Terephthalic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 October 1979 to May 17 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Older proprietary study, scientifically acceptable methods, specifically investigation urolithiasis

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was a combined repeated dose toxicity / reproduction study. Rats were exposed to the test substance in the diet for 90 days. A satellite group of rats were selected for mating at the end of the 90 days (test substance administration continued during the reproductive study). Non-reproductive study rats were sacrificed and necropsied at 30, 60 or 90 days. Pathology was limited to the urinary tract, as the study was specifically investigating TPA-induced urolithiasis.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Terephthalic acid

EC Number:

202-830-0

EC Name:

Terephthalic acid

Cas Number:

100-21-0

Molecular formula:

C8H6O4

IUPAC Name:

benzene-1,4-dicarboxylic acid

Test material form:

not specified

Details on test material:

No further details

Test animals

Species:

rat

Strain:

other: Wistar and CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Wistar and CD rats were obtained from Charles River Breeding Laboratories, aged 10-12 weeks. Fischer 344 rats were obtained from Microbiological Associates, however this strain showed serious health problems (abnormal clinical signs, weight loss) during the quarantine period thought to be indicative of Mycoplasma pulmonis infection, and were therefore not included in the study.The rats underwent a 35 day quarantine period, and were housed in separate rooms according to strain. Rats were housed in same sex groups of 3 in suspended stainless steel cages equipped with automatic waterers. Individuals were identified by metal ear tags. The rats were fed non-autoclaved NIH-31 diet (Ziegler Brothers) ad libitum, new feed was supplied weekly. Temperature was maintained at 70±2°F and relative humidity was 20-80%. Light was provided on a 12 hour light:dark cycle. Polycarbonate cages were supplied during the mating period (see section 7.8.1 for more detail).Rats were 15-17 weeks of age at the start of the study.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

NNIH-31 diet containing 0, 0.03, 0.125, 0.5, 2.0 and 5.0% TPA was prepared by Zeigler Brothers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each week 100 g feed samples were collected in glass bottles for each dietary level of TPA, purged with nitrogen for 1 minute then stored at -20°C. 2-10 g sized samples were placed in 6-ounce narrow mouth bottles and a measured amount of the internal standard (phthalic acid PA) in 2% NH4OH in methanol was added to each sample. The samples were extracted using 2% NH4OH in methanol (200 ml) by shaking vigorously in a mechanical for 1 hr at room temperature. After most of the feed particles had settled, 2 ml aliquots of the extracts were filtered through 4 µm Nucelopore membrane filters.. Aliquots of these filtered solutions were analysed using HPLC.Weekly feed samples were analysed for the first 12 weeks of the study. The 0.125% diet was found to contain consistently higher values than the nominal, and this was thought to be an error during diet formulation. Overall, the analyses indicated reasonable agreement between nominal and measured values.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuous - ad libitum in feed

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:0, 0.03, 0.125, 0.5, 2.0 and 5.0% Basis:nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:0, 30, 125, 500, 2000 and 5000 mg/kg bw/dBasis:other: approximate mean achieved intake

No. of animals per sex per dose:

30 rats/sex of each strain per dietary level

Control animals:

yes, plain diet

Details on study design:

The study was designed as a randomised complete block design. Interim necropsies were conducted on 5 rats/sex/strain/dose at 30 days and 60 days on study. At 90 days 10 rats/sex/strain/dose were necropsied. Rats were not fasted prior to necropsy. Remaining animals were then used for the one-generation reproduction study (section 7.8.1).

Positive control:

Not required.

Examinations

Observations and examinations performed and frequency:

Cageside observations were conducted twice a day 7 days a week for clinical signs, morbidity and mortality. Feed consumption and body weights were determined weekly. Physical examinations were performed at least weekly for each rat at the time time body weights were taken.

Sacrifice and pathology:

At sacrifice rats were anaesthetised with Metofane, the thoracic cavity opened, and the animals exsanguinated by cardiac puncture. The bladder was exposed and urine collected by sterile puncture. Urinary pH was determined immediately, followed by concentrations of Ca2+, Mg2+, phosphate (90 day sacrifice only), TPA and specific gravity. Various tissues were collected at necropsy for histopahtology. Any animals dying during the study were necropsied if they had not undergone significant autolysis. Necropsies were performed by Experimental Pathology Laboratories (EPL). Major internal organs were examined grossly for lesions. Both kidneys, lower urinary tract structures and tissues with gross abnormalities were fixed in 10% neutral buffered formalin. The kidneys were weighed prior to fixation. Fixed tissues were sectioned and stained with haematoxylin and eosin.

Other examinations:

Reproductive toxicity - see section 7.8.1

Statistics:

ANOVA, ANCOVA, Dunnett's t-test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

5 deaths, diarrhoea/urogenital discharge in rats fed 5% during first 4 weeks

Mortality:

mortality observed, treatment-related

Description (incidence):

5 deaths, diarrhoea/urogenital discharge in rats fed 5% during first 4 weeks

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreases in treated rats; lower terminal bodyweights and weight gains at 0.5% and above.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decreased in females at 5% level in both strains, decreased in CD females at 4 and 13 weeks at 2% level, decreased in Wistar males at 4 and 8 weeks at 5% level

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

acidic urine in treated rats, strain differences in urinary TPA levels and specific gravity

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

only kidney weights determined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

urinary calculi, hepatic cysts containing parasitic larvae

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

inflammatory lesions in the bladder and urethra

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITYWeight loss, diarrhoea or urogenital discharge were observed more frequently in rats ingesting 5% TPA during the first four weeks. No deaths occurred during the first 4 weeks. One death (1 male Wistar; 5% TPA) occurred in the 4-8 week period, and four deaths (1 male and 1 female Wistar, 3 female CD; all 5% TPA) occurred in the 8-13 week period. Trichobezoars (hairballs) were found in the gastrointestinal tract of one CD female that died. BODY WEIGHT AND WEIGHT GAINThe animals demonstrated a progressive decline in mean body weight relative to controls weeks 4, 8 and 13, with increasing concentration of TPA from 0.5 to 5.0%. The decrease in body weight (relative to controls) was significant in males and females of boths strains fed the 5% diet. The only exception was a lack of significance for the CD males at 8 weeks, however the mean weights were still lower than those of the respective controls. Body weights were also significantly reduced in the male and female CD rats fed the 2% diet at weeks 4 and 13. CD males fed the 0.5% diet had significantly lower body weights at week 4, and those fed 0.03% had significantly lower weights at week 13.Average body weight gain was calculated for intervals of 0-4 weeks, 4-8 weeks and 8-13 weeks. The average weight gain slowed or remained stable for all rats after the fourth week. Rats of both strains and sex fed the 5% diet had significantly reduced body weight gains throughout the 13 week study period. Average weight gain in CD rats fed 2.0% and 0.5% diets was also significantly reduced at 13 weeks. CD males fed 0.03% also had a sigificantly decreased average weight gain. More marked weight gain (or weight loss) was seen during the early phase of the study, consistent with an adverse effect on palatability. FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)Total food consumption was significantly decreased in females of both strains fed the 5% diet at weeks 4, 8 and 13. The CD females fed the 2% diet showed significantly decreased food consumption at 4 and 13 weeks. Males showed a tendency to decreased food consumption with increasing TPA concentration, the decrease was significant at the 5% level for Wistar males at weeks 4 and 8. URINALYSISA tendency towards decreased urinary pH at higher doses of TPA was observed at each sacrifice. Significantly increased acidity, compared to controls, was observed at the 90 day sacrifice in both strains and sexes at dietary concentrations 0.5% TPA and greater. Significantly lower pH values were noted in male CD rats fed 0.125% TPA. There was no statistically significant effect of TPA on Ca2+ at any sacrifice time-point, however a trend towards increased Ca2+ was noted in rats fed 2% and 5% TPA. No statistically significant effect of TPA on Mg2+ was noted, although there was an apparent trend toward greater Mg2+ concentrations at 5% TPA.Phosphate levels were determined at the 90 day sacrifice; there were no statistically significant differences. A tendency towards higher phosphate levels was noted at the 2% and 5% TPA levels.Lower urinary TPA levels were detected at the 30 day sacrifice compared to the 60 and 90 day sacrifices. A possible explanation offered with loss of TPA from the diluted urine due to precipitation whilst in storage. Consistent and statistically significant effects of strain on urinary TPA concentration was noted at the 90 day sacrifice at the 2% and 5% TPA dietary level in males, and at the 0.5%, 2% and 5% levels in females. The concentration of TPA in the urine of Wistar rats was 2- to 6-fold higher than that of CD rats (within treatment groups). Statistically significant effects of dose on specific gravity were observed at the 90 day sacrifice in CD males. Strain differences were observed at the higher doses, with Wistar rats generally excreting higher specific gravity urine than CD rats.ORGAN WEIGHTSThere were no differences in kidney weights between treatment groups.GROSS PATHOLOGYDistention or enlargement of the urinary bladder, calculi in the urinary bladder and distention of the cecum and colon with water contents were the most frequent gross observations that occurred in rats fed TPA. Calculi in the urinary bladder were noted in 1 male Wistar rat sacrificed at 30 days, 4 male Wistar rats sacrificed at 90 days, and 1 female CD rat sacrificed at 90 days; all rats were fed the 5% diet except 1 male Wistar (90 day sacrifice) fed 0.03% . Additional calculi were observed in the bladder during gross trimming of 1 female Wistar sacrificed at 90 days. The calculi were frequently observed in enlarged/distended bladders. Distention of the cecum and colon with watery intestinal contents was observed in male and female rats of both strains given 5.0% TPA at the 30, 60 and 90 day sacrifices. Similar changes were present in CD females fed 2% at the 60 and 90 day sacrifice, and in Wistar males fed 2% at 90 days.Tan nodules and cysts in the liver were observed in several rats in all treatment groups. The incidence of these lesions increased with length of exposure. The nodules and cysts were characterised by thick-walled cysts containing parasitic larvae, characteristic of Cysticercus fasciolaris (the larval stage of Taenia taeniaeformis). HISTOPATHOLOGY: NON-NEOPLASTICThe right kidney and right ureter were examined: Several lesions were noted, however there was no correlation between microscopic lesions and grossly observed bladder calculi. There were no other lesions that could be definitively attributed to TPA. Inflammatory and proliferative lesions of the ureter were observed only in rats given 5% TPA, however the lesions were observed in low incidence and therefore could not be definitively atrributed to treatment.The urinary bladder and urethra were examined: Inflammatory lesions were observed in the urinary bladder and urethra of several rats. Chronic cystitis and urethritis were characterised by a submucosal infiltration of mononuclear inflammatory cells. In most instances, the chronic inflammatory changes in the submucosa were associated with minimal to moderate hyperplasia of the transitional epithelium lining the bladder or urethra. Most of the rats with bladder lesions noted during necropsy had chronic cystitis and/or transitional cell hyperplasia. The incidence and severity of chronic cystitis and urethritis was greatest in rats fed 5% TPA, and lesions were more frequently observed in females than males.

Effect levels

1

Dose descriptor:

LOEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Decreased urinary pH in CD males fed diet containing 0.03% TPA

2

Dose descriptor:

NOAEL

Effect level:

1 250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced terminal body weights / weight gains at higher dose levels

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Calculated Mean TPA consumed

Strain	Sex	% TPA in diet	Total TPA consumed (mean ± SE)
Wistar	F	0	--
		0.03	0.48 ± 0.003
		0.125	2.06 ± 0.023
		0.5	7.90 ± 0.096
		2.0	31.92 ± 0.402
		5.0	75.50 ± 0.565
	M	0	--
		0.03	0.68 ± 0.005
		0.125	2.80 ± 0.016
		0.5	11.18 ± 0.127
		2.0	43.72 ± 0.526
		5.0	107.37 ± 0.900
CD	F	0	--
		0.03	0.54 ± 0.004
		0.125	2.14 ± 0.021
		0.5	8.54 ± 0.054
		2.0	33.66 ± 0.310
		5.0	78.90 ± 0.615
	M	0	--
		0.03	0.65 ± 0.005
		0.125	2.71 ± 0.024
		0.5	10.94 ± 0.128
		2.0	43.02 ± 0.452
		5.0	109.52 ± 0.630

Values calculated from mean food consumption; n = 30 weeks 0-4,

n = 25 weeks 4-8, n = 20 weeks 8-13.

Terminal bodyweights (g)

Strain	Sex	Dose level (%)
		0	0.03	0.125	0.5	2.0	5.0
Wistar	M	524.8	550.5	547.9	517.0	517.3	494.3
	F	307.4	292.2	308.7	300.9	294.2	285.7
CD	M	572.5	541.2	537.8	547.9	537.2	515.4
	F	374.9	376.3	362.3	361.6	340.3	314.0

Adjusted mean bodyweights at Week 13 (g)

Strain	Sex	Dose level (%)
		0	0.03	0.125	0.5	2.0	5.0
Wistar	M	471.6	468.0	483.0	464.1	448.9	431.6**
	F	404.7	379.7	404.7	400.8	394.6	383.9**
CD	M	485.8	447.9*	458.7	458.6	450.2*	440.5**
	F	426.8	427.1	411.4	409.6	390.6**	362.6**

*significantly different to controls (p<0.05); **p<0.01 [Dunnett's test]

Overall weight gain (g)

Strain	Sex	Dose level (%)
		0	0.03	0.125	0.5	2.0	5.0
Wistar	M	126.2	122.6	133.1	127.4	109.1	87.6
	F	46.1	41.4	45.0	42.9	38.3	22.3
CD	M	139.9	110.9	119.4	111.6	105.4	92.1
	F	70.1	70.4	59.7	50.5	41.2	14.3

Adjusted weight gains at Week 13 (g)

Strain	Sex	Dose level (%)
		0	0.03	0.125	0.5	2.0	5.0
Wistar	M	97.5	93..4	102.2	97.8	82.2	58.5**
	F	57.3	55.1	55.9	54.1	50.5	36.7**
CD	M	97.5	80.4*	88.4	76.7**	76.0**	64.2**
	F	69.9	66.9	63.0	51.3**	43.8**	19.8**

*significantly different to controls (p<0.05); **p<0.01 [Dunnett's test]

Bladder histopathology

Timepoint	Finding	Strain	Sex	Dose level (%)
				0	0.03	0.125	0.5	2.0	2.0
Day 30 (n=5)	Chronic cystitis	Wistar	M	-	-	-	-	-	1
			F	-	-	-	-	-	2
		CD	M	-	-	-	-	1	-
			F	-	-	-	-	-	-
	Transitional hyperplasia	Wistar	M	-	-	-	-	-	1
			F	-	-	-	-	-	2
		CD	M	-	-	-	-	1	-
			F	-	-	-	-	-	-
	Calculus	Wistar	M	-	-	-	-	-	-
			F	-	-	-	-	-	-
		CD	M	-	-	-	-	-	-
			F	-	-	-	-	-	-
Day 60 (n=5)	Chronic cystitis	Wistar	M	-	-	-	-	-	2
			F	-	-	-	-	-	-
		CD	M	-	-	-	-	-	-
			F	-	-	-	-	-	1
	Transitional hyperplasia	Wistar	M	-	-	-	-	-	1
			F	-	-	-	-	-	1
		CD	M	-	-	-	-	-	-
			F	-	-	-	-	-	2
	Calculus	Wistar	M	-	-	-	-	-	-
			F	-	-	-	-	-	-
		CD	M	-	-	-	-	-	-
			F	-	-	-	-	-	-
Day 90 (n=10)	Chronic cystitis	Wistar	M	-	2	-	-	-	3
			F	-	-	-	-	-	4
		CD	M	-	-	-	-	-	1
			F	1	-	-	-	-	4
	Transitional hyperplasia	Wistar	M	-	-	-	-	-	3
			F	-	-	1	-	-	5
		CD	M	-	-	-	-	-	1
			F	1	-	-	-	-	4
	Calculus	Wistar	M	-	-	-	-	-	1
			F	-	-	-	-	-	-
		CD	M	-	-	-	-	-	-
			F	-	-	-	-	-	-

Applicant's summary and conclusion

Conclusions:

TPA administration resulted in increased acidity of the urine, decreased body weights, urinary calculi and chronic inflammatory lesions on the bladder and urethra. Some small strain differences were present, but it was concluded that there is no substantial difference in TPA toxicity between CD and Wistar rats.

Executive summary:

Terephthalic acid (TPA) was fed to groups of male and female Wistar and CD rats for 90 days, to investigate TPA-induced urolithiasis. Nominal dietary concentrations were 0, 0.03, 0.125, 0.5, 2.0 and 5.0%. Sacrifices were carried out at 30, 60 and 90 days for gross pathology and histopathological examination of the urinary tract. Urine was collected at the time of sacrifice. Ten rats/sex/strain/group were not sacrificed, and instead continued onto a reproductive toxicity study (reported seperately). Toxicity was evident in rats fed diets containing 0.5% TPA and above; these rats exhibited reduced weight gain compared to controls. CD males fed 0.03% also exhibited significantly reduced weight gain. Five deaths occurred between 4 and 13 weeks in the rats fed 5% TPA; these rats did not have bladder calculi. Diarrhoea was observed in some of the rats exposed to high TPA concentrations. A small number of bladder calculi were found in rats fed the 5% TPA-diet for 90 days; calculi were more frequently observed in Wistar rats (6/20) than CD rats (1/20). TPA-administration resulted in a decrease in urinary pH. Chronic inflammatory lesions of the bladder and urethra were observed in treated rats, with the highest incidence occurring in rats fed 5% TPA. Nodules and cysts containing parasitic larvae were discovered in the liver of a number of treated rats. Some small strain differences were present, but it was concluded that there is no substantial difference in TPA toxicity between CD and Wistar rats. Based on body weight reductions, the NOAEL can be considered to be 0.125% TPA in the diet; equivalent to 1250 ppm or 125 mg/kg bw/d.