Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane- l,2-diol

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

1985-08-07 to 1985-08-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Valid scientific experiment done by a reputable laboratory.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Principles of method if other than guideline:

The methods were comparable to OECD 413

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trimellitic Anhydride

IUPAC Name:

Trimellitic Anhydride

Constituent 2

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

Test material form:

solid: flakes

Details on test material:

- Name of test material (as cited in study report): trimellitic anhydride (TMA)
- Physical state: white solid flakes
- Identification number: 5507-182
- Storage: in a tightly sealed glass jar in a ventilated cabinet at room temperature (approximately 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, obtained from Charles River Breeding Laboratories (MI). The rats were approximately 7 weeks old and weighed approximately 135 g on arrival. The rats underwent a 3 week acclimatisation period. The rats were housed in stainless steel suspended cages during inhalation exposures and the acclimatisation period. Following the exposure period the rats were housed in plastic shoe-box cages. 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 exposures.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: MMAD:
- 2 µg/m³: 1.74 ± 1.42 microns
- 15 µg/m³: 2.21 ± 1.36 microns
- 50 µg/m³: 2.18 ± 1.36 microns

Details on inhalation exposure:

Rats were exposed to a particulate aerosol of trimellitic acid, various exposure schedules were employed in the study.

Flakes of trimellitic anhydride (TMA) were ground to micron-sized particles in an air jet mill. Ground TMA was stored in a light-tight container. Solid particles of TMA were aerosolised in a modified TSI Fluidised Bed Aerosol Generator, blown by means of a Transvector Jet into a 500 litre mixing chamber, and drawn by Transvector Jets operated at various pressures into respective inhalation exposure chambers. Exposures were conducted in 2 m³ stainless steel and glass chambers, or in 1 m³ chambers in the case of some of the ancillary studies. Chamber airflow in the 2 m³ chambers varied between 325 and 850 l/min, depending on the designated exposure concentration.

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. Particles from flowmeter-measured volumes of test atmospheres were trapped onto glass fibre filter pads, extracted and injected into the HPLC column. The number of samples taken for HPLC ranged from 2 to 6 per exposure period for the test article chambers and up to 3 times per day for the control chamber.

Duration of treatment / exposure:

A-1: 13 weeks of exposure and 38 weeks of recovery
A-2: 1 - 10 weeks
A-3: 6.5 weeks
A-4: 13 weeks
A-5: 13 weeks of exposure and then held for 3 weeks without exposure
A-6: 13 weeks and then held without exposure until TMA Serum antibody levels returned to approximately background levels
B-1: 13 weeks
C-1: 2 weeks
D-1: 18 hours
E-1: 2 weeks
F-1: 2 weeks

Frequency of treatment:

A-1: 6 hours/day, five days/week
A-2: from start of test until their sacrifice
A-3: 6 hours/day, five days/week
A-4: from start of test until their sacrifice
A-5: continuous
A-6: continuous
B-1: continuous
C-1: 5 days per week
D-1: 1 treatment
E-1: 5 days/week
F-1: 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 2, 15, 50 µg/m³
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 3, 17, 55 µg/m³
Basis:
other: analytical, 6.5 wk exposure

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 2, 15, 54 µg/m³
Basis:
other: analytical, 13 wk exposure

No. of animals per sex per dose:

A-1: 6 male (50 µg/m³, 13 weeks of exposure and 38 weeks of recovery)
A-2: 14 male (50 µg/m³, 1 - 10 weeks exposure)
A-3: 40 male (10/group) (0, 2, 15 and 50 µg/m³, 6.5 weeks exposure)
A-4: 40 male and 40 female (10/sex/group) (0, 2, 15 and 50 µg/m³, 13 weeks exposure)
A-5: 48 male (12/group) (0, 2, 15 and 50 µg/m³, 13 weeks of exposure and then held for 3 weeks without exposure)
A-6: 12 male from the 50 µg/m³ group and 6 males from the 0, 2 and 15 µg/m³ group (13 weeks and then held without exposure until TMA Serum antibody levels returned to approximately background levels)
B-1: groups of 8 rats (0, 2, 15, and 50 µg/m³, 13 weeks)
C-1: 12 male rats given cyclophosphamide prior to exposure, and 12 male rats exposed (control) (100 µg/m³, 2 weeks)
D-1: male (100 µg/m³, 18 hours)
E-1: 10 male rat/group (0, 2, 15 and 50 µg/m³, 2 weeks exposure)
F-1: Forty male rats divided int 5 groups of 8 rats (100 µg/m³, 2 weeks exposure, held for 2 weeks, single challenge)

Control animals:

yes, sham-exposed

other: A-3, A-4, A-5, A-6, B-1, E-1: control run concurrently... (see attached file)

Details on study design:

The study groups were as follows:
A-1: Carried out to determine TMA-specific antibody appearance/disappearance time.
A-2: The purpose of this study was to follow the time-course of TMA toxicity development along with the possible occurence of tolerance to TMA-induced effects.
A-3: the purpose of this experiment was to evaluate more completely the effects of TMA after 6.5 weks of exposure.
A-4: The purpose of this part was to evaluate more completely the toxic effects of TMA in male and female rats after 13 weeks of exposure.
A-5: This part was carried out to determine the effects of re-exposure to TMA after a brief rest period.
A-6: This part was carried out to determine the recovery potential fro mearlier adverse effects and to determine the effects of re-exposure after serum antibody levels returned to near background levels.
B-1: The purpose of this study was to assess the effects of inhaled TMA on pulmonary function after 13 weeks of exposure.
C-1: The purpose of this experiment was to determine if TMA lung toxicity was immunologically-mediated. T and B-cell proliferation were blocked by cyclophosphamide in an attempt to eliminate or diminish focal hemorrhagis caused by TMA.
D-1: the purpose of this study was to determine if naive rats could be passively sensitised to TMA by the transfer of serum, cells, or a combination of both from TMA-sensitised rats. The naive recipient rats were then challenged by a single TMA inhalation exposure to determine if TMA lung lesions were elicited.
E-1: the purpose of this experiment was to determine the role of alveolar macrophages and other cells, as well as complement, in TMA-induced lung injury.
F-1: the purpose of this study was to determine if TMA conjugated to polyvinyl alcohol (TM-PVA) could prevent TMA-induced toxicity.

Positive control:

Not relevant

Examinations

Observations and examinations performed and frequency:

The rats were observed at least once daily for mortality and morbidity. Physical examinations were performed once prior to study initiation, rats were also observed daily during the exposure phase of the study for adverse clinical symptoms. Bodyweights were measured at study initiation, weekly thereafter, and at study termination. After the exposure period the rats were weighed at least monthly. Bodyweights were also measured after the 18 hour fasting period, immediately prior to necropsy.

Designated rats were fasted for approximately 18 hours prior to necropsy. Blood samples were obtained under anaesthesia at the time of sacrifice 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. The following haematology parameters were determined: total erythrocytecount, haemoglobin, haematocrit, total and differential leukocyte count, globulin, albumin/globulin ratio, MCV, MCH and MCHC.

Sacrifice and pathology:

Designated rats were fasted for approximately 18 hours prior to necropsy. Necropsies were performed on all designated rats and the following tisses 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 and seminal vesicles, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord, spleen, stomach, thymus, thyroids, tongue, trachea, urinary bladder, uterus, and any gross lesions. The adrenals, brain, gonads, heart, kidneys, liver, lungs and spleen were weighed at necropsy. Also, each lung lobe was counted for the number of haemorrhagic surface foci.

Other examinations:

Serum and lung lavage antibody levels.

Statistics:

ANOVA was performed on log transformed data followed by Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Details on results:

Experiment A Part 1: Male rats were exposed to 50 µg/m³ for 6 hours/day, 5 days/week for 13 weeks followed by 38 weeks of recovery. No deaths occurred during this study. There was no direct control group for these rats, however all study rats gained weight and the gains were within normal limits. TMA-specific serum antibody levels rose sharply during the first 6 weeks of exposure to a high level, declined gradually to a moderate level during the rest of the exposure, increased after the exposures ended and further declined to a low, but significant level during recovery.

Experiment A Part 2: Male rats were exposed to 50 µg/m³, two rats per time point were sacrificed at the end of weeks 1, 2, 3, 4, 6, 8 and 10. No deaths occurred during this study. There was no direct control group for these rats, however all study rats gained weight and the gains were within normal limits. TMA-specific serum antibody levels generally increased throughout the 10 weeks of the study, and bronchoaleveolar lavage antibodies increased similarly. The number of external lung haemorrhagic foci increased sharply during the first two weeks of exposure, but declined sharply thereafter.

Experiment A Part 3: male rats were exposed to 0, 2, 15 and 30 µg/m³ for 6.5 weeks (32 exposures) and sacrificed. No deaths occurred during the study. The rats from all groups exhibited the usual low incidence of adverse clinical signs without relationship to treatment. There was no effect of treatment on final absolute or cumulative bodyweights. There was a transient decrease in cumulative bodyweight gain in the 15 µg/m³ group at weeks 2 and 3, but these differences were not present at study termination. Absolute and relative lung weights and volumes were statistically significantly increased in a dose-dependent manner. Absolute kidney weight was decreased in the 15 µg/m³ but this was not considered to be biologically relevant. There were no treatment-related effects on haematology or clinical chemistry. Serum antibody levels exhibited a sharp and significant dose-response increase, reaching a maximum mean of 402 ng of TM-RSA bound/ml of serum. There were no abnormalities detected at necropsy, except for external haemorrhagic lung foci where there was a sharp and significant dose-response increase, reaching a maximum of 156 foci/lung. Histopathological examination revealed a marked dose-dependent increase in pulmonary haemorrhage among treated rats. The degree of severity of bronchopneumonia was more informative: 1.0, 1.6, 2.3 and 2.9, respectively. The incidence of multifocal, lobular bronchopneumonia was 90, 100 and 100% for the 2, 15 and 30 µg/m³ groups respectively, compared to 0% for controls. The degree of severity of bronchopneumonia was informative: 1.0, 1.6, 2.3 and 2.9, respectively. No other lesions were found that were considered to be treatment related.

Experiment A Part 4: Male and female rats were exposed to 0, 2, 15 and 50 µg/m³ for 13 weeks, then sacrificed. No deaths occurred during the study. Rats from all groups exhibited the usual low incidence of adverse clinical signs without relationship to treatment. There was no effect of treatment on bodyweight. Absolute lung weights and volumes, as well as relative lung weights, were increased in a dose-response relationship. Relative weights were also increased with dose but not significantly so. There was no effect on clinical chemistry or haematology parameters. TMA-specific serum antibody levels exhibited a significant dose-related increase in concentration to a maximum combined mean of 79 ng TM-RSA bound/ml of serum, however male rats exhibited approximately twice as much antibody as females. There was a significant dose-related increase in lung foci among males, females exhibited very few foci. No other abnormalities were detected at necropsy. Histopathological examination revealed a 30% incidence of pulmonary haemorrhage in the 50 µg/m³ male rats, compared to 0% in controls (note: no data was provided for the 2 and 15 µg/m³ doses). Females at 50 µg/m³ only had a 10% incidence. The incidence of multifocal, lobular bronchopneumonia was 100% in the 50 µg/m³ male and female rats compared to 0% in the controls.

Experiment A Part 5: Male rats were exposed to 0, 2, 15 and 50 µg/m³ for 13 weeks then held without exposure for 3 weeks. Half of each dose group then underwent a single 6 hour challenge exposure to 50 µg/m³. All rats were then sacrificed. No treatment-related deaths occurred (one rat died as a result of injury not related to exposure). Rats from all groups exhibited the usual low incidence of adverse clinical signs without relationship to treatment. There was no effect of treatment or challenge on absolute bodyweights or cumulative bodyweight gains, however there was a statistically significant decrease in absolute and cumulative body weights in the low dose challenge group, this was not thought to be biologically significant. Absolute and relative lung weights and lung volumes were generally increased in a dose related manner in both the challenge and non-challenge group. Serum antibody levels were very high and increased in a dose-related manner in both challenge and non-challenge groups. No abnormalities were detected at necropsy. Histopathological examination did not reveal an effect of treatment on pulmonary haemorrhage in either the challenge or non-challenge groups. There was a 100% incidence of multifocal lobular bronchopneumonia in the 50 µg/m³ challenge and non-challenge groups, but very little if any in the remaining groups.

Experiment A Part 6: Male rats were exposed to 0, 2, 15 and 50 µg/m³ were exposed to 13 weeks then allowed 38 weeks recovery. Half of the 50 µg/m³ group were challenged with 50 µg/m³ for 6 hours at the end of the recovery period. All rats were then sacrificed. No deaths occurred during the study. The usual incidence of clinical signs was observed. There were no effects on bodyweight. There was a statistically significant increase in relative lung weight in the challenged group. There was no effect of treatment of the number of lung foci. No treatment related effects were seen at histopathological examination.

Effect levels

1

Dose descriptor:

LOAEC

Effect level:

3 other: µg/m³

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: observation of bronchopneumonia and sensitisation. Actual doses: 3, 17 and 55 µg/m³

2

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table No. 15 - Rats sacrificed after 13 weeks of exposure and 38 weeks of recovery - summary of gross necropsy observations

	Study Group
	Filtered Air Control	Target trimellitic anhydride (TMA) exposure (µg/m^3)
		2	15	50
Tissue and Observation	N	N	N	N	C
No Gross Lesions	1	3	2	3	1
Lungs: Foci Mottled Black spots Nodule	- 1 -	1 1 -	- 2 -	2 3 -	- 4 -
Kidneys: rough surface	-	-	-	1	1
Liver: discolored	1	-	-	-	1
Stomach: dark contents	-	1	-	-	-
Small Intestine: red	-	-	-	-	1
Mandibular Salivary Gland: red	-	-	1	1	1
Urinary Bladder: Distended Contains white matter	- -	- -	- -	- 1	1 -
Respiratory Lymph Nodes: red	-	-	-	1	1
Mandibular Lymph Nodes: red enlarged	1 -	1 -	1 1	- -	1 1
Mandibular Lymph Nodes: red	-	-	1	1	1
Preputial Glands: Contain greenish material	1	-	-	-	-
Teeth Lesions	-	-	-	-	1

N- Non-challenged

C- Challenged

Table No. 16 - Rats sacrificed after 13 weeks of exposure and 38 weeks of recovery - summary of histopathological diagnoses

Tissue and Lesion	Filtered Air Control	Trimellitic anhydride (TMA) exposure levels (µg/m^3)
		2	15	50	50-C
Lungs: Perivascular cuffing Alveolar macrophages, focal Adenomatoscs Inflammation, chronic, focal	4 2 1 -	6 1 - -	5 2 - 2	4 2 - -	6 3 - 3
Calcification, pulmonary blood vessel	3	4	3	1	3
Alveolar/bronchiolar adenoma	-	-	-	-	1

Table No. 17 - D-1: Passive transfer of various agents from trimellitic anhydride (TMA)-sensitized rats into naive recipient rats subsequently given a single TMA challenge (6 rats/group)

	Donor Rats		Recipient Rats
	No. of Foci/lung	Serum Antibody	No. of Foci/lung	Serum Antibody
Sensitized Serum	228±99	65± 29	21± 41	17± 31
Spleen cells	228± 99	65± 29	4± 3	0
Sensitized Serum + 10^8Spleen cells	191± 60	111± 82	4± 4	7± 9
10^8Liver Cells	235± 93	73± 41	3± 2	0
Serum from Cyclophosphamide treated rats	1± 2	1± 2	7± 10	1± 3

Table No. 18 - Passive transfer of serum from trimellitic anhydride (TMA)-sensitized rats into naive recipient rats subsequently given a single challenge

Treatment Group	Donor Rats		Recipient Rats
	Foci/ lung	Serum Antibody	Body weight (g)	Lung volume (mL)	Lung weight (g)	Foci/ lung	Serum Antibody
Control Group (7 rats)	5± 5	5± 5	217± 2	1.36± 0.13	1.00± 0.09	4± 2	1± 1
Sensitized Group (16 rats)	112± 90	375± 129	221± 4	1.53± 0.09	1.12± 0.06	52± 89	122± 57

Table No. 19 - Effect of trimellitic anhydride (TMA) exposure on pulmonary lavage parameters of rats

							Lavage fluid
Group	Total Cells (x 10^6)	Total viable cells (x 10^6)	% viability	% macrophages	% lymphocytes	& neutrophils	Protein (mg/ml)	Lysozyme (µg/mg protein)
Control	8.29± 0.85	7.56± 0.80	94± 1	99± 1	1± 1	0± 0	0.51± 0.08	312± 57
2µg/m^3	9.16± 0.61	8.64± 0.61	94± 1	94± 3	5± 3	1± 1	0.64± 0.08	248±28
15µg/m^3	10.89± 1.52	10.36± 1.45	95± 1	89± 3	9± 2	2± 1	2.36± 0.32	81± 18
50 µg/m^3	32.36± 4.59	30.77± 4.43	95± 1	88± 2	7± 1	5± 1	3.79± 0.31	40± 4

Table No. 20 -Effect of trimellitic anhydride (TMA) exposure on protein content and functional and enzymatic parameters of lavaged alveolar macrophage

		Ectoenzymes (m.mole substrate/min/mg protein)			In vitro Phagocytosis of ingested 51Cr-CRBC (cpm)
Group	Macrophage Protein (ng/5 x 10^6cells)	5’ND	APDI	Lysozyme (µg/mg protein)
Control	0.62± 0.05	7.3± 1.5	4.6± 0.8	62.2± 6.3	16185± 1331
2µg/m^3	0.61± 0.02	8.7± 1.5	3.9± 0.5	57.3± 4.3	17288± 1494
15µg/m^3	0.61± 0.02	9.8± 1.9	3.8± 0.4	54.8±2.2	15596± 958
50µg/m^3	0.51± 0.07	9.7± 1.0	9.2± 2.0	88.8± 13.6	14591± 1037

Table No. 21 - Serum competent (CH50) levels in rats exposed to trimellitic anhydride (TMA) for two weeks

Group	Complement (CH 50/ml)
Control	463±226
2µg/m^3	668± 283
15µg/m^3	528± 115
50µg/m^3	703± 113

Table No. 22 - Serum and Lung lavage antibody levels in rats exposed to trimellitic anhydride (TMA) for two weeks

Group	Serum Antibody (ng TM-RSA Bound/ml)	Lung Lavage Antibody (ng TM-RSA Bound/ml)
Control	3.2± 3.9	0.9± 1.0
2µg/m^3	3.4± 3.7	0.3± 0.9
15µg/m^3	11.3± 9.4	1.5± 1.7
50µg/m^3	25.8± 18.7	8.5± 4.6

Applicant's summary and conclusion

Conclusions:

Trimellitic anhydride (TMA) was administered to CD rats by aerosol inhalation for various durations and with variable recovery periods after exposure. Toxicity and serum anti-TMA antibodies were assessed. TMA caused lung lesions during the first few weeks of exposure, which are consistent with aspects of the "immune complex injury" syndrome and representative of the pulmonary disease-anaemia (PD-A) syndrome seen in humans. As the exposures continued, some type of immunological tolerance developed such that long-term exposure of rats to TMA caused only minimal increases in lung weights and volumes, and bronchoalveolar pneumonia, but no indication of pulmonary fibrosis or haemorrhagic lesions. This study is informative for evaluation of the toxicity of members of the cyclic acid anhydride category, and is adequate for classification and risk assessment.

Executive summary:

Trimellitic anhydride (TMA) was administered as a particulate aerosol by inhalation at target concentrations of 0, 2, 15 and 50µg/m³to Sprague-Dawley rats for up to 13 weeks. Groups of rats representing each exposure level were sacrificed after 6.5 weeks of exposure, 13 weeks of exposure, 13 weeks of exposure plus 3 weeks of recovery and 13 weeks of exposure plus 38 weeks of recovery. Representative rats from the two recovery groups received a final TMA challenge, whereas other rats were sacrificed without challenge. Two groups of sentinel rats were included, one group was bled regularly for serum antibody determination throughout 13 weeks of exposure and 38 weeks of recovery. Representative rats from the second group of sentinels were sacrificed at regular intervals during the 13 -week exposure in order to monitor the appearance/disappearance of lung lesions.

The following additional experiments were conducted to explore the mechanism of action of TMA toxicity:

- suppressing the immune response in order to suppress or eliminate lung lesions;

- passively transferring serum from TMA-sensitised rats into naive recipients to see if such serum could sensitise the recipients

- lavaging lungs from TMA-exposed rats to examine possible cellular, enzymatic, antibdy and complement alterations;

- injecting TMA-exposed rats with various regimens of TMA-polyvinylalcohol conjugate in an attempt to inhibit the formation of lung lesions.

There were no treatment related deaths or adverse treatment-related clinical signs during the study. The lungs appeared to be the only tissue affected, treatment-related lung lesions consisted of dose-related increases in lung weights and volumes, external haemorrhagic lung foci, inflammatory cell infiltration into the lungs, and bronchoalveolar pneumonia. These lesions were pronounced after 6.5 weeks of exposure, but diminished greatly as the exposures continued. There were minimal treatment-related effects in the 13 -week groups and in the 3 week and 38 week recovery groups. TMA specific serum antibody levels increased through the first 6.5 weeks of exposure, decreased through the remainder of the 13-week exposure, increased again after the exposure was discontinued and eventually tapered off to a moderately low level through the 38 week recovery period. The presence of an antibody and TMA exposure were not sufficient to produce lesions as the exposure continued.

The results of the additional experiments confirmed that:

- if rats were immunosuppressed, then TMA did not cause lesions;

- serum from nontolerant, TMA-sensitized rats contained antibody which when passively tansferred into naive recipient rats resulted in TMA-induced lung lesions following a single TMA challenge;

- enzyme, protein and cellular analyses of lung lavage fluid from nontolerant, TMA-sensitised rats showed that TMA caused pulmonary inflammation and resultant haemorrhage into the lungs, but TMA had no effect on macrophage fraction.