Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Description of key information

Non sensitising <30%, OECD 429, Mouse LLNA, Johnson 2000
Sensitising, OECD 406, Guinea Pig Maximisation assay (M&K), Rattray 1999

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:
experimental study
Adequacy of study:
weight of evidence
Study period:
4 March - 24 April 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Guinea pig maximisation test has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adult
- Weight at study initiation: 272-345 g
- Housing: 5 per cage in cages suitable for animals of this strain and the weight range expected during the course of the study
- Diet: FD1 ad libitum
- Water: mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 16-23°C
- Humidity: 30-70%
- Air changes: minimum of 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 9 March 1999 To: 24 April 1999 (positive control study 25 January 1999- 19 February 1999)
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
3% w/v for intradermal induction, 75% w/v for topical induction, 50% w/v and 25% w/v for challenge applications
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
3% w/v for intradermal induction, 75% w/v for topical induction, 50% w/v and 25% w/v for challenge applications
No. of animals per dose:
20 test and 10 controls
Details on study design:
RANGE FINDING TESTS: Groups of 2 or 4 female guinea pigs used and up to 4 dose-levels tested per group to determine dose levels to use for each of the 3 stages of the main study. Intradermal injection: 0.3, 1, 3 and 10% tested. Topical induction: 97 and 75% tested. Challenge: 96, 75, 50 and 25% tested.

MAIN STUDY
A. INDUCTION EXPOSURE - intradermal injection
- Test groups: 3 pairs of injections (0.05-0.1 mL each) of (i) Freund's complete adjuvant (FCA) plus corn oil in ratio 1:1, (ii) 3% w/v preparation of test substance in corn oil, (iii) 3% w/v preparation of test substance in a 1:1 preparation of FCA plus corn oil
- Control group: as for test group except corn oil instead of test substance
- Site: scapular region, row of 3 injections on each side of mid-line
- Frequency of application: single

B. INDUCTION EXPOSURE - topical application (one week after injections)
- Test groups: 200-300 mg of a 75% w/v preparation in corn oil applied on filter paper (approximately 4 cm x 2 cm) under an occlusive dressing for at least 48 hours
- Control group: as for test group except corn oil only applied
- Site: scapular region
- Frequency of application: single

c. CHALLENGE EXPOSURE (two weeks after topical induction)
- Test and control groups: 0.05-0.1 mL of a 50% w/v and a 25 % w/v preparation of the test substance in corn oil were each applied to the shorn flank (50% on left, 25% on right) on a piece of filter paper (approximately 1 cm x 2 cm) under an occlusive dressing for at least 24 hours.
- Evaluation (hr after challenge): 1 and 2 days

OTHER:
Positive control substance(s):
yes
Remarks:
hexylcinnamaldehyde
Positive control results:
Following challenge of previously induced guinea pigs with undiluted hexylcinnamaldehyde, the net percentage response was 100%. Hexylcinnamaldehyde was classified as an extreme sensitiser and, therefore, confirmed the validity of the test system.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
17
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 17.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 5.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 4.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 3.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100%
No. with + reactions:
19
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 100%. No with. + reactions: 19.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100%
No. with + reactions:
19
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 100%. No with. + reactions: 19.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: negative control (positive control study)
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: negative control (positive control study). Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: negative control (positive control study)
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: negative control (positive control study). Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Table 1: Maximisation test: Number of animals with signs of allergic skin reactions

Scored after:

24 hours

48 hours

Test groups

50% w/v preparation

17/20

5/20

 

25% w/v preparation

1/20

0/20

Negative/vehicle control

50% w/v preparation

4/10

3/10

 

25% w/v preparation

0/10

0/10

Positive control

19/19 (0/10 control)

19/19 (0/10 control)

 

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The substance is considered to be a moderate skin sensitiser.
Executive summary:

The sensitisation potential of the test substance was assessed using a method based on the maximisation test described by Magnusson and Kligman. The study involved two main procedures: the potential induction of an immune response and a challenge of that response. The sensitisation response was determined 1 and 2 days after challenge by assessing the degree of erythema.

Challenge of previously-induced guinea pigs with a 50% w/v preparation of the test substance in corn oil elicited a moderate sensitisation response and challenge at 25% elicited a weak skin sensitisation response.

A positive control study using hexylcinnamaldehyde demonstrated the sensitivity of the test system.

The substance is considered to be a moderate skin sensitiser.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 April - 21 March 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca/Ola/Hsd
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adults
- Weight at study initiation: not reported
- Housing: 4 per cage (maximum)
- Diet: RM1 diet ad libitum
- Mains water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17±25°C
- Humidity: 30-70%
- Air changes: minimum of 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light)

IN-LIFE DATES: From: 2 May 2000 To: 9 May 2000
Vehicle:
dimethylformamide
Concentration:
1, 3, 10 or 30% w/v
No. of animals per dose:
4
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION
- Groups of four male mice were used. Approximately 25 µL of a 1%, 3%, 10% or 30% w/v preparation of the test substance in dimethylformamide was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using dimethylformamide alone. The procedure was repeated daily for 3 consecutive days.
- Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.
- A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA. The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to (3-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter.
- The results are expressed as a counts per minute (cpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test:control ratio for each concentration.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
not applicable
Parameter:
SI
Value:
1.43
Test group / Remarks:
1% (w/v) test substance
Parameter:
SI
Value:
1.37
Test group / Remarks:
3% (w/v) test substance
Parameter:
SI
Value:
1.46
Test group / Remarks:
10% (w/v) test substance
Parameter:
SI
Value:
0.98
Test group / Remarks:
30% (w/v) test substance
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: No effect of treatment.

The application of the test substance at concentrations of 1%, 3%, 10% and 30% w/v in dimethylformamide resulted in an isotope incorporation which was less than 3-fold at all four concentrations. Consequently, the test substance is designated as unlikely to be a moderate or strong sensitiser under the conditions of the test. However there was evidence of possible cytotoxicity at the 30% concentration. The application of hexylcinnamaldehyde at concentrations of 1%, 3% and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at the 3% and 10% w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

Table 1: Skin sensitisation potential – Test substance

Concentration of test substance (% w/v)

Number of lymph nodes assayed

Counts per minute (cpm)

cpm per lymph node (x 10 ^2)

Stimulation index

0 (vehicle only)

8

1619

2.02

N/A

1

8

2302

2.88

1.43

3

8

2210

2.76

1.37

10

8

2350

2.94

1.46

30

8

1582

1.98

0.98

N/A – not applicable

Table 2: Skin sensitisation potential – Positive control substance (hexylcinnamaldehyde)

Concentration of hexylcinnamaldehyde (% w/v)

Number of lymph nodes assayed

Counts per minute (cpm)

cpm per lymph node (x 10 ^2)

Stimulation index

0 (vehicle only)

8

987

1.23

N/A

1

8

2011

2.51

2.04

3

8

5511

6.89

3.60

10

8

9606

12.01

9.76

N/A – not applicable

Interpretation of results:
other: The substance is unlikely to be a moderate or strong skin sensitiser under the conditions of the test.
Conclusions:
The test substance is unlikely to be a moderate or strong skin sensitiser under the conditions of the test.
Executive summary:

A sample of the test substance was assessed for its skin sensitisation potential using the mouse Local Lymph Node Assay. The assay determines the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. The test substance was applied as 1%, 3%, 10% or 30% w/v preparations in dimethylformamide. The test substance did not have the capacity to cause skin sensitisation when applied as 1%, 3%, 10% or 30% w/v preparations in dimethylformamide. However there was evidence of possible cytotoxicity at the 30% concentration. In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 3% or 10% w/v preparations in acetone, confirming the validity of the protocol used for this study.

In conclusion, the test substance is unlikely to be a moderate or strong skin sensitiser under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation

Two recent studies are available, a mouse local lymph node assay (compliant with OECD 429) and a guinea pig maximisation test (according to OECD 406). The Local lymph node assay indicated that the test substance was unlikely to be a moderate skin sensitizer at concentrations of up to 30% w/v in dimethylformamide. The M&K assay indicated that the test substance was a moderate skin sensitizer. In the M&K assay there was some evidence of irritation following challenge in the control group which could raise doubts as to the validity of the study result. However the degree of response seen in the test group and the number of animals and net percentage of animals responding suggest that the test substance has the potential to be a skin sensitizer.


Justification for selection of skin sensitisation endpoint:
Two studies are available. The degree of response seen in the M&K assay test group and the number of animals and net percentage of animals responding suggest that the test substance has the potential to be a skin sensitiser. As a conservative assessment, this result is considered to override the negative result in the LLNA study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The results from the guinea pig maximisation test indicate that the test substance is a potential skin sensitizer. The substance fulfils the criteria for classification as Category 1, H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008, Annex I, Part 3, 3.4.2.2.