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Genetic toxicity in vitro

Description of key information

In vitro genetic toxicity data is available for n-hexane and structural analogue 5-80% n-hexane and presented in the dossier. This data is read across to hexane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

 

The genetic toxicity tests listed below had negative results for hexane:

 

Genetic Toxicity in vitro – Bacterial reverse mutation assay (OECD 471)

Genetic Toxicity in vitro – Mammalian Cell Gene Mutation Test (OECD TG 476)

The genetic toxicity tests listed below had negative results for 5 -80% n-hexane:

Genetic Toxicity in vitro – Mammalian Chromosome Aberration Test (OECD TG 473)

 

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because the study is well-documented and the publication which meets basic scientific principles.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 from aroclor 1254 induced male rat liver or Syrian hamster liver
Test concentrations with justification for top dose:
up to 1,000 ug/plate
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
E. coli WP2
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

The test substance was not mutagenic.
Executive summary:

This study examined the mutagenicity of the test substance n-hexane. Four strains of S. typhimurium were exposed to concentrations of test substance up to 1,000 ug/plate both with and without metabolic activation. The test substance was negative for mutagenicity both in the presence and absence of metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1989-08-24 to 1990-02-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it followed a protocol comparable to OECD Guideline 473.
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: supporting information
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
other: CHO-K1 cells
Metabolic activation:
with and without
Metabolic activation system:
S9 from aroclor induced rat liver
Test concentrations with justification for top dose:
0.015, 0.034, 0.074, 0.123, 0.416 ul/ml without S9
0.014, 0.022, 0.056, 0.118, 0.251 ul/ml with S9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: triethylenemelamine 0.5 ug/ml without S9, cyclophosphamide 50 ug/ml with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: 100 ul of dosing solutiong was added to the test medium


DURATION
- Preincubation period: 16-24 hrs at 37 degree C
- Exposure duration: 12 hrs without S9 at 37 degree in humidified air; 2 hrs with S9 at 37 degree in humidified air
- Expression time (cells in growth medium): For cells not treated with S9, two hours prior to cell harvest, treatment medium was removed and cells washed with PBS and refed with medium containing 0.1 ug/ml of Colcemid. For cells treated with S9, treatment medium was removed after exposure, cells were washed with PBS, refed, and returned to the incubator for 16 hrs. Colcemid was added at 0.1 ug/ml, and flasks incubated for two hrs.
- Fixation time (start of exposure up to fixation or harvest of cells): 14-20 hrs, collected by centrifugation



SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): 5% Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell cycle delay


OTHER EXAMINATIONS:
Chromatid and isochromatid breaks, exchange figures, chromosome breaks, fragments, pulverized chromosomes, chromatid and isochromatid gaps


OTHER:
Evaluation criteria:
In order for the test to be valid, there must be no more than 6% cells with chromosome aberrations in the negative and solvent control groups. Positive control must be statistically increased over untreated controls (p<=0.05, Fisher's exact test). A positive result is percentage of cells with aberrations was statistically increased over untreated controls (p<=0.05, Fisher's exact test).
Statistics:
Fisher's exact test was used to determine statistical significance. Cochran-Armitage test was used to test dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 0.074 ul/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity was observed at concentrations of 0.074 ul/ml or greater. No significant increase in chromosome aberrations was seen in treatment groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of CHO Chromosome Aberration Assay

Dose (µl/ml)

Mitotic Index

Cells Scored

Aberrations per cell (mean ± SD)

% Cells with Aberrations

Without S9

Negative Control

5.7

100

0.010 ± 0.100

1

Solvent Control

4.9

100

0.010 ± 0.100

1

0.015

4.8

100

0.000 ± 0.000

0

0.034

4.5

100

0.010 ± 0.100

1

0.074

2.9

100

0.010 ± 0.100

1

0.123

0.2

8

0.000 ± 0.000

0

0.416

0.0

0

Positive Control

2.2

100

0.360 ± 1.124

21

With S9

Negative Control

5.3

100

0.010 ± 0.100

1

Solvent Control

5.6

100

0.010 ± 0.100

1

0.014

5.3

100

0.010 ± 0.100

1

0.022

5.9

100

0.010 ± 0.100

1

0.056

3.1

100

0.010 ± 0.100

1

0.118

0.2

2

0.000 ± 0.000

0

0.251

0.0

0

Positive Control

2.5

100

0.840 ± 1.819

40

Conclusions:
Interpretation of results:
negative

The test substance is not clastogenic.
Executive summary:

This study examined the potential for commercial hexane to cause chromosome aberrations in Chinese Hamster Ovary (CHO) cells. CHO cells were exposed to concentrations of 0, 0.015, 0.034, 0.074, 0.123, and 0.416 ul/ml without metabolic activation and 0, 0.014, 0.022, 0.056, 0.118, and 0.251 ul/ml with metabolic activation. 0.5 ug/ml triethylenemelamine was used a positive control without metabolic activation and 50 ug/ml cyclophosphamide was used as a positive control with metabolic activation. Negative and positive controls were valid. There was no significant increase in chromosome aberrations in any test group. The test substance was cytotoxic at concentrations of 0.074 ul/ml or greater. The test substance is not clastogenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980-03-12 to 1980-07-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it was conducted in accordance with testing guideline OECD 476 and is GLP compliant.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
see below
Principles of method if other than guideline:
1. Laboratory Procedure for Assessing Specific Locus Mutations at the TK Locus in Cultured L5178Y Mouse Lymphoma Cells. D. Clive and J. Spector. Mutation Research 31_ (1975) 17-29.
2. A Mutational Assay System Using the Thymidine Kinase Locus in Mouse Lymphoma Cells. D. Clive, W.G. Flamm, M.R. Machesko and N.J. Bernheim. Mutation Research 16 (1972) 77-87.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
Experiment 1:
without activation: 80-150 µg/ml in 10 µg/ml increments
with activation: 40-180 µg/ml in 20 µg/ml increments

Experiment 2:
without activation: 105-140 µg/ml in 5 µg/ml increments
with activation: 10 µg/ml; 20-140 µg/ml in 20 µg/ml increments

Experiment 3:
without activation: 110-145 µg/ml in 5 µg/ml increments
with activation: 100-240 µg/ml in 20 µg/ml increments

Experiment 4:
without activation only: 110-200 µg/ml in 5 µg/ml increments
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): Trifluorothymidine


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 500,000 viable cells/ml (6 million precleansed TK +/- cells
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

Under the conditions of this study, the test substance was not considered mutagenic with or without activation.
Executive summary:

This study was done to determine the mutagenicity of the test substance n-hexane to mouse lymphoma cells. Mouse lymphoma L5178Y cells were exposed to concentrations of up to 200 µ g/plate of test substance both with and without metabolic activation. Under the conditions of this study, n-hexane did not exhibit a reproducible potential to induce forward mutation at the TK locus using Mouse lyphoma L5178Y cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo genetic toxicity data is available for n-hexane.

 

The genetic toxicity tests listed below had negative results for n-hexane.

 

Genetic Toxicity in vivo – Mammalian Bone Marrow Chromosome Aberration Test (OECD 475)

Genetic Toxicity in vivo – Rodent Dominant Lethal Test (equivalent/similar to OECD 478)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
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
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
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.)
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
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Soybean Oil) - Group A
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Group B
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group C
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group D
Dose / conc.:
5 000 mg/kg bw/day (nominal)
Remarks:
Group E
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)
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.
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)

N1

n

Number of cells with aberrations2

% cells with aberrations

Mitotic Index3

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)

N1

n

Number of cells with aberrations2

% cells with aberrations

Mitotic Index3

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:

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.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12/07/1988 - 19/09/1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
Only 1 dose level tested
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Vereniging van nederlandse Fabrikanten van Eetbare Olien en Vetten (VERNOF)
- Batch No.: UN nr. 1208 / 81393
- Purity: n-hexane 58.0% weight; benzene 0.060% weight (0.045% vol.)

Species:
rat
Strain:
Wistar
Remarks:
BOR; WISW (SPF, CpB)
Details on species / strain selection:
The rat is the species of choice according to regulatory requirements
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Versuchstierzucht GmbH & Co. KG, Borchen, F.R.G.
- Age at study initiation: 7 weeks
- Weight at study initiation: 170-200 g males; 125-155 g females
- Assigned to test groups randomly: yes by computer randomization and proportional to body weight
- Housing: 5 rats/cage, separated by sex in suspended stainless steel cages fitted with wire-mesh floor and front
- Diet (e.g. ad libitum): cereal-based stock diet for rats ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10 air changes/hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 19/07/1988 To: 21/07/1988

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: soya oil
- Amount of vehicle: 10.0 mL/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in soya bean oil at an appropriate concentration. The rats were dosed with 10.0 ml test solution per kg body weight. For each individual rat the dose was related to the body weight. The negative control groups were treated with the vehicle only (10.0 ml soya bean oil/kg body weight). The positive control group was treated once by intraperitoneal injection with mitomycin C (3.0 mg/kg body weight).
Duration of treatment / exposure:
1 day (single dose)
Frequency of treatment:
Single dose
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Negative Control (10.0 mL soya bean oil/kg body weight)
Dose / conc.:
5 000 mg/kg bw (total dose)
Remarks:
Test Material (Hexane)
Dose / conc.:
3 mg/kg bw (total dose)
Remarks:
Positive Control (Mitomycin C)
No. of animals per sex per dose:
15/sex (negative control and test group); 12/sex (positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: intraperitoneal injection
- Doses / concentrations: 3.0 mg/kg body weight
Tissues and cell types examined:
bone marrow cells from the femur
Details of tissue and slide preparation:
SAMPLING TIMES:

Samples were taken at 6, 24 and 48 hours after test substance administration. Two hours prior to sacrifice, animals were injected (i.p.) with colcemid (8 mg/kg body weight) to accumulate metaphase cells. Bone marrow cells were removed from the femur by flushing with phosphate buffered saline, exposed to a hypotonic solution and fixed with a 3:1 mixutre of methanol and glacial acetic acid.

DETAILS OF SLIDE PREPARATION:

The cells were spread on microscope slides, stained with a 2% Giemsa solution and embedded in DePeX. Two slides per animal were prepared. The slides were
randomized and coded.

METHOD OF ANALYSIS:

Per animal 50 well-spread metaphases, each containing 40-42 centromeres, were analysed for chromatid-type (gaps, breaks, fragments, interchanges) and chromosome-type (gaps, breaks, minutes, rings, dicentrics) aberratons and other anomalies (endoreduplication, polyploid cells, heavily damaged cells). The Vernier readings of all metaphases scored were recorded. Five hundred cells per slide were examined to determine the percentag of cells in mitosis (mitotic index).
Evaluation criteria:
A test substance producing neither a dose-related, statistically significant increase in the number of cells with structural chromosome aberrations, nor a statistically significant and reproducible positive response at any of the doses is considered non-clastogenic in this system.
Statistics:
Different types of aberrations (chromatid-type and chromosome-type) have been listed with the frequency of aberrant cells for each aberration type,
the number of cells analyzed, and the mitotic index for all animals of test and control groups.

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.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

 Table 1: Summary of chromosome aberrations in bone marrow cells of male rats

Dose

Harvest time (hours)

N1

n

Number of cells with aberrations2

% cells with aberrations

Mitotic Index3

gaps

breaks

exchanges

multiple

Incl. gaps

Excl. gaps

Soya bean oil

10 mL/kg

6

250

5

0

1

0

0

0.4

0.4

1.3

Technical hexane

5000 mg/kg

6

250

5

1

1

0

0

0.8

0.4

1.4

Mitomycin C

3 mg/kg

6

200

4

3

19***

11**

0

13.5***

10.5***

0.6

Soya bean oil

10 mL/kg

24

250

5

3

0

0

0

1.2

0.0

1.7

Technical hexane

5000 mg/kg

24

250

5

1

2

0

0

1.2

0.8

1.6

Mitomycin C

3 mg/kg

24

200

4

13*

45***

24***

1

28.5***

26.5***

1.0

Soya bean oil

10 mL/kg

48

250

5

2

1

0

0

1.2

0.4

1.9

Technical hexane

5000 mg/kg

48

250

5

2

2

0

0

1.6

0.8

1.6

Mitomycin C

3 mg/kg

48

200

4

4

13**

3

0

8.0**

7.0**

1.2

n = number of animals treated

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 2: Summary of chromosome aberrations in bone marrow cells of female rats

Dose

Harvest time (hours)

N1

n

Number of cells with aberrations2

% cells with aberrations

Mitotic Index3

gaps

breaks

exchanges

multiple

Incl. gaps

Excl. gaps

Soya bean oil

10 mL/kg

6

250

5

0

0

0

0

0.0

0.0

1.5

Technical hexane

5000 mg/kg

6

250

5

1

0

0

0

0.4

0.0

1.5

Mitomycin C

3 mg/kg

6

200

4

7*

10**

4

0

10.0***

7.0***

0.7

Soya bean oil

10 mL/kg

24

250

5

4

1

0

0

2.0

0.4

2.1

Technical hexane

5000 mg/kg

24

250

5

2

0

0

0

0.8

0.0

2.2

Mitomycin C

3 mg/kg

24

200

4

12*

58***

33***

1

36.0***

34.0***

1.0

Soya bean oil

10 mL/kg

48

250

5

2

5

0

0

2.8

2.0

1.5

Technical hexane

5000 mg/kg

48

250

5

1

0

0

0

0.4

0.0

1.8

Mitomycin C

3 mg/kg

48

200

4

6

11

0

0

7.5

5.5

2.1

n = number of animals treated

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:
Technical hexane did not induce structural chromosome aberrations in bone marrow cells of rats under the conditions employed in this study.
Executive summary:

In a chromosome aberration study, the test substance (light petroleum solvent (Technical hexane) for oil seed extraction) was administered once to Wistar derived (Bor: WISW (SPF Cpb)) rats (15/sex) via oral gavage at a dose of 5000 mg/kg in soya bean oil. Rats in the negative control group (15/sex) received a single dose of 10 mL soya bean oil/kg body weight via gavage and rats in the positive control group (12/sex) received an intraperitoneal injection of 3.0 mg mitomycin C/kg body weight. Two hours prior to sacrifice, all animals were weighed and injected with 8 mg colcemid/kg bw to accumulate metaphase cells. At 6, 24, and 48 hrs after dosing 5 rats/sex (treatment and vehicle control groups) and 4 rats/sex (positive control group) were sacrificed. 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, each containing 40-42 centromeres, were analysed for chromatid type and chromosome type aberrations and other anomalies. Technical hexane did not induce a statistically significant increase in structural chromosome aberrations at any time point examined. The positive control, mitomycin C, did induce the expected increase in structural chromosome aberrations. Under the conditions of this study, technical hexane did not induce structural chromosome aberrations in bone marrow cells of rats.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979-10-15 to 1980-05-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because although a GLP certificate was not provided the study seemed well-conducted.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Rodent Dominant Lethal Test)
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 11 weeks old
- Assigned to test groups randomly: yes


Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: filtered air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and plexiglass chambers of 0.25 cubic meter volume.
- Method of conditioning air: The chambers were operated in a dynamic mode using room air to dilute the test material.
- System of generating particulates/aerosols: The vapor was generated by bubbling dry, oil-free, breathing-quality air through a column of liquid test material in a fritted-glass cylinder gas washing bottle.
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 28.3 L per minute



TEST ATMOSPHERE
- Brief description of analytical method used: Analysis was initially performed using the Scott Model 216 Hydrocarbon Analyzer for the first two weeks. For the remainder of the study period, a Wilks Model 80 Computing Infrared Analyzer was used.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
8 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Post exposure period:
Following treatment, each male was rested for 2 days and then caged with two unexposed virgin females. At the end of 5 days, these females were removed. This weekly mating sequence was continued for 2 weeks. Each pair of mated females was transferred to a fresh cage, and approximately 14 days after the midweek of being caged with the male, these females were sacrificed. Their uterine contents were examined and scored for numbers of dead and living implants, and total implantations.
Remarks:
Doses / Concentrations:
0, 100, and 400 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
3 male mice per group (a total of 4 groups)
Control animals:
yes, concurrent vehicle
Positive control(s):
- triethylenemelamine
- Route of administration: intraperitoneal injection
Evaluation criteria:
Both pre- and post-implantation losses contribute to dominant lethality. The former is reflected in the total number of implantation sites per
pregnant female and strictly measured by the difference between the number of corpora lutea gravidus and the nunter of Implantation sites.
Toxic or physiological effects on sperm may also reduce the number of implantation sites. Therefore, unless subtle physiological effects on sperm can be discounted, pre-implantation loss is not as rigorous an indication of dominant lethality as post-Implantation loss. Corpora lutea are not evaluated in studies using mice.
Dominant lethality is typically determined from: a) a mutation index derived from the ratio of dead to total implants; or b) the number of dead Implants per pregnant female. In interpreting these values It must be remembered that the former measurement reflects both pre- and postimplantation
losses and that the ratio is affected by changes in either the numerator or the denominator. For this reason, the second parameter is perhaps a better indicator of post-implantation loss. This becomes especially so if one concurrently examines the number of living embryos per pregnant female. The two sets of data should be inversely related. In other words, if true dominant lethality is being observed, then a significant increase in the number of dead implants per pregnant female should be accompanied by a significant decrease in the number of living implants per pregnant female.
These ratios are compared with both concurrent and comparable historical control data for significant statistical differences. Dose-related
trends are also looked for, but may not always be found. For example, some compounds such as EMS tested in mice show a threshold value and
then a very steep rise. Certain portions of the response might be missed, depending on the spacing of the dose levels used.
Statistics:
A chi-square tost is used to compare each treatment group and positive control to negative control.

Armitage's trend for linear proportions is used to test whether the fertility index is linearly related to arithmetic or log dose.

The total number of implantations is evaluated by the Student's t-test to determine whether the average number of implantations per pregnant
female for ecch treatment group and the positive control group differs significantly from the negative control group. Dead implantations were evaluated in the same manner.

The average number of corpora lutea per pregnant female is evaluated by t-test to determine whether each treatment group differed significantly from the control group. A regression fit is made for both the arithmetic and logarithmic dose.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative

The test substance did not induce dominant lethal mutations in mice at the two tested dose levles. The sensitivity of the assay was confirmed by the significant induction of dominant lethal mutations in the positive control treated with TEM.
Executive summary:

This study determined the effect of inhalation exposure of n-hexane in a dominant lethal mutagenic assay. Groups of 3 male mice were exposed to 0, 100 and 400 ppm of test substance vapor for 6 hrs/day for 5 days for eight weeks. Triethylenemelamine was used as a positive control substance and administered via intraperitoneal injection. n-Hexane does not cause significant increases in either pre- or post- implantation loss of embryos when statistically compared with the negative control. The test substance did not induce dominant lethal mutations in mice at the two tested dose levels. The test substance is not mutagenic.

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity data is available for n-hexane/commercial hexane and structural analogue 5-80% n-hexane. This data is read across to hexane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

 

In Vitro

 

In vitro gene mutation study in bacteria

 

n-hexane

In a key in vitro genetic toxicity study the mutagenicity of n-hexane was examined (Dunnick, 1991; Klimisch score =1). Four strains of S. typhimurium were exposed to concentrations of test substance up to 1,000 ug/plate both with and without metabolic activation. The test substance was negative for mutagenicity both in the presence and absence of metabolic activation.

 

An in vitro genetic toxicity study (Mortelmans et al., 1986) examined the mutagenicity of the test substance hexane. Four strains of Salmonella typhimurium were exposed to concentrations of 0.000, 1.000, 3.300, 10.000, 33.000, 100.000, or 333.000 ug/plate of the test substance for 2 days.

After exposure, the number of revertant colonies was counted. Positive controls were 4-nitro-o-phenylenediamine for strain TA 98, 9-aminoacridine for strains TA 97 and TA 1537, sodium azide for strains TA 1535 and TA 100, and 2-aminoanthracene for all strains with metabolic activation. The test substance did not cause a significant increase in the number of revertant colonies as compared to negative controls. The test substance is not mutagenic.

 

An in vitro genetic toxicity study (Ishidate et al., 1984) examined the mutagenicity of the test substance hexane. Six strains of Salmonella typhimurium were exposed to concentrations of up to 10.0 mg/plate of the test substance for 2 days. After exposure, the number of revertant colonies was counted. The test substance did not cause a significant increase in the number of revertant colonies as compared to negative controls. The test substance is not mutagenic.

 

In a reverse gene mutation assay in bacteria (Phillips Petroleum Company, 1982), S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 were exposed to n-hexane in DMSO at concentrations of 22.6, 67.9, 203.7, 611.1, 1833.3, 5500 ug/plate in the presence and absence of mammalian metabolic activation using the plate-incorporation method. n-Hexane was tested up to limit concentrations (5000 μg/plate). The positive and negative controls induced the appropriate responses. There was no evidence of induced mutant colonies over background when exposed to n-hexane.

 

In vitro Chromosome Aberration in Mammalian Cells

 

5-80% n-hexane

This study examined the potential for commercial hexane to cause chromosome aberrations in Chinese Hamster Ovary (CHO) cells (Daughtrey, 1984). CHO cells were exposed to concentrations of 0, 0.015, 0.034, 0.074, 0.123, and 0.416 ul/ml without metabolic activation and 0, 0.014, 0.022, 0.056, 0.118, and 0.251 ul/ml with metabolic activation. 0.5 ug/ml triethylenemelamine was used a positive control without metabolic activation and 50 ug/ml cyclophosphamide was used as a positive control with metabolic activation. Negative and positive controls were valid. There was no significant increase in chromosome aberrations in any test group. The test substance was cytotoxic at concentrations of 0.074 ul/ml or greater. The test substance is not clastogenic.

 

In vitro Gene Mutation study in Mammalian Cells

 

n-hexane

In a key in vitro genetic toxicity study the mutagenicity of the test substance n-hexane to mouse lymphoma cells was determined (API, 1981; Klimisch score =1) . Mouse lymphoma L5178Y cells were exposed to concentrations of up to 200 µ g/plate of test substance both with and without metabolic activation. Under the conditions of this study, the test substance was not considered mutagenic with or without activation.

In a supporting in vitro genetic toxicity study the mutagenicity of n-hexane to mouse lymphoma cells was examined (Phillips Petroleum Company, 1982;Klimisch score =2). Mouse lymphoma L5178Y cells were exposed to concentrations of up to 500 ug/plate of test substance both with and without metabolic activation. A two-fold or greater increase in the number of mutations as compared to negative controls was seen at two treatment concentrations in the absence of metabolic activation. No significant increase in the number of mutations was seen in the presence of metabolic activation. Concentrations of test substance of 350 ug/plate or more were cytotoxic. The test substance is considered a weak mutagen in the absence of metabolic activation.

In Vivo

 

n-hexane/ technical hexane

In an in vivo chromosome aberration study (Til et al., 1989c), 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.

 

In a chromosome aberration study (de Vogel and Leeman, 1989), the test substance (light petroleum solvent (Technical hexane) for oil seed extraction) was administered once to Wistar derived (Bor: WISW (SPF Cpb)) rats (15/sex) via oral gavage at a dose of 5000 mg/kg in soya bean oil. Rats in the negative control group (15/sex) received a single dose of 10 mL soya bean oil/kg body weight via gavage and rats in the positive control group (12/sex) received an intraperitoneal injection of 3.0 mg mitomycin C/kg body weight. Two hours prior to sacrifice, all animals were weighed and injected with 8 mg colcemid/kg bw to accumulate metaphase cells. At 6, 24, and 48 hrs after dosing 5 rats/sex (treatment and vehicle control groups) and 4 rats/sex (positive control group) were sacrificed. 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, each containing 40-42 centromeres, were analysed for chromatid type and chromosome type aberrations and other anomalies. Technical hexane did not induce a statistically significant increase in structural chromosome aberrations at any time point examined. The positive

control, mitomycin C, did induce the expected increase in structural chromosome aberrations. Under the conditions of this study, technical hexane

did not induce structural chromosome aberrations in bone marrow cells of rats.

 

In a key in vivo genetic toxicity study the effect of inhalation exposure of n-hexane in a dominant lethal mutagenic assay was examined (API, 1980; Klimisch score =1). Groups of 3 male mice were exposed to 0, 100 and 400 ppm of test substance vapor for 6 hrs/day for 5 days for eight weeks. The test substance did not induce dominant lethal mutations in mice at the two tested dose evels. The sensitivity of the assay was confirmed by the significant induction of dominant lethal mutations in the positive control treated with TEM.

 

In an in vivo micronucleus assay (Til et al., 1989d), 20 females were administered 0 or 5000 mg/kg technical hexane by gavage, seven days a week for 13 weeks. The positive control group animals were treated once, 24 hrs prior to sacrifice, with 3.0 mg Mitomycin C/kg bw. Bone marrow smears were prepared from bone marrow cells of the femur. Polychromatic erythrocytes were examined for micronuclei and the ratio of polychromatic erythrocytes to normochromatic erythrocytes was recorded. The incidence of micronucleated polychromatic erythrocytes in the 5000 mg/kg rats was found to be slightly higher than controls, but not statistically significantly higher. Therefore, no toxicological significance is attached to this finding. There was no statistically significant difference in PE:NE ratio between the 5000 mg/kg group and the controls.

 

Commercial hexane

This study determined the effect of inhalation exposure of commercial hexane on rat bone marrow. Groups of 5 male and 5 female rats were exposed to 0, 900, 3000, and 9000 ppm of test substance vapor for 6 hrs/day for 5 days. 0.5 mg/kg triethylenemelamine was used as a positive control substance. Animals were sacrificed 3 or 21 hrs after exposure, and the bone marrow from their femurs examined for cell aberrations. There was no statistically significant increase in cell aberrations in any treatment group. The test substance is not mutagenic.

Justification for classification or non-classification

The negative results in in vitro and in vivo genotoxicity assays from n-hexane and structural analogues do not warrant the classification of n-hexane as genotoxic under the new Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).