2-Pyridinesulfonamide, 3-(2,2,2-trifluoroethoxy)-, sodium salt (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidance and good laboratory practices guidance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

sodium 2-(azanidylsulfonyl)-3-(2,2,2-trifluoroethoxy)pyridine

EC Number:

700-926-1

Cas Number:

227605-94-9

Molecular formula:

C7H6F3N2NaO3S

IUPAC Name:

sodium 2-(azanidylsulfonyl)-3-(2,2,2-trifluoroethoxy)pyridine

Constituent 2

Reference substance name:

Sodium {[3-(2,2,2-trifluoroethoxy)-2-pyridinyl]sulfonyl}azanide

IUPAC Name:

Sodium {[3-(2,2,2-trifluoroethoxy)-2-pyridinyl]sulfonyl}azanide

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): CA 3105 A (intermediate of CGA 362622)
- Substance type: Organic mono constituent substance
- Physical state: Beige crystals
- Analytical purity: 93.9%
- Purity test date: No data
- Lot/batch No.: EZ001002
- Expiration date of the lot/batch: July 2002 (reanalysis date)
- Stability under test conditions: Stable under storage conditions
- Storage condition of test material: In the original container, at room temperature (range of 20 ± 3 °C), away from direct sunlight.

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 pretest start/beginning of acclimatization period: 4-6 weeks
- Weight at pretest start (pretest groups):
Males: 303-315 g
Females: 326 g
- Weight at beginning of acclimatization period (control and test groups):
Males: 297-381 g
Females: 302-388 g
- Fasting period before study: No data
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Nafag Ecosan 845 25W4, batch no. 45/00, guinea pig breeding / maintenance diet, containing Vitamin C ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum.
- Acclimation period: One week for the control and test group under test conditions after health examination. The animals of the pretest were not acclimatized.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-21.5 °C
- Humidity (%):42.5-77% (values above 70% during cleaning process possible)
- Air changes (per hr): 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 29-08-2000 (pretest); 05-09-2000 (main study)
To: 09-10-2000

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: bi-distilled water

Induction 2

Concentration / amount:

Main test:
INDUCTION
Intradermal injection:
Test group:
1) 1:1 (v/v} mixture of Freund’s Complete Adjuvant (FCA} and physiological saline.
2) The test item at 5 % in the vehicle (bi-distilled water}.
3) The test item at 5% in a 1:1 (v/v) mixture of FCA and physiological saline
Control group:
1) 1:1 (v/v) mixture of FCA and physiological saline.
2) Bi-distilled water
3) 1:1 (w/w) mixture of the vehicle and a 1:1 (v/v) mixture of FCA and physiological saline.

Epidermal applications:
Test group: 0.3 mL of 25% test-item in bi-disitilled water
Control group: 0.3 mL of bi-distilled water

CHALLENGE
Test group and Control group:: 0.2 mL of 15% test-item in bi-disitilled water left; 0.2 mL of bi-distilled water right

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: bi-distilled water

Challenge 2

Concentration / amount:

Main test:
INDUCTION
Intradermal injection:
Test group:
1) 1:1 (v/v} mixture of Freund’s Complete Adjuvant (FCA} and physiological saline.
2) The test item at 5 % in the vehicle (bi-distilled water}.
3) The test item at 5% in a 1:1 (v/v) mixture of FCA and physiological saline
Control group:
1) 1:1 (v/v) mixture of FCA and physiological saline.
2) Bi-distilled water
3) 1:1 (w/w) mixture of the vehicle and a 1:1 (v/v) mixture of FCA and physiological saline.

Epidermal applications:
Test group: 0.3 mL of 25% test-item in bi-disitilled water
Control group: 0.3 mL of bi-distilled water

CHALLENGE
Test group and Control group:: 0.2 mL of 15% test-item in bi-disitilled water left; 0.2 mL of bi-distilled water right

No. of animals per dose:

Intradermal Pretest: 1
Epidermal Pretest: 2
Main test:
Control Group: 10
Test Group: 20

Details on study design:

RANGE FINDING TESTS:
-Intradermal Induction:
The 5 % concentration of test item selected for the induction exposure was well tolerated systemically and caused mild-to-moderate skin irritation. Higher concentrations were not tested because the 5 % concentration is the maximum concentration for intradermal induction recommended by Magnusson and Kligman (1969, p. 270).
-Epidermal Induction:
The 25 % concentration of the test item used for the induction exposure was the highest tested concentration which caused mild-ta-moderate skin irritation and was well-tolerated systemically.
-Epidermal Challenge:
The 15 % concentration of the test item used for the challenge application was non-irritant.
According to Magnusson - Kligman and to the findings observed, the test item at 25 % was considered to be the tolerated concentration to stimulate a state of immune hypersensitivity and 15 % as the highest tested non-irritating concentration to be used for the challenge.


MAIN STUDY
A. INDUCTION EXPOSURE
*Intradermal injection: test day 1: three pairs of intradermal injections (0.1mL/site)
- Test groups:
1) 1:1 (v/v} mixture of Freund's Complete Adjuvant (FCA} and physiological saline.
2) The test item at 5 % in the vehicle (bi-distilled water}.
3) The test item at 5% in a 1:1 (v/v) mixture of FCA and physiological saline
- Control group:
1) 1:1 (v/v) mixture of FCA and physiological saline.
2) Bi-distilled water
3) 1:1 (w/w) mixture of the vehicle and a 1:1 (v/v) mixture of FCA and physiological saline.

- Site: 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:
cranial
. . . . . . . . . . . . . . . . . .
. - - - - - - - - - - - - - - - - .
. - 1 1 - .
left . - 2 2 - . right
. - 3 3 - .
. - - - - - - - - - - - - - - - .
. . . . . . . . . . . . . . . . . .
caudal

. . . . . clipped area
- - - - - - area in which injections were made

* Epidermal applications: test day 8: On test day 8, the scapular region (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. Thereafter, a 2 x 4 cm patch of filter paper was saturated with the test item (25 % in bi-distilled water) and placed on the shaved area over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 mL. 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 occlusive dressings were left in place for 48 hours. This procedure ensures intensive contact of the test item. The guinea pigs of the control group were treated as described above with the vehicle only, also applied at a volume of approximately 0.3 mL.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

B. CHALLENGE EXPOSURE
Performed on test day 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area 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 test item at the highest tested non-irritating concentration of 15 % (applied to the left flank) and the vehicle only (applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle applied was approximately 0.2 mL. The dressings were left in place for 24 hours.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

Challenge controls:

The control guinea pigs were challenged two weeks after the epidermal induction application.
Hair was clipped and shaved from a 5 x 5 cm area 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 test item at the highest tested non-irritating concentration of 15 % (applied to the left flank) and the vehicle only (applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle applied was approximately 0.2 mL. The dressings were left in place for 24 hours.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole (study 905095)

Results and discussion

Positive control results:

For validation of sensitivity of the Maximization-Test of B. Magnusson and A.M. Kligman (1969) as well as the sensitivity of the test system used, a known sensitizer 2-MERCAPTOBENZOTHIAZOLE was selected as a positive control. This was performed in accordance with the recommendation of the OECD tor testing of chemicals number 406 "Skin Sensitization Test", adopted by the Council on July 17, 1992.
The study was performed with 15 (10 test and 5 control) male albino guinea pigs (GOHI), delivered by RCC Ltd, Biotechnology & Animal Breeding Division (CH-4414 Füllinsdorf, Switzerland).
The intradermal induction of sensitization in the test group was performed in the nuchal region with a 5 % dilution of the test item in mineral oil and in an emulsion of Freund's Complete Adjuvant (FCA) I physiological saline. The epidermal induction of sensitization was conducted tor 48 hours under occlusion with the test item at 15 % in mineral oil one week after the intradermal induction. The animals of the control group were intradermally induced with mineral oil and FCA/physiological saline and epidermally induced with mineral oil under occlusion. Two weeks after epidermal induction the control and test animals were challenged
by epidermal application of the test item at 1 % in mineral oil and mineral oil alone under occlusive dressing.
Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.
No toxic symptoms were evident in the guinea pigs of th e control group. No deaths occurred. All test animals showed discrete/patchy to moderate/confluent erythema after the challenge treatment with 2-MERCAPTOBENZOTHIAZOLE at 1 % (w/w) in mineral oil. No skin effect was observed in the control group.
Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 96/54/EEC, 2-MERCAPTOBENZOTHIAZOLE has to be classified and labelled as a skin sensitizer.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

15% (challenge)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15% (challenge). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15% (challenge)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15% (challenge). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

15% (challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 15% (challenge). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

15% (challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 15% (challenge). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.

Any other information on results incl. tables

Skin Reactions after the Challenge Procedure
	after 24 hours	after 48 hours
	positive / total % positive of total	positive / total % positive of total
CONTROL GROUP
CA 3105 A (Intermediate of CGA 362622), 15 % in vehicle (left flank)	0/10 0	0/10 0
Vehicle only (right flank)	0/10 0	0/10 0
TEST GROUP
CA 3105 A (Intermediate of CGA 362622), 15 % in vehicle (left flank)	0/20 0	0/20 0
Vehicle only (right flank)	0/20 0	0/20 0

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The 0 % incidence of challenge skin reactions to the test item is below the threshold (30 % for adjuvant tests) set in Commission Directive 96/54/EEC. Therefore, classification of CA 3105 A (lntermediate of CGA 362622) is not required.

Executive summary:

In order to assess the cutaneous allergenic potential of CA 3105 A (Intermediate of CGA 362622), the Maximization-Test was performed in 5 male and 5 female control and 10 male and 10 female test albino guinea pigs, in accordance with OECD Guideline No.406

and the Directive 96/54/EEC, B.6.

The intradermal induction of the test group animals was performed with 5% concentrations of the test item in the vehicle (bi-distilled water) and in Freund's Complete Adjuvant (FCA) (as 1:1 FCA in physiological saline). Control group animals were injected with the vehicle alone and FCA/physiological saline alone. Beginning on study day 8, the epidermal induction was conducted tor 48 hours with a 25% concentration of the test item in the vehicle (test group animals) or vehicle alone (control group animals). Two weeks after epidermal induction, the control and test animals were challenged by epidermal application of the test item at 15% in the vehicle and the vehicle alone. Cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.

None of the control and test animals showed skin reactions after the challenge treatment with CA 3105 A (Intermediate of CGA 362622) at 15% (w/w) in the vehicle.

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

The 0% incidence of challenge skin reactions to the test item is below the threshold (30% for adjuvant tests) set in Commission Directive 96/54/EEC. Therefore, classification of CA 3105 A (Intermediate of CGA 362622) is not required.