization. Therefore, a reaction of sensitization was noted in 15% and 10% of the animals in the area treated with the substance at 100%, 48 and 72 hours after the challenge phase. A reaction of sensitization was noted in 10% and 5% of the animals in the area treated with the substance at 50%, 48 and 72 hours after the challenge phase. In order to confirm these results, a second challenge phase was performed with the substance diluted at 25% and 10%. In the treated group (treatment dose of 25%), no macroscopic cutaneous reactions attributable to

allergy were noted after the challenge phase. In the naïve control group (associated with the treatment dose of 25%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. In the treated group (treatment dose of 10%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the naïve control group (associated with the treatment dose of 10%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. It was therefore concluded that under these experimental conditions, the substance did not produce positive skin sensitisation results and therefore does not have to be classified as a skin sensitizer.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2018 - 08 March 2018
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:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Batch: 1023R26015
Purity: 99.7%
Physical state / Appearance: yellow liquid
Expiry Date: 16 March 2018
Storage Conditions: room temperature in the dark
Species:
mouse
Strain:
CBA/Ca
Remarks:
(CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
Nulliparous and non-pregnant.
start of study weight range of 15 to 23 g.
Age at start of study 8 to 12 weeks old.
Acclimatization period of at least 5 days.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary Screening Test: 100%
Main Test: 100%, 50% or 25%
No. of animals per dose:
Preliminary Screening Test: One animal/dose
Main Test: Four animals/dose
Positive control substance(s):
other: Historical data: α-Hexylcinnamaldehyde, tech., 85%
Positive control results:
Historical data: α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Key result
Parameter:
SI
Value:
4.33
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
10.96
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
17.12
Test group / Remarks:
100%
Key result
Parameter:
EC3
Value:
21.8
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The substance is considered to be a sensitizer based on the results of this study.
Executive summary:

The skin sensitization potential of the substance was evaluated in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted according to the OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay". Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the undiluted substance or the substance as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of four animals were treated with acetone/olive oil 4:1 alone. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 25% - 4.33; 50% - 10.96; 100% - 17.12. The concentration of the substance expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 21.8% (extrapolated). Therefore the substance is considered to be a sensitizer based on the results of this study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-08 to 2009-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
April 29, 1993
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was conducted prior to LLNA being accepted as a TG.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS Albino Dunkin Hartley Guinea Pig, CRL:(HA)BR, SPF (Specific Pathogen Free)
- Source: Charles River, Kisslegg, Germany
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 538-704 g
- Housing:Individually in Makrolon type-4 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard guinea pig breeding / maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland) ad lib.
-Water (e.g. ad libitum):Community tap water ad lib.
- Acclimation period: under laboratory conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 30-70
- Air changes (per hr): at least 15/h
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 2008-11-12 to 2008-12-09
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
5% for intradermal induction, 100% for epidermal induction;
75% for epidermal challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5% for intradermal induction, 100% for epidermal induction;
75% for epidermal challenge
No. of animals per dose:
10 control animals and 20 test animals
Details on study design:
RANGE FINDING TESTS:
Intradermal pretest (together with Freund's) in two animals, 75-50-25-15-10 and 5 %. Dermal reactions were assessed 24 hours later based on the results, the test item concentration of 5 % (w/w) was selected for intradermal induction in the main study.
Epidermal pretest (after Freund's) in two animals, 100-75-50 and 25 %. Based on the results, the test item concentration of 75 % (w/w) was selected for epidermal challenge and 100% as lowest irritating concentration for epidermal induction.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1x intradermal, 1 x epicutaneous
- Exposure period: intradermal-continuous, epicutaneous: 48 h
Test group:
intradermal induction:
in parallel intradermal injection of Freund's complete adjuvant, test item (5% ) in purified water, Freund's/test item (5%) 1:1
epidermal induction:
100% test item, occlusive conditions, 48 h
- Control group:
intradermal induction:
in parallel intradermal injection of Freund's complete adjuvant, purified water, Freund's/purified 1:1
epidermal induction:
purified water, occlusive conditions, 48 h
- Site: dorsal skin from the scapular region (intradermal and epicutaneous)
- Frequency of applications: once



B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: day 22
- Exposure period: 24 h
- Test groups: test item (75%) / water
- Control group: test item (75%) / water
- Site: left flank
- Evaluation (hr after challenge): 24 h, after depilation
Challenge controls:
No re-challenge
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamaldehyde
Positive control results:
90% of positive responders in test group, 0% in control group
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the control group
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the control group.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 8.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: NLP#15 (2,2’,2’’-Nitrilotriethanol, propoxylated), 75 % in purified water . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
purified water only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No systemic toxicity and/or deaths were noted in the guinea pigs of the test group.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: purified water only . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No systemic toxicity and/or deaths were noted in the guinea pigs of the test group..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE at 1 % in PEG 300
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE at 1 % in PEG 300
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
ALPHAHEXYLCINNAMALDEHYDE at 0.1 % in PEG 300
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
discrete/patchy erythema
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
No skin sensitizing potential was identified in the guinea pig Maximization-test with the substance.
Executive summary:

The skin sensitizing potential of the substance was investigated in a guinea pig maximisation test according to OECD 406. Male guinea pigs were treated intradermally (together with Freund's adjuvant) and epicutaneously (lowest irritating concentration) with the test item. Twenty one days after first administration, the substance was applied epicutaneously at the highest non-irritating concentration. The local reactions recorded in the eight test animals (40 %) after the challenge were considered to be of irritant nature. This interpretation is supported by the fact that the reactions faded at the 48-hour reading and that 60 % of the control animals also showed a local erythema at the 24 -hour reading.

Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 2001/59/EC, the substance does not have to be classified and labelled as a skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The substance was tested for skin sensitization potential in a LLNA study and the results revealed Stimulation Indices higher than three at all dose concentrations and increasing in a dose-dependent manner. Based on these results, this substance would normally be classified as a skin sensitizer. However, there are several reasons why the validity of this LLNA result is open to question:

 

i) The chemical structure(s) do not contain any obvious features which are normally associated with skin sensitization i.e. the presence or metabolic/non-metabolic formation of protein reactive functional groups.

ii) The analogue substance Triethanolamine, propoxylated also gave a positive result in a LLNA, but a negative response in a reliable GPMT and is not classified.

iii) There is significant discussion in the toxicology literature which questions the validity of positive results obtained in the LLNA on surfactant substances such as Palm oil, propoxylated.

Based on the uncertainties listed above, the validity of the positive LLNA result is questionable. Therefore, further in vivo skin sensitization testing (GPMT) was commissioned to confirm or refute the validity of the LLNA result. The GPMT assay gave a negative result, which is being used for classification.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

There is a positive LLNA study on the substance however the results of this study are thought to be questionable. An additional in vivo study (Guinea Pig Maximisation test, GPMT) was commissioned in order to confirm or refute the LLNA result. The substance was negative for sensitisation in the reliable GPMT. A structural analogue also gave a negative result in a reliable GPMT. Based on these results classification of the substance is not justified.