Benzene-1,2,4-tricarboxylic acid 1,2-anhydride

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented, peer reviewed study forming part of the ICCVAM/NICEATM method validation of LLNA methods

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442A (Skin Sensitization: Local Lymph Node Assay: DA)

Principles of method if other than guideline:

LLNA: DA determines sensitisation potential by measuring the proliferation of lymphocytes in the auricular lymph nodes draining the site of exposure (ears). The method employs non-radioactive techniques to assess lymphocyte proliferation by measuring adenosine triphosphate (ATP) content in the lymph nodes, thus eliminating the use of tritiated thymidine or iodine-125 based measurements used by the traditional LLNA.
In the traditional LLNA, the test substance is administered on three consecutive days (days 1, 2, and 3). On day 6, tritiated thymidine or iodine-125 is administered via the tail vein and the lymph nodes are excised five hours later. A lymph node cell suspension is then prepared and tritiated thymidine or iodine-125 incorporation is determined by scintillation counting. In the LLNA: DA method the test substance is applied on days 1, 2, 3, and 7. One hour prior to each application of the test substance, 1% sodium lauryl sulfate (SLS) is applied to the dorsum of the treated ears to increase absorption of the test substance across the skin. Twenty-four to 30 hours after the last test substance application, the auricular lymph nodes are excised, a lymph node cell suspension is prepared, and the ATP content is measured by luciferin-luciferase assay.

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 11 weeks
- Weight at study initiation: 20.7 - 24.4 g
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 25 deg C
- Humidity (%): 40 - 70%
- Air changes (per hr): 8 - 10

IN-LIFE DATES: From: 2005-08-24 To: 2005-08-31

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0.1, 0.25 and 0.5%

No. of animals per dose:

3 - 4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Soluble

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA-DA
- Criteria used to consider a positive response: Si > 3

TREATMENT PREPARATION AND ADMINISTRATION: Dorsum of both ears treated with 1% sodium lauryl sulphate (SLS). One hour later 25 µL of 3 concentrations of test substance, positive control or vehicle alone applied to the dorsum of both ears. This treatment regime was followed on Days 1, 2, 3 and 7. On Day 8 (24-30 hours after last application) draining auricular lymph nodes were excised, weighed, and pooled. Lymph node cells were crushed and spread between two glass slides and then scraped and suspended in 1 mL phosphate buffered saline PBS). The cell suspension obtained was then diluted 1:100 in PBS and a 100 µL aliquot was used to measure ATP content by luciferin-luciferase assay.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean relative light unit (RLU) value for each experimental group was calculated, and the Stimulation Index (SI) calculated as the mean ATP content in the LNC suspension obtained from the test group relative to that in the LNC suspension obtained from the control group. The cut-off point for a positive result was SI ≥3 and any result with an SI <3 was considered negative.

Parameter:

SI

Remarks on result:

other: Vehicle - 1.00 TMA 0.1% - 2.46 TMA 0.25% - 3.58 TMA 0.5% - 4.96 Positive control (15% HCA) - 5.67

	Body	Lymph node weight			ATP luminescence (RLU)
Treatment	weight	Individual	Mean ± SD	SI	Individual	Mean ± SD	SI
	(g)	(mg)	(mg)
	22.6	3.30			3101
Vehicle	22.3	3.54	3.48 ± 0.51	1.00 ± 0.15	3253	3362 ± 736	1.00 ± 0.22
control	23.1	2.93			2687
	22.9	4.15			4407
	21.6	10.21			22800
Positive	24.2	9.42	9.29 ± 0.76	2.67 ± 0.22	16696	19056 ± 2636	5.67 ± 0.78
control	20.7	8.38			17973
	21.4	9.15			18757
	21.8	4.61			5681
TMA 0.1%	23.0	6.36	5.76 ± 0.99	1.65 ± 0.29	7841	8272 ± 2831	2.46 ± 0.84
	24.1	6.30			11293
	22.5	7.46			13902
TMA 0.25%	23.4	6.43	6.81 ± 0.57	1.96 ± 0.16	11270	12045 ± 1615	3.58 ± 0.48
	23.6	6.54			10963
	21.9	8.31			14361
TMA 0.5%	24.4	10.50	9.15 ± 1.18	2.63 ± 0.34	18976	16670 ± 2308	4.96 ± 0.69
	21.7	8.65			16673

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Trimellitic anhydride was sensitising in the mouse Local Lymph Node Assay.

Executive summary:

Trimellitic anhydride was sensitising in the reported variant of the mouse Local Lymph Node Assay, the authors stating an EC3 of 0.20% compared with an EC3 of 0.22% obtained with the traditional LLNA.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin Sensitisation

McCormick & Johnson (1993) investigated the sensitising effects of trimellitic anhydride in guinea pigs. 0.3 g of the test substance was applied undiluted to the backs of ten male guinea pig once per week during the three week induction period. A challenge dose of 0.3 g undiluted test substance was applied two weeks after the final induction dose. Thirteen days following this, the animals received a second challenge dose of 0.3 g of the undiluted test substance. Positive erythema reactions were not observed in any of the treated animals following either challenge dose, concluding that trimellitic anhydride did not appear to induce dermal sensitization under the exposure conditions of this study.

In a previous study, Andresenet al(1987) applied 0.3 ml of trimellitic anhydride in dimethylsulfoxide, to the backs of ten male guinea pigs once per week during the three week induction period. Two and three weeks following the induction period, the test animals received two additional challenge doses of 0.3 ml test substance in acetone, on a different site on the guinea pigs' backs. Positive erythema reactions were observed in seven of the treated guinea pigs after the first challenge, with the majority of the test animals displaying signs of positive erythema reactions following the second challenge.

Idehara reports the investigation of TMA as part of a series of 31 substances examined as part of the ICCVAM/NICEATM method validation of LLNA methods. Sensitisation potential was measured by the proliferation of lymphocytes in the auricular lymph nodes draining the site of exposure (ears). The method employs non-radioactive techniques to assess lymphocyte proliferation by measuring adenosine triphosphate (ATP) content in the lymph nodes, thus eliminating the use of tritiated thymidine or iodine-125 based measurements used by the traditional LLNA.Trimellitic anhydride was sensitising in the reported variant of the mouse Local Lymph Node Assay, the authors stating an EC3 of 0.20% compared with an EC3 of 0.22% obtained with the traditional LLNA.

Migrated from Short description of key information:
A positive result in the LLNA:DA assay show that trimellitic anhydride is a skin sensitiser. This is supported by positive responses in one of two Buehler guinea pig assays.

Justification for selection of skin sensitisation endpoint:
Best documented of the studies available

Respiratory sensitisation

Link to relevant study records

Reference

Endpoint:

respiratory sensitisation: in vivo

Type of information:

other: Research project

Adequacy of study:

weight of evidence

Study period:

Not indicated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is a research investigation that provides some information on respiratory sensitisation and the difference in sensitivity between male and female rats.

Qualifier:

no guideline available

Principles of method if other than guideline:

This is a research investigation and therefore does not follow any guideline.

GLP compliance:

no

Remarks:

This work was a research investigation and GLP is therefore not applicable.

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were male and female rats, no further information was provided in the report.

Route of induction exposure:

inhalation

Route of challenge exposure:

other: Not applicable since the study is a research project and does not follow any particular guideline.

Vehicle:

corn oil

Concentration:

500 µg/m³, the test material was ground to respirable-sized particles using a mortar and pestle.

No. of animals per dose:

5 groups of 10 rats/sex

Details on study design:

This study was designed to evaluate the sex-related differences observed between males and females exposed to trimellitic anhydride (TMA), and to identify any possible correlation between the levels of steroids (oestrogen/testosterone) or TMA-specific antibody levels and the incidence of gross external lung foci. The study was carried out using five groups of 10 rats/sex each. One of the group was exposed to filtered air and served as a control; the remaining four groups were exposed to 500 µg TMA/m³. Two groups were gonadectomised, one of which was "cross-treated" with hormones whereas the other received the vehicle corn oil for two weeks prior to exposure. The third group served as a surgery control and the fourth group remained intact and served as a TMA-exposed control. The rats were exposed 6 hours per day, 5 days a week for a total of 10 exposures

Challenge controls:

Not applicable

Positive control substance(s):

none

Negative control substance(s):

other: filtered air

Results:

Body weight and/or body weight gain were significantly elevated in the gonadectomised females and testosterone-treated ovariectomised females. Castrated oestrogen-treated males showed lower body weights when compared to controls. However, these changes were deemed to be related to steroids levels due to gonadectomy/hormone therapy and independent of TMA exposure.

Positive control results:

A positive control was not included in the study.

Negative control results:

See Table 1.

Table 1: Summary of lung parameters, antibodies and steroids following the exposure to trimellitic anhydride (TMA)

		TMA exposed
Males	Filtered air control	TMA control	Surgery control	Gonadectomised	Castrated Estrogen
Lung weights (g)	1.3 ± 0.8	2.4 ± 0.3*	2.3 ± 0.1*	2.4 ± 0.3*	1.9 ± 0.2**
Lung volume	1.9±0.1	3.0 ± 0.3*	3.0 ± 0.2*	3.1 ± 0.4*	2.4 ± 0.2**
Foci	2.5 ± 3.2	463 ± 295*	438±178*	407 ± 137*	213 ± 79**
IgG (OD)^b	0.03 ± 0.004	0.47 ± 0.24*	0.56 ± 0.22*	0.29 ± 0.19*	0.6 ± 0.2*
Testosterone (ng/ml)	2.0 ± 1.8	3.0 ± 2.6	3.8 ± 2.0	0.13 ± 0.04	0.17 ± 0.06
Estrogen (ng/ml)	0.015 ± 0.007	0.025 ± 0.008	0.039 ± 0.008	0.061 ± 0.012**	0.375 ± 0.105**
		TMA exposed
Females	Filtered air control	TMA control	Surgery control	Gonadectomised	Castrated Estrogen
Lung weights (g)	1.0 ± 0.05	1.6 ± 0.2*	1.7 ± 0.2*	1.9 ± 0.17**	1.6 ± 0.15*
Lung volume	1.9 ± 0.02	2.1 ± 1.6*	2.1 ± 0.3*	2.5 ± 0.4**	2.1 ± 0.1*
Foci	0.4 ± 0.96	161 ± 82*	188 ± 119*	278 ± 125*	243 ± 66.3*
IgG (OD)^b	0.003 ± 0.004	0.9 ± 0.3*	0.6 ± 0.3*	0.5 ± 0.3**	0.6 ± 0.29*
Testosterone (ng/ml)	0.06 ± 0.014	0.08 ± 0.33	0.08 ± 0.018	0.04 ± 0.008	36.870 ± 4.13**
Estrogen (ng/ml)	0.078 ± 0.035	0.071 ± 0.022	0.048 ± 0.017	0.026 ± 0.016**	0.042 ± 0.016

^aWt = weight

^bOD= optical density, values from hte 1:2700 dilution

^*Significantly different from filtered air control

^**Significantly different from TMA control

Interpretation of results:

sensitising

Conclusions:

Rats exposed to trimellitic anhydride (TMA) all exhibited the typical respiratory sensitisation response seen following exposure to TMA. The difference that exists between males and females is confined to the degree in which they respond. A mechanism explaining this difference could not be identified in this study. However, the difference is influenced by steroid levels, particularly oestrogen. TMA is classified as a respiratory sensitiser with the symbol Xi and is assigned the risk-phrase R42 " May cause sensitisation by inhalation" according to Directive 67/548/EEC. It is also classified as Category 1 for respiratory sensitisation with the symbol "Danger" and is assigned the hazard statement H334 "May cause allergy or asthma symptoms or breathing difficulties if inhaled".

Executive summary:

Trimellitic anhydride (TMA) is known to produce three immunologically-based syndromes in humans. Two of these syndromes were investigated using a rat model. This study was designed to evaluate the sex-related differences observed between males and females exposed to TMA, and to identify any possible correlation between the levels of steroids (oestrogen/testosterone) or TMA-specific antibody levels and the incidence of gross external lung foci. The study was carried out using five groups of 10 rats/sex each. One of the groups was exposed to filtered air and served as a control; the remaining four groups were exposed to 500 µg TMA/m³. Two groups were gonadectomised, one of which was "cross-treated" with hormones whereas the other received the vehicle corn oil for two weeks prior to exposure. The third group served as a surgery control and the fourth group remained intact and served as a TMA-exposed control. The rats were exposed 6 hours per day, 5 days a week for a total of 10 exposures.

Body weight and/or body weight gain were significantly elevated in the gonadectomised females and testosterone-treated ovariectomised females. Castrated oestrogen-treated males showed lower body weights when compared to controls. However, these changes were deemed to be related to steroids levels due to gonadectomy/hormone therapy and independent of TMA exposure. All rats exposed to TMA displayed haemorrhagic lung foci. These lesions were accompanied by an increase in lung weight, volume and TMA-specific IgG antibody levels. A significant group by sex interaction was observed (TMA exposure preferentially affected one sex over the other). The changes in lung weight, volume and foci were more pronounced in males than females. IgG levels were higher for females. It was concluded that the sex-related difference that exists between males and females appears to be confined to the degree in which they respond and unrelated to the level of serum TMA-specific IgG antibody. Higher mean levels of oestrogen appeared to result in a lower mean number of lung foci, indicating that oestrogen levels can influence the degree of response. Changes in testosterone levels did not dramatically influence the degree of response with respect to males or females. An exact mechanism explaining the differences between males and females could not be identified. However, this study confirms that TMA-exposed rats all exhibited the typical respiratory sensitisation response seen following the exposure to TMA.

TMA-exposed rats all exhibited the typical respiratory sensitisation response seen following exposure to TMA. No difference in the incidence or severity of the lesions were apparent between the TMA-exposed groups.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Respiratory Sensitisation

An investigative study by Hatoum & Ryan (1992) showed clear evidence of respiratory sensitisation (increased lung weight, volume and foci) in rats exposed for 6 hours per day, 5 days per week over two weeks. Findings indicated that males were more sensitive, however specific hormonal investigations did not further elucidate the factors underlying this sex difference. A study by Andresen (1989) demonstrated that the hydrolysis product trimellitic acid does not cause respiratory sensitisation and does not induce an immunological response in rats previously sensitised to trimellitic anhydride. In addition the study by Andresen (1989) demonstrated that trimellitic acid does not cross-react with trimellitic anhydride. Zhang et al (2006) demonstrated that inhalation exposure of rats to trimellitic anhydride induced the formation of specific IgE, airway reactivity and pulmonary pathology consistent with an allergic response.

Migrated from Short description of key information:
Animal data and experience of human exposure indicate that trimellitic anhydride is a respiratory sensitiser.

Justification for selection of respiratory sensitisation endpoint:
Best documented study of those available

Justification for classification or non-classification

Skin Sensitisation

The substance is considered to be a skin sensitiser based on the results of a positive LLNA and Buehler study. It is classified as a skin sensitiser with the symbol Xi and the risk phrase R43 " May cause sensitisation by skin contact" according to Directive 67/548/EEC. It is classified as skin sensitiser Category 1 and assigned the hazard statement H317 "May cause an allergic skin reaction" according to Regulation (EC) No 1272/2008.

Respiratory Sensitisation

TMA is classified as a respiratory sensitiser with the symbol Xi and is assigned the risk-phrase R42 " May cause sensitisation by inhalation" according to Directive 67/548/EEC. It is also classified as Category 1 for respiratory sensitisation with the symbol "Danger" and is assigned the hazard statement H334 "May cause allergy or asthma symptoms or breathing difficulties if inhaled".