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Diss Factsheets

Administrative data

Description of key information

Skin sensitisation: testing on the substance itself is waived because of the corrosivity to skin of TMAC.

Respiratory sensitisation: testing on the substance itself is waived because of the corrosivity to the respiratory tract of TMAC (EUH071)

However TMAC is considered as skin and respiratory sensitizer by read-across to Trimellitic Anhydride (TMA).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose for cross-reference:
read-across: supporting information
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.
Specific details on test material used for the study:
Trimellitic anhydride
CAS 552-30-7
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.
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)
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.
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

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).

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.

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
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Principles of method if other than guideline:
Fivee rat strains were compared for their performance in the local lymph node assay (LLNA), a promising test system for the identification of the skin-sensitizing potential of chemicals in the mouse.
The contact sensitizer 2,4-Dinitrochlorobenzene (DNCB) and the contact and respiratory sensitizer Trimellitic anhydride (TMA) were used as model chemicals and responses in rats were compared with those in BALB/c mice.
The chemicals were applied to the dorsum of both ears, once daily for three consecutive days; 2 days (for mice) or 3 days (for rats) thereafter, proliferating cells were labelled by ip injection of BrdU 2 hr before the animals were killed.
Systemic effects were subsequently assessed by determination of spleen, liver and kidrey weights, skin effects by determination of swelling and inflammatory cell infiltration of the ears, and immune effects by determination of weight and proliferative activity of the local lymph nodes (LLN).
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Specific details on test material used for the study:
Trimellitic anhydride
CAS 552-30-7
Species:
other: mice and rats
Strain:
other: Mice:BALB/c / Rats: Fischer 344 (F344), Brown Norway (BN), and Piebald Virol Glaxo (PVG), Wistar, Sprague-Dawley (SD)
Sex:
female
Details on test animals and environmental conditions:
> 10-1 l-wk-old Fischer 344 (F344), Brown Norway (BN), and Piebald Virol Glaxo (PVG) inbred rats, Wistar random-bred rats, Sprague-Dawley (SD) outbred rats
and
8-9-wk-old BALB/c inbred mice
> purchased from Charles River Wiga GmbH (Sulzfeld, Germany)
> acclimatized for at least 5 days before the start of the study
> kept under conventional laboratory conditions and received the Institute's grain-based open-formula diet and unfluoridated tap water ad lib. A
> All animal procedures were approved by the TNO Commission of Animal Welfare.
Vehicle:
other: AOO (for Acetone Olive Oil) 4:1 (v/v) solution of acetone (Merck, Darmstadt, Germany) and raffinated olive oil (Chempri BV, Raamsdonksveer, The Netherlands)
Concentration:
12.5, 25 and 50%.
No. of animals per dose:
Mice: 5 / group
Rats: 6 / group
Details on study design:
Experimental design:
Desired concentrations of the chemicals in 75 µl (rats) or 25 µl (mice) AOO (Acetone Olive Oil) were applied to the dorsum of both ears on days 0, 1 and 2.
Controls received AOO or were left untreated.
The rats were slightly anaesthetized with ether before each application, since they offered resistance to the treatment.
Ears were inspected for hyperaemia before and after each application.
Proliferating cells were labelled by ip injection of the thymidine analogue BrdU (150mg/kg body weight) on day 4 in the case of mice and (because of the assumed slower immune responsiveness of rats) on day 5 in the case of rats.
2 hr after BrdU treatment, animals were anaesthetized and bled to death through the abdominal aorta.
Ear thickness was measured immediately thereafter with an electronic pawmeter and animals were observed grossly for abnormalities.
Lymph nodes draining the skin of the ear LLN (Local Lymph Nodes), superficial cervical lymph nodes, kidneys, liver and spleen were excised, freed from fat and weighed. Next, the shells of the ears were removed as completely as possible from the head, and the dorsal ear area was calculated with the aid of calibration paper.

Immunohistochemistry:
For determination of mitotic activity, local lymph nodes were fixed in 70% ethanol, embedded in paraffin wax and 5-gm sections were prepared.
After deparaffination, the sections were immersed in 0.3% H202 (v/v) in methanol for 15 min to inhibit endogenous peroxidase, hydrolysed in 1 N HC1 (60min, 37°C) and subsequently preincubated in 25% normal goat serum for 20 min to reduce background staining.
Next, the sections were successively incubated with the monoclonal antibody anti-BrdU for 60 min, with a biotin-labelled rabbit-anti-mouse antibody with a peroxidase-conjugated streptavidin and finally incubated for 10min with the chromogen YY-diaminobenzidine-tetrahydrochloride, dissolved in phosphate buffered saline (PBS) at a concentration of 0.5 mg/ml (pH 7.4), and supplemented with 0.05% (v/v) H202. After each incubation step, sections were rinsed in PBS. The sections were slightly counterstained with haematoxylin.
Mitotic activity was determined in lymph node sections at five sites of the paracortex (a typical T-cell compartment), at three sites of the cortex (a B-cell compartment) and the medulla, without prior knowledge of the treatment.
At each site, at least 25 cells of which the nuclei were located at the crossings of the horizontal and vertical lines of a 10 x 10 intraocular grid were examined at high power magnification (x 400) to determine the mean percentage of BrdU-positive (BrdU +) cells per compartment and the average percentage of BrdU + cells per section.
As a measure of total proliferation, the average percentage of BrdU + cells per section was multiplied by lymph node weight.
For determination of inflammatory cell infiltrates, cryostat sections of ears of four rat strains that were snap-frozen on dry ice were incubated with the mouse
monoclonal antibodies MRC OXI9 (CD5; recognizing virtually all T cells) or EDI [specific for the majority of monocytes and macrophages].
Sections were then incubated with peroxid~Lse-conjugated rabbit anti-mouse lg for 30 min and finally incubated with the chlromogen 3'3'-diaminobenzidinetetrahydrochloride and H:O 2, as described above.
Control sections were processed in the same way, except for the leucocyte-specific antibodies which were replaced by PBS. The presence and composition
of inflammatory cell infiltrates were assessed on the basis of the number of OX19- and EDl-positive cells in the ear sections.
Positive control substance(s):
other: DNCB (Dinitrochlorobenzene), known as being a skin senistizer, was tested in parallel of DMA in the same study. It can be considered as the positive control.
Key result
Parameter:
SI
Remarks:
Mice BALB/c
Value:
ca. 13.3
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat fisher
Value:
ca. 14.5
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat Wistar
Value:
ca. 22
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat BN
Value:
ca. 6.8
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat SD
Value:
ca. 41.4
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat PVG
Value:
ca. 21.2
Test group / Remarks:
50% TMA

Strain-dependent systemic effects of 50% TMA (results not shown, see publication):

Three daily applications of TMA or DNCB did not affect body and kidney weights of BALB/c mice and all rat strains tested.

Relative spleen weights were significantly increased by DNCB in BALB/c mice and Fischer rats, and absolute and relative liver weights by TMA in SD and PVG rats only. The latter strain also showed a relative increase of liver weight in response to DNCB.

Strain-dependent reactions of the ears to 50% TMA (Table 1):

Rats had on average 1.8 times larger ear areas and 2.5 times thicker ears than mice, while receiving a three times larger vehicle volume than mice.

Hyperaemia of the ears was not observed in mice nor in any of the rat strains after application of TMA, whereas DNCB induced hyperaemia in three of six BN rats after the second application only.

Ear thickness generally had increased in mice and all rat strains tested; significant increases were reached in BALB/c mice, Fischer, BN and SD rats in the case of DNCB, and in BALB/c mice, Fischer and PVG rats in the case of TMA (Table 1).

No significant differences were observed between responses to DNCB and TMA in the same strain, although in BN rats the response to DNCB tended to be greater than that to TMA, whereas in BALB/c mice the reverse tendency was observed.

Immunohistochemicai examination of the ears of four rat strains revealed the presence of inflammatory cell infiltrates after DNCB or TMA treatment, but not after AOO treatment.

DNCB induced infiltrates consisting of macrophages and T lymphocytes in three of three BN rats and one of three Wistar rats; infiltrates consisting of macrophages without significant T-cell numbers were seen in two of three PVG rats and in another Wistar rat. TMA induced infiltrates of macrophages and T lymphocytes in the ears of three of three Fischer, two of three Wistar, one of three BN and one of three PVG rats.

In no instance were significant granulocyte numbers observed. The infiltrates were predominantly located in the epithelium, dermis and upper part of the subcutis and, when prominent, were accompanied by crusts and marked acanthosis.

Table 1: Groups of six mice or rats received 1% DNCB or 50% TMA in AOO or AOO alone on the dorsum of both ears on days 0, 1 and 2, and an ip injection of BrdU on day 4 (mice) or day 5 (rats). Animals were autopsied 2 hr after BrdU treatment and ear thickness was measured. Values are means + SEM (absolute values) and (for DNCB- and TMA-treated groups) as percentage increase compared with AOO-treated controls.

                 Ear thickness  
      AOO  TMA  TMA  DNCB  DNCB
 Species  Strain  x 10E-2 mm    x 10E-2 mm %    x 10E-2 mm  %
 Mouse  BALB/c  20.8 +- 034  28.0 +- 1.7*  35  23.9 +- 0.7*  15
 Rat   Fisher  50.5 +- 1.5  57.2 +- 1.1*   13  56.7 +- 1.9*  12
 Rat   Wistar  54.5 +- 1.9  58.7 +- 1.3 8   59.9 +- 2.3  10
 Rat   BN  47.9 +- 0.9  55.4 +- 2.4  16  61.0 +- 4.8*  27
 Rat   SD  53.4 +- 1.6  61.4 +- 2.2  15  63.8 +- 3.4*  19
 Rat   PVG  52.4 +- 1.0  58.2 +- 2.0*  11  56.7 +- 0.9  8

AOO: Olive Oil (vehicle)

*: significant differences from corresponding controls (*P < 0.05; ANOVA followed by pairwise t-tests).

Strain-dependent reactions of the local lymph nodes to 50% TMA (Table 2):

In rats, the lymph nodes that appeared enlarged after a 3-day application of DNCB or TMA were located dorsally to the lower poles of the submandibular glands, at the junction of the external jugular veins and the anterior and posterior facial veins. In most animals, only one node at each side was found, but occasionally two or even three nodes were observed, especially in BN rats. All nodes at the above-mentioned location were considered to be the LLN and were collected and weighed together.

In BALB/c mice as well as all rat strains tested, significantly increased mean LLN weights were observed relative to controls after TMA as well as DNCB application.

The stimulation index (SI) was highest in mice and lowest in BN rats both for TMA and DNCB application; in the other rat strains the response varied slightly. (Table 2)

Untreated rats showed slightly higher LLN weights than did vehicle-treated controls. The rather low stimulation index in BN rats may result from the high control weights in this strain.

Control LLN showed low percentages of proliferating cells, as judged by BrdU staining, in the different lymph node compartments of most strains.

Higher percentages were found in the medulla of BALB/c mice and Fischer rats, and in both the medulla and cortex of BN rats. DNCB and TMA increased proliferative activity in all lymph node compartments of all groups, but responses in the different compartments differed with the strain. In Fischer and Wistar rats, prolifi~rative activity in the paracortical lymph node area was higher than in the cortical area after DNCB treatment, and vice versa after TMA application.

In BN rats, the proliferative activity was more or less the same in cortex and paracortex after DNCB treatment, whereas a higher cortical reaction was seen after TMA application.

The mean percentage of BrdU + cells per section was significantly increased by DNCB or TMA in BALB/c mice and in all rat strains apart from the BN strain (Table 2). The stimulation index was highest in SD rats and lowest in BN rats and BALB/c mice.

Virtually the same ranking was seen when stimulation indices of the mean total proliferative activity per LLN were calculated.

Table 2: Strain-dependent effects of DNCB and TMA on weight and proliferative activity in local lymph nodes (LLN)

       LLN weight (1)    BrdU + cells   (1)(3)     BrdU + cells/LLN  (1)  
 Species Strain   Treatment  mg SI  (2)     %  SI  (2)       SI  (2)
 Mouse  BALB/c  AOO  4.0 +- 0.5  -  5.0 +- 2.1  -  0.2 +- 0.1  -
   DNCB  18.6 +- 0.7**  4.7

 15.4 +- 2.4**

  3.1  2.8 +- 0.4**  11.8

 

 

 TMA

 18.7 +- 1.6**

 4.7

 16.0 +- 0.9**

 3.2

 3.2 +- 0.3***

 13.3

 Rat

 Fisher

 AOO

 12.4 +- 1.2

 -

 3.5 +- 0.7

 -

 0.4 +- 0.1

 -

 

 

 DNCB

 51.9 +- 1.3**

 4.2

 19.6 +- 3.9***

 5.6

 10.2 +- 2.1

 22.6

 

 

 TMA

 35.7 +- 3.2**

 2.9

 18.5 +- 2.1***

 5.3

 6.5 +- 0.9**

 14.5

 Rat

 Wistar

 AOO

 19.0 +- 1.7

 -

 3.3 +- 0.8

 -

 0.6 +- 0.2

 -

 

 

 DNCB

 77.0 +- 5.6**

 4.1

 22.4 +- 2.8***

 6.8

 17.5 +- 2.8**

 28.2

 

 

 TMA

 59.1 +- 5.5**

 3.1

 22.6 +- 2.4***

 6.9

 13.7 +- 2.4**

 22.0

 Rat

 BN

 AOO

 29.6 +- 2.9

 -

 5.2 +- 1.0

 -

 1.6 +- 0.4

 -

 

 

 DNCB

 63.3 +- 4.1**

 2.1

 15.8 +- 3.2

 3.0

 10.0 +- 2.0

 6.1

 

 

 TMA

 65.6 +- 4.6*

 2.2

 15.8 +- 4.5

 3.0

 11.1 +- 3.7

 6.8
 Rat  SD  AOO  20.7 +- 2.6  -  1.6 +- 0.6  -  0.3 +- 0.1  -
     DNCB  82.6 +- 4.1**  4.0  14.4 +- 3.4*  9.0  12.4 +- 3.4*  44.1
     TMA  79.0 +- 5.5**  3.8 14.0 +- 4.9   8.7  I 1.6 +- 3.9*  41.4
 Rat  PVG  AOO  13.6 +- 1.5  -  2.8 +- 0.5  -  0.4 +- 0.1  -
     DNCB  49.3 +- 2.5**  3.6  13.9 +- 2.3**  5.0  6.7 +- 1.0***  17.6
     TMA  38.7 +- 2.0**  2.9  20.6 +- 1.5*** 7.4   8.1 +- 0.9***  21.2

Groups of five or six mice or rats received 1% DNCB or 50% TMA in AOO or AOO alone on the dorsum of both

ears on days 0, 1 and 2, and an ip injection of BrdU on day 4 (mice) or day 5 (rats).

LLN at both sites were removed 2 hr after BrdU treatment, weighed, and processed for BrdU staining to determine

the percentage or total number of BrdU + cells per LLN.

(1): Values are means + SEM.

(2): SI = Stimulation index (mean ratio of values found in LLN of chemical- and AOO-treated animals, respectively).

(3): Mean of the average percentages found in paracortex, cortex and medulla.

*, **, ***: significant differences from corresponding controls s (* P<0.05; ** P<0.01; *** P<0.001; ANOVA

followed by Dunnett's or t-test)

Dose-response study:

No clear dose-effect relationships could be noted (results not shown, see publication). All doses tested caused significant increases of all parameters in all strains and effects appeared to be of similar magnitude or tended to have an optimum at the medium dose tested.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Following application (x3) TMA, minor systemic effects were observed, as indicated by slightly elevated spleen and liver weights in a few rat strains and the mice. Skin effects were observed and were comparable with those in mice. The Stimulation Index (SI) is >= 1.9 for both mice and rats, in all strains and at all tested doses.
These skin effects observed in rats and mice (increased ear thickness and presence of mononuclear inflammatory cell infiltrates; increase of LLN weights with proliferative activity in these nodes) confirm that TMA is clearly a skin sensitiser.
Executive summary:

Five rat strains were compared for their performance in the local lymph node assay (LLNA), a promising test system for the identification of the skin-sensitizing potential of chemicals in the mouse.

The contact and respiratory sensitizer trimellitic anhydride (TMA) and the contact sensitizer Dinitrochlorobenzene (DNCB) were used as model chemicals and responses in rats were compared with those in BALB/c mice.

The chemicals were applied to the dorsum of both ears, once daily for three consecutive days; 2 days (for mice) or 3 days (for rats) thereafter, proliferating cells were labelled by ip injection of BrdU 2 hr before the animals were killed. Systemic effects were subsequently assessed by determination of spleen, liver and kidrey weights, skin effects by determination of swelling and inflammatory cell infiltration of the ears, and immune effects by determination of weight and proliferative activity of the local lymph nodes (LLN).

Following application (x3) of TMA, minor systemic effects were observed, as indicated by slightly elevated spleen and liver weights in a few rat strains and the mice. Skin effects, consisting of increased ear thickness and presence of mononuclear inflammatory cell infiltrates, were observed in all rat strains treated with TMA. LLN weights had increased, as had the proliferative activity in these nodes. The Stimulation Index (SI) is >= 1.9 for both mice and rats, in all strains and at all tested doses.

These effects observed in rats an dmice confirmed that TMA is clearly a skin senistizer.

Endpoint:
skin sensitisation: in vivo (LLNA)
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:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read-across is justified in the document 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
Parameter:
SI
Remarks:
Mice BALB/c
Value:
ca. 13.3
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat Fisher
Value:
ca. 14.5
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat Wistar
Value:
ca. 22
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat BN
Value:
ca. 6.8
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat SD
Value:
ca. 41.4
Test group / Remarks:
50% TMA
Key result
Parameter:
SI
Remarks:
Rat PVG
Value:
ca. 21.2
Test group / Remarks:
50% TMA
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation:

No data was available regarding skin sensitisation potential of TMAC.

TMA is listed as a skin senistizer (H317) in the Annex VI of the CLP regulation (EC) No 1272/2008).

In addition, TMA is clearly skin sensitizer in the LLNA study (OECD 442B) conducted both in mice ad rats.

Moreover, topical skin exposure to dry TMA powder (by patch) in rats induces production of specific antibodies, demonstrating an allergic/immunological reaction.

In a read across approach with TMA, TMAC is considered as a skin sensitizer.

Respiratory sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline followed
Principles of method if other than guideline:
This protocol uses both sensitization and challenge by the inhalation route. The Brown Norway (BN) rat model is used in the present study to examine specific airway reactivity following sensitization by the respiratory tract route.
Respiratory sensitization (dose dependent TMA-specific IgE, pulmonary inflammatory, histopathology) and airway responses (early phase airway response (EAR) and late-phase airway response (LAR)) to repeated inhalation challenge with TMA-aerosol atmospheres were examined in the Brown Norway rat model. I

Rats were exposed to 0.04, 0.4, 4, or 40 mg/m3 TMA aerosol for 10 min, once a week, over 10 weeks.
All lower exposures were, subsequently, rechallenged to 40 mg/m3 TMA aerosol.
All rats received a sham exposure 1 week prior to the first TMA exposure.
Following the sham exposure and weekly after each TMA exposure, TMA-specific IgE and both early-phase airway response (EAR) and late-phase airway response (LAR) were measured using enhanced pause (Penh).
GLP compliance:
not specified
Specific details on test material used for the study:
Trimellitic anhydride
CAS 552-30-7
Species:
rat
Strain:
Brown Norway
Sex:
female
Details on test animals or test system and environmental conditions:
- Female, inbred BN rats (150–175 g) from Charles River Laboratories (Wilmington, MA).
- Rats obtained from rooms free of ‘‘rat respiratory virus’’ and other specific pathogens.
- 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.
Route of induction exposure:
inhalation
Route of challenge exposure:
inhalation
Vehicle:
unchanged (no vehicle)
Concentration:
40, 4, 0.4, and 0.04 mg/m3
No. of animals per dose:
4 groups: 8 animals per group for the two higher doses and 4 animals per group for the two lower doses
Details on study design:
The rats were placed in the nose-only exposure chamber at concentrations of 40, 4, 0.4, and 0.04 mg/m3, respectively, starting on day 0 and every 7 days, thereafter, for 10 weeks (exposure/challenge = 1 x per week).

All rats, except for the 40 mg/m3 TMA group, were rechallenged with 40 mg/m3 of TMA aerosol, for 10 min (in the nose-only chamber), 2 weeks after the final inhalation exposure to TMA.
Chamber concentrations were measured using a real-time, continuous dust monitor and gravimetrically using a 0.45-lm, 37-mm HAWP filter.
Rats were immediately moved to whole-body plethysmograph chambers after 10 min of TMA exposure, to monitor enhanced pause (Penh), for 12–20 h. Normal Penh values (used as control) from all rats were also recorded before the day 0 TMA inhalation exposure.
Indices were recorded every 30 s. Arithmetic means of peak values from every 30 min for the first hour and every 1 h after that were used to quantify the responses. Penh area under the curve (AUC) was also determined, as well as starting point, ending point, and duration of the LAR.
EAR (earlyphase airway response) is an increase in Penh >= 0.9 (3 SDs above the mean peak Penh value) that is apparent within 30 min of challenge and is usually resolved by 1 h.
LAR (late-phase airway response) manifests 2–4 h postchallenge as an increase in Penh > 0.9 and last for several hours.

At the end of each experiment, approximately 24 h following airway challenge, rats were euthanized and bronchoalveolar lavage fluid (BALF) obtained. BALF was used for eosinophil enumeration using a Coulter Multisizer for total cell count per milliliter and microscopic differential analysis (total cell count/ml 3 percent eosinophils).
Lungs were excised, perfused, and fixed for the preparation of stained slides.
Lung sections from the left lung lobes were histopathologically examined. The severity (intensity) and distribution (extent) of changes are each evaluated with a range of 0 (none) to 5 (greatest involvement) producing a cumulative histopathology score with a potential range of 0–10 (as described by Hubbs, Hubbs et al., 1997).
Positive control substance(s):
none
Negative control substance(s):
other: Normal Penh values from all rats were recorded before the day 0 TMA inhalation exposure and were used as control.
Results:
> Specific IgE to TMA:
TMA-specific IgE was detected by day 7 in the 40 and 4 mg/m3 TMA exposure groups.
Only 1/4 rats had measurable specific IgE in the group exposed to 0.4 mg/m3 of TMA, and no specific IgE was detectable in rats from the lower exposure groups (0.04 mg/m3).

> Airway Responses after Inhalation Exposure and Final Challenge with TMA:
On day 14, 6/8 of the rats challenged with 40 mg/m3 TMA developed both EAR and LAR; however, these responses were inconsistent from week to week from each animal.
No significant respiratory changes were noted in groups before and after exposure to lower concentrations of TMA over the 10 weekly exposures.

On day 77, all the lower exposure groups were challenged with 40 mg/m3 TMA for 10 min.
All eight rats from the 4 mg/m3 TMA group and only one rat from the 0.4 mg/m3 TMA group responded with both EAR and LAR to the challenge. No airway responses were noted in
the rats from the 0.04 mg/m3 TMA group following the 40 mg/m3 TMA challenge.
The AUC of the LAR from the 4 mg/m3 TMA group challenged with 40 mg/m3 was 1120 ± 134 (the highest LAR AUC noted from each animal exposed over 10 weeks to 40 mg/m3 was 592 ± 89): the repeated high-dose exposure produced a weaker LAR than the lower dose sensitization—high-dose challenge protocol. There was no increasing or decreasing Penh AUC of the LAR with repeated exposure observed in the high dose group and, in fact, LAR Penh was inconsistently manifested following challenges in this group.
Pathologic findings: Eosinophils, but not neutrophils, were found in the lavage of animals that were sensitized with and responded to TMA challenge.
Rats exposed to 4 mg/m3 TMA (and challenged to 40 mg/m3 TMA) had significantly higher percentage of eosinophils, than that in rats exposed to 40 mg/m3 of TMA.
The total BALF eosinophils recovered was not statistically different. Lower total BALF cells in this group may be due to inflammatory changes such as activated ‘‘sticky’’ macrophages causing poor recovery of these cells.

The principal histopathologic changes in the lungs of TMA exposed rats were eosinophilic granulomatous interstitial pneumonia, eosinophil perivascular infiltrates, peribronchiolar
plasma cell infiltrates, and hyperplasia of bronchus-associated lymphoid tissue (BALT). Some rats demonstrated airway epithelial cell hypertrophy consistent with mucous metaplasia. However, the consistent and predominant histopathologic alterations in this study were not in the airways but instead were localized to the deep lung. Eosinophilic granulomatous interstitial pneumonia was characterized by interstitial infiltration by histiocytic macrophages and eosinophils. Lesser numbers of giant cells, neutrophils, and lymphocytes
contributed to the interstitial inflammation in some lungs.
Frequently, the interstitium between foci of intense inflammation was involved but to a lesser extent. At 0.4 mg/m3 TMA, two of the four rats had eosinophilic granulomatous interstitial pneumonia. All rats inhaling 4 or 40 mg/m3 had eosinophilic granulomatous interstitial pneumonia, although the severity of the response was slightly, but not significantly, reduced in rats inhaling 40 mg/m3. The pathology scores for eosinophilic granulomatous interstitial pneumonia in rats inhaling 40 mg/m3 were significantly higher than in rats inhaling 0.4 mg/m3. Perivascular eosinophils were frequently observed in TMA exposed rats and may represent vascular eosinophils migrating to the inflamed parenchyma. Peribronchiolar infiltrates of plasma cells were seen adjacent to occasional small bronchioles in all rats inhaling 40 mg/m3 and two rats inhaling 4 mg/m3. The pathology scores for these plasma cell
infiltrates were significantly greater in rats inhaling 40 mg/m3 TMA than in rats inhaling 4 or 0.4 mg/m3 TMA. These plasma cell infiltrates are unusual in the rat lung. Other evidence of antigenic stimulation seen in this study included hyperplasia of BALT. Hyperplasia of BALT was observed in all rats exposed to TMA but did not demonstrate a dose-response relationship. Eosinophilic granulomatous interstitial pneumonia, eosinophil perivascular infiltrates, peribronchiolar plasma cell infiltrates, and hyperplasia of BALT were present in TMA-exposed rats but not observed in the two controls.
Interpretation of results:
Category 1 (respiratory sensitising) based on GHS criteria
Conclusions:
An in vivo study conducted on Brown Norway rats exposed to TMA aerosol was conducted. Inhalation exposure to TMA aerosol induces the production of measurable specific IgE following a single exposure (specific IgE noted in serum prior to second 40 mg/m3 exposure), a specific airway reactivity (Penh, EAR, and LAR), pulmonary allergic inflammatory pathology and an attenuated specific IgE response with repeated high-dose aerosol exposure.
Based on these findings, the substance is considered to induce allergic pathology by inhalation.
Executive summary:

Dosed dependent TMA-specific IgE, histopathology, and airway responses after sensitization by inhalation were examined in the Brown Norway rat. Rats were exposed to 0.04, 0.4, 4, or 40 mg/m3 TMA aerosol for 10 min, once a week, over 10 weeks.

All lower exposures were, subsequently, rechallenged to 40 mg/m3 TMA aerosol.

All rats received a sham exposure 1 week prior to the first TMA exposure. Following the sham exposure and weekly after each TMA exposure, TMA-specific IgE and both early-phase airway response (EAR) and late-phase airway response (LAR) were measured using enhanced pause (Penh).

All rats sensitized by 40 mg/m3 TMA developed specific IgE, EAR, and LAR to one or more of the challenges to 40 mg/m3 TMA. TMA of 4 mg/m3 induced a much lower, but stable, specific IgE response. EAR and LAR were observed only after a 40 mg/m3 TMA rechallenge in this group, but it was much larger than that observed in the 40 mg/m3 TMA-sensitized and challenged group. Exposure-dependent histopathological changes noted included eosinophilic granulomatous interstitial pneumonia, perivascular eosinophil infiltrates, bronchial-associated lymphoid tissue hyperplasia, and peribronchiolar plasma cell infiltrates.

Endpoint:
respiratory 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:
data from handbook or collection of data
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 (TMAC)
CAS 1204-28-0
Interpretation of results:
Category 1 (respiratory sensitising) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Respiratory sensitisation:

No data was available regarding the respiratory sensitisation. And no guidelines are available to determine respiratory sensitisation.

TMA is listed as a respiratory sensitizer (H334) in the Annex VI of the CLP regulation (EC) No 1272/2008).

In addition, TMA was tested on rats exposed by inhalation. This exposure induced the production of specific IgE, with a pulmonary allergic inflammatory pathology.

Moreover, WHO concluded that cyclic acid anhydrides including Trimetillic anhydride can cause sensitization in Humans after exposure by inhalation.

In a read across approach with TMA, TMAC is considered as a respiratory sensitizer.

Justification for classification or non-classification

Harmonized classification:

Trimetillic anhydride chloride does not have an harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

No data was available regarding skin or respiratory sensitisation of TMAC.

However, in a read across approach with TMA which is a skin and a respiratory sensitiser (based on experimental data and based on its presence in the Annex VI of the CLP regulation (EC) No 1272/2008), TMAC (Trimellitic anhydride mono-chloride) should be classified as category 1 for skin sensitization (H317) and respiratory sensitisation (H334) according to Regulation (EC) No 1272/2008 (CLP) and to the UN GHS.

No sub-category is defined since the assessment is based on a read-across approach and since no data is available on the substance itself. In addition, no sub-category is defined for the analogue TMA either in the List of the European Harmonized classification (Annexe VI of CLP regulation) or in the Reach dossier.