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Toxicological information

Genetic toxicity: in vivo

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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:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP but overall good documentation, purity not specified, no positive control for every experiment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1974
Report date:
1974

Materials and methods

Principles of method if other than guideline:
Groups of  5 treated and 3 control animals were used. Animals were killed  6, 24 and 48 hours after a single administration in the acute study. In  the subacute study 5 doses, 24 hours apart, were administered and animals  were killed 6 hours after the last dose. 
GLP compliance:
no
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Adipic acid
EC Number:
204-673-3
EC Name:
Adipic acid
Cas Number:
124-04-9
Molecular formula:
C6H10O4
IUPAC Name:
adipic acid
Details on test material:
IUCLID4 Test substance: purity not specified
Specific details on test material used for the study:
compound FDA 71-50, Adipic acid, food processing quality, as supplied by the Food and Drug Administration

Test animals

Species:
rat
Strain:
not specified
Sex:
male

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
Acute study: single dosing; subacute study: once a day for 5 consecutive days
Frequency of treatment:
Acute study: single dosing; subacute study: once a day for 5 consecutive days
Post exposure period:
Animals were killed  6, 24 and 48 hours after a single administration in the acute study. In  the subacute study 5 doses, 24 hours apart, were administered and animals  were killed 6 hours after the last dose. 
Doses / concentrations
Remarks:
Doses / Concentrations:
Test 1: acute and subacute: 3.75, 37.5, 375 mg/kg bw/day; Test 2: acute 5000 mg/kg bw and subacute 2500 mg/kg bw/day
Basis:

No. of animals per sex per dose:
Groups of  5 treated and 3 control animals were used.
Control animals:
yes, concurrent vehicle
Positive control(s):
positive control animals were treated with 0.3 mg/kg bw TEM (triethylenemelamine) by intraperitoneally incection

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
Four hours after the last  compound administration, and two hours prior to killing, each animal was  given 4 mg/kg bw of colcemid intraperitoneally in order to arrest the  bone marrow cells in C-mitosis. The marrow "plug" was removed and  aspirated into Hanks' balanced salt solution. The specimen were  centrifuged and resuspended in hypotonic 0.5% KCl. The specimens were  placed in a 37 degree Celsius water bath in order to swell the cells.  Following centrifugation the cells were resuspended in a fixative (3:1  absolute methanol : glacial acetic acid) and again centrifuged. Cells were  resuspended and placed at 4 degree Celsius overnight. The following day  cells were again centrifuged and freshly prepared fixative was added. The  suspension was dropped onto a slide and ignited by an alcohol burner and  allowed to flame. Slides were stained with 5% Giemsa solution. The  preparations were examined by microscopy. The chromosomes of each cell  were counted and only diploid cells were analyzed. They were scored for  chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells  with greater than ten aberrations, polyploidy, pulverization, and other  chromosomal aberrations which were observed. Fifty metaphase spreads were  scored per animal. Mitotic indices were obtained by counting at least 500  cells and the ratio of the number of cells in mitosis / the number of  cells observed was expressed as the mitotic index. Negative and positive  (TEM) controls were run in each experiment. Two tests were performed at different time intervals.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Triethyl Melamine (TEM)

Any other information on results incl. tables

Table 1: Acute study I with single gavage dosing (3.75, 37.5 and 375 mg/kg bw):

The negative control group cells contained no aberrations. Adipic acid produced no aberrations except for one cell containing a break in the 6-hour sample of  the intermediate dose level. The expected severe chromosomal damage was observed for the positive control group (triethylene melamine treated animals). The mitotic indices  

were within normal limits. Negative and positive controls were functional.

 dosage (mg/kg bw)  time*  no. of animals no. of cells per animal  mitotic index**  % cells with breaks  % cells with other aberrations  % total cells with aberrations
 negative control saline  6/24/48  3/3/3  150  6/4/8  0/0/0  0/0/0  0/0/0
 3.75  6/24/48  5/5/5  250  7/4/6  0/0/0  0/0/0  0/0/0
 37.5  6/24/48  5/5/5  250  5/7/6  0.4/0/0  0/0/0  0/0/0
 375  6/24/48  5/5/5  250  5/6/4  0/0/0  0/0/0  0/0/0

 positive control

TEM 0.3

 48  5  250  4  0  22

 45

* time of kill after single gavage application (hours)

**percent cells in mitosis: 500 cells observed/animal

Table 2: Subacute study I (5 days) with 5 gavage dosings (3.75, 37.5 and 375 mg7kg bw/day): 

The negative control group and the low level test group contained no aberration. The intermediate level contained one cell with a reunion and one cell that  

was polyploid. The highest level contained three cells with breaks and one fragment. These were considered to be within the normal limits of the  

historical negative controls of the laboratory. Negative control was functional, no positive control.  

 dosage (mg/kg bw)#  no. of animals no. of cells per animal  mitotic index**  % cells with breaks  % cells with reunion  % cells with other aberrations % total cells with aberrations 
 negative control saline 3  150  6  0  0  0  0
 3.75 5  250  5  0  0  0  0
 37.5 5  250  5  0  0.4  0.4 (P)  0.8
 375 5  250  4  1.2  0  0.4 (f)  1.6

# dosage as gavage application 1x/day for 5 days

P - cells that have polyploidy

f - cells that have fragmentaion

** percent cells in mitosis: 500 cells observed/animal

Table 3: Acute study II: Adipic acid was administered at a single dose of 5000 mg/kg bw. The compound produced no aberrations except for 3 cells with polyploidy (2 in the 6-hour sample and 1 in the 24-hour). Neither the variety nor the number of these aberrations differed significantly from the negative controls (polyploidy observed in 4 cells). Negative and positive controls were functional.

 dosage (mg/kg bw)  time*  no. of animals no. of cells per animal  mitotic index**  % cells with breaks  % cells with other aberrations  % total cells with aberrations
 negative control saline  6/24/48  3/3/3  150  7.47/4.5/4.5  0/0/0  1pp/2pp/1pp  1 (0.66)/2 (1.33)/1 (0.66)
 5000  6/24/48  5/5/5  250  5.51/4.03/4.13  0/0/0  2pp/1pp/0  2 (0.8)/1 (0.4)/0

 positive control

TEM 0.3

24  5  250  1.62  4 (1.6)  >27 (10.8), 9f, 1pp

 86 (34.4)

P - cells that have polyploidy

f - cells that have fragmentaion

** percent cells in mitosis: at least 500 cells observed/animal

Table 4: Subacute study (5 days, 2500 mg/kg bw/day). Only 218 metaphases have been evaluated. The compound produced no aberrations except for 1 cell with polyploidy. Polyploidy was also observed in the negative control group. These are considered to be within the normal limits of the historical negative controls. Negative control was functional, no  positive 

control.

 dosage (mg/kg bw)#  no. of animals no. of cells per animal  mitotic index**  % cells with breaks  % cells with reunion  % cells with other aberrations % total cells with aberrations 
 negative control saline 3  150  5.33  0  0  1 (pp) (0.66)  1 (0.66)
 2500 5  218  2.98  0  0  1 (pp) (0.46)  1 (0.46)

P - cells that have polyploidy

f - cells that have fragmentaion

** percent cells in mitosis: 500 cells observed/animal

In summary, adipic acid can be considered non-mutagenic as measured by the cytogenetic test.

Applicant's summary and conclusion

Executive summary:

Adipic acid was not mutagenic in in vivo cytogenetic studies where groups of five male rats were gavaged with adipic acid doses up to 5000 mg/kg bw (acute studies) and with doses up to 2500 mg/kg bw/day (five-days subacute studies). 200 to 500 metaphase chromosomes of bone marrow cells per dose were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization and other chromosomal aberrations. The mitotic indices for all dose groups were considered to be within the normal limits of the controls and there was no evidence of chromosomal damage. The positive control groups, performed only during the acute studies, were functional (Litton Bionetics, Inc. 1974).

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