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

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-10-27 to 1983-11-22

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Non-GLP proprietary study, non-standard methods

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Sub-acute inhalation toxicity study in the rat with specific investigation of respiratory sensitisation effects.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

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-1,3-dihydro-2-benzofuran-5-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): Trimellitic anhydride
- Physical state: White solid in the form of flakes
- Lot/batch No.: Test Article - 128C, Identification number - 5507-133
- Storage condition of test material: Store in a tightly sealed jar in a ventilated cabinet at room temperature (approx 22°C)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were male and female Sprague-Dawley rats (Charles River Breeding Laboratories, MI). The rats were approximately 7 weeks of age and weighed approximately 135 g on arrival. The rats were acclimatised for 3 weeks. After the acclimatisation period they were housed in suspended stainless steel cages. These cages were also used for the inhalation exposure. Individuals were identified by ear tags.
The animal rooms were maintained at approximately 22°C and 40% relative humidity. Fluorescent lighting was provided on a 12 hour light/dark cycle. Purina Rodent Chow 5001 and reverse osmosis purified water were provided ad libitum, except during inhalation exposure.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The particles were reported to be micron sized.

Details on inhalation exposure:

Flakes of trimellitic anhydride were ground to micron size particles in an air jet mill. The ground test article was stored in a light-tight container under nitrogen. Rats were exposed to the test substance as a particulate aerosol at one of 4 target concentrations. Controls were exposed to filtered air only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test article was measured during each exposure period using HPLC and a continuous aerosol monitor (PCAM).

Duration of treatment / exposure:

10 days exposure (with interim necropsies performed after 5 days)

Frequency of treatment:

The rats were exposed 6 hours per day, 5 days per week.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 10, 30, 100 and 300 µg/m³
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
11.1, 37.4, 100.6 and 262.3 µg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

Five groups of rats, each consisting of 40 males and 20 females were used to assess the toxicity of trimellitic anhydride. Four of the groups were exposed to graded concentrations of the test article in air, whereas the fifth group was exposed to filtered air only and served as a control.

Control animals:

yes, concurrent no treatment

Details on study design:

The rats were exposed 6 hours per day, 5 days per week according to the following exposure regimens (Table 1):

1) Randomly selected male rats from each group were exposed for two weeks and sacrificed;
2) A second group of male and female rats were exposed for two weeks and sacrificed
3) A third group of male and female rats were exposed for two weeks and held for 12 days without exposure. Part of this group was challenged with an additional single 6-hour exposure and sacrificed, whereas the other was sacrificed without challenge
4) A fourth group of male rats were exposed for two weeks, held 12 weeks without exposure and sacrificed

Positive control:

A positive control was not included.

Examinations

Observations and examinations performed and frequency:

The rats were observed once daily for morbidity and mortality during the quarantine period. Following initiation of treatment, all rats were observed at least once daily. Rats were observed daily for adverse clinical symptoms.
Body weights were measured at study initiation, then weekly thereafter, and at study termination. After the exposure, the 12 week recovery rats were weighed biweekly. Body weights were also measured after the 18 hour fasting period, immediately prior to necropsy.
Blood samples were taken after the fasting period (prior to necropsy) for the following clinical chemistry analyses: glucose, creatinine phosphokinase, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, alkaline phosphatase, urea nitrogen, sodium, potassium, chloride, total protein and albumin. Haematology parameters determined were: total erythrocyte count, haemoglobin, haematocrit, total and differential leukocyte count.

Sacrifice and pathology:

All rats were subject to gross necropsy followed by histopathological examination. Organ weights were recorded for the adrenals, brain, gonads, heart, kidneys, liver, lungs and spleen. The following tissues were collected and fixed in 10% neutral buffered formalin: adrenals, brain, eyes, oesophagus, femur and bone marrow, gonads, heart, duodenum, jejunum, ileum, cecum, colon, kidneys, larynx, liver, lungs, lymph nodes, nasal turbinates, pancreas, parathyroids, pituitary, prostate, seminal vesicles, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord, spleen, stomach, thymus, thyroids, tongue, trachea, urinary bladder, uterus and gross lesions. Each lung lobe was counted for red brown and total number of foci.

Other examinations:

Bone marrow cellularity, serum antibodies, and specialised immunopathological techniques were utilized to determine the presence of nonspecific IgG and complement (C3) in the lungs and respiratory lymph nodes.

Statistics:

ANOVA followed by appropriate pairwise comparisons.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

: lung

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

: lung

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

: lung

Histopathological findings: neoplastic:

not examined

Details on results:

No deaths occurred during the study and no significant abnormal clinical chemistry or haematology changes were observed in the test article-treated groups. Absolute and relative lung weights were increased in a dose-related manner in the 10 day exposure, 12-day rested/non challenged and 12 day rested/challenged groups. Group pathological findings revealed dose-related focal areas of haemorrhage on the lungs in the 10 day exposure and 12 day rested/challenged groups. The male rats were much more severely affected than the female rats. Focal haemorrhages were not present in the 5 day exposure, 12 day rested/non challenged or 12 week rested groups. Histologic examination of the affected lungs showed that the foci observed grossly consisted of alveolar haemorrhage and cellular infiltrates. Immunoperoxidase measurements of nonspecific IgG and complement (C3) revealed that IgG and C3 were present in large amounts in affected areas of the lungs and in respiratory lymph nodes, although a relation between trimellitic anhydride (TMA) exposure concentration and IgG or C3 was not generally seen. Total serum antibodies specific against TMA were measured and the results indicated striking dose-response relationship across all time points examined. Statistical correlations among salient findings were highly significant between the number of lung foci and the lung weights and macrophage accumulation and serum antibody levels in groups receiving recent TMA exposure. The observed effects strongly suggest that TMA acts on the immune system in the classic immune complex injury syndrome. This syndrome involves the formation of an immune complex resulting from a chemical, an associated protein and antibody previously raised against the chemical. The complex activates complement which recruits and activates neutrophils. The neutrophils phagocytose the complex and in doing so produce superoxide radicals. The radicals cause lung parenchymal and capillary endothelial damage which results in local haemorrhage. TMA produced lung haemorrhage only after prior sensitization had taken place. Rapid recovery as measured by the disappearance of lung haemorrhages was observed following cessation of exposure.


IgG Quantification in Lungs and Respiratory Lymph Nodes:

10 day exposure groups:

The IgG score in alveolar macrophages was significantly increased in a dose response manner with the 10, 30, 100 and 300 µg/m3 groups relative to the controls. The IgG score in the alveolar walls and large airways was increased in the 10, 30 and 100 µg/m3 groups, but not in the 300 µg/m3 group. There were no differences in IgG levels between male and female rats at any time point examined. Complement (C3) Quantification in Lungs and Respiratory Lymph Nodes 10 day exposure groups: The quantity of C3 in alveolar macrophages was increased in a dose response manner in the 30, 100 and 300 µg/m3 groups. The C3 score in alveolar walls was increased in the 10, 30 and 100 µg/m3 groups. The C3 score in the large airways was increased in the 10 and 30 µg/m3 groups. The intensity of C3 in the focal lesions was increased in the 10 and 100 µg/m3 groups. The C3 score in the respiratory lymph nodes was increased in the 10 and 100 µg/m3 groups. There were no differences in C3 levels between male and female rats at any time point examined.

Serum Antibody Levels

1) 10 day exposure group: The mount of serum antibody was increased in a dose-response manner, and the increase was statistically significant in the 10, 30, 100 and 300 µg/m3 groups at the 1:10 dilution and the 30, 100 and 300 µg/m3 groups at the 1:50 dilution.

2) 12 day rested/nonchallenged group: The amount of serum antibody was increased in a dose-response manner, and the increase was statistically significant in the 10, 30, 100 and 300 µg/m3 groups at the 1:10 dilution and the 30, 100 and 300 µg/m3 groups at the 1:50 dilution. The antibody levels were greater in the rested/nonchallenged groups than in the 10 day exposure groups.

3) 12 week recovery group: The amount of serum antibody was increased in a dose-response manner and the increase was statistically significant in the 10, 30, 100 and 300 µg/m3 groups at the 1:10 dilution and the 30, 100 and 300 µg/m3 groups at the 1:50 dilution. Generally the antibody levels were greater in the 12 day rested/challenged groups than in the 12 day rested/nonchallenged groups, the 10 day exposure groups or the 12 week rested groups. There was no statistically significant difference between male and female serum antibody levels at any time point examined although the serum antibody levels appeared to be slightly greater in the male rats.

Effect levels

1

Dose descriptor:

NOAEC

Sex:

male/female

Basis for effect level:

other: Immunological effects

Remarks on result:

not determinable

Remarks:

no NOAEC identified

2

Dose descriptor:

LOAEC

Effect level:

10 other: µg/m³

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Immunological effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Clear evidence of respiratory sensitisation was seen under the conditions of this study.

Applicant's summary and conclusion

Conclusions:

A series of adverse effects were seen during the duration of the study. The observed effects strongly suggest that trimellitic anhydride (TMA) acts on the immune system in the classic immune complex injury syndrome. TMA produced lung haemorrhage only after prior sensitization had taken place. Rapid recovery as measured by the disappearance of lung haemorrhages was observed following cessation of exposure.
It can therefore be surmised that TMA caused sensitisation due to prolonged exposure rather than an irritation. No NOAEC could be identified; the LOAEC was 10 µg/m³.

Executive summary:

Trimellitic anhydride (TMA) was administered as a particulate aerosol by inhalation at target concentrations of 10, 30, 100 and 300 µg/m³ to four groups of 40 male and 20 female Sprague-Dawley rats. A filtered air control group consisting of 40 male and 20 female rats was placed in an inhalation chamber and exposed to clean filtered air for the same duration as the test article-exposed rats. The rats were exposed 6 hours per day, 5 days per week for the following exposure regimens:

1) Randomly selected male rats from each group were exposed for two weeks and sacrificed;

2) A second group of male and female rats were exposed for two weeks and sacrificed;

3) A third group of male and female rats were exposed for two weeks and held for 12 days without exposure. Part of this group was challenged with an additional single 6-hour exposure of TMA and sacrificed, whereas the other was sacrificed without challenge;

4) A fourth group of male rats were exposed for two weeks, held 12 weeks without exposure and sacrificed.

The time weighted average concentrations for test article exposure were analytically determined to be 11.1, 37.4, 100.6 and 262.3 µg/m³ for the 10, 30, 100 and 300 µg/m³ exposure groups, respectively.

No deaths occurred during the study and no significant abnormal clinical chemistry or haematology changes were observed in the test article-treated groups. Absolute and relative lung weights were increased in a dose-related manner in the 10 day exposure, 12-day rested/non challenged and 12 day rested/challenged groups. Group pathological findings revealed dose-related focal areas of haemorrhage on the lungs in the 10 day exposure and 12 day rested/challenged groups. The male rats were much more severely affected than the female rats. Focal haemorrhages were not present in the 5 day exposure, 12 day rested/non challenged or 12 week rested groups. Histologic examination of the affected lungs showed that the foci observed grossly consisted of alveolar haemorrhage and cellular infiltrates. Immunoperoxidase measurements of nonspecific IgG and complement (C3) revealed that IgG and C3 were present in large amounts in affected areas of the lungs and in respiratory lymph nodes, although a relation between TMA exposure concentration and IgG or C3 was not generally seen. Total serum antibodies specific against TMA were measured and the results indicated striking dose-response relationship across all time points examined. Statistical correlations among salient findings were highly significant between the number of lung foci and the lung weights and macrophage accumulation and serum antibody levels in groups receiving recent TMA exposure.

The observed effects strongly suggest that TMA acts on the immune system in the classic immune complex injury syndrome. This syndrome involves the formation of an immune complex resulting from a chemical, an associated protein and antibody previously raised against the chemical. The complex activates complement which recruits and activates neutrophils. The neutrophils phagocytise the complex and in doing so produce superoxide radicals. The radicals cause lung parenchymal and capillary endothelial damage which results in local haemorrhage.

TMA produced lung haemorrhage only after prior sensitization had taken place. Rapid recovery as measured by the disappearance of lung haemorrhages was observed following cessation of exposure.

It can therefore be surmised that TMA caused sensitisation due to prolonged exposure rather than an irritation. No NOAEC could be identified in this study; the LOAEC was 10 µg/m³.