Hexan-6-olide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Older proprietary study, commissioned by the FDA.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1971

Report date:

1974

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was an in vivo cytogenetic assay in rat bone marrow, acute and subacute treatment protocols were included.

GLP compliance:

no

Remarks:

: older study, pre-dates GLP

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Adipic acid

EC Number:

204-673-3

EC Name:

Adipic acid

Cas Number:

124-04-9

Molecular formula:

C6H10O4

Details on test material:

The test material, adipic acid (referred to as Compound FDA 71-50), of food processing quality, was supplied byt the Food and Drug Administration.
A read across is proposed based on structural similarities between the substances.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

The test animals were 10-12 weeks old male Sprague-Dawley CD rats, obtained from a closed colony (random bred) at Flow Laboratories. The rats had a weighed 280-350 g. They were fed a commercial 4% fat diet and water ad libitum.
Animals were held in quarantine for 4-11 days. Rats were housed singly or in groups of up to 5. Individuals were identified by ear punch.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Saline (0.85%).

Details on exposure:

Acute study 1: single doses of 0, 3.75, 37.5 and 375 mg/kg adipic acid in saline were administered by gavage to groups of rats.
Subacute study 1: doses of 0, 3.75, 37.5 and 375 mg/kg adipic acid in saline were administered daily by gavage for 5 days.

Acute study 2: single doses of 0 and 5000 mg/kg adipic acid in saline were administered by gavage to groups of rats.
Subacute study 2: doses of 0 and 2500 mg/kg adipic acid in saline were administered daily by gavage for 5 days.

Duration of treatment / exposure:

Acute study: single dose administration
Subacute study: 5 days

Frequency of treatment:

Acute study: single dose administration
Subacute study: daily for 5 days

Post exposure period:

Acute study: rats were sacrificed at 6, 24 and 48 hours after exposure.
Subacute study: rats were sacrificed 6 hours after the last dose.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 3.75, 37.5 and 375 mg/kg bw
Basis:
actual ingested
acute and subacute studies 1

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0 and 5000 mg/kg bw
Basis:
actual ingested
acute study 2

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0 and 2500 mg/kg bw
Basis:
actual ingested
subacute study 2

No. of animals per sex per dose:

Acute study 1: 5 male rats/time point (15/dose); 3/time point (9/dose) for the vehicle controls. 5 rats were treated with the positive control substance.
Subacute study 1: 5 male rats/dose, 3 negative controls.
Acute study 2: 5 male rats/time point, 3 negative controls/time point and 5 positive controls.
Subacute study 2: 5 treated rats and 3 negative controls.

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylene Melamine (TEM). 5 rats/acute study received a single dose by gavage of TEM at 0.3 mg/kg bw, and were sacrificed 48 hours later.

Examinations

Tissues and cell types examined:

Bone marrow (femur).

Details of tissue and slide preparation:

Two hours before sacrifice, each animal was given 4 mg/kg colcemid intraperitoneally in order to arrest the bone marrow cells in C-mitosis.
The rats were sacrificed using CO2 inhalation, and the adhering muscle and epiphysis of one femur were removed. The marrow was removed using a tuberculin syring and an 18 gauge needle, and aspirated into 5 ml of Hank's balanced salt solution. The samples were then centrifuged at 1500 rpm in a table-top centrifuge for 5 minutes, decanted, and 2 ml of hypotonic 0.5% KCl solution was added to resuspend the cells. The specimens were then placed in a 37°C water bath for 20 minutes to swell the cells. The sample were centrifuged again, and the supernatant decanted and 2 ml of fixative (3:1 absolute methanol:glacial acetic acid). The cells were resuspended in the fixative and placed at 4°C for 30 minutes. The samples were centrifuged a third time, decanted, 2 ml of prepared fixative was added and the cells were resuspended and placed at 4°C overnight.
The following day the samples were centrifuged, decanted and 0.3-0.6 ml of freshly prepared fixative was added to obtain a suitable density. The cellls were resuspended and 2-3 drops of the suspension were allowed to drop onto a clean, dry slide held at 15° from horizontal. As the suspension flowed to the edge of the slide, it was ignited by an alcohol burner and allowed to flame. The slides were then allowed to dry at room temperature overnight. Duplicate slides were prepared. The slides were stained using a 5% Giemsa solution for 20 minutes, rinsed in acetone, 1:1 acetone:xylene, and placed in fresh xylene for 30 minutes. The slides were mounted and covered. The slides were examined microscopically.
The chromosomes of each cell were counted and only diploid cells were analysed.

Evaluation criteria:

The cells were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than 10 aberrations, polyploidy, pulverization, and any other chromosomal aberrations which were observed. Fifty metaphases 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.

Statistics:

No statistical information available.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Acute study 1 (Table 1): The negative control groups cells contained no aberrations. The compound produced no aberrations except for one cell containing a break in the 6-hour sample of the 37.5 mg/kg bw group. The expected severe chromosomal damage was observed in the positice control group. The mitotic indices were within normal limits.

Subacute study 1 (Table 2): The negative control group and the 3.75 mg/kg bw group contained no aberrations. The 37.5 mg/kg bw group contained one cell with a reunion and one cell with that was polyploidy. The 375 mg/kg group contained three cells with breaks and one fragment. These were considered to be within normal limits of the historical negative controls.

Acute study 2 (Table 3) and subacute study 2 (Table 4): Neither the variety nor the number of chromosomal aberrations differed significantly from the negative controls.

Any other information on results incl. tables

Table 1. Metaphase summary data from the first acute study

Treatment Group	Time of kill after dosing	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks	% Cells with Reunion	% Cells other Aberrations**	% Cells with Aberrations ***
Negative (saline) Control	6	3	150	6	0	0	0	0
	24	3	150	4	0	0	0	0
	48	3	150	8	0	0	0	0
Adipic Acid 3.75 mg/kg	6	5	250	7	0	0	0	0
	24	5	250	4	0	0	0	0
	48	5	250	6	0	0	0	0
Adipic Acid 37.5 mg/kg	6	5	250	5	0.4	0	0	0.4
	24	5	250	7	0	0	0	0
	48	5	250	6	0	0	0	0
Adipic Acid 375 mg/kg	6	5	250	5	0	0	0	0
	24	5	250	6	0	0	0	0
	48	5	250	4	0	0	0	0
Positive control TEM		5	250	4	0	23	22(a)	45

* Percent of cells in mitosis: 500 cells observed/animal

**Cells that have polyploidy (p), pulverisation (pp), or greater than 10 aberrations (a)

*** Duplicate aberrations in a single cell will cause this to be a % less than a summation of the % aberration seen.

Table 2. Metaphase summary data from the first subacute study

Treatment Group	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks	% Cells with Reunion	% Cells other Aberrations**	% Cells with Aberrations
Negative (saline) Control	3	150	6	0	0	0	0
Adipic Acid 3.75 mg/kg	5	250	5	0	0	0	0
Adipic Acid 37.5 mg/kg	5	250	5	0	0.4	0.4 (p)	0.8
Adipic Acid 375 mg/kg	5	250	4	1.2	0	0.4 (f)	1.6

* Percent of cells in mitosis: 500 cells observed/animal

**Cells that have polyploidy (p), pulverisation (pp), fragments (f), or greater than 10 aberrations (a)

Table 3. Metaphase summary data from the second acute study

Treatment Group	Time of kill after dosing	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks**	% Cells with Reunion**	% Cells other Aberrations**+	% Cells with Aberrations
Adipic Acid 5000 mg/kg	6	5	250	5.51	0	0	2p (0.8)	9 (0.8)
	24	5	250	4.03	0	0	1p (0.4)	1 (0.4)
	48	5	250	4.13	0	0	0	0
Negative (saline) Control	6	3	150	7.47	0	0	1p (0.66)	1 (0.66)
	24	3	150	4.50	0	0	2p (1.33)	2 (1.33)
	48	3	150	4.50	0	0	1p (0.66)	1 (0.66)
Positive Control (TEM)	24	5	250	1.62	4 (1.6)	50 (20.0)	>27 (10.8); 9f (3.6); 1p (0.4)	86 (34.4)

* Percent of cells in mitosis: 500 cells observed/animal

**Numbers in parentheses are % aberrations per total cells counted

+ Symbols: > = greater than 10 aberrations per cell, polyploidy (p), fragments (f)

Table 4. Metaphase summary data from the second subacute study

Treatment Group	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks**	% Cells with Reunion**	% Cells other Aberrations**	% Cells with Aberrations
Adipic Acid 2500 mg/kg	5	218	2.98	0	0	1p (0.46)	1 (0.46)
Negative (saline) Control	3	150	5.33	0	0	1p (0.66)	1 (0.66)

* Percent of cells in mitosis: 500 cells observed/animal

**Numbers in parentheses are % aberrations per total cells counted

polyploidy (p)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
No evidence of clastogencitiy was seen under the conditions of this study.

Executive summary:

The cytogenic potential of adipic acid was evaluated in an in vivo chromosome aberration assay (the study was conducted in two separate experiments). The test substance was administered to male rats by gavage at single doses of 0, 3.75, 37.5, 375 and 5000 mg/kg bw, or at five consecutive daily doses of 0, 3.75, 37.5, 375 and 2500 mg/kg bw. Negative controls (saline) and positive controls (triethylene melamine) were included. Rats were sacrificed at varying intervals after dosing, and bone marrow cells were investigated microscopically for chromosome aberrations. There were no increases, relative to negative controls, in the incidence of chromosome aberrations, and all values were within normal limits. No evidence of clastogencitiy was seen under the conditions of this study.