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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 1998 to 3 December 1998.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP methodology followed and OCED guideline 406 used to performed the experiment.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1999

Materials and methods

Test guidelineopen allclose all
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
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
430-500-8
EC Name:
-
Cas Number:
204277-61-2
Molecular formula:
Hill formula: C23 H23 Cl N6 O8 CAS formula: C23 H23 Cl N6 O8
IUPAC Name:
methyl 2-({4-[2-(2-chloro-6-cyano-4-nitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)propanoate
Details on test material:
None

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Age at delivery: 4 - 6 weeks
- Age at study initiation: 4 - 6 weeks
- Body weight at pretest start: Pretest groups: 332 - 342 g
- Body weight at beginning of acclimatization period: Control and test group 332 - 430 g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Nafag Ecosan 845 25W4, batch no. 76/98 guinea pig breeding / maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum.
- Water : Community tap water from Füllinsdorf, ad libitum.
- Acclimation period: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C
- Humidity (%): relative humidity between 33-68 %
- Air changes (per hr): Air-conditioned with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light, 12-hour dark cycle. Music was played during the light period.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Concentration / amount:
- In the pre-test (Intredermal injection)during the acclimatization period of the control and test group: 5% / 3% / 1%
(According to Magnusson - Kligman and to the findings observed, the concentration selected for the main study was 5 %)

- In the pre-test (Epidermal injection)during the acclimatization period of the control and test group:10% / 15% / 25% / 30%.
According to Magnusson - Kligman and to the findings observed, the test article at 30 % was considered to be the maximally tolerated
concentration to stimulate a state of immune hypersensitivity and the highest non-irritating concentration to be used for the challenge.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Concentration / amount:
- In the pre-test (Intredermal injection)during the acclimatization period of the control and test group: 5% / 3% / 1%
(According to Magnusson - Kligman and to the findings observed, the concentration selected for the main study was 5 %)

- In the pre-test (Epidermal injection)during the acclimatization period of the control and test group:10% / 15% / 25% / 30%.
According to Magnusson - Kligman and to the findings observed, the test article at 30 % was considered to be the maximally tolerated
concentration to stimulate a state of immune hypersensitivity and the highest non-irritating concentration to be used for the challenge.
No. of animals per dose:
- Control group: 5 animals
- Test group: 10 animals
- Intradermal pretest: 1 animal
- Epidermal pretest: 2 animals
Details on study design:
PRETEST (performed during the acclimatization period of the control and test group):
- Intradermal injections:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck
of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1 % of the test article in PEG 400. The resulting dermal reactions were assessed 24 hours later

- Epidermal applications: Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete
Adjuvant/physiological saline were made. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the
application. Thereafter 4 patches of filter paper were saturated with the test article at A = 30 % (this concentration was found to be the most qualified to assure an optimum technical application procedure), B= 25%, C= 15% and D= 10% and applied to the clipped and shaved flanks. The amount of test article applied was approximately 0.2 g for the test article at 30 % and 25 % and a volume of 0.2 ml for the remaining test article concentrations.
The dressings were removed after an exposure period of 24 hours.
Approximately 21 hours after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the application site from black staining produced by the test article, so that possible erythema reactions were clearly visible at that time.

MAIN STUDY (INDUCTION):
INTRADERMAL INJECTIONS / PERFORMED ON TEST DAY 1:
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, at 5 % in PEG 400.
3) The test article at 5 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) PEG 400
3) 1:1 (w/w) mixture of PEG 400 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

EPIDERMAL APPLICATIONS/PERFORMED ON TEST DAY 8:
On test day 7 and approximately 22.5 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) of the animals of the control and test group was clipped, shaved free of hair and the test area was pretreated with a 10 % dilution of Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10 % concentration of SLS enhances sensitization by provoking a mild inflammatory reaction (Magnusson and Kligman 1970).

On test day 8, a patch of filter paper was saturated with the test article (30 % in PEG 400) and placed over the injection sites of the test animals. The amount of test article applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours.
The guinea pigs of the control group were treated as described above with PEG 400 only, applied at a volume of approximately 0.3 ml.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system
according to Draize.




Challenge controls:
CHALLENGE / PERFORMED ON TEST DAY 22:
The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs were trea
ted in the same way.
Hair was clipped and shaved on the left and right flank of each guinea pig just prior to the application. Two patches ( 3 x 3 cm) of filter paper were saturated with the highest non-irritating concentration of 30 % (left flank) and the vehicle only (PEG 400 applied to the right flank) using the same method as for the epidermal application. The amount of test article applied was approximately 0.2 g and a volume of 0.2 ml was used for the vehi
cle. The dressings were left in place for 24 hours.
Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest.
Approximately 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group (FAT 41024/B)
Dose level:
30% in PEG 400
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group (FAT 41024/B). Dose level: 30% in PEG 400. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group (FAT 41024/B)
Dose level:
30 % in PEG 400
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group (FAT 41024/B). Dose level: 30 % in PEG 400. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Vehicle control (PEG 400 only)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Vehicle control (PEG 400 only). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Vehicle control (PEG 400 only)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Vehicle control (PEG 400 only). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Test group with FAT 41024/B
Dose level:
30 % in PEG 400
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Test group with FAT 41024/B. Dose level: 30 % in PEG 400. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Test group with FAT 41024/B
Dose level:
30 % in PEG 400
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Test group with FAT 41024/B. Dose level: 30 % in PEG 400. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Test group with PEG 400
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Test group with PEG 400. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Test group with PEG 400
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Test group with PEG 400. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Main Study:

SKIN EFFECTS AFTER INTRADERMAL INDUCTION - PERFORMED ON TEST DAY 1

The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. The intradermal applications were observed with erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION - PERFORMED ON TEST DAY 8

- CONTROL GROUP: No erythematous or oedematous reaction was observed in the animals treated with PEG 400 only.

- TEST GROUP: As the test article stained the skin black, it was not possible to determine whether erythema was present or not after treatment with the test article at 30 % in PEG 400. However, no oedema was observed.

All animals of the control and test group were pretreated with 10 % SLS in paraffinum perliquidum.

SKIN EFFECTS AFTER THE CHALLENGE - PERFORMED ON TEST DAY 22

- CONTROL GROUP: No skin reactions were observed in the animals either when treated with PEG 400 only or when treated with the test article at 30 % in PEG 400.

Black discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 hours prior to challenge reading.

- TEST GROUP: No skin reactions were observed in the animals either when treated with PEG 400 only or when treated with the test article at 30 % in PEG 400.

Black discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 hours prior to challenge reading.

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

As there were no deaths during the course of the treatment period no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

One animal of the test group showed a weak loss of body weight (2 %) during the treatment period. The body weight of the other animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
FAT41024/B applied at a concentration of 30 % in PEG 400 is considered not to be a sensitizer when used under the described test conditions.
Executive summary:

In order to assess the cutaneous allergenic potential of FAT 41024/B, the Maximization-Test was performed in 15 (10 test and 5 control) female albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6.

The intradermal induction of sensitization was performed with a 5 % dilution of the test article in polyethylene glycol (PEG 400) and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under

occlusion with the test article at 30 % in PEG 400. Two weeks after the epidermal induction application the challenge was completed by epidermal application of the test article at 30 % in PEG 400 under occlusive dressing. The animals of the control group were induced with PEG 400 and FCA/physiological saline and challenged similarly to those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal of the dressing.

ERYTHEMATOUS REACTIONS AFTER THE CHALLENGE PROCEDURE

 

After 24 hours 

After 48 hours 
   (Positive/total) / (% positive of total)   (Positive/total) / (% positive of total)
  Control group: FAT 41024/B (left flank)  (0/5) / 0   (0/5) / 0
 Control group: PEG only (right flank) (0/5) / 0  
 (0/5) / 0
 Test group: FAT 41024/B (left flank)   (0/5) / 0   (0/5) / 0
 Test group: PEG 400 (right flank)   (0/5) / 0   (0/5) / 0

No toxic symptoms were evident in the guinea pigs of the control or test group.

No deaths occured.

Conclusion:

In this study none of the animals of the test group were observed with erythematous reactions after treatment with a non-irritant test article concentration of 30 % in PEG 400. No skin reactions were observed in the control group.

Therefore, the test article FAT41024/B applied at a concentration of 30 % in PEG 400 is considered not to be a sensitizer when used under the described test conditions.