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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:
13 October 1992 - 7 November 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
1234

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-dimethylbenzoyl chloride
EC Number:
413-010-9
EC Name:
3,5-dimethylbenzoyl chloride
Cas Number:
6613-44-1
Molecular formula:
C9H9ClO
IUPAC Name:
3,5-dimethylbenzoyl chloride
Details on test material:
DMBC
Appearance: clear, colourless liquid
Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England.
- Age at study initiation: Approximately 6 - 7 weeks.
- Weight at study initiation: 299 - 336 g on arrival prior to acclimatisation.
- Housing: The guinea-pigs were housed in groups of five or ten in suspended metal cages with wire mesh floors.
- Diet (e.g. ad libitum): A vitamin C enriched guinea-pig diet FD1 was provided ad libitum. Hay was given weekly.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 19 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Approximately 21 °C
- Humidity (%): 30 - 70 % relative.
- Air changes (per hr): Approximately 15.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hour period

IN-LIFE DATES: From: To: 13 October 1992 - 7 November 1992

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: intradermal and topical
Vehicle:
other: Alembicol D
Concentration / amount:
INDUCTION
Intradermal injection - 0.25 % w/v in Alembicol D.
Topical application - 30 % w/v in Alembicol D)

CHALLENGE
Topical - 5 and 2.5 % w/V in Alembicol D
Challengeopen allclose all
Route:
other: topical
Vehicle:
other: Alembicol D
Concentration / amount:
INDUCTION
Intradermal injection - 0.25 % w/v in Alembicol D.
Topical application - 30 % w/v in Alembicol D)

CHALLENGE
Topical - 5 and 2.5 % w/V in Alembicol D
No. of animals per dose:
Ten test and five control animals were used in this study.
Details on study design:
The test substance was prepared prior to each application on the day of dosing in Alembicol D.
Solutions of the test substance in Alembicol D were analysed for homogeneity, stability and target concentration. Homogeneity and stability were assessed on one occasion only prior to the start of the study for the lowest and highest concentrations that were used. Analyses of all concentrations used were performed on each occasion of formulation during the study.

TREATMENT PROCEDURE
PRELIMINARY STUDY
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.

Based on the results of the preliminary investigations, the concentration of DMBC selected for the induction was the maximum concentration that produced some irritation but did not adversely affect the animals. The concentration chosen for the challenge was the maximum concentration not giving rise to irritating effects.

MAIN STUDY
INDUCTION
Induction intradermal injections:
A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 2 x 4 cm area within the clipped area.
Injectables for the test animals were prepared as follows:
1) Freund's complete adjuvant was diluted with an equal volume of water for irrigation (Ph.Eur.).
2) DMBC, 0.25 % w/v in Alembicol D.
3) DMBC, 0.25 % w/v in a 50:50 mixture of Freund's complete adjuvant and Alembicol D.

The sites for the control animals were identical, with the exception of the omission of DMBC.

Induction topical application:
One week after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair.
A 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 mL of DMBC, 30 % w/v in Alembicol D. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape (50 mm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (50 mm width "Elastoplast") wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.
During the induction phase, the control animals were treated similarly to the test animals, with the exception that no test substance was applied either intradermally or topically.

Challenge:
The control and test animals were challenged topically two weeks after the topical induction application using DMBC, 5 and 2.5 % w/v in Alembicol D.

Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 mL of DMBC, 5 % w/v in Alembicol D) and applied to an anterior site on the flank. DMBC, 2.5 % w/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".


OBSERVATIONS
Clinical signs
All animals were observed daily for signs of ill health or toxicity.

Bodyweight
The bodyweight of each guinea-pig on the main study was recorded on Day 1 (day of intradermal injections) and on the last day observations are made of dermal responses to the challenge applications.

Dermal responses
The dermal reactions resulting from intradermal injection and topical application on the preliminary study, and topical application at the challenges were assessed using the following numerical system.

Erythema and eschar formation:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) or eschar formation (injuries in depth) preventing erythema reading 4

Oedema formation:
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (edges raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

The diameter (mm) of the dermal response at the intradermal injection sites was recorded in the preliminary study only to assist in the choice of concentrations for the main study.
Any other lesion not covered by this scoring system was described.
The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.

INTERPRETATION OF RESULTS
Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals.
A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group.
If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive.
A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in animals of the control group.
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea-pig strain used is checked periodically at the testing facility with formalin, a known sensitiser.

Results and discussion

Positive control results:
The positive control was valid, confirming formalin to be a sensitiser under the conditions of the test.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Group:
positive control
Dose level:
100%
No. with + reactions:
10
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2.5% and 5 %
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 10.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2.5% and 5 %
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 10.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Clinical signs:

No signs of ill health or toxicity were recorded.

 

Bodyweight:

Bodyweight increases were recorded for all guinea-pigs over the period of the study.

 

Induction:

Dermal reactions seen following the induction applications are summarised in Table 1.

-Intradermal injections

Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals.

Slight irritation was seen in test animals at sites receiving DMBC, 0.25 % w/v in Alembicol D) and very slight irritation was observed in control animals receiving Alembicol D.

-Topical application

Slight erythema was observed in test animals following topical application with DMBC, 30 % w/v in Alembicol D.

No erythema was seen in the control guinea-pigs.

 

Challenge:

The numerical values given to the dermal reactions elicited by the challenge application are shown in Table 2.

The dermal responses seen in all ten test animals were more marked than those of the control.

 

Table 1 Dermal Reactions Observed After Each Induction

Site

Intradermal Injection

Topical Application

Test Animals

Control Animals

Test Animals

Control Animals

1

Necrosis

Necrosis

 

Slight erythema

 

No erythema

2

Slight irritation

Very slight irritation

3

Necrosis

Necrosis

 

Table 2a: Dermal Reactions Observed After the Challenge Application with DMBC - Control animals

Animal Number

Erythema and Oedema

Score 

24 hours

48 hours

72 hours

A

P

A

P

A

P

4380

E

O

0

0

0

0

0

0

0

0

0

0

0

0

4381

E

O

0

0

0

0

0

0

0

0

0

0

0

0

4382

E

O

0

0

0

0

0

0

0

0

0

0

0

0

4383

E

O

0

0

0

0

0

0

0

0

0

0

0

0

4384

E

O

0*

0

0

0

0*

0

0*

0

0*

0

0*

0

Table 2b: Dermal Reactions Observed After the Challenge Application with DMBC - Test animals

Animal Number

Erythema and Oedema

Score 

Result

24 hours

48 hours

72 hours

A

P

A

P

A

P

4380

E

O

2

2

2

2

2*

2

2

2

2**

2

2**

1

+

4381

E

O

2

2

2

1

2*

2

2

1

2**

1

2**

1

+

4382

E

O

2

1

2

1

2

1

2

1

2**

1

L2**

0

+

4383

E

O

2

2

2

1

2*

2†

2

1

2**

1†

2**

1

+

4384

E

O

2

2

2

2

2**

2

2**

1

L2**

1

L2**

1

+

4385

E

O

2

2

2

2

2*

2

2*

2

2*

2

2*

2

+

4386

E

O

2

2

2

2

2*

2†

2*

2

2**

1†

2*

1

+

4387

E

O

2

2

2

2†

2*

2

2*

2†

2**

2

2*

2†

+

4388

E

O

2

2

2

2

2*

2

2

1

2**

1

2**

1

+

4389

E

O

L2

1

2

1

L2*

1†

2

1

L2**

1†

2**

1

+

L Localised dermal reaction (restricted to small area of the challenge site)

† Necrotic Patch

* Thickening, dryness and sloughing of the epidermis

** Dryness and sloughing of the epidermis

A Anterior site exposed to DMBC, 5 % w/v in Alembicol D

P Posterior site, exposed to DMBC, 2.5 % w/v in Alembicol D

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, DMBC was a sensitiser.
Executive summary:

A study was performed to assess the skin sensitisation potential of DMBC using the guinea-pig in accordance with EEC Methods for the determination of toxicity, Directive 841449/EEC (OJ No. L251, 19.9.84), Part B, Method B.6. Acute toxicity (skin sensitization).

 

Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected:

Intradermal injection: 0.25 % w/v in Alembicol D.

Topical application: 30 % w/v in Alembicol D.

Challenge application: 5 and 2.5 % w/v in Alembicol D.

 

In this study performed using ten guinea pigs, DMBC produced evidence of skin sensitisation (delayed contact hypersensitivity) in all ten test animals.