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Diss Factsheets

Administrative data

Description of key information

The test substance showed no evidence for sensitizing properties in guinea pigs.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 July 2014 (allocation of the animals to the preliminary phase) to 15 January 2015 (termination of the second main phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The potential of the test item, Reactive Brown DYHY 0331/0334, to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the maximisation test of Magnusson and Kligman. This test system has been used as former experience with the development of reactive dyes showed that the murine local lymph node assay (LLNA) is not a suitable test system. In the past the LLNA led to nonsensically high SI-values (up to 70) while no reactions at all were seen in the guinea pig. Hence, it was concluded that for reactive dyes, the maximisation test of Magnusson and Kligman is the relevant test to test for skin sensitising properties.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The potential of the test item, Reactive Brown DYHY 0331/0334, to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the maximisation test of Magnusson and Kligman. This test system has been used as former experience with the development of reactive dyes showed that the murine local lymph node assay (LLNA) is not a suitable test system. In the past the LLNA led to nonsensically high SI-values (up to 70) while no reactions at all were seen in the guinea pig. Hence, it was concluded that for reactive dyes, the maximisation test of Magnusson and Kligman is the relevant test to test for skin sensitising properties.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, Krenzelweg 53, Horst NL-5960 AD Host, the Netherlands
- Age at study initiation: 6 - 7 weeks (at allocation)
- Weight at study initiation: 409 - 450 g (I); 425 - 460 g (II)
- Housing: up to 5
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 2 °C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): Approximately 15 to 25 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours

IN-LIFE DATES: From: 22 July 2014 (allocation of the animals to the preliminary phase) To: 15 January 2015 (termination of the main phase II)
Route:
intradermal
Vehicle:
water
Concentration / amount:
5% in FCA or sterile water; 0.1 mL each
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
50% in sterile water; 0.4 mL
Day(s)/duration:
Day 8 - 48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
10% in sterile water; 0.2 mL
Day(s)/duration:
Day 22 - 24 h
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
10% in sterile water; 0.2 mL
Day(s)/duration:
Day 22 - 24 h - second group
Adequacy of challenge:
other: irritating concentration
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5% in sterile water; 0.2 mL
Day(s)/duration:
Day 29 - 24 h - second group
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Main phase I
Control group: 5 females
Test group: 10 females

Main phase II
Control group: 5 females
Test group: 10 females
Details on study design:
RANGE FINDING TESTS:
Intradermal injection: Six sites were selected on each animal over the shaved scapulae. Each site was injected with a single concentration of the test item. Concentrations of 50, 20, 10, 5, 1 and 0.5% in sterile water were selected.
Topical application: Seven days after the intradermal injection of emulsified Freund's complete adjuvant, the flanks of each animal, each animal was dosed with 2 concentrations of the test item, 1 on each flank such that a total of 5 concentrations (50, 20,10, 5 and 1% in sterile water) of the test item were each dosed in duplicate.

MAIN STUDY I
A. INDUCTION EXPOSURE
- No. of exposures: 1 for intradermal injection; 1 for topical application
- Exposure period: 48 hours of topical application
- Test groups: 1 of 10 animals
- Control group: 1 of 5 animals
- Site: 3 sites (anterior, median, posterior) for intradermal injection; 1 site (over the injection sites) for topical application
- Concentrations: 5% for intradermal injection; 50% for topical application.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: 24 hours
- Test groups: 1 of 10 animals
- Control group: 1 of 5 animals
- Site: centre of right or left flank
- Concentrations: 10%
- Evaluation (hr after challenge): 24 and 48 hours after the exposure period.

MAIN STUDY II
A. INDUCTION EXPOSURE
- No. of exposures: 1 for intradermal injection; 1 for topical application
- Exposure period: 48 hours of topical application
- Test groups: 1 of 10 animals
- Control group: 1 of 5 animals
- Site: 3 sites (anterior, median, posterior) for intradermal injection; 1 site (over the injection sites) for topical application
- Concentrations: 5% for intradermal injection; 50% for topical application.

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 2
- Exposure period: 24 hours
- Test groups: 1 of 10 animals
- Control group: 1 of 5 animals
- Site: centre of right or left flank
- Concentrations: 10%
- Evaluation (hr after challenge): 24 and 48 hours after the exposure period.
- Re-Challenge Concentration: 5%
- Evaluation (hr after challenge): 24 and 48 hours after the exposure period.
Positive control substance(s):
yes
Remarks:
α-HEXYLCINNAMALDEHYDE
Positive control results:
CONCENTRATION:
INDUCTION (INJECTION) - 20% in corn oil
(TOPICAL) - 50% in corn oil
CHALLENGE - 10% in acetone

RESULTS: 90% response in test group and 0% response in control group at challenge.
INTERPRETATION: Incidence at challenge acceptable. Test system regarded as valid.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Main Assay I - Slight dark coloration of the treatment sites
Remarks on result:
other: result questionable
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Main Assay I - Slight dark coloration of the treatment site not preventing evaluation of erythema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Main Assay I - Slight dark coloration of the treatment site not preventing evaluation of erythema
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Main Assay I - Slight dark coloration of the treatment site not preventing evaluation of erythema
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10% - second test group
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Remarks on result:
not determinable
Remarks:
positive reactions in negative control
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10% - second test group
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Remarks on result:
not determinable
Remarks:
positive reactions in negative control
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10% - second control group
No. with + reactions:
4
Total no. in group:
5
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Remarks on result:
not determinable
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10% - second control group
No. with + reactions:
4
Total no. in group:
5
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
5% - second test group
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
5% - second test group
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
5% - second control group
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
5% - second control group
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Main Assay II - Slight dark coloration of the treatment site not preventing evaluation of erythema
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Intradermal induction:20% in corn oil; epidermal induction: 50% in corn oil; challenge: 10% in acetone
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Intradermal induction:20% in corn oil; epidermal induction: 50% in corn oil; challenge: 10% in acetone
No. with + reactions:
9
Total no. in group:
19
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Irritation was observed at challenge with a concentration of 10% of Reactive Brown DYHY 0331/0334. At 5% test item concentration, 2/10 animals showed a slight positive reaction. However, since the incidence of the animals showing a positive reaction is lower than 30%, the test item is considered not to be a skin sensitiser and a classification is not required.
Executive summary:

The potential of the test item, Reactive Brown DYHY 0331/0334, to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the maximisation test of Magnusson and Kligman.

The concentrations of the test item used in the main study were determined by the results of preliminary screening tests. The main sensitisation test was undertaken using a test group of 10 animals and a control group of 5 animals. In an attempt to induce sensitisation, test animals were intradermally injected with an emulsion of Freund’s complete adjuvant (anterior sites), the test item at 5% concentration in the selected vehicle (sterile water) (middle sites) and the test item at 5% concentration in an emulsion of Freund’s complete adjuvant (posterior sites). One week later, animals were boosted by topical application of the test item at 50% concentration over the injection sites.

Control group animals were treated in the same manner but the selected vehicle (sterile water) was used in place of the test item. Two weeks after the second induction stage, all animals were challenged by topical application of both the vehicle (sterile water) and the test item at 10% concentration. At the challenge with the test item at concentration of 10% in sterile water, very slight reaction was observed in 3/10 animals of the test group (30%) at 24 hours after test item removal only. No response to the test item was observed in animals of the control group or in the test item treated group at 48 hours after test item removal. No reaction to the vehicle alone was observed in any animal.

In order to confirm the border-line results obtained at challenge, the test was extended to a further test group of 10 animals and control group of 5 animals. Animals of this additional phase of the study (from a new batch of guinea pigs) were treated in the same manner at the same concentrations selected for the previous phase of the study. At challenge with the test item at 10% concentration, reaction was observed in 7/10 animals of the test group (70%) at the 24 hour examination, remaining in 6/10 animals at the 48 hour examination. Reaction was also observed in 4/5 (80%) animals of the control group, indicating that irritation has occurred following dosing with the test item at 10% in sterile water. Therefore, a re-challenge was performed one week later in the same animals with the test item at a concentration of 5% in sterile water. Reaction was observed at this second challenge in 2/10 animals of the test group. No response was observed in animals of the control group.

The above results indicate that irritation was observed at challenge with a concentration of 10% of Reactive Brown DYHY 0331/0334. At 5% test item concentration, 2/10 animals showed a slight positive reaction. However, since the incidence of the animals showing a positive reaction is lower than 30%, the test item is considered not to be a skin sensitiser and a classification is not required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The potential of the test item, Reactive Brown DYHY 0331/0334, to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the maximisation test of Magnusson and Kligman.

The concentrations of the test item used in the main study were determined by the results of preliminary screening tests. The main sensitisation test was undertaken using a test group of 10 animals and a control group of 5 animals. In an attempt to induce sensitisation, test animals were intradermally injected with an emulsion of Freund’s complete adjuvant (anterior sites), the test item at 5% concentration in the selected vehicle (sterile water) (middle sites) and the test item at 5% concentration in an emulsion of Freund’s complete adjuvant (posterior sites). One week later, animals were boosted by topical application of the test item at 50% concentration over the injection sites.

Control group animals were treated in the same manner but the selected vehicle (sterile water) was used in place of the test item. Two weeks after the second induction stage, all animals were challenged by topical application of both the vehicle (sterile water) and the test item at 10% concentration. At the challenge with the test item at concentration of 10% in sterile water, very slight reaction was observed in 3/10 animals of the test group (30%) at 24 hours after test item removal only. No response to the test item was observed in animals of the control group or in the test item treated group at 48 hours after test item removal. No reaction to the vehicle alone was observed in any animal.

In order to confirm the border-line results obtained at challenge, the test was extended to a further test group of 10 animals and control group of 5 animals. Animals of this additional phase of the study (from a new batch of guinea pigs) were treated in the same manner at the same concentrations selected for the previous phase of the study. At challenge with the test item at 10% concentration, reaction was observed in 7/10 animals of the test group (70%) at the 24 hour examination, remaining in 6/10 animals at the 48 hour examination. Reaction was also observed in 4/5 (80%) animals of the control group, indicating that irritation has occurred following dosing with the test item at 10% in sterile water. Therefore, a re-challenge was performed one week later in the same animals with the test item at a concentration of 5% in sterile water. Reaction was observed at this second challenge in 2/10 animals of the test group. No response was observed in animals of the control group.

The above results indicate that irritation was observed at challenge with a concentration of 10% of Reactive Brown DYHY 0331/0334. At 5% test item concentration, 2/10 animals showed a slight positive reaction. However, since the incidence of the animals showing a positive reaction is lower than 30%, the test item is considered not to be a skin sensitiser and a classification is not required.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance was negative in in vivo skin sensitisation test and therefore no classification is required for sensitisation according to EC criteria (67/548/EEC) and CLP criteria (EC 1272/2008).