Hydrocarbons, C13-16

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 August to 21 September 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Few details on test material (no certificate of analysis)

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 vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

10 August to 21 September 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Few details on test material (no certificate of analysis)

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

No certificate of analysis

Principles of method if other than guideline:

Guideline principles

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A culture medium supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin and streptomycin, at about 37°C, in an atmosphere of about 5% C02 in air.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 from male Sprague-Dawley rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Range finding assay: half-log series of concentrations of 0.0835 to 2500 µg/mL
Main experiment:
- without metabolic activation: 3.13, 6.26, 9.35 and 12.5 µg/mL with 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL with 20-h harvest
- with metabolic activation: 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test material was insoluble in water and dimethylsulfoxide. A clear and homogeneous stock solution of 201 mg/mL with ethanol could be maintained.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: without metabolic activation: 7.25 and 17 h for 10 and 20 h assay, respectively; with metabolic activation: 2 h
- Expression time (cells in growth medium): with metabolic activation: 7.75 and 17.75 h for 20 and 10 h assay, respectively;
- Time in 0.1 µg/mL Colcemid: without metabolic activation: 1 and 0.5 h for 20 and 10 h assay, respectively; with metabolic activation: 2.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 10 h and 20 h without and without metabolic activation

STAIN (for cytogenetic assays): 5% Giemsa solution and BrdUrd (5-bromodeoxyuridine) at 10 µM

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 cells for test substance; at least 25 cells for positive controls

CYTOTOXICITY: visual observations based on confluence of monolayer and floating dead cells

Evaluation criteria:

Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 were analyzed.
The following factors were taken into account in the evaluation of the chromosomal aberrations data: the overall chromosomal aberration frequencies, the percentage of cells with any aberrations, the percentage of cells with more than one aberration, any evidence for increasing amounts of damage with increasing dose.
Chromatid and isochromatid gaps were not considered as they may be due to toxicity.

Statistics:

Fisher's exact test with an adjustment of multiple comparisons

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Range-finding without metabolic activation:
A very unhealthy cell monolayer, -70% reduction in the cell monolayer confluence, floating dead cells, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 25.0 µg/mL. Slight reductions in the number of visible mitotic cells and -15% reduction in the cell monolayer confluence were observed in the cultures dosed with 2.50 and 8.35 µg/mL.
Range-finding with metabolic activation:
An unhealthy cell monolayer, -85% reduction in the cell monolayer confluence, floating dead cells and debris, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 835 µg/mL. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 25.0 and 83.5 µg/mL.

Chromosomal aberrations assay without metabolic activation (Table 1):
In the 10 h assay, no toxicity was observed in any of the test cultures. These cultures were not analyzed for chromosomal aberrations as four dose levels were available for analysis from the 20 h assay. In the 20 h assay, an unhealthy cell monolayer, -70% and -45 % reduction in the cell monolayer confluence, floating dead cells and debris, and a severe reduction in visible mitotic cells were observed at 75.0 and 50.0 µg/mL, respectively. Toxicity was evident on the slides prepared from these cultures by the very sparse numbers of metaphases available for analysis.

Chromosomal aberration assay with metabolic activation (Tables 2 and 3):
In the 10 h assay, slight reductions in the numbers of visible mitotic cells were observed in the cultures dosed at 563 and 751 µg/mL.
In the 20 h assay, severe toxicity was exhibited on the slides prepared from the cultures dosed with 562 and 750 µg/mL by the presence of many dead cells and the sparse numbers of metaphases available for analysis. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 99.7, 187, 281, 375, 562, and 750 µg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 without metabolic activation (results from pooled duplicate cultures)

		Number and type of aberration
		Not computed		Simple	Complex	% cells with aberrations
	Concentration (µg/mL)	Chromatid gap	Chromosome gap
Negative (vehicle)	-	7	1			0.0
Positive (Mitomycin C)	0.04	7		4	7	28.0*
Test article	25.0	15	2			0.0
	37.5	7	3	1	1	0.5
	50.0	8	1		1	0.5
	75.0	19	2	4		0.5

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 2: Chromosome aberrations in CHO cells fixed 10 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

	Concentration (µg/mL)	Number and type of aberration
		Not computed		Simple	Complex	% cells with aberrations
		Chromatid gap	Chromosome gap
Negative (vehicle)	-	2				0.0
Positive (Cyclophosphamide)	25.0	1		8	13	44.0*
Test article	282	7	1			0.0
	375	3			1	0.5
	563	4			1	0.5
	751	3		3		1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 3: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

	Concentration (µg/mL)	Number and type of aberration
		Not computed		Simple	Complex	% cells with aberrations
		Chromatid gap	Chromosome gap
Negative (vehicle)	-	7		1	1	0.0
Positive (Cyclophosphamide)	12.5	1		17	31	80.0*
Test article	281	15	2		1	1.0
	375	16	6	1	1	1.0
	562	3	1	1	1	1.0
	750	10		1	1	1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Conclusions:

Interpretation of results:
negative

MRD-90-843 was found not to increase chromosome aberrations in CHO cells with and without metabolic activation.

Executive summary:

In an in vitro chromosome aberration test, Chinese Hamster Ovary cells were exposed to MRD-90-843 at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation.

Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, MRD-90-843 is not classified according to the criteria of Annex VI to Directive 67/548/EEC and the CLP Regulation (1272/2008).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

No certificate of analysis

Principles of method if other than guideline:

Guideline principles

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, < 2% aromatics

IUPAC Name:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, < 2% aromatics

Constituent 2

Reference substance name:

MRD-90-843

IUPAC Name:

MRD-90-843

Details on test material:

- Name of test material (as cited in study report): MRD-90-843
- Substance type: Petroleum product, UVCB
- Physical state: clear, colorless liquid
- Lot/batch No.: batch I
- Purity: 100% commercial product

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A culture medium supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin and streptomycin, at about 37°C, in an atmosphere of about 5% C02 in air.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 from male Sprague-Dawley rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Range finding assay: half-log series of concentrations of 0.0835 to 2500 µg/mL
Main experiment:
- without metabolic activation: 3.13, 6.26, 9.35 and 12.5 µg/mL with 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL with 20-h harvest
- with metabolic activation: 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test material was insoluble in water and dimethylsulfoxide. A clear and homogeneous stock solution of 201 mg/mL with ethanol could be maintained.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: without metabolic activation: 7.25 and 17 h for 10 and 20 h assay, respectively; with metabolic activation: 2 h
- Expression time (cells in growth medium): with metabolic activation: 7.75 and 17.75 h for 20 and 10 h assay, respectively;
- Time in 0.1 µg/mL Colcemid: without metabolic activation: 1 and 0.5 h for 20 and 10 h assay, respectively; with metabolic activation: 2.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 10 h and 20 h without and without metabolic activation

STAIN (for cytogenetic assays): 5% Giemsa solution and BrdUrd (5-bromodeoxyuridine) at 10 µM

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 cells for test substance; at least 25 cells for positive controls

CYTOTOXICITY: visual observations based on confluence of monolayer and floating dead cells

Evaluation criteria:

Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 were analyzed.
The following factors were taken into account in the evaluation of the chromosomal aberrations data: the overall chromosomal aberration frequencies, the percentage of cells with any aberrations, the percentage of cells with more than one aberration, any evidence for increasing amounts of damage with increasing dose.
Chromatid and isochromatid gaps were not considered as they may be due to toxicity.

Statistics:

Fisher's exact test with an adjustment of multiple comparisons

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Range-finding without metabolic activation:
A very unhealthy cell monolayer, -70% reduction in the cell monolayer confluence, floating dead cells, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 25.0 µg/mL. Slight reductions in the number of visible mitotic cells and -15% reduction in the cell monolayer confluence were observed in the cultures dosed with 2.50 and 8.35 µg/mL.
Range-finding with metabolic activation:
An unhealthy cell monolayer, -85% reduction in the cell monolayer confluence, floating dead cells and debris, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 835 µg/mL. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 25.0 and 83.5 µg/mL.

Chromosomal aberrations assay without metabolic activation (Table 1):
In the 10 h assay, no toxicity was observed in any of the test cultures. These cultures were not analyzed for chromosomal aberrations as four dose levels were available for analysis from the 20 h assay. In the 20 h assay, an unhealthy cell monolayer, -70% and -45 % reduction in the cell monolayer confluence, floating dead cells and debris, and a severe reduction in visible mitotic cells were observed at 75.0 and 50.0 µg/mL, respectively. Toxicity was evident on the slides prepared from these cultures by the very sparse numbers of metaphases available for analysis.

Chromosomal aberration assay with metabolic activation (Tables 2 and 3):
In the 10 h assay, slight reductions in the numbers of visible mitotic cells were observed in the cultures dosed at 563 and 751 µg/mL.
In the 20 h assay, severe toxicity was exhibited on the slides prepared from the cultures dosed with 562 and 750 µg/mL by the presence of many dead cells and the sparse numbers of metaphases available for analysis. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 99.7, 187, 281, 375, 562, and 750 µg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 without metabolic activation (results from pooled duplicate cultures)

		Number and type of aberration
		Not computed		Simple	Complex	% cells with aberrations
	Concentration (µg/mL)	Chromatid gap	Chromosome gap
Negative (vehicle)	-	7	1			0.0
Positive (Mitomycin C)	0.04	7		4	7	28.0*
Test article	25.0	15	2			0.0
	37.5	7	3	1	1	0.5
	50.0	8	1		1	0.5
	75.0	19	2	4		0.5

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 2: Chromosome aberrations in CHO cells fixed 10 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

	Concentration (µg/mL)	Number and type of aberration
		Not computed		Simple	Complex	% cells with aberrations
		Chromatid gap	Chromosome gap
Negative (vehicle)	-	2				0.0
Positive (Cyclophosphamide)	25.0	1		8	13	44.0*
Test article	282	7	1			0.0
	375	3			1	0.5
	563	4			1	0.5
	751	3		3		1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 3: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

	Concentration (µg/mL)	Number and type of aberration
		Not computed		Simple	Complex	% cells with aberrations
		Chromatid gap	Chromosome gap
Negative (vehicle)	-	7		1	1	0.0
Positive (Cyclophosphamide)	12.5	1		17	31	80.0*
Test article	281	15	2		1	1.0
	375	16	6	1	1	1.0
	562	3	1	1	1	1.0
	750	10		1	1	1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

MRD-90-843 was found not to increase chromosome aberrations in CHO cells with and without metabolic activation.

Executive summary:

In an in vitro chromosome aberration test, Chinese Hamster Ovary cells were exposed to MRD-90-843 at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation.

Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, MRD-90-843 is not classified according to the criteria of Annex VI to Directive 67/548/EEC and the CLP Regulation (1272/2008).