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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
skin sensitisation, other
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The read across justification document is attached below.
Reason / purpose for cross-reference:
read-across source
Specific details on test material used for the study:
Trimellitic anhydride chloride
CAS: 1204-28-0
Key result
Reading:
other: 2 weeks after the last exposure
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
8
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
0.30 mg
No. with + reactions:
4
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
1.25 mg
No. with + reactions:
7
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
5 mg
No. with + reactions:
8
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
20
No. with + reactions:
8
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The present study examines the ability of dry TMA powder to sensitize Brown Norway rats when applied, topically, to the skin.
A patch of hair was carefully clipped with scissors on the rat’s back. Dry TMA powder was administered on days 0, 7, 14 and 21, and the area occluded with surgical tape overnight after each application. Residual powder recovered from the occluded skin was analyzed by proton nuclear magnetic resonance and was still predominantly TMA. Circulating anti-TMA IgE and IgG were measured by ELISA.
GLP compliance:
not specified
Type of study:
patch test
Justification for non-LLNA method:
A non-LLNA method is not provided since published data in rats is avaialble. In additon, in vivo study can be waived because the substance is corrosive to skin.

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzene-1,2,4-tricarboxylic acid 1,2-anhydride
EC Number:
209-008-0
EC Name:
Benzene-1,2,4-tricarboxylic acid 1,2-anhydride
Cas Number:
552-30-7
Molecular formula:
C9H4O5
IUPAC Name:
1,3-dioxo-2-benzofuran-5-carboxylic acid
Specific details on test material used for the study:
Trimellitic anhydride
CAS 552-30-7

In vivo test system

Test animals

Species:
other: rat
Strain:
other: Brown Norway and Sprague–Dawley
Sex:
male
Details on test animals and environmental conditions:
- Male, inbred BN rats (150–175 g) and male Sprague–Dawley rats (12 weeks old) from Charles River Laboratories (Wilmington, MA).
- Animals were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International
- Rats were fed Purina rat chow, and water ad libitum
- Rats kept on a standard 12:12 light:dark cycle.
- Rats were acclimated in the facility for 1 week prior to use.

Study design: in vivo (non-LLNA)

Induction
Route:
other: patch on fur on the back
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0, 0.3, 1.25, 5 and 20 mg was applied topically to BN rats (n=8/dose)
Day(s)/duration:
duration: 20h (overnight)
Challengeopen allclose all
No.:
#1
Route:
other: patch on fur on the back
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0, 0.3, 1.25, 5 and 20 mg
Day(s)/duration:
on day 7 / duration: 20h (overnight)
No.:
#2
Route:
other: patch on fur on the back
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0, 0.3, 1.25, 5 and 20 mg
Day(s)/duration:
on day 14 / duration: 20h (overnight)
No.:
#3
Route:
other: patch on fur on the back
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0, 0.3, 1.25, 5 and 20 mg
Day(s)/duration:
on day 21 / duration: 20h (overnight)
No. of animals per dose:
8 males / dose
Details on study design:
Dry TMA powder preparation:
TMA flakes were ground in a water-cooled analytical mill. The fine powder was collected and stored in the presence of a desiccant. The particle size distribution was determined using particles suspended in balanced electrolyte solution and measured by Coulter H Multisizer II.

Topical skin exposure to dry TMA powder:
Brown Norway rats were anesthetized using 40 mg/kg, i.p., methohexital sodium.
A patch of fur on the back was carefully clipped with scissors (avoid skin irritation and trauma). Dry TMA powder was applied to the
clipped area. TMA exposed areas were occluded with a nonabrasive dermal surgical tape overnight, after which the skin area of application was washed with water to remove any residual TMA.

Controls included:
- a clipped, occluded, untreated group;
- powder Trimellitic acid ( (20 mg) treated group;
- and TMA treated but gauze occluded group (gauze was placed in between the tape and TMA treated skin to test whether the tape occlusion could alter dermal/TMA interaction).

Dose-response and antibody production time course studies following single and multiple TMA applications were conducted.
- Dose–response relationship after TMA exposure: dry TMA powder at doses of 0, 0.3, 1.25, 5 and 20 mg was applied topically to BN rats (n=8/dose) on days 0, 7, 14 and 21. Blood was collected, intracardially, after anesthetization on day 35 for serum-specific IgE and IgG analyzes.
- Time course of antibody formation after single TMA exposure: a single TMA dose (20 mg) was applied to the skin of rats (n=8) with blood collected from the tail vein on days 0, 7, 14, 21 and 28 and intracardially on day 35 for serum-specific IgE and IgG analyses.
- Time course of antibody formation after repeated TMA exposure: dry TMA powder (20 mg) was applied to the skin of rats (n=8/group) on days 0, 7, 14 and 21. Blood was collected from the tail veins on days 0, 7, 14, 21 and 28 and intracardially on day 35 for serum-specific IgE and IgG analyzes.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
other: 2 weeks after the last exposure
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
8
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
0.30 mg
No. with + reactions:
4
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
1.25 mg
No. with + reactions:
7
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
5 mg
No. with + reactions:
8
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
20 mg
No. with + reactions:
8
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 2 weeks after the last exposure
Group:
test chemical
Dose level:
20 mg
No. with + reactions:
8
Total no. in group:
8
Remarks on result:
positive indication of skin sensitisation
Remarks:
Occluded with gauze after TMA application
Reading:
other: 2 weeks after the last exposure
Group:
positive control
Dose level:
no positive control
No. with + reactions:
0
Total no. in group:
0
Remarks on result:
not measured/tested

Any other information on results incl. tables

TMA particle size:

The repeated analysis of particles suspended in the solution did not change over time suggesting that surface hydrolysis did not alter the particle size. The particle diameter sizes ranged from < 1.88 mm (lower size limit capability of instrument) to 62.15 mm, with a mean +- SD of 2.77 +- 0.94 mm. Greater than 90% of the particles had diameters < 4 mm and very few of the particles (<1%) had diameters > 10 mm.

NMR analysis:

Residual TMA powder was collected from the occlusion tape and the skin surface 20 h after application. Two chemical forms of TMA and the acid hydrolysis product (Trimellitic acid) could be identified and relative amounts quantified by NMR. At the lowest dose (1.25 mg) the predominant species recovered was Type 1 TMA and no hydrolysis product was noted. The hydrolysis product, Trimellitic acid, tended to increase with increasing dose. There was also a dose dependent shift in the type of TMA found. The specific differences between Type 1 and Type 2 TMA could not be distinguished by the NMR methodology used.

See table 1 below.

Specific antibodies

Dose–response Table 1 shows the dose-dependent relationship between TMA exposure and specific antibody response, and the antibody production in the group occluded with gauze after TMA application.

Both the concentration and prevalence (i.e., number of animals that produce TMA specific IgE and IgG) of specific antibodies) were dose-dependent. TMA specific antibodies were not detectable in a powder application control group given the TMA hydrolysis product, Trimellitic acid.

See table2 below.

Time course

TMA-specific IgE and IgG was significantly increased (P<0.001) by day 14 in sera from the BN rats given a single or multiple dermal exposures

(on days 0, 7, 14 and 21) to TMA. Antibody levels peaked after 2 weeks. Multiple TMA exposure produced higher antibody levels than a single exposure and tended to delay the peak response for approximately 1 week.

PCA

The production of TMA-specific IgE was confirmed using the PCA test. Sera from BN rats exposed to 1.25 mg, 5 mg and 20 mg TMA produced positive PCA reactions in naïve Sprague–Dawley rats. Heat treatment of the sera caused loss of PCA reactivity consistent with an IgE-mediated mast-cell activation.

(Fig. 2).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The data of this study suggest that topical skin exposure to dry TMA powder can induce allergic/immunological sensitization as demonstrated by the production of specific antibodies
Executive summary:

The present study examines the ability of dry TMA powder to sensitize Brown Norway rats when applied, topically, to the skin.

A patch of hair was carefully clipped with scissors on the rat’s back. Dry TMA powder (0.3, 1.25, 5 and 20 mg) was administered on days 0, 7, 14 and 21, and the area occluded with surgical tape overnight after each application.

Residual powder recovered from the occluded skin was analyzed by proton nuclear magnetic resonance and was still predominantly TMA.

Circulating anti-TMA IgE and IgG were measured by ELISA.

TMA elicited dose-dependent production of specific IgE and IgG. Specific antibodies were detectable 2 weeks after the first TMA exposure and peaked between 3 and 4 weeks.

In conclusion, these results suggest that topical skin exposure to dry TMA powder can induce allergic/immunological sensitization as demonstrated by the production of specific antibodies.