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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined repeated dose repro-devp. Screen
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Developmental toxicity evaluation of 1,2,3,4 butanetetracarboxylic acid in Sprague Dawley (CD®) rats
Author:
Julia D. George, Catherine J. Price, Melissa C. Marr, Christina B. Myers, Gloria D. Jahnke
Year:
2001
Bibliographic source:
Reproductive Toxicology, 15, (2001), 413–420
Reference Type:
review article or handbook
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: under a protocol approved by the RTI Institutional Animal Care and Use Committee (IACUC)
Principles of method if other than guideline:
Combined repeated dose repro-devp. study was performed to determine the oral toxic nature of 1,2,3,4 butane tetra carboxylic acid upon repeated exposure by oral route
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Butane-1,2,3,4-tetracarboxylic acid
EC Number:
216-938-0
EC Name:
Butane-1,2,3,4-tetracarboxylic acid
Cas Number:
1703-58-8
Molecular formula:
C8H10O8
IUPAC Name:
butane-1,2,3,4-tetracarboxylic acid
Test material form:
solid
Details on test material:
- Name of the test chemical: Butane-1,2,3,4-tetracarboxylic acid- IUPAC name: Butane-1,2,3,4-tetracarboxylic acid - Molecular Formula: C8H10O8- Molecular Weight: 234.159 g/mol- Smile Notation: OC(=O)C[C@H]([C@H](CC(=O)O)C(=O)O)C(=O)O- InChI : 1S/C8H10O8/c9-5(10)1-3(7(13)14)4(8(15)16)2-6(11)12/h3-4H,1-2H2,(H,9,10)(H,11,12)(H,13,14)(H,15,16)/t3-,4+- Substance type: organic
Specific details on test material used for the study:
- Name of the test chemical: 1,2,3,4 butane tetra carboxylic acid- IUPAC name: Butane-1,2,3,4-tetracarboxylic acid - Molecular Formula: C8H10O8- Molecular Weight: 234.159 g/mol- Smile Notation: OC(=O)C[C@H]([C@H](CC(=O)O)C(=O)O)C(=O)O- InChI : 1S/C8H10O8/c9-5(10)1-3(7(13)14)4(8(15)16)2-6(11)12/h3-4H,1-2H2,(H,9,10)(H,11,12)(H,13,14)(H,15,16)/t3-,4+- Substance type: organic - Lot no: Lot No. 07201 DZ- Purity: >99%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
CrI:CD® (SD)BRVAF/Plus® outbred albino rats (CD® rats)
Details on species / strain selection:
No data
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories, Inc., Raleigh, NC- Age at study initiation: Female (P) 8 to 10 weeks old plus 5 days of quarantine period- Weight at study initiation: Females (P)- 218 to 269 g on gd0- Fasting period before study: No data- Housing: The female rats were housed singly in solid bottom, polycarbonate cages with stainless steel lids and certified sani-chip hardwood cage litter- Diet (e.g. ad libitum): Purina Certified Rodent Chow, ad libitum- Water (e.g. ad libitum): tap water, Durham, NC city water, ad libitum- Acclimation period: 5 days quarantineENVIRONMENTAL CONDITIONS- Temperature (°C): 70 to 73°F (21-22 °C)- Humidity (%): 39 to 60%- Air changes (per hr): No data- Photoperiod (hrs dark / hrs light): 12 h light, 12 h darkIN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
water
Remarks:
Deionized / distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in deionized / distilled water at dose level of 0, 250, 500 or 1000 mg/Kg/day DIET PREPARATION - Rate of preparation of diet (frequency): No data - Mixing appropriate amounts with (Type of food): No data - Storage temperature of food: No data VEHICLE - Justification for use and choice of vehicle (if other than water): deionized / distilled water - Concentration in vehicle: 0, 250, 500 or 1000 mg/Kg/day - Amount of vehicle (if gavage): 5 ml/Kg - Lot/batch no. (if required): No data - Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The identity of the bulk chemical was confirmed by infrared (IR) spectroscopy and 1H nuclear magnetic resonance spectroscopy. Purity (>99%) was determined by high-performance liquid chromatography (HPLC). Bulk chemical reanalysis was performed within two weeks of study initiation. Identity was reconfirmed by IR spectroscopy and relative purity was >99% when compared to a frozen reference sample by HPLC.Dosing solutions (BTCA in deionized/distilled water) were verified to be within 92 to 103% of their theoretical concentrations by HPLC prior to and after the period of administration
Duration of treatment / exposure:
14 days (gd 6 to gd19)
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
0, 250, 500 or 1000 mg/Kg/day
No. of animals per sex per dose:
Total: 1000 mg/Kg bw: 25 females250 mg/Kg bw: 25 females500 mg/Kg bw: 25 females1000 mg/Kg bw: 25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the main study were selected on the basis of acute, oral toxicity study of BTCA [800, 1260, 1590, 2000, and 3170 mg/kg] in COX-SD rats. The BTCA LD50 for female rats was 1620 mg/kg body weight in that study. Dose range selection was also supported by a study in nonpregnant female CD rats (7 per group) exposed to BTCA [0,50,100,500,750, and 1000 mg/kg/day] for 14 consecutive days and a developmental toxicity screening study in female CD rats (14 confirmed pregnancies per group) exposed to BTCA [0, 62.5, 125, 250, 500, and 1000 mg/kg/day] on gd 6 through 19. In the screening study, maternal toxicity was noted at 1000 mg/ kg/day, including morbidity (1/15 dams; evidence of abortion at necropsy). Developmental toxicity was limited to a decreasing trend for fetal body weight (males only) with a 6% (nonsignificant) reduction at 1000 mg/kg/day. Thus, the high dose for the currently reported study was expected to cause some maternal toxicity and to satisfy the US EPA limit dose for developmental toxicity studies. The mid dose for the present study was set at one-half of the high dose, and the low dose was set at one-half of the mid dose. Doses above 1000 mg/kg/day (i.e. 200 mg/mL 3 5 ml/kg) were not considered feasible due to (a) poor solubility of BTCA at 250 mg/mL in water, (b) morbidity in 1/15 pregnant rats at 1000 mg/kg/day on gd 6 through 19 [29], and (c) mortality in 1/5 female rats given a single oral dose of 1590 mg/kg, and 5/5 female rats given a single oral dose of 2000 or 3700 mg/kg - Rationale for animal assignment (if not random): Confirmed-mated females were assigned to treatment groups by stratified randomization for body weight on gd 0, so that mean body weight on gd 0 did not differ among treatment groups. - Rationale for selecting satellite groups: No data - Post-exposure recovery period in satellite groups: No data - Section schedule rationale (if not random): No data
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: Throughout the treatment- Cage side observations checked in table [No.?] were included. The animals were observed for mortalityDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: once daily prior to initiation of treatment, and twice daily during the treatment period (i.e. at weighing/dosing and 1 to 2 h after dosing).BODY WEIGHT: Yes - Time schedule for examinations: Daily during treatment from gd 6 through 19, and on gd 20 (before and after termination)FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, feed weights were taken on gd 0, 6, 9, 12, 15, 18, 19, and 20- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specifiedFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specifiedWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes- Time schedule for examinations: Water weights were taken on gd 0, 6, 9, 12, 15, 18, 19, and 20OPHTHALMOSCOPIC EXAMINATION: Not specified- Time schedule for examinations: Not specified- Dose groups that were examined: Not specifiedHAEMATOLOGY: Not specified- Time schedule for collection of blood: Not specified- Anaesthetic used for blood collection: Not specified- Animals fasted: Not specified- How many animals:- Parameters checked in table [No.?] were examined.CLINICAL CHEMISTRY: Not specified- Time schedule for collection of blood: Not specified- Animals fasted: Not specified- How many animals: Not specified- Parameters checked in table [No.?] were examined. Not specifiedURINALYSIS: Not specified- Time schedule for collection of urine: Not specified- Metabolism cages used for collection of urine: Not specified- Animals fasted: Not specified- Parameters checked in table [No.?] were examined. Not specifiedNEUROBEHAVIOURAL EXAMINATION: Not specified- Time schedule for examinations: Not specified- Dose groups that were examined: Not specified- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not specifiedIMMUNOLOGY: Not specified- Time schedule for examinations: Not specified- How many animals: Not specified- Dose groups that were examined: Not specified- Parameters checked in table [No.?] were examined. Not specifiedOTHER: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, timed-mated females were sacrificed by C02 asphyxiation on gd 20. The body, liver, and gravid uterus of each timed-mated female were weighed. Thoracic and abdominal cavities were examined.HISTOPATHOLOGY: Yes, maternal livers were saved in 10% neutral buffered formalin for optional histopathology.
Other examinations:
No data
Statistics:
The unit for statistical measurement was the pregnant female. Quantitative continuous data were compared among treatment groups by parametric tests if Bartlett’s test for homogeneity of variance was not significant. If Bartlett’s test indicated a lack of homogeneity (p < 0.001), then nonparametric tests were applied. Statistical analyses were based on SAS software.A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed Dunnett’s Test was appropriate for maternal body and organ weight parameters, maternal feed and water consumption.Nonparametric tests for continuous variables included the Kruskal-Wallis one-way analysis of variance by ranks for among-group differences and, if significant (p < 0.05), the Mann-Whitney U test for pairwise comparisons to the vehicle control group. One- or two-tailed Mann-Whitney U tests were applicable to specific parameters, as noted above for Dunnett’s Test. Jonckheere’s test for k independent samples was used to identify significant dose response trends.Nominal scale measures were analyzed by x2 Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Proportions. None of these tests were statistically significant.The alpha level for each statistical comparison was 0.05, and the significance levels for trend tests and pairwise comparisons were reported as p < 0.05 or p < 0.01.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Clinical signs and mortality: Mortality: Treatment related mortality occurred in 4% (1/24) in 1000 mg/Kg/day females. All remaining females survived the treatmentClinical signs: Treatment related clinical signs included weight loss which occurred in all groups and showed increasing frquency and/or severity with increasing dose on individual days during the treatment period. Piloerection was observed in ≤ 4 females/day at 1000 mg/Kg/day. Rooting in the cage bedding after dosing suggested an aversion to the sensory properties of the 500 and 1000 mg/Kg/day dose formulations (≤ 3 500 mg/Kg/day females/day, and ≤ 5 1000 mg/Kg/day females/ day). Alopecia was noted with a low incidence in all groups but showed no apparent relationship to BTCA exposureBody weight and weight gain: Maternal body weight was comparable among groups on gd 0 and 6 (i.e. prior to the initiation of dosing). On gd 9, 12, 15, 18, and 19, maternal body weights at the low and mid doses were not affected (98.8 to 100.8% of concurrent controls), but maternal body weight at the high dose was significantly lower than the vehicle control on each of these days, except gd 9. On gd 20, maternal body weight at the high dose was significantly below the vehicle control. Decreases in maternal body weight became larger with repeated dosing, and the maximum reductions occurred at the end of the dosing period (gd 18, 19, and 20). Thus, the effect of BTCA on maternal body weight was restricted to the 1000 mg/Kg/day dose group and the magnitude of this effect was relatively small.Food consumption and compound intake: Maternal relative feed intake was transiently reduced at 1000 mg/kg/day from gd 6 to 9 and at 500 mg/kg/day from gd 12 to 15, although no changes were noted for the treatment period as a whole. Food efficiency: No dataWater consumption and compound intake: Maternal relative water intake was increased at 1000 mg/kg/day for gd 12 to 15, 18 to 19, 19 to 20, and the treatment period as a whole.Opthalmoscopic examination: No dataHaematology: No dataClinical chemistry: No dataUrinanalysis: No dataNeurobehaviour: No dataOrgan weights: Gravid uterine weight and maternal relative liver weight were not affected. Absolute maternal liver weight exhibited a decreasing trend but showed no significant differences between control Gross pathology: The animals that died due to the misdirected dose revealed dark red, mottled lungs and pink discharge from the vaginaHistopathology: No data

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No significant alteration were noted at the mentioned dose level w.r.t. clinical signs, mortality, body weight, food and water intake, organ weights, gross pathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Toxicity in CDt rats exposed to 1,2,3,4-butanetetracarboxylic acid on gestational days 6 through 19

Doses (mg/Kg/day)

0

250

500

1000

Total No. Assigned Females

25

25

25

25

Maternal Body Weight (g)

 

 

 

 

Gd 0

247.6±2.0

246.9±1.9

246.5±2.5

242.8±2.7

Gd 6

275.7±2.5

277. 3±2.7

278.0±3.1

269.8±2.9

Gd 20 (in life)c,e

387.7±3.9

387.4±3.8

388.8±4.7

361.1±7.0*

Maternal Weight Gain (g)

 

 

 

 

Treatment (gd 6 to 20)c

112.0±2.6

109.1±2.4

110. 8±2.9

91.8±6.1*

Treatment (gd 6 to 20)c

135.7±3.4

135.3±3.3

135. 6±3.4

113.0±6.3*

Correctedc,d,e

51.5±2.8

53.8±2.6

50.3±2.9

35.6±4.7*

Maternal Relative Feed Intake

(g/kg/d)e

 

 

 

 

Treatment (gd 6 to 20)

75.5±1.0

75.4±0.7

72.4±1.0

73.6±1.4

Maternal Relative Water Intake

(g/kg/d)e

 

 

 

 

Treatment (gd 6 to 20)

121.7±4.0

120.2±4.0

123.5±5.8

141.1±5.8*

Gravid Uterine Weight (g)

84.2±2.2

81.6±2.2

85.3±2.3

77.4±3.2

Maternal Liver Weight

 

 

 

 

Absolute (g)c

16.42±0.23

16.45±0.23

16. 19±0.33

15.21±0.40

Relative (% body weight)

4.28±0.04

4.30±0.05

4.23±0.07

4.27±0.08

aIn the high dose, one animal was removed due to a dosing error (misdirected dose). Another high-dose animal was found dead on gd 17; necropsy revealed dark red, mottled lungs and pink discharge from the vagina, but no definite evidence of a misdirected dose. This animal exhibited piloerection as the only clinical sign during treatment.

cP<0.05; test for linear trend, or Jonckheere’s Test.

dWeight gain during gestation minus gravid uterine weight.

eOne body weight in the low-dose group on gd 6 was invalid (n524 for gd 6 body weight and gd 6 to 20 weight gain). For feed and water intake n:17 to 23 due to removal of statistical outliers, spillage etc.

* P<0.05; pairwise comparison to controls, by Dunnett’s Test or Mann-Whitney U Test.

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for 1,2,3,4 butane tetra carboxylic acid is considered to be 500 mg/Kg/day when Sprague Dawley rats were exposed to the test chemical repeatedly by gavage route for 14 days.
Executive summary:

Combined repeated dose repro-devp. study was performed to determine the oral toxic nature of 1,2,3,4 butane tetra carboxylic acid upon repeated exposure by oral route. Female Sprague Dawley rats were exposed to the test chemical from gd 6 to gd 19 at dose levels of 0, 250, 500 or 1000 mg/Kg/day. The doses for the main study were selected from preliminary acute toxicity and developmental study. The treated animals were observed for mortality, clinical signs, body weight changes, food and water intake, organ weight changes and gross pathology. Treatment related mortality occurred in 4% (1/24) in 1000 mg/Kg/day females. All remaining females survived the treatment. Treatment related clinical signs included weight loss which occurred in all groups and showed increasing frquency and/or severity with increasing dose on individual days during the treatment period. Piloerection was observed in ≤ 4 females/day at 1000 mg/Kg/day. Rooting in the cage bedding after dosing suggested an aversion to the sensory properties of the 500 and 1000 mg/Kg/day dose formulations (≤ 3 500 mg/Kg/day females/day, and ≤ 5 1000 mg/Kg/day females/ day). Alopecia was noted with a low incidence in all groups but showed no apparent relationship to BTCA exposure. Maternal body weight was comparable among groups on gd 0 and 6 (i.e. prior to the initiation of dosing). On gd 9, 12, 15, 18, and 19, maternal body weights at the low and mid doses were not affected (98.8 to 100.8% of concurrent controls), but maternal body weight at the high dose was significantly lower than the vehicle control on each of these days, except gd 9. On gd 20, maternal body weight at the high dose was significantly below the vehicle control. Decreases in maternal body weight became larger with repeated dosing, and the maximum reductions occurred at the end of the dosing period (gd 18, 19, and 20). Thus, the effect of BTCA on maternal body weight was restricted to the 1000 mg/Kg/day dose group and the magnitude of this effect was relatively small. Maternal relative feed intake was transiently reduced at 1000 mg/kg/day from gd 6 to 9 and at 500 mg/kg/day from gd 12 to 15, although no changes were noted for the treatment period as a whole. Maternal relative water intake was increased at 1000 mg/kg/day for gd 12 to 15, 18 to 19, 19 to 20, and the treatment period as a whole. Gravid uterine weight and maternal relative liver weight were not affected. Absolute maternal liver weight exhibited a decreasing trend but showed no significant differences between control. Based on the observations made, the No Observed Adverse Effect Level (NOAEL) for 1,2,3,4 butane tetra carboxylic acid is considered to be 500 mg/Kg/day when Sprague Dawley rats were exposed to the test chemical repeatedly by gavage route for 14 days.

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