[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30/08/1988 - 01/02/1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30/08/1988 - 01/02/1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: The test material did not induce structural chromosome aberrations.

Additional information on results:

Both sexes examined for all dose levels except for the top dose (5000 mg/kg bw) where only males were examined. Data on body weight, food intake and water intake obtained from the rats of the positive control group for the cytogenic study were essentially the same as those obtained in the negative controls

RESULTS OF DEFINITIVE STUDY

I] Chromosome Aberration Test:
No statistically significant differences in the number of bone marrow cells with structural chromosome aberrations were observed in any of the test groups when compared to the vehicle controls. The mitotic index obtained for the various groups did not show a dose-related difference from the vehicle control value while the positive control substance, mitomycin C, showed the expected statistically significant increase in the number of cells with structural chromosome aberrations.

Table 2. Summary of chromosome aberrations in bone marrow cells of male rats

Dose (per kg/bw)	Harvest time (days)	N^1	n	Number of cells with aberrations^2				% cells with aberrations		Mitotic Index^3
				gaps	breaks	exchanges	multiple	Incl. gaps	Excl. gaps
Soya bean oil 10 mL/day	90	500	10	13	4	0	0	3.4	0.8	2.3
Technical hexane 40 mg/day	90	500	10	16	5	0	0	4.0	1.0	1.8
Technical hexane 200 mg/day	90	400	8	10	4	0	0	3.5	1.0	2.0
Technical hexane 1000 mg/day	90	500	10	13	5	0	0	3.2	1.0	1.8
Technical hexane 5000 mg/day	90	350	7	12	9	0	0	5.1	2.6	1.9
Mitomycin C 3 mg	1	250	5	32***	60***	27***	0	32.4***	28.8***	1.6

n = number of animals examined

Statistics: Fisher’s exact probability test (two-sided): * p<0.05; ** p<0.01; *** p<0.001

1. N = number of metaphases analysed (50 metaphases per animal)

2. gaps: include chromatid and isochromatid (chromosome) gaps; breaks: include chromatid and isochromatid (chromosome) breaks, interstitial deletions (minutes) and acentric fragments not associated with any obvious exchange process; exchanges: include chromatid and chromosome inter- and intrachanges; multiple aberrations: more than 10 aberrations (excl. gaps) per metaphase

3. mean percentage of metaphases determined in 1000 nuclei per animal

 

Table 3. Summary of chromosome aberrations in bone marrow cells of female rats

Dose (per kg/bw)	Harvest time (days)	N^1	n	Number of cells with aberrations^2				% cells with aberrations		Mitotic Index^3
				gaps	breaks	exchanges	multiple	Incl. gaps	Excl. gaps
Soya bean oil 10 mL/day	90	400	8	12	2	0	0	3.5	0.5	2.2
Technical hexane 40 mg/day	90	500	10	18	4	0	0	4.2	0.8	2.0
Technical hexane 200 mg/day	90	500	10	11	7	0	0	3.6	1.4	2.6
Technical hexane 1000 mg/day	90	450	9	8	3	1	0	2.7	0.9	2.3
Mitomycin C 3 mg	1	150	3	16	52***	19***	0	43.3***	40.7***	1.2

n = number of animals examined

Statistics: Fisher’s exact probability test (two-sided): * p<0.05; ** p<0.01; *** p<0.001

1. N = number of metaphases analysed (50 metaphases per animal)

2. gaps: include chromatid and isochromatid (chromosome) gaps; breaks: include chromatid and isochromatid (chromosome) breaks, interstitial deletions (minutes) and acentric fragments not associated with any obvious exchange process; exchanges: include chromatid and chromosome inter- and intrachanges; multiple aberrations: more than 10 aberrations (excl. gaps) per metaphase

3. mean percentage of metaphases determined in 1000 nuclei per animal

Conclusions:

There was no evidence that technical hexane induced structural chromosome aberrations in bone marrow cells of rats in vivo.

Executive summary:

This data is being read across from the source study that tested technical hexane based on analogue read across.

In an in vivo chromosome aberration study,the test material (Technical hexane (hexane food grade) containing 58% n-hexane) was administered once daily to Wistar derived (Bor: WISW (SPF Cpb)) rats (10/sex/dose, except for the highest dose group where only males were examined) via oral gavage at doses of 0, 40, 200, 1000 or 5000 mg/kg/bw/day in soya bean oil for 13 weeks.The positive control group animals (5/sex) were treated once, 24 hrs prior to sacrifice, with 3.0 mg mitomycin C/kg bw. Two hours prior to sacrifice, all animals were weighed and injected with 8 mg colcemid/kg bw to accumulate metaphase cells. Animals were sacrificed via decapitation. Bone marrow cells obtained from the femur were fixed in 3:1 methanol and glacial acetic acid, spread on microscope slides, stained with 2% Giemsa solution and embedded in DePeX. Per animal, two slides were prepared and 50 metaphases containing 40-42 centromeres were examined. The slides were examined for chromatid- and chromosome-type aberrations and for other anomalies. Five hundred cells per slide were examined to determine the mitotic index. There was no evidence that technical hexane induced structural chromosome aberrations in bone marrow cells of rats in vivo.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

minor deviations from the protocol are not considered to have influenced the integrity or outcome of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N-hexane

EC Number:

203-777-6

EC Name:

N-hexane

Cas Number:

110-54-3

Molecular formula:

C6H14

IUPAC Name:

hexane

Details on test material:

- Analytical purity:
Technical grade
- Composition of test material, percentage of components:
n-hexane - 58%; benzene 0.060% weight (0.045% vol.)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Shell, Amsterdam, The Netherlands
- Purity: n-hexane 58.0% weight; benzene 0.060% weight (0.045% vol.)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar derived, Bor:WISW (SPF Cpb)

Details on species / strain selection:

The rat was used because this species is considered the most suitable for this type of study and is usually required by regulatory agencies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Institute for the Breeding of Laboratory Animals GmbH & Co. KG, Borchen, F.R.G.
- Age at study initiation: 3-4weeks upon arrival
- Weight at study initiation: 66.6 g (males)/ 63.0 g (females)
- Housing: groups of 5 separated by sex, in suspended, stainless steel cages fitted with wire screen bottom and front
- Diet (e.g. ad libitum): cereal-based open-formula stock diet provided ad libitum
- Water (e.g. ad libitum): tap water provided ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 50-70 %
- Air changes (per hr): 10 air changes/hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 30 August 1988 To: 2 December 1988

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: soya oil
- Concentration of test material in vehicle: 0.4, 2, 10 or 50% (w/v)
- Amount of vehicle: 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test substance in soybean oil containing 0.4, 2, 10 or 50% (w/ v) were freshly prepared once every week. The test solutions were stored in closed glass bottles in a refrigerator at about 5°C.

Each of the rats was given daily a single dose (10 ml/kg body weight) of the appropriate solution into the stomach by means of a syringe. The controls (groups A and F) received 10 ml soybean oil/kg body weight/day. The amount of test substance administered to each rat was adapted once a week to the change in body weight .

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (Soybean Oil) - Group A

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Group B

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group C

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group D

Doses / concentrations 5

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

Group E

Doses / concentrations 6

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group F (Positive Control)

No. of animals per sex per dose:

10/sex/dose based on number of surviving animals in each dose group (See Table 1)

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Route of administration: intraperitoneal injection
- Doses / concentrations: 3.0 mg/kg body weight (1 injection 24 hrs prior to sacrifice)

Examinations

Tissues and cell types examined:

Bone marrow cells from the femur

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The number of surviving animals in each group of those designated for cytogenicity examination are given in Table 1. All these animals were used for metaphase chromosomal analysis. Group F group was added to the experiment, and used as a positive control in the cytogenetic study.

The rats of the positive control group were treated once, 24 hours prior to sacrifice, by intraperitoneal injection of 3.0 mg mitomycin C/ kg body weight to examine the sensitivity of the strain of rats used in this chromosome analysis test.

Two hours prior to sacrifice, the animals of the test groups and the positive and negative controls were weighed and then injected (i.p.) with 8 mg colcemid/kg body weight to accumulate metaphase cells. The animals were killed by decapitation. Then, the femurs were freed from adherent tissues. Bone marrow cells were removed from the femur by flushing with phosphate-buffered saline (pH 7.4), exposed to a hypotonic solution (0.075 M KCl) and fixed with 3:1 mixture of methanol and glacial acetic acid.

DETAILS OF SLIDE PREPARATION:

Cells were spread on microscope slides, stained with a 2% Giemsa (Merck, Darmstadt, F.R.G.) solution and embedded in DePeX. Two slides per animal were prepared for chromosome aberration analysis. The slides were randomized and coded to enable "blind" scoring.

METHOD OF ANALYSIS:

Per animal, 50 well-spread metaphases each containing 40-42 centromeres, were analysed for chromatid-type aberrations (gaps, breaks, fragments, interchanges) and chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics), and for other anomalies (endoreduplication, polyploid cells, heavily damaged cells). The Vernier readings of all metaphases scored were recorded. Five hundered cells per slide were examined to determine the percentage of cells in mitosis (mitotic index).

Evaluation criteria:

A test substance producing neither a statistically significant dose-related increase in the number of cells with structural chromosome aberrations, nor a statistically significant and reproducible positive response at any of the doses was considered non-clastogenic in this system.

Statistics:

Chromosome Aberration test:
For statistical analysis of data aberration classes were combined into the general categories of chromatid- and chromosome type deletions (breaks), chromatid exchanges and chromosome exchanges. Data were analysed using Fisher's exact probability test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: The test material did not induce structural chromosome aberrations.

Additional information on results:

Both sexes examined for all dose levels except for the top dose (5000 mg/kg bw) where only males were examined. Data on body weight, food intake and water intake obtained from the rats of the positive control group for the cytogenic study were essentially the same as those obtained in the negative controls

RESULTS OF DEFINITIVE STUDY

I] Chromosome Aberration Test:
No statistically significant differences in the number of bone marrow cells with structural chromosome aberrations were observed in any of the test groups when compared to the vehicle controls. The mitotic index obtained for the various groups did not show a dose-related difference from the vehicle control value while the positive control substance, mitomycin C, showed the expected statistically significant increase in the number of cells with structural chromosome aberrations.

Any other information on results incl. tables

Table 2. Summary of chromosome aberrations in bone marrow cells of male rats

Dose (per kg/bw)	Harvest time (days)	N^1	n	Number of cells with aberrations^2				% cells with aberrations		Mitotic Index^3
				gaps	breaks	exchanges	multiple	Incl. gaps	Excl. gaps
Soya bean oil 10 mL/day	90	500	10	13	4	0	0	3.4	0.8	2.3
Technical hexane 40 mg/day	90	500	10	16	5	0	0	4.0	1.0	1.8
Technical hexane 200 mg/day	90	400	8	10	4	0	0	3.5	1.0	2.0
Technical hexane 1000 mg/day	90	500	10	13	5	0	0	3.2	1.0	1.8
Technical hexane 5000 mg/day	90	350	7	12	9	0	0	5.1	2.6	1.9
Mitomycin C 3 mg	1	250	5	32***	60***	27***	0	32.4***	28.8***	1.6

n = number of animals examined

Statistics: Fisher’s exact probability test (two-sided): * p<0.05; ** p<0.01; *** p<0.001

1. N = number of metaphases analysed (50 metaphases per animal)

2. gaps: include chromatid and isochromatid (chromosome) gaps; breaks: include chromatid and isochromatid (chromosome) breaks, interstitial deletions (minutes) and acentric fragments not associated with any obvious exchange process; exchanges: include chromatid and chromosome inter- and intrachanges; multiple aberrations: more than 10 aberrations (excl. gaps) per metaphase

3. mean percentage of metaphases determined in 1000 nuclei per animal

 

Table 3. Summary of chromosome aberrations in bone marrow cells of female rats

Dose (per kg/bw)	Harvest time (days)	N^1	n	Number of cells with aberrations^2				% cells with aberrations		Mitotic Index^3
				gaps	breaks	exchanges	multiple	Incl. gaps	Excl. gaps
Soya bean oil 10 mL/day	90	400	8	12	2	0	0	3.5	0.5	2.2
Technical hexane 40 mg/day	90	500	10	18	4	0	0	4.2	0.8	2.0
Technical hexane 200 mg/day	90	500	10	11	7	0	0	3.6	1.4	2.6
Technical hexane 1000 mg/day	90	450	9	8	3	1	0	2.7	0.9	2.3
Mitomycin C 3 mg	1	150	3	16	52***	19***	0	43.3***	40.7***	1.2

n = number of animals examined

Statistics: Fisher’s exact probability test (two-sided): * p<0.05; ** p<0.01; *** p<0.001

1. N = number of metaphases analysed (50 metaphases per animal)

2. gaps: include chromatid and isochromatid (chromosome) gaps; breaks: include chromatid and isochromatid (chromosome) breaks, interstitial deletions (minutes) and acentric fragments not associated with any obvious exchange process; exchanges: include chromatid and chromosome inter- and intrachanges; multiple aberrations: more than 10 aberrations (excl. gaps) per metaphase

3. mean percentage of metaphases determined in 1000 nuclei per animal

Applicant's summary and conclusion

Conclusions:

There was no evidence that technical hexane induced structural chromosome aberrations in bone marrow cells of rats in vivo.

Executive summary:

In an in vivo chromosome aberration study,the test material (Technical hexane (hexane food grade) containing 58% n-hexane) was administered once daily to Wistar derived (Bor: WISW (SPF Cpb)) rats (10/sex/dose, except for the highest dose group where only males were examined) via oral gavage at doses of 0, 40, 200, 1000 or 5000 mg/kg/bw/day in soya bean oil for 13 weeks.The positive control group animals (5/sex) were treated once, 24 hrs prior to sacrifice, with 3.0 mg mitomycin C/kg bw. Two hours prior to sacrifice, all animals were weighed and injected with 8 mg colcemid/kg bw to accumulate metaphase cells. Animals were sacrificed via decapitation. Bone marrow cells obtained from the femur were fixed in 3:1 methanol and glacial acetic acid, spread on microscope slides, stained with 2% Giemsa solution and embedded in DePeX. Per animal, two slides were prepared and 50 metaphases containing 40-42 centromeres were examined. The slides were examined for chromatid- and chromosome-type aberrations and for other anomalies. Five hundred cells per slide were examined to determine the mitotic index. There was no evidence that technical hexane induced structural chromosome aberrations in bone marrow cells of rats in vivo.