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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1971-08-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1972
Report date:
1972

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Repeated dose oral toxicity; administered in the diet.
GLP compliance:
no
Remarks:
Study performed prior to the inception of the Principles of GLP
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Isophthalic acid
EC Number:
204-506-4
EC Name:
Isophthalic acid
Cas Number:
121-91-5
Molecular formula:
C8H6O4
IUPAC Name:
isophthalic acid
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Isophthalic acid
- Molecular formula (if other than submission substance): C6H4(COOH)2
- Physical state: white crystalline solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isophthalic acid
- Molecular formula (if other than submission substance): C6H4(COOH)2
- Physical state: white crystalline solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Laboratory Derived Stock: The rats (Wistar derived) were selected from litters weaned at 28 days of age from the breeding colony. They were assigned to achieve equal sex and weight distribution.
- Housing: Individually in mesh bottom cages
- Diet ( ad libitum): Purina Laboratory Chow, prepared biweekly.
- Water (ad libitum): fresh tap water

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Isophthalic acid was added to Purina Laboratory Chow at levels of 0.5, 1.6 and 5%. Diets were prepared biweekly and offered ad libitum.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily/continous - ad libitum in diet.
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 other: % nomianl in diet
Dose / conc.:
1.6 other: % nominal in diet
Dose / conc.:
3 other: % nominal in diet
Remarks:
5% nominal in the diet for the first week.
No. of animals per sex per dose:
25 males and 25 females per dose
Control animals:
yes, plain diet
Details on study design:
Dose levels were initially chosen based on previously obtained data. The highest concentration was lowered from 5 % to 3 % after a one week exposure as it was felt that the rats would not survive for the full 13 week study at that dose.
After 30 and 60 days on test, 3 animals per sex per group were terminated and gross pathological examinations were performed. At 90 days of the study, all surviving animals were euthanized and submitted to detailed necropsies
Positive control:
A positive control was not included - the study included two test compounds for a comparison of the results (dimethyl terephthalate and isophthalic acid).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Animals observed daily for any deviations from normal appearance or behaviour which would indicate an adverse effect of the treatments.

BODY WEIGHT: Yes
Body weight was recorded weekly

FOOD CONSUMPTION
Recorded Weekly

HAEMATOLOGY: Yes
Clinical determinations were made on 5 male and 5 female rats of each group prior to administration of the test materials and at 30, 60 and 90 days after initiation of the program. Haematology – total erythrocytes, haemoglobin, hematocrit, total and differential leukocyte counts

CLINICAL CHEMISTRY: Yes
Clinical determinations were made on 5 male and 5 female rats of each group prior to administration of the test materials and at 30, 60 and 90 days after initiation of the program. Biochemistry – blood urea nitrogen, blood glucose, serum alkaline phosphatase and serum glutamic pyruvic transaminase (SGPT).

URINALYSIS: Yes
Clinical determinations were made on 5 male and 5 female rats of each group prior to administration of the test materials and at 30, 60 and 90 days after initiation of the program. Urine – pH, specific gravity, qualitative tests for albumin, glucose, ketones, occult blood and microscopic examination of the centrifuged sediment.

OTHER: DETERMINATION OF PHTHALATES
Blood and urine samples were collected from representative rats in each group at 7, 30, 60 and 90 days after the initiation of test feeding. The urine samples were collected for 24 hours in a metabolism cage on the day prior to sacrifice and blood was obtained by cardiac puncture at autopsy. At the 7th and 90th day, 5 rats per sex per group were sacrificed. At the 30th and 60th day, 3 per sex per group were terminated.

Preparatory to analysis the blood samples were diluted with twice the volume of distilled water and the pH adjusted to 9.5 with NaOH. After complete haemolysis of the cells, the sample was dehydrated from the frozen state to provide a stable storage form until analysis could be completed. Urine samples were also adjusted to pH and freeze-dried.

Total phthalate content of the blood and urine samples were determined by a proprietary gas chromatographic method. Basically, the procedure depends on trans-esterification with trimethylphosphate in pyridine, extraction of the esters in chloroform and injection of an aliquot into a precalibrated gas chromatograph. A Model 7260 Hewlet-Packard Research chromatograph using the dual hydrogen flame detector and a 6 ft by 2 mm glass column packed with 3 % OV-101 on AWDMCS Chromosorb G. The column temperature was programmed at 15 °C rise per minute starting at 110 ° and ending at 265 °C with a 4 minute hold. The injection port temperature was 290 °C and the detector was maintained at 295 °C. The helium flow was 60 mL per minute. Under these conditions, the internal standard peak appeared at 2.9 minutes and the phthalate peaks at 7.8. Column calibration was done with the same test materials fed to the animals.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After 30 and 60 days on test, 3 animals per sex per group were terminated and gross pathological examinations were performed. At 90 days of the study, all surviving animals were euthanized and submitted to detailed necropsies. The organs examined were liver, spleen, stomach, small and large intestine, pancreas, kidney, ureters, urinary bladder, adrenals, gonads, thyroid, pituitary, thymus, salivary gland, lymph nodes, heart, lungs, bone marrow, skin, skeletal muscle, brain and eye. The following organs were weighed: liver, spleen, kidneys, heart, brain, gonads, adrenals, thyroid and pituitary. Subsequently, the organ to body weight and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
Microscopic examinations were performed on the liver, spleen, stomach, small intestine, large intestine, pancreas, kidneys, ureters, urinary bladder, adrenals, gonads, thyroids, pituitary, thymus, salivary, gland, lymph nodes, heart, lungs, bone marrow, skin, skeletal muscle and brain from 3 animals per sex per group at 1 and 2 months and from 5 animals per sex per group after the 3 month examination. For the 2 and 3 month examinations, thymus, salivary gland and lymph nodes were deleted from the histopathological studies. In addition, histopathological examinations were made of the bladders of all animals sacrificed terminally as well as any tissues which appeared to be grossly abnormal at time of autopsy.
Other examinations:
No other examinations reported.
Statistics:
No information available.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
: reduced at the highest dose level
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
slightly reduced at the highest dose level
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:
effects observed, treatment-related
Description (incidence and severity):
haematuria, crystalluria
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
: increased kidney weights
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
kidney, bladder
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities occurred during the study.

BODY WEIGHT AND WEIGHT GAIN
Examination of the weight gains of all rats at the end of the first week made it appear unlikely that the 5% level would allow growth, or even survival of the rats. The dose level was therefore dropped to 3%.

FOOD CONSUMPTION AND COMPOUND INTAKE
There was no effects on food intake. Food efficiencies were slightly reduced in the high dose groups.

HAEMATOLOGY
Results of haematological examinations performed in these animals revealed no adverse responses associated with the ingestion of IA on total or differential leukocyte counts, total erythrocyte counts, haemoglobin or hematocrit levels.

CLINICAL CHEMISTRY
The results from the Blood chemistry investigations showed no adverse effects associated with the ingestion of IA on blood urea nitrogen, fasting blood glucose, serum glutamic pyruvic transaminase or serum alkaline-phosphatase levels.

URINALYSIS
No major differences were seen between the control and various test groups with regard to pH or specific gravity in either male or female rats. Qualitative tests for albumin, glucose and ketones were essentially similar among all groups of animals. However, microscopic sediment showed evidence of red blood cells in the urine of animals given IA. The incidence appeared to be somewhat higher among males than females. In addition, crystals apparently similar to the test materials were found in all groups of animals given IA. On the basis of this data, it is evident that the only clinical observation which may be correlated with the occurrence of the reported pathology was that of hematuria or crystalluria. It is apparent that while there is not a one-to-one correspondence between these two semi-qualitative observations, the tendency toward increasing frequency with time is obvious. There is also a general correlation with dosage.

ORGAN WEIGHTS
There was no striking effect on organ weights in any group at any time. However, the weight for all organs except kidneys was lowered. This is probably due to decreased body weights. The increase in kidney weight, although statistically significant (p<0.01) is not physiologically impressive.

GROSS PATHOLOGY
There was no notable gross pathology in any of the rats which could be associated treatment. The organs examined were liver, spleen, stomach, small and large intestine, pancreas, kidney, ureters, urinary bladder, adrenals, gonads, thyroid, pituitary, thymus, salivary gland, lymph nodes, heart, lungs, bone marrow, skin, skeletal muscle, brain and eye. The most remarkable finding arising from this series of examination was the complete absence of significant pathology in all tissues examined except the kidney and bladder. The incidence of renal and urinary bladder calculi found at autopsy has been summarised as a list of animal numbers having positive findings. Grossly, the stones occurred almost exclusively in the bladder with three exceptions and then only after 90 days on the test diets. They ranged in colour from off-white to dark brown and in size from fine sand to stones several millimetres in diameter. The bladder contents of all rats having calculi at 90 days have been retained in the laboratory and are available for subsequent examination or analysis if desired. It should be noted that the data in Table 6 is skewed by the fact that only 3 rats per sex were examined at the 30 and 60 day intervals. Nevertheless, it seems apparent that calculus formation was a relatively slow process and that it increased in frequency as the dose level increased. Aside from the urinary tract findings, there was no notable gross pathology in any of the rats which could be associated treatment.

HISTOPATHOLOGY:
No evidence of neoplastic change.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
0.5 other: % in diet
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Bladder and/or kidney stones were seen in all treated groups of males
Dose descriptor:
NOAEL
Effect level:
1.6 other: % in diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Bladder stones bodyweight effects and increased kidney weights at the highest concentration of 3%

Target system / organ toxicity

Critical effects observed:
not specified
Lowest effective dose / conc.:
0.5 other: % in the diet
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Examination of the weight gains of all rats at the end of the first week made it appear unlikely that the 5% level would permit growth or even survival of rats fed with IPA. The 5 % level was then reduced to 3%.

Weight gains (all rats) and food consumption for weeks 1 and 13

Test Group

Dose Level

Males

Females

Week 1

Week 13

Week 1

Week 13

Net Gain

Food Intake

Net Gain

Food Intake*

Net Gain

Food Intake

Net Gain

Food Intake*

Control

0

34

72

307

156

24

69

169

112

IPA

Low

31

80

290

163

28

74

180

127

Mid

30

76

310

170

26

76

175

126

High**

17

57

278

156

18

60

167

128

Control

0

31

82

294

164

24

74

170

117

* Food intake for 13th Week only

** Dietary levels were 5.0 % for the 1stWeek and 3.0 % thereafter

All weights in grams.

Kidney weights

 

Male

Female

Control

Low

Mid

High

Control

Low

Mid

High

1 month

Kidney (g)

2.32, 2.24

2.20

2.40

2.45

1.60, 1.50

1.61

1.69

1.66

Kidney (%)

0.89, 0.93

0.92

0.93

1.02

0.94, 0.88

0.92

0.95

0.98

Kidney (%brain)

1.40, 1.33

1.30

1.50

1.36

1.01, 1.04

1.03

1.11

1.07

2 months

Kidney (g)

2.55, 2.81

2.31

2.67

2.13

1.66, 1.81

1.68

1.77

1.65

Kidney (%)

0.82, 0.90

0.77

0.85

0.80

0.80, 0.83

0.83

0.89

1.01

Kidney (%brain)

1.49, 1.57

1.53

1.60

1.25

1.02, 1.07

1.02

1.11

1.08

3 months

Kidney (g)

2.76. 2.79

2.60**

2.91**

2.73

1.89, 1.94

2.14

2.04

2.12

Kidney (%)

0.75, 0.76

0.75

0.80

0.80

0.83, 0.85

0.90

0.88

0.95**

Kidney (%brain)

1.49, 1.51

1.44

1.61

1.48

1.13, 1.13

1.25

1.21

1.25

Identification of Animals Showing Urinary Occult Blood

Level

30 Days

60 Days

90 Days

Males

None

-

-

-

IPA, Low

-

-

-

IPA, Mid

11135

11037

-

IPA, High

-

11162

11169

None

-

-

-

 

Females

None

-

-

11233

IPA, Low

-

-

-

IPA, Mid

-

-

-

IPA, High

-

-

11395

None

-

-

-

- = No animals displayed symptoms

Identification of Animals Showing Urinary RBCs in Centrifuged Sediment

Level

30 Days

60 Days

90 Days

Males

None

-

-

-

IPA, Low

-

-

-

IPA, Mid

11129, 11135, 11137

11137

11146

IPA, High

11160, 11162

11162

11169

None

-

-

-

 

Females

None

-

-

-

IPA, Low

-

-

-

IPA, Mid

-

-

-

IPA, High

-

-

11395

None

-

-

11433

- = No animals displayed symptoms

Crystalluria does occur occasionally in normal urine. This became a constant finding by the 60thday, although it was not observed at the first urine collection period at 30 days. Microscopically, the crystals did not resemble any of the normal sedimentary components of rat urine but appeared grossly similar to the test materials. This finding did not correlate closely with the appearance of red blood cells or occult blood.

Blood levels of the test compounds were determined as total phthalate after trans-esterification and temperature programmed gas chromatography as described earlier. The average values obtained at each experimental period are summarised in the table below. The most striking feature of this data is the correlation with dose level with respect to both blood concentration and urinary output. At the low and middle dose levels, blood values of IA remained fairly constant or tended to decrease whereas the corresponding urinary outputs remained constant or tended to increase. At the high dosage level, IA tended to decrease in blood and increase in urine output concurrently. At the moderate levels of intake, the compounds achieve a steady state or equilibrium condition with respect to metabolism and/or excretion. However, faced with an overload, it appears that the rat may respond by utilizing an alternate pathway which may even involve enzyme induction or at least different intermediate steps in the degradation of the phthalate molecule. It should be noted that any reactions which would lead to partially decarboxylated metabolites would go undetected by the analytical methods employed for the blood and urine determinations.

Phthalate Blood Levels

Level

7 day2

30 day3

60 day3

90 day2

Males

Females

Males

Females

Males

Females

Males

Females

None

-

-

ND

ND

ND

ND

ND

ND

IPA Low

-

-

8.75

7.51

26.0

27.1

3.4

ND

IPA Mid

29.0

37.1

16.3

25.3

9.6

24.6

17.5

21.3

IPA High

97.5

114.0

32.0

30.0

17.9

31.2

26.3

40.8

None

-

ND

ND

ND

ND

ND

ND

ND

Rats were not fasted before blood drawing

2 – Average of five rats per sex per group

3 – Average of 3 rats per sex per group

ND = None detected at sensitivity of 5.0 mcg per ml.

Incidence of kidney (K) and urinary bladder (B) calculi at necropsy

Level

30 Daya

60 Daya

90 Days

Kidney

Bladder

Kidney

Bladder

Kidney

Bladder

Males

None

-

-

-

-

-

-

IPA Low

11087

-

-

-

-

11118

IPA Mid

-

-

-

-

-

11144

IPA High

 

-

-

11175

-

11153

11162

None

-

-

-

-

-

-

Females

None

-

-

-

-

-

-

IPA Low

-

-

-

-

-

-

IPA Mid

-

-

-

-

-

-

IPA High

-

-

-

-

11385

-

None

-

-

-

-

-

-

A= 3 rats per sex

Applicant's summary and conclusion

Conclusions:
Findings in this study were limited to reduced weight gain at the highest dose level, with haematuria, crystalluria and associated increases in kidney weights seen in treated groups.
Executive summary:

Groups of rats (25/sex) were administered isophthalic acid in the diet at dose levels of 0.5, 1.6 and 3% for up to 90 days. The highest dose level was reduced from 5% to 3% after one week due to effects on bodyweight. Animals were observed for mortality and clinical signs; bodyweights were recorded at intervals. Blood samples were taken for the assessment of clinical chemistry and haematological parameters; urinalysis was also performed. Animals were investigated at necropsy (30, 60 and 90 days) for gross and microscopic findings; organ weights were also recorded. Blood levels of phthalates were also investigated.

 

No deaths occurred and there were no signs of toxicity. Bodyweights were markedly reduced at the highest dose level of 5/3%. No effects were seen on haematological and clinical chemistry parameters; urinalysis revealed the presence of red blood cells and crystals in the urine of treated animals. Findings were associated with increased kidney weights but did not have any gross or microscopic pathological correlates. A LOAEL of 0.5% (5000 ppm, equivalent to 500 mg/kg bw/d) can be determined for male rats in this study, based on the incidence of bladder/kidney stones in all treated groups. A LOAEL of 1.6% (16000 ppm, equivalent to 1600 mg/kg bw/d) can be determined for female rats in this study based on the incidence of bladder stones, bodyweight effects and kidney weight effects at the highest dose level.