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

Endpoint summary

Description of key information

Trimellitic anhydride and trimellitic acid (rapidly generated by the hydrolysis of trimellitic anhydride) have been investigated in a number of oral studies in the rat and dog and are shown to be of low toxicity.  In contrast, trimellitic anhydride is shown to cause immunological reactions in rats following inhalation exposure to low concentrations.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study appears to be well conducted, however only the results and statistical analyses are presented in this report (no methodological information is available).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

13 week repeated dose toxicity study

GLP compliance:

not specified

Limit test:

no

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 is available.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test substance was mixed into the diet at three concentration levels and fed to the rats for 13 weeks.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The duration of the treatment was thirteen weeks.

Frequency of treatment:

Daily in diet

Remarks:

Doses / Concentrations:
1000, 5000 and 10000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

The study included data from rats in a control and three treatment groups consisting of 10 males and 10 females per group.

Control animals:

yes, concurrent no treatment

Details on study design:

No further information available.

Positive control:

A positive control was not included.

Observations and examinations performed and frequency:

The rats were observed for clinical signs. Raw data consisted of weekly body weights, weekly food consumption and haematology data (half the animals or 5 rats/sex/group) for three time points (i.e., initial or baseline, week 7 and week 13) consisting of haemoglobin, haematocrit, white blood cells and differential white cell counts consisting of EOS, BASO, MYE, JUV, BAND, SEG, LYMP and MONO. Cumulative body weight gains, daily food consumption and change in haematology parameters from baseline to study end were calculated from the data. Statistical analysis of the differential white cell count did not include BASO, MYE, JUV or BAND, as these were all zero in all groups.

Sacrifice and pathology:

Gross necropsy was performed followed by histopathological examination in the control and high dose groups.

Other examinations:

No other examinations reported.

Statistics:

Quantitative data were log-transformed (except body weight gains and changes in haematology from baseline to study termination) and analysed by univariate and multivariate two-factor, fixed-effects analysis of variance (ANOVA) across time points.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical observations noted included primarily wheezing and some slight emaciation. Incidences of wheezing were 6/20, 7/19, 7/19 and 4/19 in the control, 1000, 5000 and 10,000 ppm treated groups, respectively. Emaciation was not seen in the controls but was observed in one or two animals per sex in each of the groups treated with trimellitic anhydride (TMA).

Gross necropsy observations included lung lesions, consisting of congestion or caseous abscesses, and kidney lesions, usually dilated pelvis of one or both kidneys. Gross lung lesions were observed in 6/20, 2/19, 5/19 and 6/19 in the control, 1000, 5000 and 10,000 ppm treated groups, respectively. Kidney lesions were similarly evenly distributed among the groups at 5/20, 4/19, 3/19 and 2/19 respectively.

Microscopic examination of tissues was performed on 16 animals. Microscopic lung lesions consisted of bronchiectasis, bronchitis, peribronchitis and/or focal pneumonia. Incidences of microscopic lung lesions were 5/8 in the controls examined and 8/8 in the 10,000 ppm TMA-treated group.
Thus, there were no significant differences in the incidences of clinical observations, gross necropsy and histopathological findings between the treated and control groups.

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant differences in the incidences of clinical observations, gross necropsy and histpathological findings between the treated and control groups.

Critical effects observed:

not specified

Table 1: Thirteen-week oral (diet) toxicity study of trimellitic anhydride (TMA) in rats

Study group
Parameter	Control			1000			5000			10000
	M	F	T	M	F	T	M	F	T	M	F	T
Number of animals	10	10	20	9	10	19	10	9	19	9	10	19
Clinical Observations
Wheezing	4	2	6	4	3	7	5	2	7	3	2	5
Emaciation	0	0	0	1	2	3	1	1	2	1	1	2
Hypersensitivity	0	1	1	1	0	1	0	0	0	0	0	0
Miscellaneous Lesions	2	0	3	1	3	4	0	1	1	1	0	1
Gross Necropsy Findings Lungs
Caseous Abscess	2	1	3	0	2	2	3	1	4	3	2	5
Congestion	5	0	5	0	1	1	0	0	0	0	0	0
Free Blood	0	0	0	0	0	0	0	1	1	0	1	1
Kidneys
Dilated Pelvis	2	3	4	2	2	4	2	1	3	2	0	2
Nodule	1	0	1	0	0	0	0	0	0	0	0	0
Liver
Abscess	0	0	0	1	0	1	0	0	0	0	0	0
Thymus
Haemorrhage	0	0	0	0	0	0	1	0	1	0	0	0
Histopathology
Number Examined	4	4	8	..	..	..	..	..	..	4	4	8
Lungs
Bronchitis	0	2	2	..	..	..	..	..	..	4	4	8
Peribronchitis	1	2	3	..	..	..	..	..	..	0	0	0
Pneumonia	1	2	3	..	..	..	..	..	..	1	1	2
Bronchiectasis	1	0	1	..	..	..	..	..	..	1	2	3
Free Blood	1	01		..	..	..	..	..	..	0	1	1

Table 2: Mean male body weights (g)

Study Group
		TMA (ppm)
Week	Control	1000	5000	10000
0	66± 9.8	73± 6.3	70± 9.7	72± 9.2
1	109± 17.5	110± 12.3	102± 20.2	98± 14.5
2	168± 29.6	160± 21.9	145± 42.1	148± 20.7
3	226± 28.7	212± 39.9	199± 38.6	203± 25.2
4	279± 28.1	272± 33.2	257±37.8	254± 25.7
5	328± 28.8	325± 27.4	310±38.7	300± 21.5
6	366± 29.6	368± 25.9	352± 43.4	341± 26.8
7	388± 29.5	394± 26.3	384± 46.7	373± 29.3
8	417± 31.6	426± 30.4	419±52.9	403± 30.2
9	443± 30.7	452± 30.9	447± 55.8	426± 30.5
10	461± 32.2	471± 33.1	467± 55.1	442±31.3
11	476 ± 34.8	489± 34.9	491± 60.8	464± 36.5
12	476 ± 32.1	503± 34.2	504± 66.9	477± 37.5
13	487± 31.8	506± 40.8	507± 70.3	487± 46.1

Table 3: Mean female body weights (g)

	Study Group
		TMA (ppm)
Week	Control	1000	5000	10000
0	65± 9.2	66± 6.7	66± 11.7	68± 5.0
1	100± 9.5	93± 13.0	98± 16.2	94±16.6
2	144± 11.3	129± 20.9	138± 23.7	128± 19.4
3	179± 15.2	155± 30.2	168± 22.0	162±14.0
4	192± 17.8	178± 24.2	190± 20.9	185± 12.9
5	215± 17.6	199±23.9	211± 24.6	207± 12.5
6	233± 20.6	213± 24.3	229± 23.2	224± 14.3
7	243± 20.7	226± 21.4	242± 24.8	237± 15.3
8	255± 23.6	242±23.9	253± 27.7	246 ± 16.4
9	266± 28.0	249± 23.9	262± 29.7	255± 17.2
10	275± 25.0	260± 22.1	275± 34.2	261± 16.8
11	278±23.8	265± 19.8	279± 34.9	268± 16.3
12	281±26.7	271±22.7	280± 34.2	272±17.9
13	286±27.1	274± 25.6	281± 33.3	276± 19.8

Table 4: Mean male haematocrit and white blood cell differential values sampled at study initiation

	Study Group
		TMA (ppm)
Week	Control	1000	5000	10000
HGB	11.0± 1.1	11.9± 0.3	10.7± 0.6	11.3 ± 0.8
HCT	43± 2.4	48± 2.2	43 ± 1.1	43 ± 1.6
WBC	5.9± 0.7	6.3± 2.1	3.6 ± 1.6	6.6±2.0
EOS	0± 0.5	0± 0.4	0± 0.0	0± 0.0
BASO	0± 0.0	0± 0.0	0± 0.0	0± 0.0
MYE	0± 0.0	0± 0.0	0± 0.0	0± 0.0
JUV	0± 0.0	0± 0.0	0± 0.0	0± 0.0
BAND	0± 0.0	0± 0.0	0± 0.0	0± 0.0
SEGS	30± 7.8	19± 14.6	30 ± 12.7	18± 3.5
LYM	70± 8.3	81± 15.0	70 ± 12.7	82± 3.5
MONO	0± 0.0	0± 0.0	0 ± 0.0	0± 0.0

Table 5: Mean female haematocrit and white blood cell differential values sampled at study initiation

	Study Group
		TMA (ppm)
Week	Control	1000	5000	10000
HGB	11.6 ± 0.5	11.8± 1.0	11.2± 0.6	11.5± 1.7
HCT	44± 2.8	45± 3.5	43± 1.3	44±1.3
WBC	4.3± 1.3	4.7±1.0	2.3± 0.3	4.8± 1.1
EOS	0 ±0.5	0 ± 0.0	0 ± 0.0	0 ± 0.0
BASO	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0
MYE	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0
JUV	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0
BAND	0 ± 0.0	0 ± 0.0	0 ± 0.0	0 ± 0.0
SEGS	23± 11.4	24± 10.1	16±7.6	18±12.7
LYM	76± 11.1	76± 10.1	83± 7.4	82± 12.7
MONO	0 ± 0.4	0± 0.0	0± 0.4	0± 0.0

Conclusions:

There were no significant differences in the incidences of clinical observations, gross necropsy and histopathological findings between the treated and control groups, therefore the NOAEL was 10,000 ppm.

Executive summary:

Trimellitic anhydride (TMA) was administered in the diet to groups of 10 male and 10 female rats at dose levels of 0, 1000, 5000 and 10000 ppm. Investigations included weekly body weights, weekly food consumption and haematology data (5 rats/sex/group) for three time points (i.e., initial or baseline, week 7 and week 13) consisting of haemoglobin, haematocrit, white blood cells and differential white cell counts consisting of EOS, BASO, MYE, JUV, BAND, SEG, LYMP and MONO. Cumulative body weight gains, daily food consumption and change in haematology parameters from baseline to study end were calculated from the data. Statistical analysis of the differential white cell count did not include BASO, MYE, JUV or BAND, as these were all zero in all groups. The data sheet for each animal also had nominations of clinical observations and gross necropsy findings.

Clinical observations noted included primarily wheezing and some slight emaciation. Incidences of wheezing were 6/20, 7/19, 7/19 and 4/19 in the control, 1000, 5000 and 10000 ppm treated groups, respectively. Emaciation was not seen in the controls but was observed in one or two animals per sex in each of the treated groups. Gross necropsy observations included lung lesions, consisting of congestion or caseous abscesses, and kidney lesions, usually dilated pelvis of one or both kidneys. Gross lung lesions were observed in 6/20, 2/19, 5/19 and 6/19 in the control, 1000, 5000 and 10000 ppm treated groups, respectively. Kidney lesions were similarly evenly distributed among the groups at 5/20, 4/19, 3/19 and 2/19 respectively. Microscopic examination of tissues was performed on 16 animals. Microscopic lung lesions consisted of bronchiectasis, bronchitis, peribronchitis and/or focal pneumonia. Incidences of microscopic lung lesions were 5/8 in the controls examined and 8/8 in the 10000 ppm TMA-treated group. There were no significant differences in the incidences of clinical observations, gross necropsy and histopathological findings between the treated and control groups, therefore the NOAEL was 10000 ppm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

900 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A number of studies are available, all indicating similar outcome with regard to systemic toxicity

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

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: Proprietary study, methodology similar to current guidelines.

Reason / purpose for cross-reference:

reference to same study

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

Remarks:

no GLP certificate/statement included in report

Limit test:

no

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.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

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 anhydride, 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

Remarks:

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

Remarks:

Doses / Concentrations:
0, 2, 15, 54 µg/m³
Basis:
analytical conc.

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 into 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 occurrence 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 weeks 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 from earlier 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 haemorrhages 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

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 erythrocyte count, 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 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 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.

Clinical signs:

no effects observed

Description (incidence and severity):

in 13 week study

Mortality:

no mortality observed

Description (incidence):

in 13 week study

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:

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 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. 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. 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 increase 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.

Dose descriptor:

LOAEC

Effect level:

ca. 2 other: µg/m³

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: observation of adverse effects and sensitisation

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

See the attached file 'Result Tables.pdf'.

Conclusions:

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.

Executive summary:

Trimellitic anhydride (TMA) was administered as a particulate aerosol by inhalation at target concentrations of 0, 2, 15 and 50 µg/m³ to 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, antibody 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 transferred 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEC

50 µg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

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: Proprietary study, methodology similar to current guidelines.

Reason / purpose for cross-reference:

reference to same study

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

Remarks:

no GLP certificate/statement included in report

Limit test:

no

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.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

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 anhydride, 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

Remarks:

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

Remarks:

Doses / Concentrations:
0, 2, 15, 54 µg/m³
Basis:
analytical conc.

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 into 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 occurrence 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 weeks 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 from earlier 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 haemorrhages 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

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 erythrocyte count, 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 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 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.

Clinical signs:

no effects observed

Description (incidence and severity):

in 13 week study

Mortality:

no mortality observed

Description (incidence):

in 13 week study

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:

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 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. 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. 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 increase 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.

Dose descriptor:

LOAEC

Effect level:

ca. 2 other: µg/m³

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: observation of adverse effects and sensitisation

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

See the attached file 'Result Tables.pdf'.

Conclusions:

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.

Executive summary:

Trimellitic anhydride (TMA) was administered as a particulate aerosol by inhalation at target concentrations of 0, 2, 15 and 50 µg/m³ to 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, antibody 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 transferred 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

2 µg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated Dose Toxicity: Oral

Studies with trimellitic anhydride (TMA)

Estep & Teske (1969a) report a 13-week dietary study in rats exposed to concentrations of 1000, 5000 and 10000 ppm. No effects of treatment were seen in this study. Plank (1970) also reports no effects of treatment in rats exposed to 10000 ppm in the diet for 90 days. Hatoum (1991) also reports a NOAEL of 10000 ppm in a 90 -day dietary study in rats using dose levels of 0, 1000, 5000 and 10000 ppm. In a thirteen week oral toxicity study, Estep et al. (1969) exposed 16 Beagle dogs (2/sex/group) to trimellitic anhydride at concentrations of 1000, 10000 and 20000 ppm and a control group. The results of this study indicate that there were no significant differences between the control and the treatment groups.

Read-across studies with trimellitic acid (TMLA)

In a 28 -day study, Hankinson & Sakal (1991) gavaged groups of five rats/sex with trimellitic acid at dose levels of 0, 100, 300 or 1000 mg/kg bw/d on 5 days/week for 28 days. Treatment-related effects in this study were limited to diarrhoea and fluid caecal contents at the highest dose level; there was no systemic toxicity and findings are not of relevance to the risk assessment. A NOAEL of 1000 mg/kg bw/d was determined for this study.

McRae et al (2003) exposed three groups of 20 Sprague Dawley rats (10 per sex) to dietary concentrations of 1000, 5000 and 20000 ppm trimellitic acid for 90 days. Treatment-related effects in this study were limited to diarrhoea, reduced food consumption and bodyweight secondary to effects on dietary palatability and local gross and macroscopic effects on the caecum. A NOAEL of 1000 ppm (equivalent to 82 and 86 mg/kg bw/d in males and females respectively), was derived for this study based on the effects at 5000 ppm. However the findings at the highest dose level in this study of 20000 ppm (equivalent to 1594 and 1716 mg/kg bw/d) are of questionable relevance to the occupational risk assessment.

The results of these studies clearly indicate that trimellitic anhydride and its hydrolysis product, trimellitic acid, are of very low toxicity following oral (dietary or gavage) administration. No evidence of systemic toxicity was seen in any study with trimellitic anhydride; findings related to treatment with trimellitic acid were either secondary to poor dietary palatability or were a local effect on the caecum and are therefore not considered to be of relevance to the risk assessment.

A NOAEL of 900 mg/kg bw/day is carried forward to the risk assessment.

Repeated Dose Toxicity: Dermal

No data are available; based on the results of the oral toxicity studies, very low toxicity is also predicted by this route of exposure. A waiver is proposed on scientific grounds and no additional studies are proposed using this route of exposure.

Repeated Dose Toxicity: Inhalation

Studies with trimellitic anhydride (TMA)

Leach et al (1985) administered trimellitic anhydride to 4 groups of 40 male and 20 female Sprague Dawley rats at concentrations of 10, 30, 100 and 300 µg/m3 for 14 days. The results of the study indicated respiratory sensitisation at all exposure concentrations with males more severely affected; the LOAEC for this study is therefore 10 µg/m3.

Bernard (1981) prepared a review of selected histopathology slides from a two-week inhalation toxicity study in rats. No definitive conclusion could be determined on completion of this review due to the presence of lesions in the lungs of the control rats.

Bishop et al (1976) exposed 30 rats to an average concentration of 0.20mg/m3 for 14 days. At the time of sacrifice, there were no adverse effects observed for body weight in the test article treated group compared to the control. Lung weight data from the final sacrifice revealed significantly elevated lung weights for male and female test animals when compared to the controls. In addition to this, examination of the lungs of treated and control rats revealed small red foci in the lungs of the treated rats, which were approximately three times as numerous as foci in the lungs of control rats. Histopathologic examination of the respiratory tract of treated and control animals did not disclose any tissue changes directly attributable to the effects of the test material. A LOAEC of 0.2 mg/m3 can therefore be determined for this study.

In an additional 14-day study (Benson, 1981), six groups of albino rats were exposed at concentrations ranging from 0.01 to 0.30 µg/L. A further group served as the control. The results of the study indicated that the lung was the primary organ affected with a statistically significant increase in the lung and trachea weight of the treatment groups. In addition, small foci were evident at necropsy and an increase in the number of macrophages in the alveolar spaces and septa was observed in the treated animals.

The NOAEC determined for this study was less than 0.005 µg/L.

Horath et al (1975a) exposed 2 groups of albino rats to either trimellitic anhydride or trimellitic anhydride and synthetic silica for two weeks at concentrations of 3.82 and 12.49 mg/m3 respectively. Compared to the untreated control group, there were no significant effects of the treated rats compared to the controls when body weights, lung weights and lung histopathology were examined, however increased lung weight was apparent in males (a statistical analysis of the data was not carried out). The same authors (Horath et al, 1975b) conducted another study evaluating the effects of trimellitic anhydride on 30 albino rats exposed to the test substance for 6 hours per day over the course of a 2 week exposure period, at a concentration of 0.054 mg/m3. Lung weights and ratios for the test animals were greater than untreated control animals. The number of grossly visible red foci present in the lungs of the test animals was greater than that of the controls. Histologic examination revealed no significant differences between test and control rats. The NOAEC for this study is therefore <0.054 mg/m³.

Grapenthien et al (1971) conducted a pilot inhalation toxicity study on 30 albino rats (15/sex), exposed to the dust of trimellitic anhydride at a concentration of 0.12 mg/m3. No abnormal or untoward reaction were observed in any test animal. Lung weights were comparable with controls. On gross pathological examination, focal hyperaemia was observed on the surface of the lungs of the treated animals. The NOAEC for this study is therefore <0.12 mg/m3.

Hathaway et al (1970) evaluated the effects of ground trimellitic anhydride on 30 albino rats (15/sex) exposed to the test substance at an average concentration of 17mg/m3. Gross pathologic examination of the lungs of both test article treated and control rats revealed focal hyperaemia on the lung surface of the test article treated rats. In addition to this, histopathologic findings showed significant changes in the lungs of treated rats, specifically, hyperaemia with extravasation of erythrocytes into the alveoli, acute, organising and chronic inflammatory changes and vacuolated macrophages in the alveoli. The NOAEC for this study is therefore <17 mg/m3.

In a thirteen week inhalation toxicity study, Leach et al (1988) evaluated the effects of trimellitic anhydride on 4 groups of rats exposed at concentrations of 0, 2, 15 and 50 µg/m3. The scheduling for sacrifice was after 6.5 weeks exposure, 13 weeks exposure, 13 weeks exposure and 3 weeks recovery and 13 weeks exposure and 38 weeks recovery. The results of this study indicate that trimellitic anhydride caused lung lesions during the first few weeks of exposure which were consistent with known-aspects of the immune complex injury syndrome. However, as exposures continued, some type of immunologic tolerance developed, meaning long-term exposure of rats to trimellitic anhydride caused only minimal increases in lung weights and volumes. Findings were not seen in immunosuppressed rats. No additional effects of treatment were seen in this study.

Read-across studies with trimellitic acid (TMLA)

Leach et al (1985) administered trimellitic acid to 2 groups of 30 male Sprague-Dawley rats at concentrations of 30 and 300 µg/m3 for 14 days. In addition to these 2 groups, a control group was used and additional rats were exposed for 2 weeks, held for 12 days without exposure and either challenged with an additional 6 hour exposure and sacrificed or sacrificed with no challenge. On completion of the study, there were no significant differences observed between the test article treated rats and the control group. In addition to this, there were no effects seen on repeat (challenge) exposure to the test article.

In a thirteen week inhalation toxicity study of the read-across substance trimellitic acid in rats (Hatoum et al, 1989), the test substance was administered to 4 groups of Sprague-Dawley rats (20 rats/sex) at concentrations of 0, 50, 100 and 300 µg/m3. There were no statistically significant differences observed between the test article treated rats and the control group of rats.

In a 14-day inhalation toxicity study with trimellitic acid (Horath et al, 1975c), increased relative lung weight was seen at the single exposure concentration of 9 mg/m3.

The results of the studies indicate that the only effect of inhalation exposure to trimellitic anhydride relates to respiratory sensitisation; no additional effects were seen in any study, findings were not apparent in immunocompromised animals and were not seen following exposure to the hydrolysis product trimellitic acid.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Most recent and best documented of the available studies.
NOAEL of 900 mg/kg bw/day calculated from reported dietary concentration of 10000 ppm and using a default conversion factor of 0.09 as described by "Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data (EFSA Journal 10(3): 2579, 2012).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Single study investigating markers of sensitisation

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: lung

Justification for classification or non-classification

Oral studies demonstrate that trimellitic anhydride is of very low toxicity; similar low toxicity is predicted for dermal exposure. Following inhalation exposure, the only observed effect related to treatment is respiratory sensitisation. Additional classification is not required.