Carboxylic acids, di-, C4-6

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Principles of method if other than guideline:

AGS mixtures were administered in a single oral dose to SD rats. Animals were sacrificed 6, 18 or 30 h after treatment. A positive control group was included (Cyclophosphamide). 2 hours before scheduled sacrifice animals were administered colchicine at to arrest cells in metaphase. Animals were sacrificed and both femurs were removed from each animal and metaphase slides were prepared. Slides were stained, coded and scored for chromosomal aberrations (50 metaphase bone marrow cells/animal were evaluated.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, di-, C4-6

EC Number:

271-678-5

EC Name:

Carboxylic acids, di-, C4-6

Cas Number:

68603-87-2

Molecular formula:

C5H8O4, C4H6O4, C6H10O4

IUPAC Name:

Carboxylic acids, C4-6 di-

Details on test material:

Adipic acid: 6-9%
Glutaic acid 30-35%
Succinic acid 8-11%
Nitric acid 0.5 - 3%
Lot: FIT-85-45,46,47; white solid powder

Specific details on test material used for the study:

test material: AGS mixture was used as a 50% aqueous solution.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

breeder: Charles River Lab., Wilmington, Massachsusetts

Sex:

male/female

Details on test animals or test system and environmental conditions:

body weight at randomization. 239-264 g for males and 178-208 g for females

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

deionized water

Details on exposure:

all animals were fasted overnight prior to treatment; AGS mixture (50% aqueous solution), solvent or positive controls were administered as single oral doses at 5 mL/kg bw.
2 to 3 hours prior to sacrifice each animal was given a single intraperitoneal dose of colchicine at 4 mg/kg bw to arrest dividing cells in metaphase. Colchicine was dosed at 10 mL/kg bw and was prepared in distilled water.

Justification for doses:
AGS mixture (50%) was evaluated in a preliminary study at doses of 1375, 2750 and 5500 mg/kg bw. Due to the mortality and pharmacotoxic signs observed at 5500 mg/kg bw for males and 2750 mg/kg bw for females, doses selected for evaluation in the Metaphase Analysis Assay were 2750 mg/kg bw for males and 1375 mg/kg bw for females as an estimation of the maximum tolerated dose.
5500 mg/kg bw: both treated females died within four hours post dosing; one male died 24 hours post dosing and the other exhibited severe pharmacotoxic signs through day 7
2750 mg/kg bw: one female died 24 hours post dosing and the second female died by day 3; both males survived the treatment and exhibited mild to severe pharmacotoxic signs through day 7
Additional tests with higher dilutions (same doses) showed similar effects.

Duration of treatment / exposure:

6, 18, 30 h

Frequency of treatment:

single treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 375 mg/kg bw (total dose)

Remarks:

dose for females, sacrifice 6, 18, and 30 hours after treatment

Doses / concentrations 2

Dose / conc.:

2 750 mg/kg bw (total dose)

Remarks:

dose for males, sacrifice 6, 18, and 30 hours after treatment

No. of animals per sex per dose:

vehicle control (water): 7 males and 5 females (6 hour treatment), 5 males and 5 females (each 18 hour and 30 hour treatment)
AGS mixture (50%), 2750 mg/kg bw: 8 males (6 hour treatment), 5 males (each 18 hour and 30 hour treatment)
AGS mixture (50%): 1375 mg/kg bw: 5 females (each 6, 18 hour and 30 hour treatment)
Cyclophosphamide: 20 mg/kg bw: 5 males and 5 females (18 hour treatment)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:

Animals were sacrificed and both femurs were removed from each animal and metaphase slides were prepared.
A total of 500 well spread metaphase cells with a minimum of
overlapping chromosomes, were scored for the presence of chromosome aberration
per experimental treatment point (50 per animal) by two investigators (25 each
per animal). A total of 600 metaphases (12 animals) were scored the 6 hour
solvent control group. Cells were located by systematic searching of the
slide under low power (20X-40X) magnification. Cells judged acceptable for
analysis based on cell morphology and total chromosome nurnber (± 2 of the
normal diploid no. of 42) were then further analyzed with lOOX oil immersion
objective where abnormalities were detected and classified. Vernier
coordinates were recorded for the first and last metaphase scored, as well as
for any abnormal rnetaphases (including gaps) observed. The centromere number
was recorded on all cells analyzed.

Evaluation criteria:

Cytogenetic abnormalities were classified on a standard
scoring sheet according to chromosome or chromatid aberrations and further
according to type of aberration (see Legend on page 12). Aberrations were
classified according to the nomenclature cf Buckton and Evans, 1973 and
Savage, 1975.

Statistics:

Mean aberrations per cell per rat (50 cells per rat)
were analyzed for statistically significant increases in chromosome aberration
by one-tailed t tests. Each treatment group was analyzed separately as
compared to its concurrent negative control group. The mean and standard
deviation of aberrations/cell were also determined for each group of rats (500
cells~ 50 cells per rat). The number of aberrant metaphases was analyzed by
Chi-square analysis for statistically significant increases. Statistical
significance was determined at the p >= 0.05 probability level.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

death in high dose group

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Doses selected for evaluation in the Metaphase Analysis Assay were 2750 mg/kg bw for males and 1375 mg/kg bw for females as an estimation of the maximum tolerated dose.

General toxicity in the main assay:

Three males died before sacrifice; most of the remaining males and females had decreased body tone and activity, body drop, abnormal gait, ptosis and piloerection. In addition, some animals had tremors and vocalization on touch.

6 Hour Group - (observed approximately 4 hours after dosing):
One male was dead before colchicine administration. The remaining males had decreased body tone and activity, body drop, abnormal gait, ptosis and piloerection. In addition, two males bad tremors. All females showed bad decreased body tone, piloerection and vocalization on touch. A second male died before harvest. The sponsor was notified and requested that 5 extra male rats be dosed, two with the solvent control and the other three with AGS mixture (50%) at 2750 mg/kg to ensure two available rats to replace the dead ones. One male died before colchicine administration. The other two males exhibited moderate pharmacotoxic signs.

18 Hour Group - (observed approximately 16 hours after dosing):
All rats had piloerection. Three males and three females also had decreased body tone. Four males and three fernales made vocalizations when touched. One male bad abnormal gait and one female abnormal stance.

30 Hour Group - (observed approximately 28 hours after dosing):
All rats had decreased body tone and piloerection. All males had abnormal stance. One male and two females made vocalizations when touched. Two fernales had body drop.

Any other information on results incl. tables

No statistically significant increase in the incidence of aberrations or the number of cells with one or more aberrations were observed in the animals treated with dicarboxylic acid mixture at any dose and at any of the three sampling time points. (see Table 1 and attachment)

 

Table 1: in vivo bone marrow cytogenetics - proportion of cells with one ore more aberrations

 

compound	dose (mg/kg bw)	harvest time (hrs)	sex	no. of rats	no. of cells with one or more aberrations	no. of normal cells	% aberrant cells/group
water	5 mL/kg bw	6	M	7	1	349	0.3
water	5 mL/kg bw	6	F	5	1	249	0.4
AGS mix (50%)	2750	6	M	5	1	249	0.4
AGS mix (50%)	1375	6	F	5	0	250	0.0
water	5 mL/kg bw	18	M	5	2	248	0.8
water	5 mL/kg bw	18	F	5	4	246	1.6
AGS mix (50%)	2750	18	M	5	1	249	0.4
AGS mix (50%)	1375	18	F	5	0	250	0.0
cyclophosphamide	20	18	M	5	123	127	49.2**
cyclophosphamide	20	18	F	5	128	122	51.2**
water	5 mL/kg bw	30	M	5	0	250	0.0
water	5 mL/kg bw	30	F	5	1	249	0.4
AGS mix (50%)	2750	30	M	5	2	248	0.8
AGS mix (50%)	1375	30	F	5	1	249	0.4

** significant in Chi-square at p >= 0.01

Applicant's summary and conclusion

Executive summary:

The potential of dicarboxylic acid mixture (50%) to induce structural chromosomal aberrations in the hemopoetic cells of the rat bone marrow were investigated in vivo after oral gavage. Dicarboxylic acid mixture (2750 mg/kg bw for males and 1375 mg/kg bw for females) was administered in single oral doses to SD rats and animals were sacrificed 6, 18 and 30 hours after dosing. A positive control group (cyclophosophamide; 20 mg/kg) was sacrificed at the 18 hours time point. 2 Hours before sacrifice animals were administered with colchinine to arrest cells in metaphase. Both femurs were removed from each animal and metaphase slides were prepared. A total of 50 metaphase  cells were analysed for each animal for the presence of chromatid and chromosome type aberrations. The positive control group resulted in a significant increase in chromosomal aberrations.

Two rats dosed with 2750 mg/kg died. The surviving animals in all test groups exhibited from mild to severe pharmacotoxic signs and the authors concluded that the test item was evaluated at or near the MTD. No statistically significant increase in the incidence of aberrations or the number of cells with one or more aberrations were observed in the animals treated with dicarboxylic acid mixture at any dose and at any of the three sampling time points. Therefore, in vivo the test item was not clastogenic to hempoietic cells of the rat bone marrow.