Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted sicentifica standards, well documented and acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
Sensitization of Guinea Pigs to Alpha-Chloroacetophenone (CN) and Ortho-Chlorobenzylidenemalononitrile (CS), Tear Gas Chemicals.
Author:
Chung CW and Giles AL
Year:
1972
Bibliographic source:
The Journal of Immunology 109 (2): 284-293

Materials and methods

Principles of method if other than guideline:
Three intradermal injections of the test substance preparation in vehcile vof Freund's Complete adjuvant:water 1:1 on three different days. Two challenge doses were applied to a previously untreated site.
The test was performed according to the method described by Maguire and Chase (1967) with some alterations. Skin effects were scored according to the method described in detail by Chung et al. (1970).
Maguire and Chase (1967). J. Invest. Dermatol. 49: 460.
Chung et al. (1970). J. Invest. Dermatol. 55: 306.
GLP compliance:
no
Type of study:
Freund's complete adjuvant test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): alpha-Chloroacetophenone (CN)
- Analytical purity: no data
- Supplier: Aldrich Chemical Co., Milwaukee, Wis.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hartley albino
- Source: Zartman Farm, Douglasville, Pa.
- Weight at study initiation: 300 - 400 g


ENVIRONMENTAL CONDITIONS
no details given

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: acetone
Concentration / amount:
0.5 or 1 % solution in acetone
For intradermal injection, the 1 % acetone solution was further diluted in saline.
Challengeopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: acetone
Concentration / amount:
0.5 or 1 % solution in acetone
For intradermal injection, the 1 % acetone solution was further diluted in saline.
No. of animals per dose:
Main experiment: 7 animals/dose
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- Freund's complete adjuvant (FCA) containing heat-killed Myobacterium tuberculosis, H37Ra, was diluted to 0.5 mg net bacterial dry weight/mL with Freund's incomplete adjuvant. A volume of 0.1 mL was injected into each footpad. This footpad method was preferred because it enabled the intact whole neck and back region to be used for other simultaneous tests.
- For induction of sensitization by topical administration, a volume of 0.2 mL of 1 or 0.5 % acetone solution of the compound was applied to the closely clipped neck region (corresponding to doses of 2000 and 1000 µg).
- For intradermal immunization, a volume of 0.5 mL containing 10 to 25 µg of compound was given in five sites in the clipped neck region within a few hrs after injection of adjuvant as described in the procedure by Maguire & Chase (1967).
- The number of induction procedures was not specified by the authours.

Challenge was performed 3 to 4 weeks later, when any skin damage resulting from these procedures was completely healed.


B. CHALLENGE EXPOSURE
- For topical challenge a volume of 0.1 mL of 0.2 to 1.0 % acetone solution of CN was used, corresponding to doses of 200 - 1000 µg.
- For intradermal challenge a volume of 0.1 mL of a saline solution containing 1 to 10 µg/mL was injected.
- When only one compound was used for challenge, four sites were employed for a single dose and two sites per dose for several doses per animal.

The authours did not report any skin reactions at the lowest challenge dose caused by the irritating properties of the the substance. It can be assumed that the substance was not irritating at the lowest dose used for topical challenge, i.e. 200 µg.


C. CROSS REACTION STUDIES
- For cross-reaction studies a volume of 0.1 mL of equimolar solutions was used (12.94 µmol/mL in acetone and 0.1294 µmol/mL in saline containing acetone at 1 % or less for topical and intradermal challenges, respectively) and only one site was used per chemical. Each animal received eight chemicals including CN.
Challenge controls:
In each challenge, untreated animals were used as controls. The adjuvant controls were treated with adjuvant only at the time when the immunized animals were treated.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Results
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.2 mg at challenge (topical induction & challenge)
No. with + reactions:
7
Total no. in group:
7
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.2 mg at challenge (topical induction & challenge). No with. + reactions: 7.0. Total no. in groups: 7.0.

Any other information on results incl. tables

1. Main experiment:

 

The influence of routes of induction and challenge on elicitation response in guinea pigs sensitized to alpha-chloroacetophenone:

 

 

 

No. of positive animals / no. of total animals

Challenge

Induction route

Route

Dose

topical

intradermal

topical

1.0 mg

7/7

7/7

0.5 mg

7/7

7/7

0.2 mg

7/7

7/7

The results were from skin reactions read at 48 hrs after the first challenge.

 

 

 

2. Additional experiment:

 

The effect of route and dosage of substance administration on the sensitization of guinea pigs to alpha-chloroacetophenone:

 

 

 

 

 

Skin reaction

Induction

Challenge

1st challenge

2nd challenge

 

 

 

 

24 hrs

120 hrs

24 hrs

Route

Dose [µg]

Route

Dose [µg]

FR

Net AI

AATI

FR

Net AI

AATI

FR

Net AI

AATI

Control

none

i.d.

2

0/2

1.5

0

0/2

0

0

0/7

0.9

0

i.d.

5

1/2

2.5

84

0/2

0

0

-

-

-

i.d.

10

2/2

3.0

135

1/2

2.0

84

-

-

-

top.

1000

2/2

4.0

-

0/2

1.0

-

8/8

4.7

-

i.d.

10

i.d.

2

8/8

2.6

170

2/8

2.0

200

4/4

5.1

391

i.d.

5

8/8

2.5

400

7/8

2.6

274

-

-

-

i.d.

10

8/8

2.8

626

8/8

2.9

433

-

-

-

top.

1000

-

-

-

-

-

-

4/4

1.0

-

i.d.

25

i.d.

2

8/8

2.5

172

0/8

1.1

0

4/4

5.1

438

i.d.

5

8/8

2.5

453

5/8

2.1

306

-

-

-

i.d.

10

8/8

2.8

635

8/8

2.8

400

-

-

-

top.

1000

-

-

-

-

-

-

4/4

1.4

-

i.d.

50

i.d.

2

6/6

2.8

174

4/6

2.5

167

4/4

5.1

450

i.d.

5

6/6

2.8

512

5/6

5.0

374

-

-

-

i.d.

10

6/6

3.0

705

6/6

3.7

545

-

-

-

top.

1000

-

-

-

-

-

-

2/2

1.4

-

i.d. & top.

25; 1000

i.d.

2

-

-

-

-

-

-

4/4

5.1

199

top.

1000

8/8

1.8

-

8/8

4.0

-

4/4

0.4

-

top.

2000

i.d.

2

-

-

-

-

-

-

4/4

4.6

354

top.

1000

8/8

1.5

-

8/8

1.6

-

4/4

0.9

-

i.d. intradermal

top. topical

FR: Fraction of positive responses or fractional response.

Net FR: The difference between the FRs of sensitized and unimmunized controls.

AI: Average intensity of the numerical scores of skin reactions.

Net AI: The difference between the AI values of immunized and untreated controls.

AATI: Average of area times reaction intensity of positive skin reactions.

AATI ratio: The ratio of AATI of immunized to that of untreated control animals.

The number of animals in each immunization dose group was six or eight and all animals received either an i.d.or a topical challenge in the first challenge. In the second challenge they were divided into two groups. One group was challenged by the i.d route with 1.0 µg CN/site, the lowest dose in the first challenge, and the other received the topical challenge.

The blank spaces indicate that the animals were not tested.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information